JP2013535956A - Formation of alcohol esters and in situ product removal during alcohol fermentation. - Google Patents

Abstract

  Ester of product alcohol in a fermentation medium containing a carboxylic acid (eg, fatty acid) and a catalyst (eg, lipase) that can esterify a product alcohol such as butanol with the carboxylic acid to form an alcohol ester Alcohol fermentation processes and compositions comprising the formation of alcohol esters by crystallization. The alcohol ester can be extracted from the fermentation medium and the product alcohol can be recovered from the alcohol ester. The carboxylic acid can also act as an extractant for removing alcohol esters from the fermentation medium.

Description

  This application is based on US Provisional Patent Application No. 61 / 356,290, filed Jun. 18, 2010; US Provisional Patent Application No. 61 / 368,451, filed Jul. 28, 2010; 2010. US Provisional Patent Application 61 / 368,436, filed July 28, US Provisional Patent Application 61 / 368,444, filed July 28, 2010; filed July 28, 2010 US Provisional Patent Application No. 61 / 368,429; US Provisional Patent Application No. 61 / 379,546, filed September 2, 2010; and US Provisional Patent, filed February 7, 2011. No. 61 / 440,034; claims the benefit of US Patent Application No. 13 / 160,766, filed June 15, 2011; the entire contents of which are hereby incorporated by reference. Be used in

  The sequence listing relevant to this application is submitted in electronic form via EFS-Web, which is incorporated herein by reference in its entirety.

  The present invention relates to a fermentation production of alcohols including ethanol and butanol, and a method for improving alcohol fermentation using all related by-products and in situ product removal methods.

  Alcohol has a variety of industrial and scientific uses, such as beverages (ie, ethanol), fuels, reagents, solvents, and disinfectants. For example, butanol is an important industrial chemical and drop having a variety of uses, including as a renewable fuel additive, as a raw chemical in the plastics industry, and as a food grade extractant in the food and fragrance industry It is an alcohol that is a component of drop-in fuel. Therefore, there is a high demand for alcohols such as butanol, as well as efficient and environmentally friendly production methods.

  Alcohol production using microbial fermentation is one such environmentally friendly production method. In the production of butanol, in particular, some microorganisms that produce butanol in high yields also have a low butanol toxicity threshold. Removing butanol from the fermentor as butanol is produced is a means of managing these low butanol toxicity thresholds. Accordingly, there is a continuing need to develop efficient methods and systems for producing butanol in high yield despite the low butanol toxicity threshold of butanol producing microorganisms in fermentation media.

  In situ product removal (ISPR) (also called extractive fermentation) is used to remove butanol (or other fermented alcohol) from the fermentor as it is produced, thereby allowing the microorganisms to produce butanol in high yield. May be possible to generate. One ISPR method for removing fermented alcohol described in the art is liquid-liquid extraction (Patent Document 1). In general, for butanol fermentation, the fermentation medium containing the microorganism is contacted with the organic extractant at a time before the butanol concentration reaches a toxic level. The organic extractant and the fermentation medium form a two-phase mixture. Butanol separates into the organic extractant phase, reducing the concentration of butanol in the aqueous phase containing microorganisms, thereby reducing microbial exposure to inhibitory butanol. Because it is technically and economically feasible, liquid-liquid extraction is a contact between the extractant and the fermentation broth for efficient mass transfer of the product alcohol into the extractant; Requires extraction agent phase separation (during and / or after fermentation); efficient recovery and reuse of extractant; and minimal reduction of extractant partition coefficient over long periods of operation.

  The extractant can be contaminated over time with each reuse, for example, due to the accumulation of lipids present in the biomass supplied to the fermenter as a raw material for hydrolysable starch. As an example, during the conversion of glucose to butanol, a liquefied corn mash that is filled into a fermenter with 30 wt% dry corn solids is subjected to simultaneous saccharification and fermentation (addition of glucoamylase to produce glucose during fermentation) Can result in a fermentation broth containing about 1.2 wt% corn oil produced by saccharification of the liquefied mash. As corn oil lipids dissolve in oleyl alcohol (OA), which acts as an extractant during ISPR, the lipid concentration increases with each OA reuse, and as the lipid concentration in the OA increases with each OA reuse, The partition coefficient for the product alcohol in OA can be reduced.

  Furthermore, the presence of undissolved solids during extractive fermentation can adversely affect the efficiency of alcohol production. For example, the presence of undissolved solids may reduce the mass transfer coefficient inside the fermenter, may interfere with phase separation in the fermentor, and reduce corn oil accumulation from undissolved solids in the extractant. Resulting in a decrease in extraction efficiency over time, increasing solvent loss because the solvent is trapped in solids and eventually removed as soluble cereal-containing dry cereal distillers (DDGS) May slow the detachment of extractant droplets from the fermentation broth and / or reduce fermenter volumetric efficiency.

  Some methods for reducing the reduction in the partition coefficient of the extractant used for extractive fermentation have included wet grinding, fractionation, and solid removal. Wet milling is a costly multi-step process that separates biomass (eg, corn) into its main components (germ, seed coat fiber, starch, and gluten) to gain value from each by-product separately. This process results in a purified starch stream; however, this process is costly and involves separating the biomass into its non-starch components that are not required for fermentation alcohol production. Fractionation removes fibers and germs that contain most of the lipids present in ground whole corn, resulting in fractionated corn with a higher starch (endosperm) content. Dry fractionation is less expensive than wet milling because it does not separate the germ from the fiber. However, the fraction does not remove the entire fiber or germ and does not completely remove the solid. In addition, some starch is lost during fractionation. While wet grinding of corn is even more expensive than dry fractionation, dry fractioning is more expensive than dry grinding of unfractionated corn. For example, as described in co-pending and co-pending US Provisional Patent Application No. 61 / 356,290 filed June 18, 2010, prior to use in fermentation. When the solid containing the germ containing lipid is removed from the liquefied mash, the undissolved solid can be substantially removed. However, it would be advantageous to be able to suppress the reduction in extractant partition coefficient caused by lipid contamination without substantially fractionating or even removing undissolved solids. Conversion of lipids present in the liquefied mash to extractants that can be used for ISPR is, for example, co-pending US Provisional Patent Application No. 61 / 368,436 owned by the same applicant and In another way to reduce the amount of lipid fed to the fermentor, as described in US Provisional Patent Application No. 61 / 368,444 (both filed on July 28, 2010). is there.

US Patent Application Publication No. 2009/030305370

  As a means to reduce the toxic effects of product alcohols such as butanol on microorganisms, it is not necessary to separate the fermentation medium and ISPR extractant, and it is also possible to suppress a decrease in the partition coefficient of the fermentation product extractant There is a continuing need for new extractive fermentation methods.

  The conversion of alcohols such as butanol produced from microorganisms in the fermentation medium to substances that are less toxic to microorganisms can increase the production of alcohols such as butanol for a given fermentor volume. Alcohol esters can be formed by contacting the alcohol in the fermentation medium with a carboxylic acid (eg, a fatty acid) and a catalyst capable of esterifying the alcohol with the carboxylic acid. In addition, the carboxylic acid can act as an ISPR extractant from which the alcohol ester separates. Carboxylic acid can be supplied to the fermentor and / or derived from biomass that supplies fermentable carbon feed to the fermentor. Lipids present in the raw material can be catalyzed to carboxylic acid, and the same catalyst (eg, enzyme) can esterify the carboxylic acid with an alcohol (eg, butanol); Direct transesterification can produce alcohol esters. The catalyst can be supplied to the raw material before fermentation, or can be supplied to the fermentor before or simultaneously with the supply of raw material. When the catalyst is fed to the fermentor, the alcohol ester is obtained by hydrolyzing the lipid to a carboxylic acid and simultaneously esterifying the carboxylic acid with butanol present in the fermentor; Direct transesterification with butanol can produce the alcohol ester. Carboxylic acids and / or natural oils not derived from the above raw materials can also be fed directly to the fermentor, where the natural oils are hydrolyzed to carboxylic acids. Carboxylic acids and / or natural oils not derived from the above raw materials can be fed to the fermenter in sufficient quantities so that a two-phase mixture comprising an organic phase and an aqueous phase is formed. Thus, in certain embodiments, any carboxylic acid that is not esterified with an alcohol can serve as an ISPR extractant or part thereof. The extractant containing the alcohol ester can be separated from the fermentation medium and the alcohol can be recovered from the extractant. The extractant can be reused in the fermenter. Thus, for example, in the case of butanol production, conversion of butanol to an ester reduces the free butanol concentration in the fermentation medium and protects the microorganisms from the toxic effects of increasing butanol concentrations. In addition, separating the lipids in the unfractionated cereal, since the lipids can be catalyzed to carboxylic acid, thereby reducing the rate of increase of lipids in the ISPR extractant. And can be used as a raw material.

  The present invention is a method for producing a butyl ester, wherein butanol produced in a fermentation process is esterified with at least one carboxylic acid and butanol to form a butyl ester of a carboxylic acid. Contacting with at least one catalyst capable of; comprising a step wherein the carboxylic acid in the fermentation process is present in a concentration sufficient to produce a biphasic mixture. In one embodiment, butanol and butyl ester are produced simultaneously or sequentially. In one embodiment, the fermentation process feedstock comprises one or more fermentable sugars. In another embodiment, the raw material for the fermentation process is corn kernels, corn cobs, corn hulls, crop residues such as corn stover, grass, wheat, rye, wheat straw, barley, barley straw, Grass, rice straw, switchgrass, waste paper, sugarcane pomace, sorghum, sugarcane, soybean, ingredients obtained from grinding grains, cellulosic materials, lignocellulosic materials, trees, branches, roots, leaves, wood chips, sawdust One or more fermentable sugars derived from shrubs and shrubs, vegetables, fruits, flowers, animal manure, and mixtures thereof. In one embodiment, the method further comprises providing a natural oil and converting at least a portion of the natural oil to a carboxylic acid by contacting the oil with one or more enzymes. In one embodiment, the carboxylic acid includes a fatty acid. In another embodiment, the carboxylic acid contains 12-22 carbons. In one embodiment, the carboxylic acid is a mixture of carboxylic acids. In another embodiment, the butyl ester of a carboxylic acid is a butyl ester of a fatty acid. In one embodiment, the catalyst is an enzyme capable of esterifying a carboxylic acid with butanol to form a butyl ester of the carboxylic acid. In another embodiment, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.

  The present invention is a method for producing butanol and butyl esters from raw materials: (a) providing the raw material; (b) liquefying the raw material to produce liquefied biomass containing oligosaccharides And (c) separating the raw slurry to produce a water stream comprising oligosaccharides, an oil stream, and a product comprising solids; and (d) adding the water stream to a fermentor comprising a fermentation broth; (E) saccharifying the oligosaccharides in the water stream; (f) fermenting the oligosaccharide saccharification products present in the water stream to produce butanol and simultaneously converting the butanol into at least one carboxylic acid; And contacting with at least one catalyst capable of esterifying the carboxylic acid with butanol to form a butyl ester of the carboxylic acid, wherein the carboxylic acid is a two-phase mixture To generate to and a step present in sufficient concentration; optionally, step (e) and (f) is also relates to a process to be performed simultaneously. In one embodiment, the method further comprises obtaining oil from the oil stream and converting at least a portion of the oil to carboxylic acid. In one embodiment, the raw slurry is decanter bowl centrifuge, tricanter centrifuge, disc stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, pressure filtration, sieving filtration, sieving. Separation, grating, porous grid, flotation, hydroclone, filter press, screw press, gravity settling, vortex separator, or combinations thereof. In another embodiment, the carboxylic acid includes a fatty acid. In one embodiment, the carboxylic acid contains 12-22 carbons. In one embodiment, the method further comprises adding oil to the fermentor prior to converting at least a portion of the oil to carboxylic acid. In one embodiment, the method further comprises adding additional carboxylic acid to the fermentor. In one embodiment, after the step of adding additional carboxylic acid, the oil is converted to carboxylic acid. In another embodiment, the carboxylic acid is corn oil fatty acid, soybean oil fatty acid, or a mixture of corn oil fatty acid and soybean oil fatty acid. In one embodiment, the oil obtained from the oil stream comprises glycerides and the one or more catalysts hydrolyze the glycerides into fatty acids. In another embodiment, the butyl ester of a carboxylic acid is a butyl ester of a fatty acid. In one embodiment, the catalyst is an enzyme capable of esterifying a carboxylic acid with butanol to form a butyl ester of the carboxylic acid. In one embodiment, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. In one embodiment, the method further comprises washing the solid with a solvent. In one embodiment, the solvent is selected from petroleum distillates such as hexane, isobutanol, isohexane, ethanol, petroleum ether, or mixtures thereof. In another embodiment, the solid is processed to form an animal feed product. In one embodiment, the solid is processed to form an animal feed product. In one embodiment, the animal feed product comprises one or more crude protein, crude fat, triglyceride, fatty acid, fatty acid isobutyl ester, lysine, neutral detergent fiber (NDF), and acidic detergent fiber (ADF). In another embodiment, the animal feed product further comprises one or more vitamins, minerals, flavors, or colorants. In one embodiment, the animal feed product comprises 20-35% crude protein, 1-20% crude fat, 0-5% triglyceride, 4-10% fatty acid, and 2-6% by weight. Of fatty acid isobutyl ester. In one embodiment, the step of separating the solid from the raw slurry increases the efficiency of butanol production by increasing the liquid-liquid mass transfer coefficient of butanol from the fermentation broth to the extractant; increases the extraction efficiency of butanol by the extractant. Increase the efficiency of butanol production; increase the efficiency of butanol production by increasing the rate of phase separation between the fermentation broth and the extractant; butanol production by increasing the recovery and reuse of the extractant Increase the efficiency of butanol production; or by reducing the extractant flow rate.

  The present invention is a method for producing butanol and butyl esters from raw materials: (a) providing the raw material; (b) liquefying the raw material to produce liquefied biomass containing oligosaccharides And (c) separating the raw slurry to produce a stream comprising oligosaccharides and oil and solids; (d) adding the stream to a fermentor comprising fermentation broth; and (e) a stream. Saccharifying the oligosaccharides of: (f) fermenting the oligosaccharide saccharification product present in the stream to produce butanol and simultaneously converting the butanol into at least one carboxylic acid and butanol together with the carboxylic acid. Contacting with at least one catalyst capable of esterification to form a butyl ester of a carboxylic acid, wherein the carboxylic acid forms a biphasic mixture. And a step present in sufficient concentration; optionally, step (e) and (f) is also relates to a process to be performed simultaneously. In one embodiment, the method further comprises converting at least a portion of the oil to carboxylic acid. In one embodiment, the raw slurry is decanter bowl centrifuge, tricanter centrifuge, disc stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, pressure filtration, sieving filtration, sieving separation, lattice Or by a porous grid, flotation method, hydroclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof. In another embodiment, the carboxylic acid includes a fatty acid. In one embodiment, the carboxylic acid contains 12-22 carbons. In one embodiment, the method further comprises adding oil to the fermentor. In one embodiment, the method further comprises adding additional carboxylic acid to the fermentor. In one embodiment, after the step of adding additional carboxylic acid, the oil is converted to carboxylic acid. In one embodiment, the carboxylic acid is corn oil fatty acid, soybean oil fatty acid, or a mixture of corn oil fatty acid and soybean oil fatty acid. In one embodiment, the oil comprises glycerides and the one or more catalysts hydrolyze the glycerides into fatty acids. In one embodiment, the butyl ester of a carboxylic acid is a butyl ester of a fatty acid. In one embodiment, the catalyst is an enzyme capable of esterifying a carboxylic acid with butanol to form a butyl ester of the carboxylic acid. In one embodiment, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. In one embodiment, the method further comprises washing the solid with a solvent. In one embodiment, the solvent is selected from petroleum distillates such as hexane, isobutanol, isohexane, ethanol, petroleum ether, or mixtures thereof. In one embodiment, the solid is processed to form an animal feed product. In one embodiment, the solid is processed to form an animal feed product. In certain embodiments, the animal feed product comprises one or more crude proteins, crude fats, triglycerides, fatty acids, fatty acid isobutyl esters, lysine, neutral detergent fibers (NDF), and acidic detergent fibers (ADF). In certain embodiments, the animal feed product further comprises one or more vitamins, minerals, flavors, or colorants. In certain embodiments, the animal feed product comprises 20-35% crude protein, 1-20% crude fat, 0-5% triglyceride, 4-10% fatty acid, and 2-6% by weight. Of fatty acid isobutyl ester. In some embodiments, the step of separating solids from the raw slurry increases the efficiency of butanol production by increasing the liquid-liquid mass transfer coefficient of butanol from the fermentation broth to the extractant; increases the extraction efficiency of butanol by the extractant. Increase the efficiency of butanol production; increase the efficiency of butanol production by increasing the rate of phase separation between the fermentation broth and the extractant; butanol production by increasing the recovery and reuse of the extractant Increase the efficiency of butanol production; or by reducing the flow rate of the extractant.

  The present invention is a method for producing butanol, wherein (a) butanol produced in a fermentation process is esterified with at least one carboxylic acid, and carboxylic acid together with butanol to produce a butyl ester of carboxylic acid. Contacting with at least one catalyst that can be formed; the carboxylic acid in the fermentation process is present in a concentration sufficient to produce a biphasic mixture comprising an aqueous phase and a butyl ester-containing organic phase It also relates to a process comprising: (b) separating the butyl ester-containing organic phase from the aqueous phase; and (c) recovering butanol from the butyl ester. In certain embodiments, recovering butanol from the butyl ester comprises hydrolyzing the ester to a carboxylic acid and butanol. In certain embodiments, the butyl ester is hydrolyzed in the presence of a hydrolysis catalyst. In certain embodiments, the butyl ester is hydrolyzed in the presence of water, and the hydrolysis catalyst comprises an acid catalyst, an organic acid, an inorganic acid, a water-soluble acid, or a water-insoluble acid. In certain embodiments, the hydrolysis catalyst comprises an enzyme capable of hydrolyzing butyl esters to form carboxylic acids and butanols. In certain embodiments, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. In certain embodiments, the enzyme reaction conditions favor enzyme hydrolysis over esterification. In certain embodiments, the enzymatic reaction conditions include a co-solvent. In certain embodiments, fatty acid butyl ester, fatty acid, isobutanol, and water are soluble in a co-solvent and free fatty acids do not react with the co-solvent. In certain embodiments, the co-solvent is selected from acetone, tert-butanol, 2-Me-2-butanol, 2-Me-2-pentanol, and 3-Me-3-pentanol. In certain embodiments, the enzymatic reaction conditions include final product removal. In certain embodiments, the final product is isobutanol or a fatty acid. In certain embodiments, isobutanol is removed by vacuum distillation, pervaporation, permselective filtration, or gas sparging. In certain embodiments, the fatty acids are removed by precipitation, permselective filtration, or electrophoresis. In certain embodiments, the hydrolysis reaction occurs in a reaction vessel. In certain embodiments, recovering butanol from the butyl ester comprises transesterifying the butyl ester into butanol and a fatty acid alkyl ester or acylglyceride. In certain embodiments, the fatty acid alkyl ester comprises a fatty acid methyl ester, a fatty acid ethyl ester, or a fatty acid propyl ester. In certain embodiments, the method further comprises providing a natural oil and converting at least a portion of the natural oil to a carboxylic acid by contacting the oil with one or more enzymes. In certain embodiments, the enzyme is an enzyme capable of hydrolyzing or transesterifying a butyl ester to form butanol. In certain embodiments, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. In certain embodiments, the carboxylic acid includes a fatty acid. In certain embodiments, the carboxylic acid has a carbon chain length in the range of 12-22 carbons. In certain embodiments, at least about 10% of butanol is recovered from the butyl ester. In certain embodiments, at least about 50% of butanol is recovered from the butyl ester. In certain embodiments, at least about 90% of butanol is recovered from the butyl ester. In certain embodiments, the carboxylic acid is recovered from the butyl ester. In certain embodiments, the method further comprises removing butanol from the fermentor as an extractant stream; and adding the extractant stream to two or more distillation columns. In certain embodiments, the distillation column is a super-atmospheric distillation column with a steam heated reboiler. In certain embodiments, the method further comprises recovering water and solvent from the distillation column; recycling water and solvent. In certain embodiments, the method further comprises recovering heat from the distillation process; and reusing heat to evaporate the water.

  The present invention is a method for producing butanol from a raw material, comprising: (a) a step of providing the raw material; (b) a step of liquefying the raw material to produce a raw material slurry; and (c) a raw material slurry. Separating to produce a product comprising a water stream, an oil stream, and a solid; (d) adding the water stream to a fermentor containing the fermentation broth; (e) saccharifying the water stream; ) Fermenting the saccharified water stream to produce butanol, at the same time, butanol can be esterified with at least one carboxylic acid and with butanol to form a butyl ester of carboxylic acid. Contacting with a seed catalyst, wherein the carboxylic acid is present at a concentration sufficient to form a biphasic mixture; (g) separating the butyl ester-containing organic phase from the aqueous phase; Extent and; (h) and the step of recovering the butanol from butyl ester; optionally, step (e) and (f) is also relates to a process to be performed simultaneously. In certain embodiments, the method further comprises obtaining oil from the oil stream and converting at least a portion of the oil to carboxylic acid. In certain embodiments, the raw slurry is separated by centrifugation, filtration, or decantation. In certain embodiments, the carboxylic acid includes a fatty acid. In certain embodiments, the carboxylic acid has a carbon chain length in the range of 12-22 carbons. In certain embodiments, the method further comprises adding oil to the fermentor prior to converting at least a portion of the oil to carboxylic acid. In certain embodiments, the method further comprises adding additional carboxylic acid to the fermentor. In certain embodiments, after the step of adding additional carboxylic acid, the oil is converted to carboxylic acid. In certain embodiments, the carboxylic acid is corn oil fatty acid, soybean oil fatty acid, or a mixture of corn oil fatty acid and soybean oil fatty acid. In certain embodiments, the oil obtained from the oil stream comprises glycerides and the one or more catalysts hydrolyze the glycerides into fatty acids. In certain embodiments, the butyl ester of a carboxylic acid is a butyl ester of a fatty acid. In certain embodiments, the catalyst is an enzyme capable of esterifying a carboxylic acid with butanol to form a butyl ester of the carboxylic acid. In certain embodiments, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. In certain embodiments, the solid is processed to form an animal feed product. In certain embodiments, recovering butanol from the butyl ester comprises hydrolyzing the ester to a carboxylic acid and butanol. In certain embodiments, the butyl ester is hydrolyzed in the presence of a hydrolysis catalyst. In certain embodiments, the butyl ester is hydrolyzed in the presence of water, and the hydrolysis catalyst comprises an acid catalyst, an organic acid, an inorganic acid, a water-soluble acid, or a water-insoluble acid. In certain embodiments, the hydrolysis catalyst comprises an enzyme capable of hydrolyzing butyl esters to form carboxylic acids and butanols. In certain embodiments, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. In certain embodiments, the hydrolysis reaction occurs in a reaction vessel. In certain embodiments, recovering butanol from the butyl ester comprises transesterifying the butyl ester into butanol and a fatty acid alkyl ester or acylglyceride. In certain embodiments, the fatty acid alkyl ester comprises a fatty acid methyl ester, a fatty acid ethyl ester, or a fatty acid propyl ester. In certain embodiments, the method further comprises providing a natural oil and converting at least a portion of the natural oil to a carboxylic acid by contacting the oil with one or more enzymes. In certain embodiments, the enzyme is an enzyme capable of hydrolyzing or transesterifying a butyl ester to form butanol. In certain embodiments, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.

  In another embodiment, the fermentation method comprises providing an aqueous feed stream obtained from biomass, the aqueous feed stream comprising water, a fermentable carbon source derived from biomass, and oil; Contacting the stream with a catalyst, whereby at least a portion of the oil is hydrolyzed to free fatty acids to form a feed stream treated with a catalyst comprising free fatty acids and the catalyst; Contacting the raw material stream with the fermentation broth in the fermentor; fermenting the fermentable carbon source in the fermentor to produce product alcohol; and free fatty acid and product alcohol in the fermenter Contacting the product alcohol with free fatty acid and catalyst during fermentation to catalyze esterification to produce an alcohol ester of a fatty acid. In certain embodiments, the steps of contacting the feed stream with the catalyst and fermentation broth and contacting the fermentation and product alcohol with the free fatty acid and the catalyst can occur simultaneously. In certain embodiments, the product alcohol is butanol and the alcohol ester of a fatty acid is a butyl ester of a fatty acid.

  The present invention is a method for removing alcohol from a fermentation medium during fermentation, comprising: providing a fermentation medium comprising microorganisms that produce alcohol in the fermentation medium; , Carboxylic acid, and esterifying the alcohol with the carboxylic acid to contact with a catalyst capable of forming the alcohol ester. In certain embodiments, the alcohol produced by the microorganism is butanol and the alcohol ester is a butyl ester. In certain embodiments, the fermentation medium is contacted with a carboxylic acid that is substantially insoluble in the fermentation medium and a catalyst capable of esterifying the alcohol with the carboxylic acid to form an alcohol ester.

  The present invention is a method for producing an alcohol ester of a fatty acid during a fermentation process comprising: providing a fermentation medium comprising alcohol, a fermentable carbon source, and free fatty acids; Contacting the free fatty acid with one or more enzymes capable of being esterified, whereby the free fatty acid is esterified with an alcohol to form an alcohol ester of the fatty acid. In certain embodiments, the fermentable carbon source is derived from biomass. In certain embodiments, the microorganism in the fermentation medium is a recombinant microorganism. In certain embodiments, the alcohol is butanol and the alcohol ester of a fatty acid is a butyl ester of a fatty acid.

  In another embodiment, the method for producing product alcohol comprises providing a biomass feedstock comprising water, a fermentable carbon source, and oil, wherein the oil comprises an acylglyceride; Liquefying to produce liquefied biomass containing oligosaccharides; one or more enzymes capable of converting at least a portion of acylglycerides into free fatty acids from the biomass feedstock or liquefied biomass; A step in which the free fatty acid forms an extractant, thereby allowing the one or more enzymes to esterify the free fatty acid along with the product alcohol into an alcohol ester of the fatty acid. Contacting the liquefied biomass with a saccharifying enzyme capable of converting oligosaccharides into fermentable sugars; Contacting the liquefied biomass with a recombinant microorganism capable of converting fermentable sugars to product alcohol, thereby producing a fermentation product comprising the product alcohol; free fatty acids and product alcohol Contacting the product alcohol with a free fatty acid and one or more enzymes so as to catalyze esterification of the fatty acid to produce an alcohol ester of the fatty acid; and contacting the fermentation product with an extractant. obtain. In an embodiment, contact with the extractant forms a two-phase mixture comprising an aqueous phase and an extractant phase, and the fatty acid alcohol ester separates into the extractant phase to form an ester-containing extractant phase. In certain embodiments, the product alcohol is butanol and the alcohol ester of a fatty acid is a butyl ester of a fatty acid.

  In another embodiment, the method for producing product alcohol comprises providing a biomass feedstock comprising water, a fermentable carbon source, and oil, wherein the oil comprises an acylglyceride; Liquefying to produce a liquefied biomass comprising oligosaccharides; and a composition comprising one or more enzymes capable of converting the liquefied biomass to at least a portion of acylglycerides into free fatty acids. Contacting with one or more enzymes, wherein one or more enzymes can esterify free fatty acids along with product alcohols into alcohol esters of fatty acids; liquefied biomass, fermenting oligosaccharides Contacting with a saccharifying enzyme that can be converted into a sucrose; and, when fermenting the saccharified biomass, fermentable saccharide is a product alcohol Contacting a recombinant microorganism capable of being converted into a fermentation medium comprising a product alcohol; and contacting the fermentation medium with a carboxylic acid extractant during fermentation comprising: The fermentation medium may comprise a step including one or more enzymes capable of esterifying free fatty acids with the product alcohol to form alcohol esters of fatty acids. In a further embodiment of the method, the fermentation medium is contacted with a carboxylic acid that is substantially insoluble in the fermentation medium and a catalyst capable of esterifying the alcohol with the carboxylic acid to form an alcohol ester. In other embodiments of the method, the alcohol produced by the microorganism is butanol and the alcohol ester is a butyl ester.

  The present invention is a method for producing a product alcohol from a raw material: liquefying starch or fermentable carbon source in the raw material to produce a slurry having oligosaccharides; centrifuging the raw slurry (I) producing an aqueous layer comprising an oligosaccharide, (ii) an oil layer, and (iii) producing a centrifuged product comprising a solid; supplying the aqueous layer to a fermentor comprising a fermentation broth; Fermenting the aqueous layer to produce product alcohol. The product alcohol is then contacted with a carboxylic acid and a catalyst, whereby the carboxylic acid is esterified with the product alcohol to form an alcohol ester. In certain embodiments, the oil is a plant derived oil. In other embodiments, the product alcohol is butanol and the alcohol ester of the carboxylic acid is a butyl ester of a fatty acid.

  In certain embodiments, a method for producing product alcohol comprises a fractionated biomass feedstock comprising water, starch, and / or a fermentable carbon source, and a small residual amount of oil remaining after biomass fractionation. Providing residual oil containing acylglycerides; liquefying the fractionated biomass feedstock to produce liquefied fractionated biomass containing oligosaccharides; Contacting the fractionated biomass with a composition comprising one or more enzymes capable of converting at least some of the residual acylglycerides into free fatty acids, wherein the one or more enzymes are A process that can esterify free fatty acids with the product alcohol to form alcohol esters of fatty acids; and liquefied, fractionated bio Contacting the sugar with a saccharifying enzyme capable of converting oligosaccharides into fermentable sugars; and converting the saccharified biomass into fermentable sugars during fermentation. Contacting a recombinant microorganism, thereby producing a fermentation medium containing a product alcohol; contacting the fermentation medium with a carboxylic acid extractant during fermentation, wherein the fermentation medium is a product alcohol And a process comprising one or more enzymes capable of esterifying free fatty acids to form alcohol esters of fatty acids. In a further embodiment of the method, the fermentation medium is contacted with a carboxylic acid in the fermentation medium and a catalyst capable of esterifying an alcohol with the carboxylic acid to form an alcohol ester. In a further embodiment, the carboxylic acid can be substantially insoluble in the fermentation medium. In other embodiments of the method, the alcohol produced by the microorganism is butanol and the alcohol ester is a butyl ester.

  The present invention relates to a mash formed from biomass and containing water and fermentable sugar; free fatty acids along with alcohol can be esterified to fatty acid alkyl esters, and optionally acylglycerides are hydrolyzed to free fatty acids. Also provided is a composition comprising a decomposable catalyst; an alcohol; a free fatty acid; and a fatty acid alcohol ester formed in situ from the esterification of the free fatty acid by the catalyst with the alcohol. In certain embodiments, the alcohol is butanol and the fatty acid alcohol ester is a fatty acid butyl ester.

  The present invention also provides a fermentation broth comprising a recombinant microorganism capable of producing alcohol; a fermentable carbon source; and a fatty acid alcohol ester, wherein the fatty acid alcohol ester is produced during fermentation. To do. In certain embodiments, the recombinant microorganism is capable of producing butanol. In certain embodiments, the fatty acid alcohol ester is a fatty acid butyl ester. In certain embodiments, the fermentable carbon source includes sugar. In certain embodiments, the fermentable carbon source includes methane, the recombinant microorganism is capable of producing methanol, and the fatty acid alcohol ester is a fatty acid methyl ester.

  Recombinant yeast cells useful for the production of product alcohol are also provided herein. In embodiments, the recombinant host cells disclosed herein can be any bacterial, yeast or fungal host useful for genetic recombination and recombinant gene expression. In other embodiments, the recombinant host cell is a Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Enterococcus, Enterococcus Klebsiella), Paenibacillus genus (Paeniba) illus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Kluyveromyces, It can be a member of the genus Pichia, Candida, Hansenula, Issatchenkia, or Saccharomyces. In other embodiments, the host cell may be Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces cerevisiae Kleberomyces cerevisiae, , Kluyveromyces marxianus, Candida glabrata, Candida albicans, Pichia stipitiria Yiparipo ipolytica), Escherichia coli (E.coli), or L. Plantarum (L. plantarum). In yet other embodiments, the host cell is a yeast host cell. In certain embodiments, the host cell is a member of the genus Saccharomyces. In certain embodiments, the host cell is Kluyveromyces lactis, Candida glabrata or Schizosaccharomyces pombe. In certain embodiments, the host cell is Saccharomyces cerevisiae. S. S. cerevisiae yeast is known in the art and includes the American Type Culture Collection (Rockville, MD), the Central BureaureVort Stimures, CBS Available from a variety of sources including, but not limited to, Ferm Solutions, North American Bioproducts, Martrex, and Lalland. S. Examples of S. cerevisiae include, but are not limited to, BY4741, CEN. PK 113-7D, Ethanol Red® yeast, Gert Prestige Turbo yeast, Ferm Pro ™ yeast, Bio-Ferm® XR yeast, Gert Strand Distillers yeast, FerMax ™ Grex yeast, FerMax (Trademark) Gold yeast, Thermosacc (registered trademark) yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.

  A method for producing isobutanol comprising the steps of providing a recombinant host cell comprising an isobutanol biosynthetic pathway, the enzyme or isobutyraldehyde catalyzing the substrate-product conversion of α-ketoisovaleric acid to isobutyraldehyde At least one of the enzymes that catalyze the substrate-product conversion from to isobutanol is encoded by a heterologous polynucleotide integrated into the chromosome; contacting the recombinant host cell with a fermentable carbon source, Forming a fermentation broth under conditions where butanol is produced is also provided. In certain embodiments, the method further comprises adding an extractant to form a biphasic mixture. In other embodiments, the extractant comprises a carboxylic acid. In certain embodiments, the extractant includes a fatty acid. In other embodiments, the method further comprises adding an esterifying enzyme capable of catalyzing the esterification of isobutanol with a carboxylic acid.

  Providing a fermentation medium comprising a product alcohol, water, a fermentable carbon source, and a microorganism that produces the product alcohol; during fermentation, the fermentation medium is contacted with an extractant to provide an aqueous phase and an organic phase. Also provided herein is a method comprising forming a biphasic mixture comprising; contacting the fermentation medium with a carboxylic acid and an enzyme capable of esterifying the carboxylic acid with the product alcohol. In certain embodiments, the extractant comprises a carboxylic acid. In certain embodiments, carboxylic acids are produced from biomass feedstocks by oil hydrolysis. In certain embodiments, the fermentable carbon and carboxylic acid are derived from the same biomass source. In certain embodiments, the carboxylic acid comprises a saturated, monounsaturated, polyunsaturated carboxylic acid having 12 to 22 carbons, and mixtures thereof. In certain embodiments, the steps of contacting the fermentation medium with the extractant and the carboxylic acid and enzyme are performed simultaneously. In certain embodiments, the microorganism is a genetically modified microorganism (eg, a host cell such as a recombinant microorganism or a recombinant yeast cell).

  Also provided herein are compositions comprising a PNY1504, PNY2205, or recombinant host cell; an extractant; and optionally an esterifying enzyme. Also provided herein are compositions comprising PNY1504, PNY2205, or a recombinant host cell and a butyl ester.

  Further provided herein is the use of PNY1504, PNY2205, or other recombinant yeast cells and compositions comprising recombinant yeast cells for the production of isobutanol.

  The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate the invention and, together with the description, explain the principles of the invention and allow those skilled in the relevant art to understand the invention. And make it available for use.

1 schematically illustrates an exemplary method and system of the present invention in which a catalyst for alcohol esterification is fed to a fermentor with a carboxylic acid and / or natural oil. 1 schematically illustrates an exemplary method and system of the present invention in which natural oil is converted to a carboxylic acid using a catalyst and the carboxylic acid and catalyst are fed to a fermentor. Fig. 4 schematically illustrates an exemplary method and system of the present invention in which liquefied biomass is contacted with a catalyst for lipid hydrolysis prior to fermentation. FIG. 4 schematically illustrates an exemplary method and system of the present invention in which liquefied and saccharified biomass is contacted with a catalyst for lipid hydrolysis prior to fermentation. A predetermined amount of lipids and undissolved solids are removed from the liquefied biomass prior to fermentation, the removed lipids are converted to carboxylic acid using a catalyst, and the carboxylic acid and catalyst are fed to the fermentor. 1 schematically illustrates an exemplary method and system of the present invention. 2 shows the aqueous and solvent phase concentrations of isobutanol produced by fermentation with sucrose as a carbon source. The water phase titer (panel A) is reported in units of g / L and the solvent phase species (isobutanol, panel B and isobutanol as FABE, panel C. Panel D is the total isobutanol in the solvent phase. Are reported in units of weight percent. The effective titer (g / L) of isobutanol over time is shown. The effective titer in this example was calculated as described in the text based on the initial volume of aqueous fermentation broth after inoculation. Shows saccharide consumption reported in glucose equivalents over time.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions, will control. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety for all purposes.

Unless otherwise specified, when the following abbreviations are used herein, they have the following meanings:
ADH Alcohol dehydrogenase ALS Acetolactate synthase AQ Aqueous fraction BuO-COFA Butyl ester of corn oil fatty acid CALB Candida antarctica lipase B
COFA Corn oil fatty acid DDGS Soluble-containing dry cereal distiller DG Diglyceride DHAD Dihydroxy acid dehydratase EOR Execution end EtOH Ethanol EtO-COFA Ethyl ester FABE Fatty acid butyl ester FAEE Fatty acid ethyl ester FAME Fatty acid methyl ester FFA Free fatty acid FOA Fluoro Orotic acid HADH Horse liver alcohol dehydrogenase IBA Isobutanol i-BuOH Isobutanol i-BuO-COFA Isobutyl ester of corn oil fatty acid i-BuO-oleate Oleic acid iso-butyl i-PrOH Isopropanol i-PrO-COFA Isopropyl of corn oil fatty acid Ester ISPR In-situ product removal KARI Ketolate reduct Somerase KivD Ketoisovalerate decarboxylase MAG Monoacylglyceride MeBOH 2-Methyl-1-butanol MeBO-COFA 2-methyl-1-butyl ester of corn oil fatty acid MeOH Methanol MeO-COFA Methyl ester of corn oil fatty acid MG Monoglyceride n-BuOH n-butanol OA oleyl alcohol ORG organic fraction PenOH 1-pentanol PenO-COFA 1-pentyl ester of corn oil fatty acid PrOH 1-propanol PrO-COFA 1-propyl ester of corn oil fatty acid SOFA soybean oil fatty acid SSF simultaneous saccharification and fermentation t -BuOH tert-butyl alcohol TG triglyceride 3M3P 3-Me-3-pentanol

  In order to further define the invention, the following terms and definitions are provided herein.

  As used herein, “comprises”, “comprising”, “includes”, “including”, “has”, “having” , “Contains”, or “containing”, or any other conjugation thereof, includes the integer or group of integers described, but any other integer or It will be understood that it means not to exclude groups of integers. For example, a composition, mixture, process, method, article, or device that includes an enumeration of elements is not necessarily limited to only those elements, and is not explicitly listed or such a composition, It may include mixtures, processes, methods, articles, or other elements specific to the device. Further, unless expressly stated to the contrary, “or” refers to an inclusive “or” rather than an exclusive “or”. For example, condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) And B is true (or exists), both A and B are true (or exist).

  Also, the indefinite articles “a” and “an” preceding an element or component of the invention are not intended to be limited regarding the number of instances, ie, the number of occurrences of the element or component. Thus, “a” or “an” should be read to include one or at least one, and the singular form of an element or component is plural unless the number is explicitly meant to be singular. Including.

  The terms “invention” or “invention” as used herein are non-limiting terms and are not intended to refer to any one embodiment of a particular invention, Includes all possible embodiments described.

  As used herein, the term “about”, which modifies the amount of the inventive component or reactant used, is typical of, for example, used to make concentrates or solutions in the real world. Due to physical measurements and liquid processing procedures; due to inadvertent errors in these procedures; the quantity of components that can occur due to differences in the manufacture, source, or purity of the components used to make the composition or perform the method Refers to fluctuations. The term “about” encompasses different amounts due to different equilibrium conditions for compositions obtained from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In one embodiment, the term “about” means within 10% of the reported numerical value, or within 5% of the reported numerical value.

  “Biomass” as used herein refers to corn, sugarcane, wheat, cellulosic or lignocellulosic materials and cellulose, hemicellulose, lignin, starch, oligosaccharides, disaccharides and / or monosaccharides, and their It refers to natural products containing hydrolyzable polysaccharides that provide any saccharides derived from natural resources such as materials including mixtures and fermentable sugars including starch. Biomass can also include additional components such as proteins and / or lipids. The biomass may be derived from one source, or the biomass may include a mixture derived from two or more sources. For example, the biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, paper sludge, garden waste, sugarcane waste, wood waste and forestry waste. Examples of biomass include, but are not limited to, corn kernels, corn cobs, corn hulls, crop residues such as corn stover, grass, wheat, rye, wheat straw, barley, barley straw , Grass, rice straw, switchgrass, waste paper, sugarcane pomace, sorghum, sugarcane, soy, ingredients obtained from grinding grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and shrubs, vegetables, fruits , Flowers, animal manure, and mixtures thereof. For example, mash, juice, molasses, or hydrolyzate is formed from the biomass by any process known in the art for processing biomass for fermentation, such as by grinding, processing, and / or liquefaction. And contains fermentable sugars and may contain water. For example, cellulosic and / or lignocellulosic biomass can be treated to obtain a hydrolyzate containing fermentable sugars by any method known to those skilled in the art. Particularly useful are the low ammonia pretreatments disclosed in US 2007/0031918 A1, which is incorporated herein by reference. Enzymatic saccharification of cellulosic and / or lignocellulosic biomass typically involves the degradation of cellulose and hemicellulose to produce hydrolysates containing sugars including glucose, xylose, and arabinose. Is used. (Saccharifying enzymes suitable for cellulosic and / or lignocellulosic biomass are reviewed in Lynd, et al. (Microbiol. Mol. Biol. Rev. 66: 506-577, 2002).

  The mash, juice, molasses, or hydrolyzate can include the raw material 12 and raw slurry 16 described herein. The aqueous feed stream can be derived or formed from the biomass by any process known in the art for processing biomass for fermentation, such as by grinding, processing, and / or liquefaction, such as a fermentable carbon substrate (e.g., Sugar) and water. The aqueous feed stream may include feed 12 and feed slurry 16 as described herein.

  “Biomass yield” as used herein refers to grams of biomass produced (ie, cell biomass production) per gram of carbon substrate produced.

  As used herein, “raw material” is a raw material in a fermentation process that contains a fermentable carbon source with or without undissolved solids, and where applicable, the fermentable carbon source is starch. Means a raw material that contains a fermentable carbon source either before or after it has been released from or obtained from the decomposition of complex saccharides by further processing, such as by liquefaction, saccharification, or other processes. The raw material includes or is derived from biomass. Suitable raw materials include, but are not limited to, rye, wheat, corn, corn mash, sugar cane, sugar cane mash, barley, cellulosic material, lignocellulosic material, or mixtures thereof. When referring to “feed oil”, it will be understood that this term encompasses oil produced from a given feed.

As used herein, a “fermentation medium” refers to water, sugars, dissolved solids, optionally microorganisms that produce alcohol, product alcohol, and alcohol, water, and dioxide produced by microorganisms present. By means of a mixture of all the other ingredients of the material retained in the fermentor in which the product alcohol is made by the reaction of sugars to carbon (CO ₂ ). In some cases, the terms “fermentation broth” and “fermented mixture” as used herein may be used interchangeably with “fermentation medium”.

  A “fermentable carbon source” or “fermentable carbon substrate” as used herein is a carbon source that can be metabolized by the microorganisms disclosed herein to produce fermented alcohol. Means. Suitable fermentable carbon sources include, but are not limited to, monosaccharides such as glucose or fructose; disaccharides such as lactose or sucrose; oligosaccharides; polysaccharides such as starch or cellulose; C5 saccharides such as xylose and arabinose. Monocarbon substrates containing methane; and mixtures thereof.

  “Fermentable sugar” as used herein refers to one or more saccharides that can be metabolized by the microorganisms disclosed herein to produce fermented alcohol.

  A “fermentor” as used herein means a tank in which a fermentation reaction takes place, whereby a product alcohol such as butanol is made from sugars.

  As used herein, “liquefaction tank” means a tank in which liquefaction is performed. Liquefaction is a process in which oligosaccharides are released from raw materials. In certain embodiments where the feedstock is corn, the oligosaccharide is released from the corn starch content during liquefaction.

  As used herein, “saccharification tank” means a tank in which saccharification (ie, decomposition of oligosaccharides into monosaccharides) takes place. When fermentation and saccharification are performed simultaneously, the saccharification tank and the fermentation tank may be the same tank.

  “Sugar” as used herein refers to oligosaccharides, disaccharides, monosaccharides, and / or mixtures thereof. The term “sugar” also includes starch, dextran, glycogen, cellulose, pentosan, and carbohydrates including sugars.

  As used herein, a “saccharifying enzyme” refers to one or more polysaccharides and / or oligosaccharides such as glycogen or starch that can hydrolyze α-1,4-glucoside bonds. Means an enzyme. Saccharifying enzymes may also include enzymes capable of hydrolyzing cellulosic or lignocellulosic materials.

  As used herein, “non-dissolved solid” means non-fermentable portions of raw materials, such as germs, fibers, and gluten. For example, the non-fermentable portion of the feed includes the portion that remains as a solid of the feed and can absorb liquid from the fermentation broth.

  Soluble-containing dry cereal distillers (DDGS) as used herein are by-products or by-products from fermentation of raw materials or biomass (eg, fermentation of cereals or cereal mixtures to produce product alcohol). Point to. In certain embodiments, DDGS also refers to animal feed products produced from methods of making product alcohols (eg, butanol, isobutanol, etc.).

“Product alcohol” as used herein refers to any alcohol that can be produced by a microorganism in a fermentation process that uses biomass as a source of fermentable carbon substrate. Product alcohols include, but are not limited to, C ₁ -C ₈ alkyl alcohols. In certain embodiments, the product alcohol is an alkyl alcohol of _C 2 -C _8. In other embodiments, the product alcohol is an alkyl alcohol of C ₂ -C _5. It will be appreciated that C ₁ -C ₈ alkyl alcohols include, but are not limited to, methanol, ethanol, propanol, butanol, and pentanol. Similarly, the alkyl alcohol C ₂ -C _8, but are not limited to, ethanol, propanol, butanol, and pentanol. “Alcohol” is also used herein in connection with the product alcohol.

  “Butanol” as used herein is notably the butanol isomers 1-butanol (1-BuOH), 2-butanol (2-BuOH), tert-butanol (t-BuOH), and / or isoforms. Butanol (iBuOH or i-BuOH or I-BUOH, also known as 2-methyl-1-propanol) is referred to individually or as a mixture thereof. Optionally, when referring to esters of butanol, the terms “butyl ester” and “butanol ester” can be used interchangeably.

  “Propanol” as used herein refers to the propanol isomer isopropanol or 1-propanol.

  “Pentanol” as used herein refers to the pentanol isomers 1-pentanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 2,2-dimethyl-1-propanol, It refers to 3-pentanol, 2-pentanol, 3-methyl-2-butanol, or 2-methyl-2-butanol.

  The term “alcohol equivalent” as used herein refers to the weight of alcohol that would be obtained by complete hydrolysis of the alcohol ester and subsequent recovery of the alcohol from a given amount of alcohol ester.

  The term “water phase titer” as used herein refers to the concentration of a particular alcohol (eg, butanol) in the fermentation broth.

  As used herein, the term “effective titer” refers to a specific alcohol produced by fermentation (eg, butanol) or an alcohol ester alcohol produced by alcohol esterification per liter of fermentation medium. Refers to the total amount of equivalents. For example, the effective titer of butanol in a unit volume of fermentation is: (i) the amount of butanol in the fermentation medium; (ii) the amount of butanol recovered from the organic extractant; (iii) where gas stripping is used The amount of butanol recovered from the gas phase; and (iv) the alcohol equivalent of the butyl ester in the organic or aqueous phase.

  The term “effective rate” as used herein is the value obtained by dividing the effective titer by the fermentation time.

  The term “effective yield” as used herein is the total grams of product alcohol produced per gram of glucose consumed.

  As used herein, “in situ product removal (ISPR)” refers to biological, such as fermentation, to control product concentration in a biological process as the product is produced. It means selectively removing a specific fermentation product from the process.

  “Extractant” or “ISPR extractant” as used herein is used to extract any product alcohol, such as butanol, or produced catalytically from product alcohol and carboxylic acid or lipid. Means any organic solvent used to extract any product alcohol ester. In some cases, the term “solvent” as used herein may be used interchangeably with “extractant”. In the methods described herein, the extractant is water immiscible.

  The term “water-immiscible” or “insoluble” refers to a chemical component such as an extractant or solvent that cannot be mixed with an aqueous solution, such as a fermentation broth, to form one liquid phase.

  The term “aqueous phase” as used herein refers to the aqueous phase of a two-phase mixture obtained by contacting a fermentation broth with a water-immiscible organic extractant. In one embodiment of the process described herein involving a fermentation extraction, the term “fermentation broth” in this case specifically refers to the aqueous phase in a biphasic fermentation extraction.

  The term “organic phase” as used herein refers to the non-aqueous phase of a two-phase mixture obtained by contacting a fermentation broth with a water-immiscible organic extractant.

  The term “carboxylic acid” as used herein refers to the general chemical formula —COOH, where a carbon atom is bonded to an oxygen atom by a double bond to form a carbonyl group (—C═O). And any organic compound represented by a single bond to a hydroxyl group (—OH). The carboxylic acid may be in the form of a protonated carboxylic acid, a salt of a carboxylic acid (eg, an ammonium salt, a sodium salt, or a potassium salt), or a mixture of a protonated carboxylic acid and a salt of a carboxylic acid. There may be. The term carboxylic acid can refer to a single chemical species (eg, oleic acid) or a mixture of carboxylic acids that can be produced by hydrolysis of, for example, biomass-derived fatty acid esters or triglycerides, diglycerides, monoglycerides, and phospholipids.

As used herein, the term “fatty acid” refers to a C _{4 to} C ₂₈ carbon atom (most commonly a C _{12 to} C ₂₄ carbon atom) that is either saturated or unsaturated. The carboxylic acid (for example, aliphatic monocarboxylic acid) which has. The fatty acids may also be branched or unbranched. Fatty acids may be derived from animal or vegetable fats, oils, or waxes, or included in esterified forms therein. Fatty acids may be naturally occurring in the form of glycerides in fats and fatty oils, or are obtained by hydrolysis of fats or synthetically. The term fatty acid can refer to a single chemical species or a mixture of fatty acids. Furthermore, the term fatty acid also encompasses free fatty acids.

The term “fatty alcohol” as used herein refers to an alcohol having an aliphatic chain of C _{4 to} C ₂₂ carbon atoms, either saturated or unsaturated.

The term “fatty aldehyde” as used herein refers to an aldehyde having an aliphatic chain of C _{4 to} C ₂₂ carbon atoms, either saturated or unsaturated.

The term “fatty amide” as used herein refers to an amide having an aliphatic long chain of C _{4 to} C ₂₂ carbon atoms, either saturated or unsaturated.

The term “fatty acid ester” as used herein refers to an ester having an aliphatic long chain of C _{4 to} C ₂₂ carbon atoms, either saturated or unsaturated.

  “Natural oil” as used herein refers to lipids obtained from plants (eg, biomass) or animals. A “plant-derived oil” as used herein refers to a lipid, particularly obtained from plants. In some cases, “lipid” can be used interchangeably with “oil” and “acylglyceride”. Natural oils include, but are not limited to, tallow, corn oil, canola oil, capric acid / caprylic acid triglyceride, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard, linseed oil, cow leg oil, yew deer Oil, palm oil, peanut oil, rapeseed oil, rice oil, safflower oil, soybean oil, sunflower oil, kiri oil, jatropha oil, and vegetable oil blends.

  As used herein, the term “separation” is synonymous with “recovery” and removes chemical compounds from the initial mixture to give a purity or higher concentration than the purity or concentration of the compounds in the initial mixture. To obtain a compound of

  The term “butanol biosynthetic pathway” as used herein refers to an enzymatic pathway for producing 1-butanol, 2-butanol, or isobutanol.

  The term “1-butanol biosynthetic pathway” as used herein refers to an enzymatic pathway for producing 1-butanol from acetyl-coenzyme A (acetyl-CoA).

  The term “2-butanol biosynthetic pathway” as used herein refers to an enzymatic pathway for the production of 2-butanol from pyruvate.

  The term “isobutanol biosynthetic pathway” as used herein refers to an enzymatic pathway for the production of isobutanol from pyruvate.

  The term “gene” refers to a nucleic acid fragment that can be expressed as a particular protein, optionally including regulatory sequences preceding (5 ′ non-coding sequences) and subsequent regulatory sequences (3 ′ non-coding sequences) of the coding sequence. . “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene (ie, has been modified from its native state or derived from another source), including regulatory and coding sequences that are not found together in nature. Thus, a chimeric gene may comprise regulatory and coding sequences that are derived from different sources or derived from the same source, but are arranged in a manner different from that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign gene” or “heterologous gene” refers to a gene that is not normally found as a natural gene in the host organism, but is introduced into the host organism by gene transfer. A foreign gene can include a natural gene or a chimeric gene inserted into a non-native organism.

  The term “coding region” as used herein refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequence” refers to a nucleotide sequence located upstream of a coding sequence (5 ′ non-coding sequence), a nucleotide sequence located internally, or a nucleotide sequence located downstream (3 ′ non-coding sequence) and related Affects the transcription, RNA processing or stability, or translation of the coding sequence. Regulatory sequences include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem loop structures.

  The term “codon optimization” refers to the typical codons of a host organism without changing the polypeptide encoded by the DNA when referring to the genes or coding regions of the nucleic acid molecule for transformation of various hosts. Refers to a codon modification in the gene or coding region of a nucleic acid molecule to reflect its use. Codon optimization is within the skill of one of ordinary skill in the art.

  The term “polynucleotide” is intended to encompass a nucleic acid as well as a plurality of nucleic acids, and refers to a nucleic acid molecule or construct, eg, messenger RNA (mRNA) or plasmid DNA (pDNA). A “gene” as used herein is a polynucleotide. A polynucleotide may comprise the nucleotide sequence of a full-length cDNA sequence or a fragment thereof, including untranslated 5 'and 3' sequences and a coding sequence. A polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA (eg, heterologous DNA). For example, a polynucleotide can be a single-stranded and double-stranded DNA, a DNA that is a mixture of single-stranded and double-stranded regions, a single-stranded and double-stranded RNA, and a mixture of single-stranded and double-stranded regions. It can consist of a single component, a single component, or more typically a mixed component comprising DNA and RNA, which can be double stranded or a mixture of single and double stranded regions. “Polynucleotide” embraces chemically, enzymatically or metabolically modified forms.

  A polynucleotide sequence may be referred to as being “isolated” removed from its natural environment. For example, a heterologous polynucleotide encoding a polypeptide or polypeptide fragment having dihydroxy acid dehydratase activity contained in a vector is considered isolated for the purposes of the present invention. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells in solution or (partially or substantially) purified polynucleotides. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. An isolated polynucleotide fragment in the form of a polymer of DNA can include one or more segments of cDNA, genomic DNA, or synthetic DNA.

  The term “polypeptide” as used herein is intended to encompass a single “polypeptide” as well as a plurality of “polypeptides”, and an amide bond (also known as a peptide bond). Refers to a molecule composed of monomers (amino acids) linearly linked by The term “polypeptide” refers to any one or more chains of two or more amino acids, and does not refer to a product of a particular length. Thus, a peptide, dipeptide, tripeptide, oligopeptide, “protein”, “amino acid chain” or any other term used to refer to one or more chains of two or more amino acids is “polypeptide” The term “polypeptide” can be used in place of any of these terms, ie, synonymously with these terms. A polypeptide may be derived from a natural biological source or produced by recombinant techniques, but is not necessarily translated from a designated nucleic acid sequence. Polypeptides can be produced by any method including chemical synthesis.

  By “isolated” polypeptide or fragment, variant or derivative thereof is intended a polypeptide that is not in its natural environment. No specific level of purification is required. For example, an isolated polypeptide can be removed from its natural or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells appear to be natural or recombinant polypeptides that are separated, fractionated, or partially or substantially purified by any suitable technique. It is considered isolated for the purposes of the present invention.

  A “recombinant microorganism” as used herein refers to recombinant DNA technology by modifying a host cell to include a biosynthetic pathway, such as, for example, a biosynthetic pathway for producing an alcohol such as butanol. Refers to microorganisms such as bacteria or yeast that are modified by use.

  The present invention requires an alternative extractive fermentation process that does not require separating the product alcohol into a fermentation medium and ISPR extractant as a means of reducing the toxic effects of product alcohol (such as butanol) on microorganisms. Satisfy sex. The present invention is a method for producing an alcohol, such as butanol, wherein the product alcohol is converted to an alcohol ester that can be less toxic to microorganisms, where the partition coefficient of the fermentation product extractant is reduced. Of the fermentation product by providing a method that results in an improved production yield of alcohol (as a combination of a free alcohol and an alcohol ester that can be converted back to alcohol after separation from the fermentation medium). It also meets the need to suppress a decrease in ISPP extractant partition coefficient. In addition, the present invention provides another alcohol production so that the methods herein can be combined with existing methods (eg, solids removal) to increase product removal in an economical and environmentally beneficial manner. Provides a solution to the disadvantages of the object removal method. Accordingly, the present invention provides further related advantages as will become apparent from the description of the embodiments below.

  The present invention is a method for removing alcohol from a fermentation medium by esterifying the alcohol with a carboxylic acid and extracting the resulting alcohol ester from the fermentation medium, after which the alcohol is recovered from the alcohol ester. Provide a way to The acid may be added directly to the fermentation medium as a free fatty acid or may be derived from oil. The present invention hydrolyzes oils derived from raw materials into carboxylic acids that can be used for esterification of alcohols and / or can serve as ISPR extractants or ISPR extractants components to extract alcohol esters This also provides a method for removing or reducing oil from the alcohol fermentation process.

  Reduction of the amount of water present in the reaction system, or the use of a reaction system that uses one or more non-aqueous solvents, is usually required for esterification of alcohols with carboxylic acids when catalyzed by enzymes such as lipases It has been said. The surprising discovery that a lipase enzyme can effectively catalyze the esterification of a product alcohol by a carboxylic acid during fermentation of a fermentable carbon source to produce a product alcohol is described herein. ing. The surprising discovery that fermentation performance can be improved by esterification of the product alcohol with carboxylic acid during fermentation is also described herein. It effectively reduces the concentration of product alcohol in the aqueous phase, for example, by capturing product alcohol (eg, butanol) produced in ester form, thus producing for glucose consumption and product production Reduce the toxic effects of alcohol.

  The invention will now be described with reference to the figures. FIG. 1 illustrates an exemplary process flow diagram for the production of a fermented alcohol such as ethanol or butanol, according to one embodiment of the present invention. As shown in the figure, the raw material 12 can be introduced into the inlet of the liquefaction tank 10 and liquefied to produce a raw material slurry 16. The raw material 12 contains a hydrolyzable polysaccharide that provides a fermentable carbon substrate (eg, a fermentable sugar such as glucose) and can be a biomass such as, but not limited to, rye, wheat, sugar cane or corn. Or it can be derived from biomass. In some embodiments, feedstock 12 can be one or more components of fractionated biomass, and in other embodiments, feedstock 12 can be crushed, unfractionated biomass. In certain embodiments, the feedstock 12 can be corn, such as dry-milled, unfractionated corn kernels, and the undissolved particles can include germ, fiber, and gluten. The undissolved solid is a non-fermentable part of the raw material 12. For purposes of the description herein with reference to the embodiment shown in the figure, the feedstock 12 may be described as being ground, unfractionated corn from which undissolved solids are not separated. Many. However, it will be appreciated by those skilled in the art that the exemplary methods and systems described herein can be modified for a variety of ingredients, whether fractionated or not. Furthermore, as can be appreciated by those skilled in the art, maximizing the feed content (eg, corn content) can maximize the sugar content as well as the product titer. In certain embodiments, feedstock 12 can be high oleic corn, so the corn oil derived therefrom is a high oleic corn oil having an oleic acid content of at least about 55% by weight oleic acid. In certain embodiments, the oleic acid content in high oleic corn oil can be up to about 65% by weight compared to the oleic acid content in normal corn oil that is about 24% by weight. High oleic oil can provide several advantages for use in the method of the present invention because hydrolysis of the oil provides free fatty acids with high oleic acid content for contact with the fermentation broth.

  The process of liquefying the raw material 12 is a conventional process, including hydrolyzing the polysaccharide in the raw material 12 into saccharides including, for example, dextrin and oligosaccharides. Any known liquefaction process commonly used in the art, including but not limited to acid processes, acid-enzyme processes, or enzyme processes, as well as corresponding liquefaction tanks may be used. Such processes can be used alone or in combination. In certain embodiments, an enzymatic process can be used and a suitable enzyme 14, such as α-amylase, is introduced into the inlet of the liquefaction tank 10. Water can also be introduced into the liquefaction tank 10. In certain embodiments, saccharification enzymes, such as glucoamylase, may also be introduced into the liquefaction tank 10. In a further embodiment, lipase can also be introduced into the liquefaction tank 10 to catalyze the conversion of one or more components of the oil to free fatty acids.

  The raw material slurry 16 generated from liquefying the raw material 12 includes a fermentable carbon substrate (for example, sugar), oil, and undissolved solids derived from the raw material. The raw material slurry 16 can be discharged from the outlet of the liquefaction tank 10. In some embodiments, the feed slurry 16 is a corn mash slurry because the feed 12 is corn or corn seed. In an embodiment, since the raw material 12 is a lignocellulosic raw material, the raw material slurry 16 may be a lignocellulosic hydrolyzate. In some embodiments, ingredient 12 is sugar cane.

  The raw material slurry 16 is introduced into the fermenter 30 together with the microorganisms 32. The fermenter 30 is configured to ferment the slurry 16 to produce alcohol. In particular, the microorganism 32 metabolizes the fermentable sugar in the slurry 16 and discharges the product alcohol. The microorganism 32 is selected from the group consisting of bacteria, cyanobacteria, filamentous fungi, and yeast. In certain embodiments, the microorganism 32 may be a bacterium such as E. coli. In certain embodiments, the microorganism 32 can be a fermentable recombinant microorganism. The slurry may include, for example, a sugar in the form of an oligosaccharide, and water, and in certain embodiments, less than about 20 g / L monomer glucose, less than about 10 g / L, or less than about 5 g / L monomer glucose. Can be included. Suitable methods for determining the amount of monomeric glucose are well known in the art. Such suitable methods known in the art include HPLC.

  In certain embodiments, slurry 16 is subjected to a saccharification process to break down complex saccharides (eg, oligosaccharides) in slurry 16 into monosaccharides that can be readily metabolized by microorganisms 32. Any known saccharification process commonly used in the art may be used, including but not limited to acid processes, acid-enzyme processes, or enzyme processes. In certain embodiments, simultaneous saccharification and fermentation (SSF) can be performed inside fermentor 30, as shown in FIG. In certain embodiments, an enzyme 38, such as glucoamylase, may be introduced at the inlet of the fermentor 30 to break down the starch or oligosaccharide into glucose that can be metabolized by the microorganism 32.

  Carboxylic acid 28 and / or natural oil 26 is introduced into fermentor 30 along with catalyst 42. The catalyst 42 can be introduced before, after, or simultaneously with the enzyme 38. Thus, in certain embodiments, the addition of enzyme 38 and catalyst 42 can be gradual (eg, catalyst 42, then enzyme 38, or vice versa) or nearly simultaneously (ie, once by a person or machine). Add the other catalyst / enzyme immediately after one enzyme / catalyst in exactly the same time as it takes to add at the same time as the time it takes for a person or machine to add twice) It can be. The catalyst 42 can esterify the product alcohol with the carboxylic acid 28 to form an alcohol ester. For example, in the case of butanol production, catalyst 42 can esterify butanol with carboxylic acid 28 to form a butyl ester.

  When natural oil 26 is fed to fermentor 30, at least a portion of the acylglycerides in oil 26 can be hydrolyzed to carboxylic acid 28 by contacting oil 26 with catalyst 42. The acid / oil composition resulting from the hydrolysis of oil 26 is typically at least about 17% by weight carboxylic acid 28 (as a free fatty acid). In certain embodiments, the acid / oil composition resulting from the hydrolysis of oil 26 comprises at least about 20% by weight carboxylic acid, at least about 25% by weight carboxylic acid, at least about 30% by weight carboxylic acid, at least about 35%. % By weight carboxylic acid, at least about 40% by weight carboxylic acid, at least about 45% by weight carboxylic acid, at least about 50% by weight carboxylic acid, at least about 55% by weight carboxylic acid, at least about 60% by weight carboxylic acid. At least about 65% by weight carboxylic acid, at least about 70% by weight carboxylic acid, at least about 75% by weight carboxylic acid, at least about 80% by weight carboxylic acid, at least about 85% by weight carboxylic acid, at least about 90% by weight % Carboxylic acid, at least about 95% by weight carboxylic acid, or at least about 99 weight Is the percent of a carboxylic acid. In certain embodiments, the resulting acid / oil composition comprises monoglycerides and / or diglycerides from partial hydrolysis of acylglycerides in the oil. In certain embodiments, the resulting acid / oil composition comprises glycerol, a by-product of acylglyceride hydrolysis. In certain further embodiments, the resulting acid / oil composition comprises lysophospholipids from partial hydrolysis of phospholipids in the oil.

  In certain embodiments, after hydrolysis of the acylglycerides in oil 26, the residual acylglycerides from oil 26 is from about 0% to at least about 2% by weight of the fermentation broth composition. In certain further embodiments, after hydrolysis of the acylglycerides in oil 26, the residual acylglycerides from oil 26 is at least about 0.5% by weight of the fermentation broth composition. Thus, in certain embodiments, the acylglycerides from oil 26 can be catalytically hydrolyzed to carboxylic acid 28 using catalyst 42, which can also esterify carboxylic acid 28 with the product alcohol. In certain embodiments, a second catalyst (not shown) can be introduced into the fermentor for acylglyceride hydrolysis. Furthermore, the acylglycerides in the oil derived from feedstock 12 and present in slurry 16 can also be hydrolyzed to carboxylic acid 28 '(see, eg, the embodiment of FIG. 3). In certain embodiments, the concentration of carboxylic acids (such as fatty acids) in the fermenter exceeds the solubility limit in the aqueous phase, producing a two-phase fermentation mixture comprising an organic phase and an aqueous phase. In certain embodiments, the concentration of carboxylic acid in the fermentation broth is typically about 0.8 g / L or less and is limited by the solubility of the carboxylic acid in the broth.

  In certain embodiments, catalyst 42 and second catalyst, when used, can be one or more enzymes, such as lipase enzymes. In certain embodiments, the catalyst 42 can be one or more enzymes, for example, hydrolytic enzymes such as lipase enzymes. The lipase enzymes used are, for example, Absidia, Achromobacter, Aeromonas, Alcaligenes, Alternaria, Aspergillus, Aspergillus, Aspergillus Achromobacter, Aureobasidium, Bacillus, Beauveria, Brochothrix, Candida, Chromobacter genus (Chromobacter genus) ), Fusarium, Geotrichum (G otricum, Hansenula, Humicola, Hyphozyma, Lactobacillus, Metarhidium, Mucor, Nectria p, Nectria p ), Pecilomyces, Penicillium, Pseudomonas, Rhizoctonia, Rhizomucor, Rhizodum ), Saccharomyces (Saccharomyces), Wild Boar (Sus), Sporoboromyces, Thermomyces, Thiarosporella, Trichoderma, Verticirium and Verticillium From any source, including strains of Yarrowia). In a preferred embodiment, the source of lipase is Absidia blackesleena, Absidia corymbifera, Achromobacter terridium Alsp. ), Aspergillus flavus, Aspergillus niger, Aspergillus tubingensis, Aureobasidium pullans (Aureobasidium pullus) lans, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis and Brochothria cerdosa (Candida rugosa), Candida paralipolytica, Candida Antarctica lipase A, Candida antarctica (Candida antarctica) lipase C, Candida antarctica da ernobii), Candida deformans, Chromobacter viscosum, Coprius cinerius, Fusarium oxysporum (Fusarium oxysporum) Pisi (Fusarium solani pisi), Fusarium roseum culmorum, Geotricum penicillatum, Hansenula anomala (Hansenula anomala) brevispora), Humicola brevis variant thermoidea (Humicola brevis var. thermoidea, Humicola insolens, Lactobacillus curiumus, Rhizopus oryzae, Penicillium cyclopium (Penicillium citrus) expansum, Penicillium sp. I, Penicillium sp. II, Pseudomonas aeruginosa, Pseudomonas alcalis enes), Pseudomonas cepacia (synonymous with Burkholderia cepacia), fluorescent bacteria (Pseudomonas fluorescens), Pseudomonas fluorscens, Pseudomonas phrodisto Pseudomonas mendocina, Pseudomonas mephitica lipolytica, Pseudomonas alcaligenes, Pseudomonas alcaligenes, Pseudomonas planta monas plantarii), Pseudomonas shoe de alkali monocytogenes (Pseudomonas pseudoalcaligenes), Pseudomonas putida (Pseudomonas putida), Pseudomonas Sutsuttsueri (Pseudomonas stutzeri), and Pseudomonas Wisuko Nshi Nene cis (Pseudomonas wisconsinensis), sheath blight fungus (Rhizoctonia solani), Rhizomucor・ Rhizomucor miehei, Rhizopus japonicus, Rhizopus microsporus, Rhizopus nodosus (Rhizopus nodosus) ), Rhodes toruloides (Rhodosporidium toruloides), Rhodotorula Guruchinisu (Rhodotorula glutinis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), scan polo rags My Seth Shibatanusu (Sporobolomyces shibatanus), wild boar (Sus scrofa), Thermomyces lanuginosus (Thermomyces lanuginosus (formerly Humicola lanuginosa), Thiarosporella phaseolina, Trichoderma harzianum, Rikoderuma reesei (Trichoderma reesei), and is selected from the group consisting of Yarrowia lipolytica (Yarrowia lipolytica). In a further preferred embodiment, the lipase is a Thermomyces lanuginosus lipase, an Aspergillus sp. Lipase, an Aspergillus sp. Candida antarctica lipase B, Pseudomonas sp. Lipase, Penicillium roqueforti lipase, Penicillium camembert lipase, Le Corban lipase Candida deformans lipase, Lipase A and B derived from Geotricum candidum, Neurospora crassa lipase, Nectria haematococa (Nectriaco haematoporumto) usarium heterosporum) lipase, Rhizopus Derema (Rhizopus delemar) lipase is selected Rhizomucor miehei (Rhizomucor miehei) lipase, Rhizopus Arizusu (Rhizopus arrhizus) lipase, and from the group consisting of Rhizopus oryzae (Rhizopus oryzae) lipase. Suitable commercially available lipase preparations suitable as catalyst 42 include, but are not limited to, Lipolase® 100 L, Lipex® 100 L, Lipoclean® 2000T, available from Novozymes, Lipozyme® CALB L, Novozyme® CALA L, and Palatase 20000L, or available from Sigma Aldrich, Pseudomonas fluorescens, Pseudomonas cemu Porcine pancreas, Candida cylindracea, Rhizopus nibeus (R Hizzopus niveus, Candida antarctica, Candida rugosa, Rhizopus arrhizus or Aspergillus from Aspergillus.

  Phospholipases are enzymes that hydrolyze the ester bonds of phospholipids, but many phospholipases can also hydrolyze triglycerides, diglycerides, and monoglycerides (lipid acyl hydrolase (LAH) activity). The term “phospholipase” as used herein has any phospholipase activity and cleaves, for example, a glycerol phosphate bond in an oil such as crude oil or vegetable oil (glycerol phosphate ester). Enzymes that catalyze the hydrolysis of bonds. The phospholipase activity of the present invention can produce phosphorylated bases and diglycerides that can be extracted with water. Phospholipase activity may be phospholipase C (PLC) activity; phospholipase A (PLA) activity such as PI-PLC activity, phospholipase A1 or phospholipase A2 activity; lysophospholipase (LPL) activity and / or lysophospholipase-transacylase (LPT A) activity Phospholipase B (PLB) activity such as phospholipase B1 or phospholipase B2 activity; phospholipase D (PLD) activity such as phospholipase D1 or phospholipase D2 activity; and / or patatin activity or any combination thereof.

The term “phospholipase” also encompasses enzymes having lysophospholipase activity, where the two substrates of the enzyme are 2-lysophosphatidylcholine and H ₂ O, the two products of which are glycerophosphocholine and Carboxylate. The phospholipase A1 (PLA1) enzyme removes the 1-position fatty acid to produce free fatty acid and 1-lyso-2-acyl phospholipid. The phospholipase A2 (PLA2) enzyme removes the 2-position fatty acid to produce free fatty acid and 1-acyl-2-lysophospholipid. PLA1 and PLA2 enzymes can be intracellular or extracellular, membrane bound or soluble. The phospholipase C (PLC) enzyme removes the phosphate moiety to produce 1,2 diacylglycerol and phosphate ester. The phospholipase D (PLD) enzyme produces 1,2-diacylglycerophosphate and a base group. Phospholipases useful in the present invention can be derived from a variety of biological sources such as, but not limited to: F. culmorum, F. Heterosporum, F. F. solani, or F. solani Filamentous fungi within Fusarium, such as strains of F. oxysporum; or Aspergillus awagi, Aspergillus fosidus sp., Aspergillus sp. from filamentous fungal species within the genus Aspergillus, such as strains of Niger or Aspergillus oryzae. Commercially available Thermomyces lanuginosus phospholipase variants such as Lecitase® Ultra (Novozymes A'S, Denmark) are also useful in the present invention. One or more phospholipases can be applied as a lyophilized powder, immobilized or dissolved in an aqueous solution.

  Alcohol (eg, butanol) produced by fermentation of one or more fermentable sugars can be converted to a carboxylic acid ester by an enzyme-catalyzed reaction in which the carboxylic acid is esterified with the alcohol. Enzymes such as lipases, phospholipases, and lysophospholipases could catalyze this reaction; these enzymes, however, are not limited to the following: hydrodynamic shear or inactivation at gas-liquid and liquid-liquid interfaces It can be inactivated by one or more factors including. In fermentations where the oligosaccharide is further converted to one or more fermentable sugars, the enzyme that converts the oligosaccharide to a fermentable sugar (eg, glucoamylase) may also be impaired by one or more of these above factors. Can be activated.

  Inactivation of enzymes at the gas-liquid interface resulting from aeration of the fermentation broth and / or produced by the generation of gaseous carbon dioxide in the broth during the fermentation of one or more fermentable sugars (e.g. , Which can occur at the air bubble interface by the fermentation broth) is well known in the art. Inactivation of Thermomyces lanuginosus lipase produced in chicken egg white lysozyme and Aspergillus oryzae (Novozymes Lipolase®) It was observed at the gas-liquid interface of the equipped stirred tank (no gas sparging aeration) and falling film (Ghadge, et al., Chem. Eng. Sci. 58: 5125-5134, 2003). Produced in Thermomyces lanuginosus lipase (Aspergillus oryzae) at the gas-liquid interface in a baffled stirred tank reactor (no gas sparging aeration); Novozymes 100 ) Has been reported (Patil, et al., AIChE J. 46: 1280-1283, 2000).

  Stahmmann, et al. (Eur. J. Biochem. 244: 220-225, 1997), Ashbya gossippi lipase is used in a gas / water, trioleoylglycerol / water or oleic acid / water mixture with stirring. It was reported that it was deactivated within a few minutes due to the deactivation of the interface at either the liquid or liquid / liquid interface. Elias, et al. (Adv.Biochem.Engineering/Biotechnology 59: 47-71, 1998): (i) some enzymes are inactivated by hydrodynamic shear even in the absence of a gas-liquid interface; (ii) fluid For enzymes that are inactivated by mechanical shear, the rate of inactivation increases in the presence of a gas-liquid interface; (iii) some enzymes may or may not be applied with or without hydrodynamic shear In the absence of a gas-liquid interface, it is not inactivated; (iv) for enzymes that require a gas-liquid interface for inactivation, the rate of inactivation increases with increasing hydrodynamic shear Reported. Ross, et al. (J. Mol. Catal. B: Enzymatic 8: 183-192, 2000), α-chymotrypsin dissolved in various aqueous / organic solvent mixtures by passing solvent droplets through an aqueous enzyme solution in a bubble column apparatus. , Β-chymotrypsin, papain, and porcine liver esterase interface inactivation have been described. The kinetics and mechanism of shear inactivation of Candida cylindracea lipase in a stirred tank reactor has also been reported, where the mechanism of inactivation is a gas-liquid interface effect due to shear. (Lee, et al., Biotechnol. Bioeng. 33: 183-190, 1989).

  Under the fermentation conditions used in some of the methods described herein, hydrodynamic shear and gas-liquid and liquid-liquid interfaces, respectively, exist during fermentation and cause enzyme inactivation. Can do. One of the enzymes (eg, glucoamylase, lipase, phospholipase, and lysophospholipase) present in the two-phase mixture (eg, fermentation broth and carboxylic acid) during fermentation under the conditions described herein. The potential impact of each of these factors on more than one stability and activity could not be predicted based on the prior art. Each of these factors could result in inactivation of one or more enzymes in the fermentation mixture, but sufficient to catalyze the esterification of the product alcohol with the carboxylic acid to produce the carboxylic acid ester. Enzymatic activity was maintained during fermentation. Glucoamylase also maintained sufficient enzymatic activity (ie, to convert oligosaccharides to fermentable sugars) in the reactions present in the two-phase fermentation mixture of fermentation broth and carboxylic acid.

  The carboxylic acid 28 can be any carboxylic acid that can be esterified with a product alcohol such as butanol or ethanol to produce an alcohol ester of the carboxylic acid. For example, in certain embodiments, the carboxylic acid 28 can be a free fatty acid, and in certain embodiments the carboxylic acid or free fatty acid can be 4 to 28 carbons, in other embodiments 4 to 22 carbons, etc. 8-22 carbons in other embodiments, 10-28 carbons in other embodiments, 7-22 carbons in other embodiments, 12-22 carbons in other embodiments, other implementations In embodiments, it has 4-18 carbons, in other embodiments 12-22 carbons, and in still other embodiments 12-18 carbons. In certain embodiments, the carboxylic acid 28 includes the following fatty acids: azelaic acid, capric acid, caprylic acid, castor oil, coconut oil (ie, for example, lauric acid, myristic acid, palmitic acid, caprylic acid, capric acid, stearic acid). , As a naturally occurring combination of fatty acids including caproic acid, arachidic acid, oleic acid, and linoleic acid), isostearic acid, lauric acid, linseed oil, myristic acid, oleic acid, palm oil, palmitic acid, palm kernel oil, One or more of pelargonic acid, ricinoleic acid, sebacic acid, soybean oil, stearic acid, tall oil, tallow, and # 12 hydroxystearic acid. In certain embodiments, carboxylic acid 28 is one or more of diacids.

  In certain embodiments, the carboxylic acid 28 can be a mixture of two or more different fatty acids. In certain embodiments, carboxylic acid 28 comprises free fatty acids derived from hydrolysis of acylglycerides by any method known in the art, including chemical or enzymatic hydrolysis. In certain embodiments described above, carboxylic acid 28 may be derived from natural oil 26 by enzymatic hydrolysis of oil glycerides using an enzyme as catalyst 42. In certain embodiments, the fatty acids or mixtures thereof include unsaturated fatty acids. The presence of unsaturated fatty acids lowers the melting point and provides advantages in handling. Among the unsaturated fatty acids, those that are monounsaturated, that is, those that have one carbon-carbon double bond, have advantages regarding the melting point without sacrificing suitable thermal and oxidative stability during process studies. Can provide.

  In some embodiments, natural oil 26 is tallow, corn, canola oil, capric / caprylic triglyceride, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard, linseed oil, cow leg oil, boar leg oil, palm oil. Oil, peanut oil, rapeseed oil, rice oil, safflower oil, soybean oil, sunflower oil, kiri oil, jatropha oil, pumpkin oil, grape seed oil, and vegetable oil blends (or higher concentrations of different chain lengths and levels of unsaturation) (Ie, an oil that can be refined to 18: 1). In certain embodiments, natural oil 26 is a mixture of two or more natural oils, such as a mixture of palm oil and soybean oil. In certain embodiments, natural oil 26 is a plant derived oil. In certain embodiments, the plant-derived oil can be derived from biomass that can be used in the fermentation process, but not necessarily. Biomass can be the same or different source from which raw material 12 is obtained. Thus, for example, in certain embodiments, oil 26 can be derived from corn, while feedstock 12 can be sugar cane. For example, in certain embodiments, oil 26 may be derived from corn and the biomass source of feedstock 12 is also corn. Any conceivable combination of different biomass sources of oil 26 for feed 12 can be used, as should be apparent to those skilled in the art. In certain embodiments, oil 26 is derived from biomass used in the fermentation process. Thus, in one embodiment, oil 26 is derived directly from feed 12 as oil 26 ', as described below with reference to FIG. For example, when the raw material 12 is corn, the oil 26 'is corn oil which is a component of the raw material.

  Optionally, ethanol 33 can be fed to the fermentor 30 to be included in the fermentation broth. In certain embodiments, when a recombinant microorganism having a butanol biosynthetic pathway and / or reduced or eliminated expression of pyruvate decarboxylase is used as microorganism 32, microorganism 32 survives and grows. May require supplementation with a 2-carbon substrate, such as ethanol. Thus, in certain embodiments, ethanol 33 can be supplied to the fermenter 30.

  However, the method of the present invention in which a carboxylic acid such as a fatty acid is present in the fermentor can reduce the amount of ethanol 33 normally supplied to a given recombinant microorganism without compromising the vitality of the recombinant microorganism. I found it unexpectedly. Furthermore, in certain embodiments of the method of the present invention, the rate of alcohol (eg, butanol) production without ethanol supplementation can be made comparable to the production rate that can be achieved when ethanol 33 is supplemented. As further shown by the comparative examples shown in Examples 1-14 below, the butanol production rate when there is no fatty acid in the fermenter but no ethanol is the butanol production when there is no fatty acid and no ethanol in the fermenter. Can be equal to or higher than speed. Thus, in certain embodiments, the amount of ethanol 33 replenishment is reduced compared to conventional processes. For example, a typical amount of ethanol added to a fermentor for microorganisms that require supplementation of 2-carbon substrates is about 5 g / L absolute ethanol (ie, 5 g absolute ethanol per liter of fermentation medium). . In certain embodiments, the fermentation is not supplemented with any ethanol 33. In the latter case, the entire flow of ethanol 33 is excluded from the fermentor. Thus, in certain embodiments of the present invention, the costs associated with supplementing ethanol 33 as well as microorganisms that may require supplementation of a 2-carbon substrate to store or survive and grow large amounts of ethanol 33 and butanol. It is possible to reduce or eliminate the disadvantages associated with supplying ethanol to the fermenter during other alcohol fermentations used.

  Furthermore, regardless of ethanol supplementation, in certain embodiments, the methods of the present invention can provide a higher rate of glucose uptake by microorganism 32 due to the presence of fatty acids during fermentation. Fatty acids can be introduced into the fermentor 30 as carboxylic acid 28, can be hydrolyzed from the supplied oil 26, and / or can be obtained from hydrolysis of biomass oil as a component of the slurry 16. A method for producing product alcohol from a fermentation process in which free fatty acids are produced in the process and contacted with the microbial medium in the fermentor to improve microbial growth rate and glucose consumption is by reference in its entirety. No. 61 / 368,451, filed Jul. 28, 2010 and owned by the same assignee and owned by the same applicant.

  In the fermenter 30, the alcohol produced by the microorganism 32 is esterified with the carboxylic acid 28 using the catalyst 42 to form an alcohol ester. For example, in the case of butanol production, butanol produced by microorganism 32 is esterified with carboxylic acid 28 using catalyst 42 to form a butyl ester. In situ product removal (ISPR) can be used to remove alcohol esters from the fermentation broth. As shown herein, using a catalyst to form an ester with ISPR can improve the performance of the fermentation. In certain embodiments, the use of a catalyst to form an ester with an ISPR (eg, liquid-liquid extraction, etc.) is at least compared to the effective titer in a similar fermentation using ISPR without the catalyst forming the ester. Only about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% The effective titer can be increased. Similarly, in some embodiments, the use of a catalyst to form an ester with an ISPR (eg, liquid-liquid extraction, etc.), the effective rate (eg, performance) in a similar fermentation using ISPR without the catalyst forming the ester Example 9 and 11-14, see Table 3) at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70 %, At least about 80%, at least about 90%, or at least about 100%. In certain embodiments, the effective yield is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In certain embodiments, the fermentation broth obtained after alcohol esterification may comprise free (ie, non-esterified) alcohol, and in certain embodiments, if the product alcohol is butanol, fermentation after alcohol esterification. The concentration of free alcohol in the broth is 1, 3, 6, 10, 15, 20, 25, 30, 25, 40, 45, 50, 55, or 60 g / L, or the product alcohol is ethanol. In some cases, the concentration of free alcohol in the fermentation broth after alcohol esterification is 15, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90. , 95, or 100 g / L or less. In certain embodiments, the ratio of alcohol ester to alcohol in the fermentor can be about 1: 1. In certain embodiments, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the effective potency of the alcohol. % Is converted to the alcohol ester.

  In certain embodiments, filling of the cereal with water at a concentration sufficient to obtain a final effective titer of at least about 50 g / L, at least about 75 g / L, or at least about 100 g / L may result in alcohol such as butanol. It can be used for cereal mash fermentations containing microorganisms that can be produced. In other embodiments, cereal mash fermentation may use simultaneous saccharification and fermentation (SSF), and the concentration of glucose may remain relatively low, for example, L of glucose in the fermentation broth phase during fermentation. At least about 75 g.

  In certain embodiments, the fatty acid may be added to the fermenter in an amount less than about 70% of the fermenter volume, less than about 50% of the fermenter volume, or less than about 30% of the fermenter volume. The amount of fatty acid added to the fermenter can be a means for maintaining the aqueous phase titer of butanol during fermentation. In other embodiments, the aqueous phase titer of butanol may be maintained at a level of less than about 35 g per L fermentation broth, less than about 25 g per L fermentation broth, or less than about 20 g per L fermentation broth. In other embodiments, the amount of active esterifying enzyme in the fermentation broth can be less than about 100 ppm, less than about 50 ppm, or less than about 10 ppm active enzyme. In certain embodiments, the cell mass used in the fermentation broth can be less than about 50 g dcw / L, less than about 20 g dcw / L, or less than about 10 g dcw / L. In other embodiments, the fermentation process can be performed for at least about 30 hours to at least about 100 hours, at least about 40 hours to at least about 80 hours, or at least about 50 hours to at least about 70 hours.

  In certain embodiments, a final effective titer of at least about 30 g butanol per liter of fermentation broth phase, at least about 45 g butanol per liter of fermentation broth phase, or at least about 60 g butanol per liter of fermentation broth phase is obtained. Brix in water at a sufficient concentration can be used for sugarcane fermentation containing microorganisms capable of producing butanol. In certain embodiments, the fatty acid may be added to the fermenter in an amount less than about 70% of the fermenter volume, less than about 50% of the fermenter volume, or less than about 30% of the fermenter volume. The amount of fatty acid added to the fermenter can be a means for maintaining the aqueous phase titer of butanol during fermentation. In other embodiments, the aqueous phase titer of butanol can be maintained at a level of less than about 35 g per L of fermentation broth, less than about 25 g per L of fermentation broth, or less than about 15 g per L of fermentation broth. In other embodiments, the amount of active esterifying enzyme in the fermentation broth can be less than about 200 ppm, less than about 100 ppm, or less than about 20 ppm active enzyme. In certain embodiments, the cell mass used in the fermentation broth may initially be at least about 100 g of cells per liter of broth, with an initial loading that accounts for at least about 30% of the volume of the fermenter. After 3-7 hours of fermentation, the cell mass can be diluted to at least about 25 g cells per liter of fermentation broth by adding sugarcane ingredients. Cells can continue to grow to at least about 30 g cells per liter of fermentation broth over 8-15 hours of the total fermentation time.

In certain embodiments, the fermentation broth is contacted with the extractant during fermentation to form a two-phase mixture comprising an aqueous phase and an organic phase. In such embodiments, ISPR including liquid-liquid extraction can be conveniently performed. Liquid-liquid extraction can be performed according to the method described in US Patent Application Publication No. 2009/030305370, the entire disclosure of which is incorporated herein by reference. US Patent Application Publication No. 2009/030305370 describes a method for producing and recovering butanol from fermentation broth using liquid-liquid extraction, which method makes the fermentation broth immiscible with water. Contacting with an extractant to form a biphasic mixture comprising an aqueous phase and an organic phase. Usually, the extraction agent is a saturated, esters of monounsaturated, polyunsaturated (and mixtures _thereof) fatty alcohols of C 12 _{-C 22,} fatty acids C 12 _{-C 22,} fatty acids C 12 _{-C 22,} C It can be an organic extractant selected from the group consisting of _{12 to} C ₂₂ fatty aldehydes, C _{12 to} C ₂₂ fatty amides, and mixtures thereof. Extractant also saturated, esters of monounsaturated, polyunsaturated (and mixtures _thereof) fatty alcohols of C 4 _{-C 22,} fatty acids C 4 _{-C 28,} fatty acids _{C 4} ~C _28, C 4 fatty aldehydes -C _22, and may be an organic extractant selected from the group consisting of a mixture thereof. When used with the methods described herein, the catalytic esterification of carboxylic acid 28 with an alcohol extractant avoids consumption of carboxylic acid 28 in fermentor 30, thereby producing less carboxylic acid. The extractant for ISPR is usually a non-alcohol extractant, as would be available for esterification with physical alcohols. As further shown in Example 24 below, for example, when oleyl alcohol is used as the ISPR extractant, an oleyl alcohol ester of a carboxylic acid can be produced in the fermentor due to the presence of the active catalyst 42. .

  With reference to the embodiment of FIG. 1, carboxylic acid 28 may also serve as ISPR extractant 28 or a component thereof. As noted above, when natural oil 26 is fed to fermentor 30, carboxylic acid 28 can be fed and / or formed in situ and / or raw material 16 can be hydrolyzed when feed 16 is hydrolyzed. Formed in position. In certain embodiments, ISPR extractant 28 includes free fatty acids. In certain embodiments, ISPR extractant 28 comprises corn oil fatty acid (COFA). In certain embodiments, oil 26 is corn oil, whereby ISPR extractant 28 is COFA. ISPR extractant (carboxylic acid) 28 contacts the fermentation broth and forms a two-phase mixture comprising an aqueous phase 34 and an organic phase. The product alcohol ester formed in the fermentor is preferentially separated into the organic phase to form an ester-containing organic phase 36. That is, the product alcohol ester is produced at a concentration that exceeds the equilibrium concentration of the alcohol ester present in the aqueous phase 34 and thus preferentially separates into the organic phase. Any free product alcohol in the fermentation broth is also preferentially separated into the ester-containing organic phase. The biphasic mixture can be removed from the fermenter 30 as stream 39 and introduced into the vessel 35 where the ester-containing organic phase 36 is separated from the aqueous phase 34. Separation of the two-phase mixture 39 into the ester-containing organic phase 36 and the aqueous phase 34 includes, but is not limited to, siphoning, inhalation, decantation, centrifugation, use of gravity settlers, membrane assisted phase separation, hydrocyclone This can be done using any method known in the art including All or a portion of the aqueous phase 34 is reused or discarded in the fermenter 30 as a fermentation medium (shown) and replaced with fresh medium or removed residual product alcohol. Can then be processed and then recycled to the fermenter 30.

  Referring to FIG. 1, an ester-containing organic phase 36 is introduced into a tank 50 where the alcohol ester is reacted with one or more substances 52 to recover the product alcohol 54. Product alcohol 54 may be recovered using any method known in the art for obtaining alcohol from alcohol esters. For example, in certain embodiments, the product alcohol can be recovered from the alcohol ester by hydrolysis with a base followed by acidification. In other embodiments, the product alcohol ester may be hydrolyzed with water in the presence of a hydrolysis catalyst as material 52. For example, in certain embodiments, hydrolysis of the product alcohol ester to an alcohol and a carboxylic acid 28 (eg, a fatty acid when the carboxylic acid 28 is a fatty acid) may include lipase, water-soluble acid, inorganic acid as the substance 52. , Organic acids, or solid acid catalysts. For example, sulfuric acid can be used as an inorganic acid catalyst for alcohol ester hydrolysis. Some suitable hydrolysis catalysts include lipase enzymes; esterase enzymes; strong inorganic acids such as sulfuric acid, hydrochloric acid, or phosphoric acid; strong organic acids such as toluene sulfonic acid or naphthalene sulfonic acid; or Amberlyst® sulfonation Solid acid catalyst such as polystyrene resin, or zeolite. In certain embodiments, hydrolysis of the alcohol ester may be performed using steam as material 52 by increasing the temperature and / or applying pressure. In certain embodiments, hydrolysis of the alcohol ester can be performed in a column such as, for example, a reactive distillation column. Examples 45-54 and 56-58 illustrate several methods for recovering the product alcohol from the alcohol ester. In certain embodiments, by-product 56 is obtained from the recovery of product alcohol 54. By-product 56 does not contain carboxylic acid 28 that can be recovered from hydrolysis of the alcohol ester.

  In certain embodiments, the hydrolysis of the fatty acid alcohol ester present in the ester-containing organic phase 36 to the product alcohol and free fatty acid is in a ratio of fatty acid to water of about 10: 1 to about 1:10, or In other embodiments, it is performed at a fatty acid to water ratio of about 100: 1 to about 1: 100. In certain embodiments, the alcohol ester of a fatty acid is hydrolyzed with water at a temperature less than about 100 ° C. In certain embodiments, the hydrolysis is performed at a temperature greater than 100 ° C, greater than 150 ° C, greater than 200 ° C, or greater than 250 ° C.

For example, in certain embodiments, the alcohol ester can be transesterified to produce the product alcohol 54, and in certain embodiments, a secondary alcohol ester 56, such as a fatty acid alkyl ester, can also be a byproduct. 56 may be generated. To perform such transesterification, the alcohol ester can be contacted with a catalyst capable of transesterifying the alcohol ester to release butanol. In certain embodiments, the alcohol ester can be transesterified with glycerol to produce the product alcohol 54 and acylglycerides as by-products 56. The acylglycerides produced can include mono- and diacylglycerides. Some suitable catalysts for the transesterification reaction include, for example, lipase enzymes, especially alkoxide salts of secondary alcohols, soluble inorganic acids such as alkyl titanates, sulfuric acid and phosphoric acid, toluenesulfonic acid and naphthalenesulfonic acid, etc. Soluble organic acids and solid acids such as Amberlyst® sulfonated polystyrene resins, or zeolites. Suitable lipases for transesterification or hydrolysis include, but are not limited to, Burkholderia cepacia, Thermomyces lanuginosa, or Candida antartica. Originating lipase is mentioned. In certain embodiments, the lipase is immobilized on a soluble or insoluble carrier using methods well known to those skilled in the art (eg, Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, NJ, USA, 1997). Enzyme immobilization is: 1) binding of the enzyme to a porous or non-porous carrier carrier by covalent support, physical adsorption, electrostatic binding, or affinity binding; 2) bifunctional or polyfunctional reagents Can be performed using a variety of techniques including: 3) gel matrix, polymer, emulsion, or confinement in some form of membrane; and 4) any combination of these methods. In other embodiments, the lipase may not be immobilized. In certain embodiments, the lipase is soluble. The fatty acid alkyl ester 56 may include, for example, a fatty acid methyl ester. Other fatty acid alkyl esters 56 may include, for example, C _{2 to} C ₁₂ linear, branched, and cyclic alcohol esters. The product alcohol 54 can then be separated from the reaction mixture containing the by-product 56 using any separation means known in the art such as, for example, distillation. Other suitable separation mechanisms can include, for example, extraction and membrane separation.

  In certain embodiments, at least about 5% of the product alcohol, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least About 80%, at least about 90%, or at least about 99% is recovered from the alcohol ester.

  ISPR extractant (carboxylic acid) 28 can be separated from the alcohol ester prior to reaction of the alcohol ester to recover product alcohol 54. Alternatively, after the reaction of the alcohol ester, ISPR extractant 28 can be separated from the product alcohol and any by-products. The resulting recovered dilute extractant 27 is then typically derived from the fresh make-up extractant 28 (if supplied, oil 26 for further production and / or extraction of alcohol esters. In combination) can be recycled back to the fermenter 30. Alternatively, fresh extractant 28 (or oil 26) can be continuously added to the fermentor to replace the extractant removed in the two-phase mixed stream 39.

  In certain embodiments, the catalyst 42 can be recovered from the two-phase mixture 39 and reused in steps during the fermentation process such as fermentation itself or product alcohol recovery.

  In certain embodiments, one or more additional ISPR extractants 29 (see FIG. 2) can be introduced into the fermentor 30 to form a two-phase mixture comprising an aqueous phase and an organic phase. In such an embodiment, ISPR extractant 29 is oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, 1-undecanol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, It can be an external organic extractant such as methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal, and mixtures thereof. However, for the reasons described above, ISPR extractant 29 is preferably not an alcohol. Rather, ISPR extractant 29 is preferably a carboxylic acid (eg, a free fatty acid). In certain embodiments, ISPR extractant 29 is COFA. In certain embodiments, ISPR extractant 29 is a fatty acid derived from linseed oil fatty acid, soybean oil fatty acid, jatropha oil fatty acid, or palm oil, castor oil, olive oil, coconut oil, peanut oil, or any seed oil. In certain embodiments, ISPR extractant 29 is a co-pending and co-assigned US provisional patent application filed July 28, 2010, which is incorporated herein by reference. Fatty acids, fatty alcohols, fatty amides, fatty acid esters (especially 1-8 carbons in the alcohol portion) obtained from chemical conversion of natural oils such as biomass lipids as described in 61 / 368,436 It may be a fatty acid extractant selected from the group consisting of those containing atoms, such as fatty acid methyl esters and lower alcohol esters of fatty acids), fatty acid glycol esters, hydroxylated triglycerides, and mixtures thereof. In certain embodiments, ISPR extractant 29 is a free fatty acid obtained by chemical hydrolysis of biomass lipids. In certain embodiments, ISPR extractant 29 is a co-pending and co-assigned US provisional patent application filed July 28, 2010, which is incorporated herein by reference. It may be a free fatty acid produced from the enzymatic hydrolysis of natural oils such as biomass lipids as described in 61 / 368,444.

  In-situ product removal can be performed in the fermenter 30 in batch mode or continuous mode. In continuous mode of in-situ product removal, product is continuously removed from the tank (or reactor). In a batch mode of in-situ product removal, a volume of organic extractant is added to the fermentor and the extractant is not removed during the process. In situ product removal, the organic extractant can be contacted with the fermentation medium at the start of fermentation to form a two-phase fermentation medium. Alternatively, the organic extractant can be contacted with the fermentation medium after the microorganism has achieved the desired amount of growth (which can be determined by measuring the optical density of the medium). In addition, the organic extractant can be contacted with the fermentation medium when the product alcohol level in the fermentation medium reaches a preselected level. In the case of butanol production according to an embodiment of the present invention, at a point before the butanol concentration reaches a toxic level, the carboxylic acid extractant is contacted with the fermentation medium to esterify butanol with the carboxylic acid to produce a butyl ester. In certain embodiments, a biphasic mixture comprising an aqueous phase and an organic phase comprising a butyl ester can be produced. Thus, the concentration of butanol in the fermentor is reduced, and as a result, the toxic effects of butanol on microorganisms are minimized. Next, after the desired effective titer of butyl ester has been achieved, the ester-containing organic phase can be removed from the fermentor (and separated from the fermentation broth that constitutes the aqueous phase). For example, in certain embodiments, after the effective titer of butyl ester exceeds about 10 g per kg of fermentation broth, the ester-containing organic phase can be separated from the fermentation broth. In other embodiments, the effective titer of butyl ester is greater than about 230 g per kg fermentation broth, greater than about 300 g per kg fermentation broth, greater than about 400 g per kg fermentation broth, greater than about 500 g per kg fermentation broth, Alternatively, after greater than about 600 grams per kg of fermentation broth, the ester-containing organic phase can be separated from the fermentation medium. In another embodiment, after the% conversion of COFA is at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%, the ester-containing organic phase is It can be separated from the medium. In certain embodiments, the ester-containing organic phase is separated from the aqueous phase after the fermentation of available fermentable sugars in the fermentor is substantially complete.

  In the example embodiment described in FIG. 1, the alcohol ester is extracted in situ from the fermentation broth and the separation of the two-phase mixture 39 occurs in a separate tank 35. In certain embodiments, the separation of the two-phase mixture can be performed in a fermentor, as shown in the example embodiments of FIGS. 3 and 4 described below, in which the ester-containing organic phase stream 36 is Get out of fermenter 30 directly. The aqueous phase stream 34 can also exit the fermenter 30 directly and can be treated to remove any remaining alcohol ester or product alcohol, reused, or discarded and replaced with fresh fermentation medium. Extraction of the alcohol ester and product alcohol with the organic extractant can be performed with or without removal of the microorganisms 32 from the fermentation broth. Microorganism 32 may be removed from the fermentation broth by means known in the art, including but not limited to filtration or centrifugation. For example, the aqueous phase stream 34 may include microorganisms 32 such as yeast. The microorganisms 32 can be easily separated from the aqueous phase flow, for example, in a centrifuge (not shown). Thereafter, the microorganisms 32 can be reused in the fermenter 30, and the fermenter 30 can increase the production rate of alcohol production with the passage of time, thereby increasing the efficiency of alcohol production.

  As should be apparent to those skilled in the art, in one embodiment, the system and method of FIG. 1 allows the simultaneous saccharification and fermentation in fermentor 30 to be replaced with a separate saccharification tank 60 before fermentor 30. (See, for example, the embodiment of FIG. 4).

  In still other embodiments, for example, as shown in the example embodiment of FIG. 2, natural oil 26 is fed to a tank 40 that is also fed with a catalyst 42 (instead of being fed directly to the fermenter 30). , Thereby hydrolyzing at least a portion of the acylglycerides in oil 26 to form carboxylic acid 28. Next, the product stream from the tank 40 containing the carboxylic acid 28 and the catalyst 42 is introduced into the fermenter 30. Carboxylic acid 28 and catalyst 42 are in contact with the product alcohol produced in the fermentation medium, whereby the alcohol ester of the product alcohol is produced in the same manner as described above with reference to FIG. Formed in situ from the catalyzed esterification of carboxylic acids by. Carboxylic acid 28 can also serve as an ISPR extractant, and in certain embodiments, sufficient carboxylic acid 28 and / or one or more additional ISPR extractants 29 are introduced into fermentor 30 to provide aqueous and organic phases. A biphasic mixture containing phases can be formed and the alcohol ester separates into the organic phase. The remaining process operations of the embodiment of FIG. 2 are the same as FIG. 1 and will not be described again in detail.

  In certain embodiments of the invention, for example, as shown in the embodiment of FIG. 3, catalyst 42 may be added to feed slurry 16 that includes oil 26 ′ derived from biomass that forms feed 12. In the embodiment shown, catalyst 42 can hydrolyze glycerides in oil 26 'to free fatty acids 28'. Therefore, after the introduction of the catalyst 42 into the raw slurry 16, at least a part of the glycerides in the oil 26 'is hydrolyzed to obtain the raw slurry 18 having the free fatty acid 28' and the catalyst 42. For example, when the raw material 12 is corn, the oil 26 'is corn oil which is a component of the raw material, and the free fatty acid 28' is corn oil fatty acid (COFA).

  The raw material slurry 18 is introduced into the fermenter 30 together with the alcohol-producing microorganism 32 so as to be included in the fermentation medium. In certain embodiments, an enzyme 38, such as glucoamylase, may also be introduced into the fermentor to simultaneously saccharify the sugars in the slurry 18 and ferment the alcohol within the fermentor 30. The presence of catalyst 42 (introduced via slurry 18) in the fermentor catalyzes the esterification of alcohol by free fatty acid 28 '(introduced via slurry 18), see FIG. In the same way as described above, the fatty acid alcohol ester is formed in situ. In one embodiment, for butanol production, butanol producing microorganisms 32 are introduced into the fermentor 30 along with the raw slurry 18. Catalyst 42 in the fermenter (introduced via slurry 18) catalyzes the esterification of butanol with free fatty acid 28 '(introduced via slurry 18) to produce fatty acid butyl ester (FABE). Form in position. Free fatty acid 28 'can also act as an ISPR extractant. For example, if the free fatty acid 28 'is COFA, an alcohol ester of COFA is formed in situ, and COFA acts as an ISPR extractant or part thereof.

  In certain embodiments, one or more additional ISPR extractants 29 can be introduced into the fermentor 30 to preferentially separate the alcohol ester (and any free alcohol) from the aqueous phase. In certain embodiments, ISPR extractant 29 can be carboxylic acid 28 described with reference to the embodiments of FIGS. In certain embodiments, ISPR extractant 29 is introduced into fermentor 30 as oil 26 and then hydrolyzed to fatty acids by catalyst 42 to become ISPR extractant 29. In certain embodiments, oil 26 is corn oil, whereby ISPR extractant 29 is COFA. In certain embodiments, as described above with reference to the embodiments of FIGS. 1 and 2, ISPR extractant 29 may be a fatty acid, fatty alcohol, fatty amide, fatty acid ester (particularly 1-8 carbon atoms in the alcohol moiety). A fatty acid extractant selected from the group consisting of fatty acid methyl esters and lower alcohol esters of fatty acids), fatty acid glycol esters, hydroxylated triglycerides, and mixtures thereof. In still other embodiments, ISPR extractant 29 can be a free fatty acid obtained by chemical or enzymatic hydrolysis of biomass lipids. In such an embodiment, the biomass lipids for producing the extractant 29 may be derived from the same or different biomass sources from which the feedstock 12 is obtained. For example, in certain embodiments, the biomass lipid for producing the extractant 29 may be derived from soybeans, but the biomass source of feedstock 12 is corn. Any possible combination of different biomass sources for the extractant 29 relative to the feedstock 12 can be used, as should be apparent to those skilled in the art. The remaining process operations of the embodiment of FIG. 2 are the same as FIG. 1 and will not be described again in detail.

  As a non-limiting data-proven example, referring to the embodiment of FIG. 3, ground whole corn (as raw material 12) that can typically contain about 4% by weight corn oil. )) Can be treated with amylase (as liquefying enzyme 14) at about 85 ° C. to 120 ° C. for 30 minutes to 2 hours, and the resulting liquefied mash 16 can be treated at 65 ° C. to 30 ° C. At a pH of 4.5 to 7.5 (in certain embodiments, a pH of 5.5 to 6.5) and a conversion of available fatty acid content in the lipid to free fatty acids at least 30% to at least Treated with 0.1 ppm to 10 ppm (in one embodiment, 0.5 ppm to 1.0 ppm) lipase (as catalyst 42) for a time sufficient to be as high as 99%. That. The liquefied and lipase-treated mash 18 can be cooled to about 30 ° C. (eg, using a heat exchanger) and charged to the fermenter 30 with about 25% to 30% by weight dry corn solids. Glucose can be produced by saccharification of the liquefied mash 18 during fermentation by the addition of glucoamylase (as saccharifying enzyme 38). The resulting fermentation broth uses liquefied mash that has not been treated with lipase 42 to produce an amount significantly less than the amount of corn oil that may be present in the broth (eg, about 1.2 wt% corn oil). May be contained. In particular, lipase 42 treatment converts corn oil lipid 26 '(triglyceride (TG)) to COFA 28' (and some diglyceride (DG) or monoglyceride (MG)) in COFA ISPR extraction solvent 28 'or 29. The rate of accumulation of lipid 26 'can be reduced. The lipase 42 treatment can also convert the butanol produced during fermentation to the butyl ester of COFA, where the butyl ester of COFA is about dissolution in the COFA phase 36 during liquid-liquid extraction ISPR. Has a high distribution coefficient. At the end of the fermentation, the COFA phase 36 containing the butyl ester of COFA can be separated from the fermentation broth (in tank 30/35) and butanol 54 can be, for example, without limitation, lipase 52, solid acid catalyst 52 or The butanol 54 and COFA 27 can be produced from this organic mixture 36 (in tank 50) using one of several methods including ester hydrolysis using steam 52.

  In still other embodiments, for example, as shown in the embodiment of FIG. 4, the system and method of FIG. It can be changed to be replaced by the tank 60. FIG. 4 includes a separate saccharification tank 60 that receives the enzyme 38 and is substantially identical to FIG. 3 except that the catalyst 42 is introduced into the liquefied and saccharified feed stream 62. The raw material slurry 16 is introduced into the saccharification tank 60 together with an enzyme 38 such as glucoamylase, whereby the saccharides in the form of oligosaccharides in the slurry 16 can be decomposed into monosaccharides. The liquefied and saccharified raw material stream 62 exits the saccharification tank 60 and the catalyst 42 is introduced into it. Feed stream 62 includes monosaccharides derived from the feed, and oil 26 'and undissolved solids. The oil 26 ′ is hydrolyzed by the introduction of the catalyst 42, and a liquefied and saccharified raw material slurry 64 having the free fatty acid 28 ′ and the catalyst 42 is obtained.

  Alternatively, in some embodiments, similar to the introduction of enzyme 38 and catalyst 42 into fermenter 30 for SSF in the embodiment of FIG. 1, catalyst 42 is added with saccharifying enzyme 38 to produce glucose. At the same time, the oil lipid 26 'can be hydrolyzed to the free fatty acid 28'. The addition of enzyme 38 and catalyst 42 can be gradual (eg, catalyst 42, then enzyme 38, or vice versa) or simultaneous. However, the addition of the catalyst 42 to the fermentor 30 during SSF is also in contrast to the embodiment of FIG. No alcohol ester is formed until it is introduced. Alternatively, in some embodiments, slurry 62 can be introduced into fermentor 30 and catalyst 42 is added directly to fermentor 30.

  In the embodiment of FIG. 4, slurry 64 is introduced into fermenter 30 along with alcohol-producing microorganisms 32 that metabolize monosaccharides to produce product alcohol. The presence of catalyst 42 (introduced via slurry 64) in the fermentor catalyzes the esterification of alcohol by free fatty acid 28 '(introduced via slurry 62), see FIG. In the same way as described above, the fatty acid alcohol ester is formed in situ. Free fatty acid 28 'can also serve as an ISPR extractant to preferentially separate the alcohol ester (and any free alcohol) from the aqueous phase. In certain embodiments, one or more additional ISPR extractants 29 can also be introduced into the fermentor 30 as described above with reference to FIG. The remaining process operations of the embodiment of FIG. 4 are the same as FIG. 3 and will not be described again in detail.

  In certain embodiments, including any of the embodiments described above with reference to FIGS. 1-4, undissolved solids may be removed from the raw slurry 16 before being introduced into the fermenter 30. For example, as shown in the embodiment of FIG. 5, raw slurry 16 is introduced into the inlet of a separator 20 that is configured to discharge undissolved solids as a solid phase or wet cake 24. For example, in some embodiments, the separator 20 can be a filter press, vacuum filtration, mechanical pressure filtration, or centrifuge (eg, decanter centrifuge) for separating undissolved solids from the raw slurry 16. May be included. In certain embodiments, any conventional centrifuge used in the industry, including, for example, a decanter bowl centrifuge, a tricanter centrifuge, a disk stack centrifuge, a filtration centrifuge, or a decanter centrifuge Can be used to separate undissolved solids. In certain embodiments, removal of undissolved solids from the raw slurry 16 may include filtration, vacuum filtration, belt filter, pressure filtration, sieving filtration, sieving separation, grate or lattice, porous lattice, It can be done by flotation, hydroclone, filter press, screw press, gravity settling, vortex separator, or any method that can be used to separate solids from liquids. Optionally, in some embodiments, separator 20 can be configured to remove some or substantially all of oil 26 ′ present in feed slurry 16. In such embodiments, separator 20 removes oil from aqueous feed streams, including but not limited to, siphoning, decantation, centrifugation, use of gravity settling, membrane assisted phase separation, and the like. Can be any suitable separator known in the art. The remaining raw material containing sugar and water is discharged to the fermenter 30 as a water stream 22.

  For example, in certain embodiments, separator 20 includes an aqueous layer containing sugar and water (ie, stream 22), a solid layer containing undissolved solids (ie, wet cake 24), and an oil layer (ie, oil stream). A tricanter centrifuge 20 that agitates or rotates the raw slurry 16 to produce a centrifuge product comprising 26 '). In such cases, catalyst 42 can be contacted with removed oil 26 'to produce a stream of free fatty acids 28' and catalyst 42. Next, a stream of free fatty acid 28 'and catalyst 42 can be introduced into fermentor 30 for contact with the fermentation medium, thereby catalyzing the esterification of the product alcohol in the fermentation medium to a fatty acid alcohol ester. Can be done in situ in the same manner as described above with reference to FIG.

  The free fatty acid 28 ′ can act as an ISPR extractant 28 ′ and one or more additional ISPR extractants 29 can also be introduced into the fermenter 30. Thus, the feedstock 26 'can be catalytically hydrolyzed to carboxylic acids, thereby reducing the amount of lipid present in the ISPR extractant, while also producing an ISPR extractant. The ester-containing organic phase 36 can be separated from the aqueous phase 34 of the two-phase mixture 39 in the tank 35 and the product alcohol can be recovered from the alcohol ester in the tank 50 (see FIG. 1). The remaining process operations of the embodiment of FIG. 5 are the same as FIG. 3 and will not be described again in detail.

  When the wet cake 24 is removed by the centrifuge 20, in some embodiments, when the raw material is corn, some of the oil from the raw material 12, such as corn oil, remains in the wet cake 24. After the aqueous solution 22 has been drained from the centrifuge 20, the wet cake 24 can be washed with additional water in the centrifuge. By washing the wet cake 24, sugar (eg, oligosaccharide) present in the wet cake is recovered, and the recovered sugar and water can be reused in the liquefaction tank 10. After washing, the wet cake 24 can be combined with solubles by any suitable known method and then dried to form a soluble-containing dried cereal distillation koji (DDGS). There are several advantages to forming DDGS from the wet cake 24 formed in the centrifuge 20. Because non-dissolved solids are not sent to the fermentor, DDGS does not capture product alcohol such as extractant and / or butanol, and DDGS is not subjected to fermentor conditions and microorganisms present in the fermentor Do not contact with. All these advantages make it easier to process and sell DDGS as animal feed, for example. In certain embodiments, the oil 26 'is not drained separately from the wet cake 24, and the oil 26' is included as part of the wet cake 24 and is ultimately present in the DDGS. In such cases, the oil can be separated from DDGS and converted to ISPR extractant 29 for subsequent use in the same or different alcohol fermentation processes. A method and system for removing undissolved solids from a raw slurry 16 by centrifugation is a co-pending, identical application filed June 18, 2010, which is incorporated herein by reference in its entirety. It is described in detail in US Provisional Patent Application No. 61 / 356,290 owned by a human.

  As described above, oil 26 'can be separated from DDGS using any suitable known method including, for example, a solvent extraction process. In one embodiment of the present invention, DDGS is filled into an extraction vessel and washed with a solvent such as hexane to remove oil 26 '. Other solvents that can be used include, for example, petroleum distillates such as isobutanol, isohexane, ethanol, petroleum ether, or mixtures thereof. After extraction of oil 26 ', the DDGS can be treated to remove any residual solvent. For example, the DDGS can be heated to evaporate any residual solvent using any method known in the art. After removing the solvent, the DDGS can be subjected to a dry process to remove any residual water. Treated DDGS can be used as a supplementary feed for animals such as poultry, livestock, and pets.

  After extraction from DDGS, the resulting oil 26 'and solvent mixture can be collected to separate oil 26' from the solvent. In one embodiment, the oil 26 '/ solvent mixture may be processed by evaporation so that the solvent can be evaporated, collected and reused. The recovered oil can be converted to ISPR extractant 29 for subsequent use in the same or different alcohol fermentation processes.

  In addition to solid recovery, it may be desirable to recover other by-products of the fermentation process. In one embodiment, for example, fatty acid esters (eg, fatty acid isobutyl esters) can be recovered to increase the yield of carbohydrate to product alcohol (eg, butanol). This involves, for example, using a solvent to extract fatty acid isobutyl esters from the by-product formed by mixing several by-product streams in combination and drying the product of the combined and mixed process Can be done. Such a solvent-based extraction system for recovering corn oil triglycerides from DDGS is described in US 2010/0092603, the teachings of which are incorporated herein by reference. The

  In one embodiment of solvent extraction of fatty acid esters, solids can be separated from the total distillation effluent (“separated solids”), which is the most fatty acid ester in the by-product stream whose streams are not combined. It is because it may contain a proportion. These separated solids can then be fed to an extractor and washed with a solvent. In one embodiment, the separated solid is rotated at least once to ensure that all sides of the separated solid are washed with solvent. After washing, the resulting mixture of lipid and solvent, known as Misella, is collected for separation of the extracted lipid from the solvent. For example, the resulting mixture of lipid and solvent can be deposited in a separator for further processing. During the extraction process, as the solvent washes the separated solids, the solvent not only puts lipids into solution, but also collects solid particulates. These “particulates” are generally undesirable impurities in the micelles, and in one embodiment, the micelles can be discharged from the extractor or separator by a device that separates or scrubs the particulates from the micelles. .

  In order to separate the lipids and solvents contained in the miscella, the miscella can be subjected to a distillation step. In this process, the miscella can be processed, for example, by an evaporator, which is high enough to evaporate the solvent, but not high enough to adversely affect or evaporate the extracted lipids. Heat the miscella to temperature. As the solvent evaporates, the solvent can be collected, for example, in a condenser and reused for future use. Separation of the solvent from the miscella results in the accumulation of crude lipids, which can be further processed to separate water, fatty acid esters (eg, fatty acid isobutyl esters), fatty acids, and triglycerides.

  After lipid extraction, the solid can be sent out of the extractor and subjected to a stripping process to remove residual solvent. Recovery of residual solvent is important for process economy. In one embodiment, the wet solid may be sent to a vapor tight environment for storing and collecting the solvent that temporarily evaporates from the wet solid as the solvent is sent to the desolventizer. As the solid enters the desolventizer, the solid can be heated to evaporate and remove the residual solvent. To heat the solid, the desolvator may include a mechanism for distributing the solid to one or more trays, where the solid is heated directly, such as by direct contact with heated air or steam. Or heated indirectly such as by heating a tray carrying food. To facilitate the movement of solids from one tray to another, the tray carrying the solids can include openings that allow the solids to be transported from one tray to the next. From the desolvator, the solid may optionally be sent to a mixer, where the solid is mixed with other by-products before being sent to the dryer. An example of solid extraction is described in Example 63. In this example, the solid is fed to a desolvator where the solid is contacted by steam. In one embodiment, the vapor and solid streams in the desolvator may be retrograde. The solid may then exit the desolvator and be fed to a dryer or optionally a mixer, where various by-products can be mixed. The vapor exiting the desolvator can be condensed, optionally mixed with miscella, and then fed to the decanter. The water-enriched phase leaving the decanter may be fed to a distillation column where hexane is removed from the water-enriched stream. In one embodiment, the hexane-depleted water enriched stream may exit the distillation column bottom and be recycled back to the fermentation process, for example, to slurry ground corn solids. Can be used. In another embodiment, the top and bottom products can be reused in the fermentation process. For example, the lipid-enriched tower bottom can be added to the feed of the hydrolyzer. The top can be condensed, for example, and fed to a decanter. The hexane-enriched stream exiting this decanter can optionally be used as part of the solvent feed to the extractor. The water-enriched phase exiting this decanter can be fed to a column that removes hexane from the water. As those skilled in the art can appreciate, the method of the present invention can be modified in various ways to optimize the fermentation process for the production of product alcohols such as butanol.

  In another embodiment of the fatty acid ester solvent extraction, the solids can be separated from the beer and solvent discharged from the fermentation before being introduced into the preflash column as a heterogeneous mixture. These solid wet cakes can be formed using a separation device such as a sieving filter or a centrifuge. The solid screened cake can be displacement washed with hydrous isobutanol to remove the fatty acid ester retained in the wet solid. Alternatively, the solid centrifuged cake can be repulped in hydrous isobutanol and separated again to effect removal of the fatty acid ester retained in the wet solid. An example of this embodiment of solid extraction is described in Example 63.

  In a further embodiment, by-products (or by-products) are obtained from the mash used in the fermentation process. For example, corn oil can be separated from the mash, and the corn oil can contain triglycerides, free fatty acids, diglycerides, monoglycerides, and phospholipids (see, eg, Example 66). Corn oil may optionally be added to other by-products (or by-products) at various rates, thus creating, for example, the ability to change the amount of triglycerides in the resulting by-products. Thus, the fat content of the resulting by-product is controlled to provide a lower fat, higher protein animal feed that would, for example, better meet the needs of dairy cows compared to higher fat products. obtain.

  In one embodiment, the crude corn oil separated from the mash may be further processed into an edible oil for consumer use, or its high triglyceride content can provide an excellent source of metabolic energy, so animal feed It can also be used as a component of In another embodiment, the crude corn oil can also be used as a feedstock for biodiesel or renewable diesel.

  In one embodiment, the extractant by-product may be used in whole or in part as a component of animal feed by-product, or may be used as a raw material for biodiesel or renewable diesel.

  In further embodiments, the solid may be separated from the mash and may include triglycerides and free fatty acids. These solids (or streams) may be used as animal feed and are recovered either as a drain from centrifugation or after drying. The solid (or wet cake) may be particularly suitable as a ruminant (eg, dairy) feed due to the high content of available lysine and bypass or non-ruminal protein. For example, these solids can be particularly useful in high protein, low fat feeds. In another embodiment, these solids may be used as a base, i.e. other by-products such as syrup are added to this solid to form a product that can be used as animal feed. May be. In another embodiment, various amounts of other by-products may be added to the solid to adjust the properties of the resulting product to meet the needs of a particular animal species.

  The solid composition separated from the total distillation effluent described in Example 62 can include, for example, crude protein, fatty acid, and fatty acid isobutyl ester. In one embodiment, the composition (or by-product) may be used wet or dry as animal feed, in which case, for example, high protein (eg, high lysine), low fat, and high Fiber content is desired. In another embodiment, if a higher fat, low fiber animal feed is desired, fat may be added to the composition, eg, from another byproduct stream. In one embodiment, this higher fat, low fiber animal feed can be used in pigs or poultry. In further embodiments, non-aqueous compositions of Condensed Distillers Soluble (CDS) (see, eg, Example 66) include, for example, proteins, fatty acids, and fatty acid isobutyl esters and salts and carbohydrates, etc. Other dissolved and suspended solids can be included. The CDS composition may be used, for example, in a wet or dry state as animal feed, in which case a high protein, low fat, high inorganic salt feed component is desired. In one embodiment, the composition may be used as a component of dairy cow feed.

  In another embodiment, oil from the fermentation process can be recovered by evaporation. The non-aqueous composition may include a fatty acid isobutyl ester and a fatty acid (see, eg, Example 66), and this composition (or stream) is passed to a hydrolyzer to recover isobutanol and fatty acid. Can be supplied. In a further embodiment, this stream can be used as a feedstock for biodiesel production.

  The various streams produced by the production of alcohol (eg, butanol) by a fermentation process can be combined in many ways to produce several by-products. For example, if crude corn from mash is used to produce fatty acids that are used as extractants and the lipids are extracted by an evaporator for other purposes, the remaining streams are combined to produce crude protein, crude It can be processed to produce a byproduct composition comprising fat, triglyceride, fatty acid, and fatty acid isobutyl ester. In one embodiment, the composition comprises at least about 20-35% crude protein, at least about 1-20% crude fat, at least about 0-5% triglyceride, at least about 4-10% by weight. Fatty acids, and at least about 2-6% by weight fatty acid isobutyl esters may be included. In one particular embodiment, the byproduct composition comprises about 25% crude protein, about 10% crude fat, about 0.5% triglyceride, about 6% fatty acid, and about 4% by weight. Fatty acid isobutyl ester may be included.

  In another embodiment, the lipid is extracted by an evaporator, the fatty acid is used for other purposes, about 50% by weight of the crude corn from the mash and the remaining stream are combined, processed and obtained. The resulting by-product composition can include crude protein, crude fat, triglyceride, fatty acid, and fatty acid isobutyl ester. In one embodiment, the composition comprises at least about 25-31% crude protein, at least about 6-10% crude fat, at least about 4-8% triglyceride, at least about 0-2% by weight. Fatty acids, and at least about 1-3% by weight fatty acid isobutyl esters may be included. In one particular embodiment, the byproduct composition comprises about 28% crude protein, about 8% crude fat, about 6% triglyceride, about 0.7% fatty acid, and about 1% by weight. Fatty acid isobutyl ester may be included.

  In another embodiment, 50% by weight of the corn oil extracted from the mash and solids separated from total distillate waste is combined and the resulting by-product composition is crude protein, crude fat, triglyceride, fatty acid, fatty acid isobutyl Esters, lysines, neutral detergent fibers (NDF), and acidic detergent fibers (ADF) can be included. In one embodiment, the composition comprises at least about 26-34% crude protein, at least about 15-25% crude fat, at least about 12-20% triglyceride, at least about 1-2% by weight. Fatty acids, at least about 2-4% by weight fatty acid isobutyl ester, at least about 1-2% by weight lysine, at least about 11-23% by weight NDF, and at least about 5-11% by weight ADF. In one particular embodiment, the by-product composition comprises about 29% crude protein, about 21% crude fat, about 16% triglyceride, about 1% fatty acid, about 3% fatty acid isobutyl ester. About 1% by weight lysine, about 17% by weight NDF, and about 8% by weight ADF. The high fat, high triglyceride, and high lysine content and lower fiber content of this by-product composition may be desirable for pig and poultry feed.

  As noted above, the various streams generated by the production of alcohol (eg, butanol) by a fermentation process are often used to produce by-product compositions that include crude protein, crude fat, triglycerides, fatty acids, and fatty acid isobutyl esters. Can be combined in the following manner. For example, a composition comprising at least about 6% crude fat and at least about 28% crude protein can be used as an animal feed product for dairy animals. A composition comprising at least about 6% crude fat and at least about 26% crude protein can be used as an animal feed product for feedlot cattle, while at least about 1% crude fat and at least about 27% crude protein. The containing composition can be used as an animal feed product for wintering cattle. Compositions comprising at least about 13% crude fat and at least about 27% crude protein can be used as poultry animal feed products. Compositions comprising at least about 18% crude fat and at least about 22% crude protein can be used as monogastric animal feed products. Thus, the various streams can be combined to customize the feed product for a particular animal species.

  In one embodiment, one or more streams produced by the production of alcohol (eg, butanol) by a fermentation process produces a composition comprising at least about 90% COFA that can be used as a fuel source, such as biodiesel. Can be combined in many ways.

  As an example of one embodiment of the method of the present invention, ground cereal (eg, corn treated with a hammer mill) and one or more enzymes are combined to produce a slurryed cereal. This slurryed cereal is cooked, liquefied and optionally flushed with flash steam to obtain a cooked mash. The cooked mash is then filtered to remove suspended solids and produce a wet cake and filtrate. Filtration can be performed by several methods, such as centrifugation, sieving, or vacuum filtration, which can remove at least about 80% to at least about 99% suspended solids from the mash.

  The wet cake is reslurried with water and refiltered to remove additional starch and produce a washed filter cake. The reslurry process may be repeated several times, for example 1-5 times. The water used to reslurry the wet cake may be recycled water produced during the fermentation process. The filtrate produced by the reslurry / refiltration process can be returned to the initial mixing step to form a slurry with the milled cereal. The filtrate can be heated or cooled prior to the mixing step.

  The washed filter cake can be reslurried with beer at several stages during the production process. For example, the washed filter cake can be reslurried with beer after the fermenter, before the preflash tower, or at the feed point to the cereal dryer of the still. The washed filter cake may be dried separately from other by-products or used directly as a wet cake for the production of DDGS or animal feed products.

  The filtrate produced as a result of the initial mixing step can be further processed as described herein. For example, the filtrate can be heated using steam or process to process heat exchange. A saccharification enzyme may be added to the filtrate, and the dissolved starch in the filtrate may be partially or fully saccharified. The saccharified filtrate can be cooled by several means such as interprocess exchange, cooling water exchange, or cold water exchange.

  The cooled filtrate can then be added to a fermentation apparatus as well as a recombinant yeast capable of producing a microorganism suitable for alcohol production, such as butanol. In addition, ammonia and a recycle stream can also be added to the fermenter. The process may include at least one fermenter, at least two fermenters, at least three fermenters, or at least four fermenters. In order to reduce atmospheric emissions (eg, butanol atmospheric emissions) and increase product yield, the carbon dioxide produced during fermentation can be vented to a scrubber.

  The solvent may be added to the fermentor via a recycling loop or may be added directly to the fermentor. The solvent can be one or more organic compounds that can dissolve or react with an alcohol (eg, butanol) and can have limited solubility in water. The solvent may be removed from the fermenter in succession as one liquid phase or as two liquid phase materials, or the solvent may be withdrawn batchwise as one or two liquid phase materials.

  The beer can be degassed. The beer can be heated prior to degassing, for example, by inter-process exchange with a hot mash or pre-flash tower overhead. Vapor may be exhausted to a condenser and then to a scrubber. The degassed beer can be further heated, for example, by inter-process heat exchange with other streams in the distillation zone.

  Preheated beer and solvent may enter a preflash tower that can be modified from the beer tower of a conventional dry ground fuel ethanol plant. The column is operated at a pressure below atmospheric pressure and can be driven by water vapor taken from a series of evaporators or from a mash cooking process. The top of the preflash tower can be condensed by heat exchange by several combinations of cooling water and interprocess heat exchange including heat exchange with the preflash tower feed. The liquid condensate can be sent to an alcohol / water decanter (eg, butanol / water decanter).

  The bottom of the preflash tower can be advanced to a solvent decanter. The bottom of the preflash tower can be substantially free of free alcohol (eg, butanol). The decanter can be a still well, a centrifuge, or a hydroclone. Water is substantially separated from the solvent phase in the decanter, producing an aqueous phase. The aqueous phase containing suspended and dissolved solids can be centrifuged to produce a wet cake and dilute distillate. The wet cake can be combined with other streams and dried to produce DDGS, which can be dried and sold separately from other streams that produce DDGS, or it can be sold as a wet cake . The aqueous phase can be split to provide a backset that is partially used to reslurry the filter cake. The split also provides a dilute distillation waste that can be injected into the evaporator for further processing.

  The organic phase produced in the solvent decanter can be an ester of an alcohol (eg, butanol). The solvent can be hydrolyzed to regenerate the reactive solvent and recover additional alcohol (eg, butanol). Alternatively, the organic phase can be filtered and sold as a product. The hydrolysis is driven by heat and can be obtained with a homogeneous catalyst or with a heterogeneous catalyst. Hydrolysis can also occur by enzymatic reactions. The heat injection into this process can be a furnace, hot oil, electrical heat injection, or high pressure steam. The water added to drive hydrolysis can come from recycled water streams, fresh water, or steam.

  The cooled and hydrolyzed solvent may be injected into a solvent tower at a pressure below atmospheric pressure, where the solvent can substantially remove alcohol (eg, butanol) by steam. This steam may be water vapor from the evaporator, may be steam from the flash step of the mash process, or may be steam from a boiler (e.g., incorporated herein by reference). U.S. Patent Application Publication No. 2009/0171129). A rectifying tower from a conventional dry grinding ethanol plant may be suitable as a solvent tower. The rectifying tower can be modified to act as a solvent tower. The bottom of the solvent tower can be cooled, for example, by cooling water or interprocess heat exchange. The cooled bottoms may be decanted to remove residual water, and this water may be reused for other steps including this process, or may be reused for the mash step. .

  The top of the solvent column may be cooled by exchange with cooling water or by inter-process heat exchange, and the condensate may be evacuated alcohol / water decanter (e.g., which can be shared with the preflash tower top. Butanol / water decanter). Other mixed water and alcohol (eg, butanol) streams, including the scrubber tower bottoms and condensate from the degassing step, may be added to the decanter. Exhaust gas containing carbon dioxide may be sent to the water scrubber. The decanter aqueous layer may also be fed to a solvent column or alcohol (eg, butanol) may be removed in a small dedicated distillation column. The aqueous layer may be preheated by interprocess exchange with preflush tower tops, solvent tower tops, or solvent tower bottoms. This dedicated tower can be retrofitted from a conventional dry ground fuel ethanol process side stripper.

  The organic layer of alcohol / water decanter (eg, butanol / water decanter) can be injected into an alcohol (eg, butanol) tower. This tower may be an overpressure tower and may be driven by vapor condensation in the reboiler. In order to reduce the energy demand for operating the tower, the feed to the tower can be heated by inter-process heat exchange. The inter-process heat exchanger may include a preflash tower partial condenser, a solvent tower partial condenser, a hydrolyzer product, water vapor from the evaporator, or a butanol tower bottom. The alcohol (eg, butanol) tower vapor condensate may be cooled and returned to the alcohol / water decanter (eg, butanol / water decanter). The bottoms of an alcohol (eg, butanol) tower may be cooled by inter-process heat exchange including exchange with alcohol (eg, butanol) tower feed, further cooled with cooling water, filtered, and product alcohol ( For example, it can be sold as butanol).

  The dilute waste liquor produced from the bottom of the preflash column described above can be sent to a multi-effect evaporator. The evaporator can have two, three, or more stages. The evaporator may have a four body configuration with two effects, similar to the conventional design of a fuel ethanol plant, may have a three bodies with three effects, or other configurations You may have. The dilute waste liquor can enter any of the plurality of utility parts. At least one of the first effect body may be heated by steam from an overpressure alcohol (eg, butanol) tower. Steam can be withdrawn from the lowest pressure utility and heat in the form of water vapor can be provided to preflash and solvent towers at pressures below atmospheric pressure. Syrup from the evaporator may be added to the cereal dryer of the still.

  Carbon dioxide emissions from fermenters, degassers, alcohol / water decanters (eg, butanol / water decanters) and other sources can be sent to a water scrubber. The water supplied to the top of the scrubber may be fresh makeup water or recycled water. The recycled water can be treated (eg, biologically digested) and quenched to remove volatile organic compounds. The scrubber tower bottom may be sent to an alcohol / water decanter (eg, butanol / water decanter), solvent tower, or used with other recycled water to reslurry the wet cake. Also good. Condensate from the evaporator can be processed by anaerobic biological digestion or other processes to purify the water before reuse to re-slurry the filter cake.

  When corn is used as a source of ground cereal, corn oil can be separated from the process stream in any of several ways. For example, a centrifuge may be operated to produce a corn oil stream after filtration of the cooked mash, or a preflush tower water phase centrifuge is operated to produce a corn oil stream. May be. The intermediate concentrated syrup for the final syrup can be centrifuged to produce a corn oil stream.

  In another example of an embodiment of the method of the present invention, the material discharged from the fermenter is processed in a separation system including devices such as a centrifuge, a settler, a hydrocyclone, etc., and combinations thereof, A concentrated form of live yeast can be recovered that can be reused for re-use in the next fermentation batch, either directly or after some reconditioning. This separation system can also produce an organic stream containing fatty acid esters (eg, isobutyl fatty acid esters) and alcohols (eg, isobutanol) produced from fermentation and an aqueous stream containing a slight amount of immiscible organics. This stream of water can be used before or after removing the alcohol (eg, isobutanol) content to repulp and inject the washed low starch solids separated from the liquefied mash. This has the advantage of avoiding what could otherwise be a long belt driven transport system to move these solids from the liquefaction zone to the grain drying and syrup mixing zone. Furthermore, this total distillation waste obtained after the alcohol (eg, isobutanol) has been removed needs to be separated into a dilute distillation waste and a wet cake fraction using existing or new separation equipment. This dilute waste liquor will partially form a back flow to combine with cooking water to prepare a new batch of fermentable mash. Another advantage of this embodiment is that a portion of any residual dissolved starch retained in the solid moisture separated from the liquefied mash can be captured and recovered through this backflow. Alternatively, the yeast contained in the solid stream can be considered non-viable and can be redistributed into the water stream, and this combined stream is freed of any alcohol (eg, butanol) content remaining from the fermentation by distillation. Non-viable organics can be further separated for use as nutrients in the propagation process.

  In another embodiment, the multiphase material exits the bottom of the preflash tower and can be processed in the separation system as described above. The concentrated solids can be redistributed into a water stream, and this combined stream can be separated from the liquefied mash and used to repulp and inject the washed low starch solids.

  The above process as well as other processes described herein may be demonstrated using computer modeling such as Aspen modeling (see, eg, US Pat. No. 7,666,282). For example, the commercially available modeling software Aspen Plus® (Aspen Technology, Inc., Burlington, Mass.) Is used by the American Institute to create Aspen models for integrated butanol fermentation, purification, and water management processes. of Chemical Engineers, Inc. (New York, NY) can be used with a physical property database such as DIPPR available. This process modeling can perform many basic engineering calculations such as mass and energy balance, gas / liquid equilibrium, and reaction rate calculations. To generate an Aspen model, information input includes, for example, experimental data, moisture content and ingredient composition, temperature for cooking and flushing mash, saccharification conditions (eg, enzyme ingredient, starch conversion, temperature, pressure) , Fermentation conditions (eg, microbial raw materials, glucose conversion, temperature, pressure), deaeration conditions, solvent towers, preflash towers, condensers, evaporators, centrifuges, and the like.

  The present invention provides systems and methods for producing fermentation products, such as product alcohols, by fermentation while increasing the process productivity and cost effectiveness of biomass. In certain embodiments, the product alcohol is butanol. The raw material can be liquefied to produce a raw slurry, where the raw slurry includes soluble sugars and undissolved solids. If the raw slurry is fed directly to the fermentor, the undissolved solids can prevent efficient removal and recovery of product alcohol, such as butanol, from the fermentor. In particular, when liquid-liquid extraction is used to extract butanol from the fermentation broth, the presence of undissolved particles reduces the mass transfer rate of butanol to the extractant by preventing contact between the extractant and the fermentation broth. Reducing; producing an emulsion in the fermentor, thereby preventing good phase separation of the extractant and fermentation broth; to a solid where at least a portion of the extractant and butanol is ultimately removed as DDGS Reducing the efficiency of recovering and reclaiming the extractant because it is “trapped”; because there is a solid that occupies volume in the fermenter, and because the extraction agent is more slowly removed from the fermentation broth Lower volumetric efficiency of the system; and non-existence of systems including, but not limited to, shortening of extractant life due to contamination with corn oil It can lead to rate. All these effects increase capital and operating costs. In addition, extractants that are “trapped” by DDGS can deviate from DDGS values and requirements for selling as animal feed. Therefore, to avoid and / or minimize these problems, at least some of the undissolved particles (or solids) are removed from the raw slurry before adding the sugar present in the raw slurry to the fermentor. Is done. When the extraction activity and efficiency of butanol formation is performed in a fermentation broth containing an aqueous solution from which undissolved particles have been removed, compared to an extraction performed in a fermentation broth containing an aqueous solution from which undissolved particles have not been removed Enhanced.

  Extractive fermentation without the presence of undissolved solids results in a higher mass transfer rate of product alcohol from the fermentation broth to the extractant, better phase separation of the extractant from fermentation inside or outside the fermenter, and higher It may result in a lower hold of the extractant as a result of the extractant drop rise rate. Also, for example, extractant droplets that are retained in the fermentation broth during fermentation will disengage faster and more completely from the fermentation broth, thereby reducing the free extractant in the fermentation broth and during the process. The amount of extractant lost can be reduced. Further, for example, the microorganisms can be reused, and further equipment in downstream processing can be omitted, such as, for example, some or all of the beer tower and / or total distillation waste centrifuge. Furthermore, for example, the possibility of the extractant being lost in DDGS is eliminated. Also, for example, the ability to recycle microorganisms increases the overall rate of product alcohol production, reduces total titer requirements, and / or reduces aqueous titer requirements, thereby healthier microorganisms and higher The production rate can be brought about. In addition, for example, omitting the agitator in the fermenter to reduce capital costs; because the extractant retained is minimized and the volume is used more efficiently because there are no undissolved solids It may be possible to improve the productivity of the fermenter; and / or to use continuous fermentation or a smaller fermenter in an unconstructed plant.

  Examples of improved extraction efficiencies include, for example, a stabilized partition coefficient, improved (eg, faster or more complete) phase separation, improved liquid-liquid mass transfer coefficient, operation at lower titers, improved Process stream recyclability, improved fermentation volumetric efficiency, improved feedstock (eg corn) loading, improved butanol titer tolerance of microorganisms (eg recombinant microorganisms), water reuse, energy reduction, extractant There may be increased reuse and / or microbial reuse.

  For example, the volume occupied by solids in the fermenter is reduced. Therefore, the effective volume of the fermentation apparatus available for fermentation can be increased. In certain embodiments, the volume of fermenter available for fermentation is increased by at least about 10%.

  For example, there can be stabilization of the distribution coefficient. Since the corn oil in the fermenter can be reduced by removing solids from the raw slurry prior to fermentation, the extractant is exposed to less corn oil. Corn oil can be combined with an extractant to reduce the partition coefficient when present in sufficient amounts. Therefore, the distribution coefficient of the extractant phase in the fermenter becomes more stable due to the reduction of corn oil introduced into the fermenter. In certain embodiments, the partition coefficient is reduced by less than about 10% over 10 fermentation cycles.

  For example, there is a higher mass transfer rate of the product alcohol from the fermentation broth to the extractant (eg, in the form of a higher mass transfer coefficient), thereby resulting in improved efficiency of product alcohol production. There may be an improvement in the extraction efficiency of butanol by the agent. In certain embodiments, the mass transfer coefficient is increased at least 2-fold (see Examples 4 and 5).

  Furthermore, there may be an improved phase separation between the fermentation broth and the extractant, which reduces the likelihood of emission formation, thereby resulting in improved efficiency of product alcohol production. For example, phase separation can occur faster or can be more complete. In certain embodiments, phase separation can occur when no appreciable phase separation has been observed in advance for 24 hours. In certain embodiments, phase separation occurs at least about 2 times faster, at least about 5 times faster, or at least about 10 times faster than phase separation when no solids are removed (Examples 6 and 7). ).

  Furthermore, there may be an increase in extractant recovery and reuse. The extractant is not “trapped” in a solid that can eventually be removed as DDGS, thereby providing improved efficiency of product alcohol production (see Examples 8 and 9). Also, the dilution of the extractant with corn oil can be reduced and the extractant can be less degraded (see Example 10).

  Also, since the flow rate of the extractant can be reduced, operating costs are reduced, thereby providing improved efficiency of product alcohol production.

  Furthermore, retention of the extractant is reduced as a result of the extractant droplets rising at a higher rate, thereby providing improved efficiency of product alcohol production. Reduction in the amount of undissolved solids in the fermenter also results in improved efficiency of product alcohol production.

  Furthermore, since there is no longer a need to suspend undissolved solids, the stirrer can be removed from the fermenter, thereby reducing capital costs and energy and increasing the efficiency of product alcohol production.

  FIGS. 1-5 provide various non-limiting embodiments of methods and systems that include fermentation processes in which alcohol esters are produced in situ, extracted from the fermentation medium, and reacted to recover the product alcohol. . 1-5 also provide various non-limiting embodiments of methods and systems using carboxylic acids that can be esterified with the product alcohol and at the same time serve as ISPR extractants. FIGS. 1-5 also provide various non-limiting embodiments of methods and systems for converting lipids in the feed to carboxylic acids that can be esterified with the product alcohol and at the same time serve as an ISPR extractant.

  In certain embodiments, including any of the above-described embodiments described with reference to FIGS. 1-5, the fermentation broth in fermentor 30 is butanol via a biosynthetic pathway from at least one fermentable carbon source. At least one recombinant microorganism 32 that has been genetically modified (ie, genetically engineered) to produce In particular, the recombinant microorganism can be grown in a fermentation broth containing a suitable carbon substrate. Additional carbon substrates include, but are not limited to, monosaccharides such as fructose and galactase; oligosaccharides such as lactose, maltose, or sucrose; polysaccharides such as starch or cellulose; or mixtures thereof, and whey permeate , Corn steep liquor, sugar beet molasses, and unrefined mixtures from renewable raw materials such as barley malt. Other carbon substrates include ethanol, lactate, succinate, or glycerol.

  In addition, the carbon substrate can be a monocarbon substrate, such as carbon dioxide or methanol, that has been shown to be metabolically converted to a major biochemical intermediate. In addition to single and dual carbon substrates, methylotrophic organisms are also known to use several other carbon-containing compounds such as methylamine, glucosamine, and various amino acids for metabolic activity. For example, methylotrophic yeast is known to form trehalose or glycerol using carbon from methylamine (Bellion, et al., Microb. Growth C1 Compd., [Int. Symp.], 7th ( 1993), 415-32, Editor (s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida metabolize alanine or oleic acid (Sulter, et al., Arch. Microbiol. 153: 485-489, 1990). Accordingly, it is believed that the carbon source used in the present invention may include a wide variety of carbon-containing substrates and is limited only by the choice of organism.

  Although all of the above carbon substrates and mixtures thereof are considered suitable, in certain embodiments, the carbon substrate is glucose, fructose, and sucrose, or yeast modified to use C5 sugars with these. Mixtures with C5 sugars such as xylose and / or arabinose for cells. Sucrose can be derived from renewable sugar sources such as sugar cane, sugar beet, cassava, sweet sorghum, and mixtures thereof. Glucose and dextrose can be derived from renewable cereal sources by saccharification of starch-based ingredients including cereals such as corn, wheat, rye, barley, oats, and mixtures thereof. In addition, fermentable sugars can be regenerated from cellulosic systems by pretreatment and saccharification processes, such as described in US Patent Application Publication No. 2007/0031918 A1, which is incorporated herein by reference. Alternatively, it can be derived from lignocellulosic biomass. In addition to a suitable carbon source (from water stream 22), the fermentation broth is suitable minerals, salts known to those skilled in the art suitable for promoting the enzymatic pathway for growth of the culture and production of product alcohol. Must contain cofactors, buffers and other ingredients.

  From the above description and examples, those skilled in the art can ascertain the essential characteristics of the present invention and various modifications of the present invention to adapt to various applications and conditions without departing from the invention. And can make changes. For example, in certain embodiments, the esterification and extraction of alcohols according to the present invention can be used prior to fermentation, that is, during seed culture of microorganism 32 prior to fermentation in fermentor 30. As is known in the art, typically microorganisms 32 such as yeast can be harvested and grown from the seed medium to the desired cell concentration before being inoculated into the fermentor 30.

  Since carbon source feedstock is an important cost factor in microbial production such as yeast production, biomass yield on sugars is an important optimization criterion. Since the ATP yield from alcohol fermentation is much less than that from respiration sugar catabolism, the occurrence of alcohol fermentation will adversely affect biomass yield and should be avoided during yeast production (ie, seed culture) It is said that. Nevertheless, cultivation of microorganisms in a seed medium can produce a predetermined amount of fermentation product containing alcohol. For example, S.M. In S. cerevisiae yeast, alcohol fermentation and respiration occur simultaneously whenever the specific growth rate (μ) and / or the sugar concentration in the aerobic medium exceeds a reference value (see, for example, van Hoek, et al. Biotechnol. Bioeng. 68: 517-523, 2000). In order to obtain a high biomass yield, the growth of yeast is usually controlled by respiration conditions using, for example, fed-batch fermentation techniques for seed culture. For example, sugar is fed at a low rate, resulting in low sugar concentrations in the medium and low sugar uptake so that sugar metabolism can be substantially respiratory. Under these conditions, high biomass yields can be obtained and the accumulation of toxic products can be minimized. In practice, in large scale fed-batch industrial processes, concentration gradients can occur in cells due to inefficient mixing (eg, Enfors, et al., J. Biotechnol. 85: 175-185, 2001). See). Fermentation by-product production and resorption can be one of the reasons for the reduced biomass yield per glucose in large scale bioreactors compared to the laboratory scale.

  However, when butanol-producing yeast is cultured under these conditions, for example, fermentation products containing butanol are not reabsorbed and can accumulate in a medium that can be toxic to microorganisms at high concentrations. If product accumulation exceeds a limiting cytostatic concentration (eg, cell growth is lower than growth that can be suppressed by the feedstock), loss of fed-batch control can occur. According to the present invention, removal of butanol from the medium using esterification and extraction of alcohol allows the fed-batch fermentation to proceed despite problems with inefficient mixing and butanol toxicity. .

  Thus, according to one embodiment, the seed medium is contacted with catalyst 42 and carboxylic acid 28 to produce alcohol esters by esterification of the product alcohol and ultimately biomass per glucose in a large scale bioreactor. It can lead to improved yield. Furthermore, since the concentration of the product alcohol in the medium is controlled by the esterification of the alcohol, the adverse effects of the product alcohol on the microorganism can be minimized or avoided. In certain embodiments, the alcohol ester can be extracted from the seed medium and the alcohol can be recovered from the alcohol ester in the same manner as described above with respect to extraction of the alcohol ester from the fermenter 30 and recovery of the product alcohol 54. In certain embodiments, the esterification of alcohols according to the present invention can be used to esterify product alcohols in both seed and fermentation media. In such embodiments, generally not only alcohol esters (and free product alcohol) are recovered from the fermentation medium, but also alcohol esters produced during seed culture (eg, alcohol esters and By recovering the product alcohol), higher yields of product alcohol can be achieved for the fermentation process. In certain embodiments, the alcohol esterification according to the present invention can be used prior to fermentation for removal of alcohol from the seed medium, while the conventional ISPR of the product alcohol is used during fermentation in the fermentor 30. Can be used to remove product alcohol.

  Thus, it should be apparent that the esterification and extraction of alcohols according to the present invention can be used at various stages of the alcohol fermentation process without departing from the present invention.

  The alcohol product produced by the method of the present invention has several uses, for example as reagents, solvents and fuels. Butanol produced by the claimed method is a fuel used to produce diesel or biodiesel fuels, raw chemicals in the plastics industry, components of formulated products such as cosmetics, and esters that can be used as chemical intermediates (Eg biofuel), fuel additive, alcohol can be used directly. Butanol can also be used as a solvent for paints, coatings, varnishes, resins, gums, dyes, fats, waxes, resins, shellacs, gums, and alkaloids. Thus, the present invention provides an alternative method for producing alcohols containing butanol that can support the high demand for these industrial chemicals.

Recombinant microorganisms and butanol biosynthetic pathways While not wishing to be bound by theory, the methods described herein produce alcohols at any alcohol-producing microorganism, particularly at potencies that exceed their resistance levels. It is considered useful in connection with recombinant microorganisms.

Alcohol-producing microorganisms are known in the art. For example, the fermentation oxidation of methane by a methane-utilizing bacterium (for example, Methylosinus trichosporum) produces methanol, and methanol (C ₁ alkyl alcohol) is esterified with carboxylic acid and methanol together with carboxylic acid. By contacting with a catalyst that can be converted, a methanolic ester of carboxylic acid is formed. Yeast strain CEN. PK113-7D (CBS 8340, the Central Blue room Scheme; van Dijken, et al., Enzyme Microb. Techno. 26: 706-714, 2000) can produce ethanol, which, together with carboxylic acid and ethanol The ethyl ester is formed by contacting the acid with a catalyst capable of esterification (see, eg, Example 36).

  Recombinant microorganisms that produce alcohol are also known in the art (eg, Ohta, et al., Appl. Environ. Microbiol. 57: 893-900, 1991; Underwood, et al., Appl. Environ. Microbiol. 68: 1071-1081, 2002; Shen and Liao, Metab. Eng.10: 312-320, 2008; Hahnai, et al., Appl. Environ.Microbiol.73: 7814-7818, 2007; U.S. Pat. No. 5,712,133; PCT application publication number WO 1995/028476; Feldmann, et al., Appl. Biobiol.Biotechnol.38: 354-361,1992; Zhang, et al., Science 267: 240-243, 1995; U.S. Patent Application Publication No. 2007/0031918 A1; U.S. Patent No. 7,223,575 U.S. Patent No. 7,741,119; U.S. Patent Application Publication No. 2009/0203099 A1; U.S. Patent Application Publication No. 2009/0246846 A1; No. 075241, which are all incorporated herein by reference).

  Suitable recombinant microorganisms capable of producing butanol are known in the art, and specific suitable microorganisms capable of producing butanol are described herein. Recombinant microorganisms that produce butanol via the biosynthetic pathway include Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, and Erwinia. , Klebsiella genus, Shigella genus, Rhodococcus genus, Pseudomonas genus, Bacillus genus, Lactobacillus genus L , Klebsiella, Paenibatil Genus (Paenibacillus), Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kryveres, Kluyvermyeso ), Pichia, Candida, Hansenula, Issatchenkia, or Saccharomyces. In one embodiment, the recombinant microorganism is Escherichia coli, Lactobacillus plantarum, Kluyveromyces lactis, Kluyveromyces marciane, Kluyveromyces cerevisiax, (Saccharomyces cerevisiae) may be selected. In one embodiment, the recombinant microorganism is yeast. In one embodiment, the recombinant microorganism is selected from the genus Saccharomyces, Zygosaccharomyces, Schizosaccharomyces, Dekkera, Toltopres iss, Torulopsis is, Crabtree positive yeast selected from several species of the genus (Candida). Examples of crabtree positive yeast species include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe (Schizosaccharomyces pombe).・ Siccharomyces mikatae, Saccharomyces paradoxus, Zygosaccharomyces rouxii, and Candida glabrata ata).

  In certain embodiments, the host cell is Saccharomyces cerevisiae. S. S. cerevisiae yeast is known in the art and includes the American Type Culture Collection (Rockville, MD), the Central BureaureVort Stimures, CBS Available from a variety of sources including, but not limited to, Ferm Solutions, North American Bioproducts, Martrex, and Lalland. S. Examples of S. cerevisiae include, but are not limited to, BY4741, CEN. PK 113-7D, Ethanol Red (registered trademark) yeast, Ferm Pro (registered trademark) yeast, Bio-Ferm (registered trademark) XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strb yeast yeast, Gert Strb yeast FerMax ™ Green yeast, FerMax ™ Gold yeast, Thermosacc® yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.

  Production of butanol using fermentation by microorganisms, as well as microorganisms that produce butanol, are disclosed, for example, in US Patent Publication No. 2009/0305370, which is incorporated herein by reference. In certain embodiments, the microorganism comprises a butanol biosynthetic pathway. In certain embodiments, at least one, at least two, at least three, or at least four polypeptides that catalyze pathway substrate-product conversion are encoded by heterologous polynucleotides in the microorganism. In certain embodiments, all polypeptides that catalyze pathway substrate-product conversion are encoded by heterologous polynucleotides in the microorganism. In certain embodiments, the microorganism comprises a decrease or disappearance of pyruvate decarboxylase activity. Microorganisms that are substantially free of pyruvate decarboxylase activity are described in US Patent Application Publication No. 2009/030305363, which is incorporated herein by reference. Microorganisms substantially free of enzymes having NAD-dependent glycerol-3-phosphate dehydrogenase activity, such as GPD2, are also described in this document.

  Suitable biosynthetic pathways for the production of butanol are known in the art and certain suitable pathways are described herein. In certain embodiments, the butanol biosynthetic pathway comprises at least one gene that is heterologous to the host cell. In certain embodiments, the butanol biosynthetic pathway comprises two or more genes that are heterologous to the host cell. In certain embodiments, the butanol biosynthetic pathway includes a heterologous gene that encodes a polypeptide corresponding to every step of the biosynthetic pathway.

  Certain suitable proteins having the ability to catalyze specified substrate-product conversion are described herein, and other suitable proteins are provided in the art. For example, U.S. Patent Application Publication Nos. 2008/0261230, 2009/0163376, and 2010/0197519, which are incorporated herein by reference, are acetohydroxy acid isomeroreductases. US Patent Application Publication No. 2010/0081154, incorporated by reference, describes dihydroxy acid dehydratase; US Patent Application Publication, alcohol dehydrogenase, incorporated herein by reference. No. 2009/0269823.

  Many levels of sequence identity are useful for identifying polypeptides from other species, and such polypeptides have the same or similar function or activity and are described herein. It is well understood by those skilled in the art that it is suitable for use with recombinant microorganisms. Useful examples of percent identity include, but are not limited to, 75%, 80%, 85%, 90%, or 95%, or 75%, 76%, 77%, 78%, 79 %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Any integer percentage between 75% and 100%, such as 96%, 97%, 98% or 99% may be useful in describing the present invention.

1-butanol biosynthetic pathways 1-butanol and suitable polypeptides that can be used and biosynthetic pathways for the production of polynucleotides encoding such polypeptides are described in Donaldson, et al., Incorporated herein by reference. al. U.S. Patent Application Publication No. 2008/0182308 A1. This biosynthetic pathway involves the following substrate-product conversion:
a) For example, acetyl-CoA to acetoacetyl-CoA, which can be catalyzed by acetyl-CoA acetyltransferase;
b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, which can be catalyzed, for example, by 3-hydroxybutyryl-CoA dehydrogenase;
c) 3-hydroxybutyryl-CoA to crotonyl-CoA, which can be catalyzed, for example, by crotonase;
d) Crotonyl-CoA to butyryl-CoA, which can be catalyzed, for example, by butyryl-CoA dehydrogenase;
e) butyryl-CoA to butyraldehyde, which can be catalyzed by, for example, butyraldehyde dehydrogenase; and f) butyraldehyde to 1-butanol, which can be catalyzed by, for example, 1-butanol dehydrogenase.

  In certain embodiments, the 1-butanol biosynthetic pathway comprises at least one gene, at least two genes, at least three genes, at least four genes, or at least five genes that are heterologous to a yeast cell. In certain embodiments, the recombinant host cell comprises a heterologous gene for each substrate-product conversion of the 1-butanol biosynthetic pathway.

2-Butanol Biosynthetic Routes 2-Butanol and suitable polypeptides that can be used and the biosynthetic pathways for the production of polynucleotides encoding such polypeptides are all incorporated herein by reference, Donaldson , Et al. U.S. Patent Application Publication Nos. 2007/0259410 A1 and 2007/0292927 A1, and PCT Application Publication Number WO 2007/130521. One 2-butanol biosynthetic pathway involves the following substrate-product conversion:
a) α-acetolactic acid from pyruvate, which can be catalyzed, for example, by acetolactate synthase;
b) α-acetolactic acid to acetoin, which can be catalyzed, for example, by acetolactate decarboxylase;
c) 2,3-butanediol from acetoin, which can be catalyzed, for example, by butanediol dehydrogenase;
d) 2,3-butanediol to 2-butanone, which can be catalyzed, for example, by butanediol dehydratase; and e) 2-butanone to 2-butanol, which can be catalyzed, for example, by 2-butanol dehydrogenase.

  In certain embodiments, the 2-butanol biosynthetic pathway comprises at least one gene, at least two genes, at least three genes, or at least four genes that are heterologous to the yeast cell. In certain embodiments, the recombinant host cell comprises a heterologous gene for each substrate-product conversion of the 2-butanol biosynthetic pathway.

Isobutanol Biosynthetic Routes Isobutanol and suitable polypeptides that can be used and biosynthetic pathways for the production of polynucleotides encoding such polypeptides are hereby incorporated by reference. No. 2007/0092957 A1 and PCT application publication number WO 2007/050671. One isobutanol biosynthetic pathway involves the following substrate-product conversion:
a) pyruvate to acetolactate, which can be catalyzed, for example, by acetolactate synthase;
b) 2,3-dihydroxyisovaleric acid from acetolactate, which can be catalyzed, for example, by acetohydroxy acid reductoisomerase;
c) 2,3-dihydroxyisovaleric acid to α-ketoisovaleric acid, which can be catalyzed, for example, by acetohydroxyacid dehydratase;
d) α-ketoisovaleric acid to isobutyraldehyde, which can be catalyzed by, for example, branched chain keto acid decarboxylase; and e) isobutyraldehyde to isobutanol, which can be catalyzed by, for example, branched chain alcohol dehydrogenase.

  A suitable polypeptide sequence encoding an enzyme that catalyzes the substrate-product conversion of the isobutanol biosynthetic pathway as well as an E. coli corresponding to an enzyme suitable for the specified pathway step. C. Numbers include, but are not limited to, those listed in Tables AA and BB. Given E. C. Suitable enzymes associated with the numbers are readily available to those skilled in the art, for example by the BRENDA database (http://www.brenda-enzymes.org/).

  Provided herein is a recombinant microorganism comprising an isobutanol biosynthetic pathway comprising steps a) -e) (above), wherein the recombinant microorganism comprises an enzyme that catalyzes step c) and an enzyme that catalyzes step e). At least one of the selected enzymes is encoded by a heterologous polynucleotide that is integrated into the chromosome of the microorganism. In certain embodiments, the enzyme that catalyzes step c) is encoded by a heterologous polynucleotide that is integrated into the chromosome of the microorganism, and the enzyme that catalyzes step e) is encoded by the heterologous polynucleotide that is integrated into the chromosome of the microorganism. Is done.

  Provided herein are polynucleotides suitable for recombinant microorganisms that include a butanol biosynthetic pathway, such as the isobutanol biosynthetic pathway. Such polynucleotides include the coding region of the alsS gene derived from Bacillus subtilis (nt positions 457 to 2172 of SEQ ID NO: 1) and the coding region of the ilvC gene derived from Lactococcus lactis (sequence) Nt 3634-4656) as well as plasmids containing either or both. For expression of ALS, the alsS gene from Bacillus subtilis expressed from the yeast CUP1 promoter (nt 2 to 449 of SEQ ID NO: 1) followed by CYC1 terminator (nt 2181 to 2430 of SEQ ID NO: 1) For the expression of KARI and the chimeric gene having the coding region (nt positions 457 to 2172 of SEQ ID NO: 1), and the KARI for expression of the yeast ILV5 promoter (2433 to 3626 of SEQ ID NO: 1) followed by the ILV5 terminator (nt of SEQ ID NO: 1) 4682-5304), a chimeric gene having the coding region of the ilvC gene derived from Lactococcus lactis (nt 3634-4656 of SEQ ID NO: 1), and one or both of the chimeric genes Plasmids are also suitable.

  Suitable polynucleotides include the coding region of the ilvD gene from Streptococcus mutans (nt positions 3313 to 4849 of SEQ ID NO: 2), the coding region of codon-optimized horse liver alcohol dehydrogenase (SEQ ID NO: 2 Nt 6286-7413), the coding region of the codon optimized kivD gene from Lactococcus lactis (nt 9249-10895 of SEQ ID NO: 2) and any or all or any combinations thereof Containing plasmids. For the expression of DHAD, S. IlvD gene from Streptococcus mutans expressed from S. cerevisiae FBA1 promoter (nt 2109-3105 of SEQ ID NO: 2) followed by FBA1 terminator (nt 4858-5857 of SEQ ID NO: 2) A chimeric gene having the coding region (nt positions 3313 to 4849 of SEQ ID NO: 2); Code derived from codon-optimized horse liver alcohol dehydrogenase expressed from the S. cerevisiae GPM1 promoter (nt 7425-8181 of SEQ ID NO: 2) followed by ADH1 terminator (nt 5962-6277 of SEQ ID NO: 2) A chimeric gene having the region (nt 6286-7413 of SEQ ID NO: 2); and for expression of KivD, a TDH3 promoter (nt 10896-11918 of SEQ ID NO: 2) followed by a TDH3 terminator (nt 8237-9235 of SEQ ID NO: 2) A chimeric gene having a coding region of codon-optimized kivD gene derived from Lactococcus lactis (nt 9249-10895 of SEQ ID NO: 2) expressed from Chimeric any gene, which plasmid also suitable, including all or any combination, such as. Furthermore, suitable polynucleotides include polynucleotides having at least about 75% identity to the coding region and the defined chimeric gene, as well as to plasmids containing such polynucleotides.

  In certain embodiments, the isobutanol biosynthetic pathway comprises at least one gene, at least two genes, at least three genes, or at least four genes that are heterologous to the yeast cell. In certain embodiments, the recombinant host cell comprises a heterologous gene for each substrate-product conversion of the isobutanol biosynthetic pathway.

  Suitable strains include those described in the specific application cited and incorporated herein by reference as well as US Provisional Patent Application No. 61 / 380,563, filed Sep. 7, 2010. Is mentioned. Creation of certain suitable strains, including those used in the examples, is provided herein.

Preparation of Saccharomyces cerevisiae strain BP1083 ("NGCI-070"; PNY1504) Strain BP1064 is a CEN. PK 113-7D (CBS 8340; Centralbureau voor Schulmelculures (CBS) Fungal Biodiversity Centre, Netherlands), including deletions of the following genes: URA3, HIS3, PDC1, PDC5, PDC5, PDC5, PDC5, PDC5 BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 1, described in US Provisional Patent Application No. 61 / 246,844) and pLH468 (SEQ ID NO: 2) to obtain strain NGCI-070 (BP1083, PNY1504). Was generated.

  Homologous recombination with PCR fragments containing homologous regions upstream and downstream of either the G418 resistance marker for selection of target genes and transformants or the URA3 gene created deletions that completely removed the entire coding sequence. . The G418 resistance marker flanked by the loxP site was removed using Cre recombinase. The URA3 gene was removed by homologous recombination to create a scarless deletion or, if the loxP site is flanked, removed using Cre recombinase.

  The procedure for scarless deletion is described in Akada, et al. , (Yeast 23: 399-405, 2006). In general, a PCR cassette for each scarless deletion was made by combining the four fragments, ABUC, by overlapping PCR. The PCR cassette, along with a promoter (250 bp upstream of the URA3 gene) and a terminator (150 bp downstream of the URA3 gene), is a native CEN. It contained a selectable / counter-selectable marker, URA3 (fragment U), consisting of the PK 113-7D URA3 gene. Fragments A and C, each 500 bp in length, corresponded to 500 bp (fragment A) immediately upstream of the target gene and 3'500 bp (fragment C) of the target gene. Fragments A and C were used for integration of the cassette into the chromosome by homologous recombination. When a direct repeat of the sequence corresponding to fragment B was generated upon integration of the cassette into the chromosome, fragment B (500 bp in length) corresponds to 500 bp immediately downstream of the target gene, which Used to excise the URA3 marker and fragment C from the chromosome by genetic recombination. Using the PCR product ABUC cassette, the URA3 marker was first integrated into the chromosome and then excised from the chromosome by homologous recombination. Upon initial integration, the gene was deleted except for 3'500 bp. At the time of excision, the 3'500 bp region of the gene was also deleted. In gene integration using this method, the gene to be integrated was contained in the PCR cassette between fragments A and B.

Deletion of URA3 To delete the endogenous URA3 coding region, the ura3 :: loxP-kanMX-loxP cassette was amplified by PCR from pLA54 template DNA (SEQ ID NO: 3). pLA54 is K.I. The loxP site is flanked, containing the K. lactis TEF1 promoter and the kanMX marker, allowing recombination by Cre recombinase and removal of the marker. PCR was performed using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) And primers BK505 and BK506 (SEQ ID NOs: 4 and 5). The URA3 portion of each primer was obtained from the 5 ′ region upstream of the URA3 promoter and the 3 ′ region downstream of the coding region so that incorporation of the loxP-kanMX-loxP marker resulted in substitution of the URA3 coding region. Using standard genetic recombination techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), the PCR product is CEN. Transformants were selected into YK containing G418 (100 μg / mL) at 30 ° C. transformed into PK 113-7D. Primers LA468 and LA492 (SEQ ID NOs: 6 and 7) and the designated CEN. Transformants were screened by PCR using PK 113-7D Δura3 :: kanMX to confirm correct integration.

HIS3 Deletion Four fragments of a PCR cassette for scarless HIS3 deletion were obtained from Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) And Gentra® Puregene®. Trademark: Yeast / Bact, CEN. As a template, prepared using a kit (Qiagen, Valencia, CA). Amplified using PK 113-7D genomic DNA. HIS3 fragment A was amplified using primer oBP452 (SEQ ID NO: 14) and primer oBP453 (SEQ ID NO: 15) containing a 5 ′ tail having homology to the 5 ′ end of HIS3 fragment B. Primer oBP454 (SEQ ID NO: 16) containing a 5 'tail having homology to the 3' end of HIS3 fragment A, and a 5 'tail having homology to the 5' end of HIS3 fragment U Was amplified using a primer oBP455 (SEQ ID NO: 17). Primer oBP456 (SEQ ID NO: 18) containing a 5 'tail having homology to the 3' end of HIS3 fragment B, and HIS3 fragment U, 5 'tail having homology to the 5' end of HIS3 fragment C Was amplified using a primer oBP457 (SEQ ID NO: 19) containing HIS3 fragment C was amplified using primer oBP458 (SEQ ID NO: 20) containing a 5 ′ tail with homology to the 3 ′ end of HIS3 fragment U, and primer oBP459 (SEQ ID NO: 21). The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA). HIS3 fragment AB was generated by overlapping PCR by mixing HIS3 fragment A and HIS3 fragment B and amplifying with primers oBP452 (SEQ ID NO: 14) and oBP455 (SEQ ID NO: 17). HIS3 fragment UC was generated by overlapping PCR by mixing HIS3 fragment U and HIS3 fragment C and amplifying with primers oBP456 (SEQ ID NO: 18) and oBP459 (SEQ ID NO: 21). The resulting PCR product was purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, CA). The HIS3 ABUC cassette was generated by overlapping PCR by mixing HIS3 fragment AB and HIS3 fragment UC and amplifying with primers oBP452 (SEQ ID NO: 14) and oBP459 (SEQ ID NO: 21). The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA).

  CEN. Competent cells of PK 113-7D Δura3 :: kanMX were prepared and transformed with HIS3 ABUC PCR cassette using Frozen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, Calif.). The transformation mixture was plated at 30 ° C. in uracil-deficient synthetic complete medium supplemented with 2% glucose. Gentra (R) Puregene (R) Yeast / Bact. Transformants with his3 knockout were screened by PCR with primers oBP460 (SEQ ID NO: 22) and oBP461 (SEQ ID NO: 23) using genomic DNA prepared using the kit (Qiagen, Valencia, CA). Accurate transformants were obtained from strain CEN. Selected as PK 113-7D Δura3 :: kanMX Δhis3 :: URA3.

KanMX marker removal from Δura3 site and URA3 marker removal from Δhis3 site Using the Frozen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, Calif.), PRS423 :: PGAL1-cre (SEQ ID NO: 66, USA) Provisional patent application 61 / 290,639) and CEN. The KanMX marker was removed by transforming PK 113-7D Δura3 :: kanMX Δhis3 :: URA3 and plating in histidine and uracil deficient synthetic complete medium supplemented with 2% glucose at 30 ° C. Transformants were grown in YP supplemented with 1% galactose for about 6 hours at 30 ° C. to induce Cre recombinase and KanMX marker excision and YPD (2% glucose at 30 ° C. for recovery. ) Plated on plate. Isolates grown overnight in YPD and plated at 30 ° C. in synthetic complete medium containing 5-fluoro-orotic acid (5-FOA, 0.1%) to lose URA3 marker Selected. 5-FOA resistant isolates were grown in YPD and plated to remove the pRS423 :: PGAL1-cre plasmid. Isolates were examined for loss of KanMX marker, URA3 marker, and pRS423 :: PGAL1-cre plasmid by assaying growth on YPD + G418 plates, plates of uracil-deficient synthetic complete medium, and plates of histidine-deficient synthetic complete medium. An accurate isolate that was sensitive to G418 and auxotrophic for uracil and histidine was obtained from strain CEN. Selected as PK 113-7D Δura3 :: loxP Δhis3 and represented as BP857. Gentra (R) Puregene (R) Yeast / Bact. Using genomic DNA prepared using the kit (Qiagen, Valencia, CA), primers oBP450 (SEQ ID NO: 24) and oBP451 (SEQ ID NO: 25) for Δura3, and primer oBP460 (SEQ ID NO: 22) for Δhis3 and Deletion and marker removal were confirmed by PCR and sequencing with oBP461 (SEQ ID NO: 23).

PDC6 Deletion Four fragments of a PCR cassette for scarless PDC6 deletion were obtained from Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) And Gentra® Puregene®. Trademark) Yeast / Bact. CEN. As a template prepared using a kit (Qiagen, Valencia, CA). Amplified using PK 113-7D genomic DNA. PDC6 fragment A was amplified using primer oBP440 (SEQ ID NO: 26) and primer oBP441 (SEQ ID NO: 27) containing a 5 ′ tail having homology to the 5 ′ end of PDC6 fragment B. Primer oBP442 (SEQ ID NO: 28) containing a 5 'tail having homology to the 3' end of PDC6 fragment A, and 5 'tail having homology to the 5' end of PDC6 fragment U Amplification was performed using primer oBP443 (SEQ ID NO: 29). Primer oBP444 (SEQ ID NO: 30) containing a 5 'tail having homology to the 3' end of PDC6 fragment B, and 5 'tail having homology to the 5' end of PDC6 fragment C Was amplified using a primer oBP445 (SEQ ID NO: 31) containing PDC6 fragment C was amplified using primer oBP446 (SEQ ID NO: 32) containing a 5 ′ tail with homology to the PDC6 fragment U3 ′ end and primer oBP447 (SEQ ID NO: 33). The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA). PDC6 fragment AB was generated by overlapping PCR by mixing PDC6 fragment A and PDC6 fragment B and amplifying with primers oBP440 (SEQ ID NO: 26) and oBP443 (SEQ ID NO: 29). PDC6 fragment UC was generated by overlapping PCR by mixing PDC6 fragment U and PDC6 fragment C and amplifying with primers oBP444 (SEQ ID NO: 30) and oBP447 (SEQ ID NO: 33). The resulting PCR product was purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, CA). A PDC6 ABUC cassette was generated by overlapping PCR by mixing PDC6 fragment AB and PDC6 fragment UC and amplifying with primers oBP440 (SEQ ID NO: 26) and oBP447 (SEQ ID NO: 33). The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA).

  CEN. Competent cells of PK 113-7D Δura3 :: loxP Δhis3 were prepared and transformed with the PDC6 ABUC PCR cassette using Frozen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, Calif.). The transformation mixture was plated at 30 ° C. in uracil-deficient synthetic complete medium supplemented with 2% glucose. Transformants with pdc6 knockout were obtained from Gentra® Puregene® Yeast / Bact. Screening was performed by PCR with primers oBP448 (SEQ ID NO: 34) and BP449 (SEQ ID NO: 35) using genomic DNA prepared using the kit (Qiagen, Valencia, CA). Accurate transformants were obtained from strain CEN. Selected as PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 :: URA3.

  CEN. PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 :: URA3 was grown overnight in YPD and plated at 30 ° C. in synthetic complete medium containing 5-fluoro-orotic acid (0.1%) Isolates that lost the URA3 marker were selected. Gentra (R) Puregene (R) Yeast / Bact. Deletions and marker removal were confirmed by PCR and sequencing with primers oBP448 (SEQ ID NO: 34) and oBP449 (SEQ ID NO: 35) using genomic DNA prepared using the kit (Qiagen, Valencia, CA). Negative PCR results using primers specific for the coding sequences of PDC6, oBP554 (SEQ ID NO: 36) and oBP555 (SEQ ID NO: 37) demonstrated the absence of the PDC6 gene from the isolate. The correct isolate was obtained as strain CEN. PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 was selected and expressed as BP891.

PDC1 deletion ilvDSm integration The PDC1 gene was deleted and Streptococcus mutans ATCC No. Substitution with the ilvD coding region from 700610. A fragment, followed by the IlvD coding region from Streptococcus mutans of the PCR cassette for PDC1 deletion-ilvDSm integration, the Phusion® High Fidelity PCR Master Mix (New England BioLabs. , Ipswich, Mass.) And Gentra® Puregene® Yeast / Bact. Amplification was performed using NYLA83 genomic DNA as a template prepared using a kit (Qiagen, Valencia, CA). NYLA83 is a strain having a PDC1 deletion-ilvDSm integration (incorporated herein by reference in its entirety) as described in US Patent Application Publication No. 2009/030363, which is hereby incorporated by reference in its entirety. US Patent Application Publication No. 2011/0124060). Proliferation of PDC1 fragment A-ilvDSm (SEQ ID NO: 141) with primer oBP513 (SEQ ID NO: 38) and primer oBP515 (SEQ ID NO: 39) containing a 5 ′ tail having homology to the 5 ′ end of PDC1 fragment B did. PDC1 deletion—B, U, and C fragments of the PCR cassette for ilvDSm integration were obtained from Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) And Gentra® Puregene ( (Registered trademark) Yeast / Bact. CEN. As a template prepared using a kit (Qiagen, Valencia, CA). Amplified using PK 113-7D genomic DNA. Primer oBP516 (SEQ ID NO: 40) containing a 5 'tail with homology to the 3' end of PDC1 fragment A-ilvDSm, and PDC1 fragment B with homology to the 5 'end of PDC1 fragment U 'Amplified with primer oBP517 (SEQ ID NO: 41) containing tail. Primer oBP518 (SEQ ID NO: 42) containing a 5 'tail having homology to the 3' end of PDC1 fragment B, and 5 'tail having homology to the 5' end of PDC1 fragment C Was amplified using a primer oBP519 (SEQ ID NO: 43). PDC1 fragment C was amplified using primer oBP520 (SEQ ID NO: 44) containing a 5 ′ tail having homology to the 3 ′ end of PDC1 fragment U, and primer oBP521 (SEQ ID NO: 45). The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA). PDC1 fragment A-ilvDSm-B is generated by overlapping PCR by mixing PDC1 fragment A-ilvDSm and PDC1 fragment B and amplifying with primers oBP513 (SEQ ID NO: 38) and oBP517 (SEQ ID NO: 41) did. PDC1 fragment UC was generated by overlapping PCR by mixing PDC1 fragment U and PDC1 fragment C and amplifying with primers oBP518 (SEQ ID NO: 42) and oBP521 (SEQ ID NO: 45). The resulting PCR product was purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, CA). PDC1 A-ilvDSm-BUC cassette by duplicating PCR by mixing PDC1 fragment A-ilvDSm-B and PDC1 fragment UC and amplifying with primers oBP513 (SEQ ID NO: 38) and oBP521 (SEQ ID NO: 45) (SEQ ID NO: 142) was generated. The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA).

  CEN. Competent cells of PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 were made and PDC1 A-UlvDSm cassette with Fzen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, Calif.) Converted. The transformation mixture was plated at 30 ° C. in uracil-deficient synthetic complete medium supplemented with 2% glucose. Transformants with pdc1 knockout ilvDSm integration were obtained from Gentra® Puregene® Yeast / Bact. Screening was performed by PCR with primers oBP511 (SEQ ID NO: 46) and oBP512 (SEQ ID NO: 47) using genomic DNA prepared using the kit (Qiagen, Valencia, CA). Negative PCR results using primers specific for the coding sequences of PDC1, oBP550 (SEQ ID NO: 48) and oBP551 (SEQ ID NO: 49) demonstrated the absence of the PDC1 gene from the isolate. Accurate transformants were obtained from strain CEN. Selected as PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm-URA3.

  CEN. PK 113-7D Δura3 :: loxP Δhis3 Δpdc6: pdc1 :: ilvDSm-URA3 is grown overnight in YPD and plated in synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30 ° C. Thus, isolates that lost the URA3 marker were selected. Deletion of PDC1, integration of ilvDSm, and marker removal were performed according to Gentra® Puregene® Yeast / Bact. The genomic DNA prepared using the kit (Qiagen, Valencia, CA) was used to confirm by PCR and sequencing with primers oBP511 (SEQ ID NO: 46) and oBP512 (SEQ ID NO: 47). The correct isolate was obtained as strain CEN. PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm was selected and expressed as BP907.

PDC5 deletion sadB integration The PDC5 gene was deleted and replaced with a sadB coding region derived from Achromobacter xylosoxidans. A segment of the PCR cassette for PDC5 deletion-sadB integration was first cloned into the plasmid pUC19-URA3MCS.

  pUC19-URA3MCS is based on pUC19 and contains the sequence of the URA3 gene derived from Saccharomyces cerevisiae located at the multiple cloning site (MCS). pUC19 contains the pMB1 replicon and a gene encoding β-lactamase for replication and selection in Escherichia coli. In addition to the coding sequence for URA3, upstream and downstream sequences of this gene were included for expression of the URA3 gene in yeast. The vector can be used in the cloning process and can be used as a yeast integration vector.

  Saccharomyces cerevisiae CEN. DNA containing the URA3 coding region along with 250 bp upstream and 150 bp downstream of the URA3 coding region derived from PK 113-7D genomic DNA was added to the Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, MA). Was amplified using primers oBP438 (SEQ ID NO: 12) containing BamHI, AscI, PmeI, and FseI restriction sites, and oBP439 (SEQ ID NO: 13) containing XbaI, PacI, and NotI restriction sites. Genomic DNA was obtained from Gentra® Puregene® Yeast / Bact. Prepared using kit (Qiagen, Valencia, CA). The PCR product and pUC19 (SEQ ID NO: 150) were ligated with T4 DNA ligase after digestion with BamHI and XbaI to generate the vector pUC19-URA3MCS. The vector was confirmed by PCR and sequencing with primers oBP264 (SEQ ID NO: 10) and oBP265 (SEQ ID NO: 11).

  The coding sequences of sadB and PDC5 fragment B were cloned into pUC19-URA3MCS to generate the sadB-BU portion of the PDC5 A-sadB-BUC PCR cassette. PLH468-sadB (sequence) as template with primer oBP530 (SEQ ID NO: 50) containing an AscI restriction site and primer oBP531 (SEQ ID NO: 51) containing a 5 'tail having homology to the 5' end of PDC5 fragment B No. 67) was used to amplify the coding sequence of sadB. PDC5 fragment B was amplified using primer oBP532 (SEQ ID NO: 52) containing a 5 'tail with homology to the 3' end of sadB, and primer oBP533 (SEQ ID NO: 53) containing a PmeI restriction site. The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA). The sadB-PDC5 fragment B was generated by overlapping the PCR by mixing the sadB and PDC5 fragment B PCR products and amplifying with the primers oBP530 (SEQ ID NO: 50) and oBP533 (SEQ ID NO: 53). The resulting PCR product was digested with AscI and PmeI and, after digestion with the appropriate enzyme, ligated into the corresponding site of pUC19-URA3MCS using T4 DNA ligase. The resulting plasmid was subjected to sadB-fragment B-fragment U using primers oBP536 (SEQ ID NO: 54) and oBP546 (SEQ ID NO: 55) containing a 5 'tail having homology to the 5' end of PDC5 fragment C. Used as a template for amplification of PDC5 fragment C was amplified using primer oBP547 (SEQ ID NO: 56) containing a 5 ′ tail with homology to the 3 ′ end of PDC5 sadB-fragment B-fragment U, and primer oBP539 (SEQ ID NO: 57). . The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA). PDC5 sadB-fragment B-fragment U and PDC5 fragment C are mixed by overlapping PCR and amplified with primers oBP536 (SEQ ID NO: 54) and oBP539 (SEQ ID NO: 57) to obtain PDC5 sadB-fragment B- Fragment U-fragment C was generated. The resulting PCR product was purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, CA). Primer oBP542 (SEQ ID NO: 58) containing a 5 'tail with homology to 50 nucleotides immediately upstream of the native PDC5 coding sequence, and oBP539 (SEQ ID NO: 57) were used to produce PDC5 sadB-fragment B-fragment U- By amplifying fragment C, a PDC5 A-sadB-BUC cassette (SEQ ID NO: 143) was generated. The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA).

  CEN. Competent cells of PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm were prepared, and the Frozen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, CA) was used with C-U using PD as C-U5-C). Transform with PCR cassette. The transformation mixture was plated at 30 ° C. in uracil deficient synthetic complete medium supplemented with 1% ethanol (not glucose). Transformants with pdc5 knockout sadB integration were obtained from Gentra® Puregene® Yeast / Bact. Screening was performed by PCR with primers oBP540 (SEQ ID NO: 59) and oBP541 (SEQ ID NO: 60) using genomic DNA prepared by the kit (Qiagen, Valencia, CA). Negative PCR results using primers specific for the coding sequences of PDC5, oBP552 (SEQ ID NO: 61) and oBP553 (SEQ ID NO: 62) demonstrated the absence of the PDC5 gene from the isolate. Accurate transformants were obtained from strain CEN. Selected as PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB-URA3.

  CEN. PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB-URA3 is grown overnight in YPE (1% ethanol), supplemented with ethanol (not glucose) and at 30 ° C. Isolates that lost the URA3 marker were selected by plating in synthetic complete medium containing 5-fluoro-orotic acid (0.1%). Deletion of PDC5, sadB integration, and marker removal were performed as described in Gentra® Puregene® Yeast / Bact. It was confirmed by PCR with primers oBP540 (SEQ ID NO: 59) and oBP541 (SEQ ID NO: 60) using genomic DNA prepared using the kit (Qiagen, Valencia, CA). The correct isolate was obtained as strain CEN. PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB was selected and expressed as BP913.

Deletion of GPD2 To delete the endogenous GPD2 coding region, PCR was performed using gpd2 :: loxP-URA3-loxP cassette (SEQ ID NO: 151) using loxP-URA3-loxP (SEQ ID NO: 68) as template DNA. Amplified with loxP-URA3-loxP contains a URA3 marker derived from the flanking loxP recombinase site (ATCC No. 77107). PCR was performed using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) And primers LA512 and LA513 (SEQ ID NOs: 8 and 9). The GPD2 portion of each primer was derived from the 5 ′ region upstream of the GPD2 coding region and the 3 ′ region downstream of the coding region, such that incorporation of the loxP-URA3-loxP marker resulted in substitution of the GPD2 coding region. The PCR product was transformed into BP913 and transformants were selected in uracil-deficient synthetic complete medium supplemented with 1% ethanol (not glucose). Transformants were screened to confirm correct integration by PCR using primers oBP582 and AA270 (SEQ ID NOs 63 and 64).

  The URA3 marker was reused by transformation with pRS423 :: PGAL1-cre (SEQ ID NO: 66) and plating in synthetic complete medium lacking histidine supplemented with 1% ethanol at 30 ° C. Transformants are streaked in synthetic complete medium supplemented with 1% ethanol and containing 5-fluoro-orotic acid (0.1%) and cultured at 30 ° C. to lose the URA3 marker Selected isolates. 5-FOA resistant isolates were grown in YPE (1% ethanol) for removal of the pRS423 :: PGAL1-cre plasmid. Deletion and marker removal were confirmed by PCR with primers oBP582 (SEQ ID NO: 63) and oBP591 (SEQ ID NO: 65). The correct isolate was obtained as strain CEN. PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB Δgpd2 :: loxP was selected and expressed as PNY1503 (BP1064).

  BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 1) and pLH468 (SEQ ID NO: 2) to generate strain NGCI-070 (BP1083; PNY1504).

Preparation of Saccharomyces cerevisiae strain PNY2205 Strain PNY2205 was obtained from PNY1503 (BP1064) described above.

  Homologous recombination with PCR fragments containing homologous regions upstream and downstream of the URA3 gene for selection of target genes and transformants generally created deletions with the entire coding sequence removed. The URA3 gene was removed by homologous recombination to create a scarless deletion. Gene integration was similarly generated.

  The procedure for scarless deletion is described in Akada et al. , (Yeast, 23: 399, 2006). In general, a PCR cassette for each scarless deletion was made by combining the four fragments, ABUC, by overlapping PCR. In some cases, individual fragments were first cloned into a plasmid before the entire cassette was amplified by PCR for the deletion / integration procedure. The PCR cassette is composed of native CEN. With a promoter (250 bp upstream of the URA3 gene) and terminator (150 bp downstream of the URA3 gene) region. It contained a selectable / counter-selectable marker, URA3 (fragment U), consisting of the PK 113-7D URA3 gene. Fragments A and C, each generally 500 bp in length, corresponded to 500 bp (fragment A) immediately upstream of the target gene and 3'500 bp (fragment C) of the target gene. Fragments A and C were used for integration of the cassette into the chromosome by homologous recombination. When a direct repeat of the sequence corresponding to fragment B was generated upon integration of the cassette into the chromosome, fragment B (500 bp in length) corresponds to 500 bp immediately downstream of the target gene, which Used to excise the URA3 marker and fragment C from the chromosome by genetic recombination.

  Using the PCR product ABUC cassette, the URA3 marker was first integrated into the chromosome and then excised from the chromosome by homologous recombination. Upon initial integration, the gene was deleted except for 3'500 bp. At the time of excision, the 3'500 bp region of the gene was also deleted. In gene integration using this method, the gene to be integrated was contained in the PCR cassette between fragments A and B.

FRA2 deletion The FRA2 deletion was designed to delete 250 nucleotides from the 3 ′ end of the coding sequence, leaving the first 113 nucleotides of the FRA2 coding sequence intact. An in-frame stop codon was present at 7 nucleotides downstream of the deletion. Four fragments of the PCR cassette for scarless FRA2 deletion were obtained from Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) And Gentra® Puregene® Yeast / Bact. CEN. As a template prepared using a kit (Qiagen, Valencia, CA). Amplified using PK 113-7D genomic DNA. FRA2 fragment A was amplified using primer oBP594 (SEQ ID NO: 152) and primer oBP595 (SEQ ID NO: 153) containing a 5 'tail having homology to the 5' end of FRA2 fragment B. Primer oBP596 (SEQ ID NO: 154) containing a 5 'tail having homology to the 3' end of FRA2 fragment A, and 5 'tail having homology to the 5' end of FRA2 fragment U Was amplified using a primer oBP597 (SEQ ID NO: 155) containing Primer oBP598 (SEQ ID NO: 156) containing a 5 'tail having homology to the 3' end of FRA2 fragment B, and 5 'tail having homology to the 5' end of FRA2 fragment C Was amplified using a primer oBP599 (SEQ ID NO: 157). FRA2 fragment C was amplified using primer oBP600 (SEQ ID NO: 158) containing a 5 'tail with homology to the 3' end of FRA2 fragment U, and primer oBP601 (SEQ ID NO: 159). The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA). The FRA2 fragment AB was generated by overlapping the PCR by mixing FRA2 fragment A and FRA2 fragment B and amplifying with primers oBP594 (SEQ ID NO: 152) and oBP597 (SEQ ID NO: 155). The FRA2 fragment UC was generated by overlapping the PCR by mixing FRA2 fragment U and FRA2 fragment C and amplifying with primers oBP598 (SEQ ID NO: 156) and oBP601 (SEQ ID NO: 159). The resulting PCR product was purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, CA). The FRA2 ABUC cassette was generated by duplicating PCR by mixing FRA2 fragment AB and FRA2 fragment UC and amplifying with primers oBP594 (SEQ ID NO: 152) and oBP601 (SEQ ID NO: 159). The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA).

  PNY1503 competent cells were generated and transformed with FRA2 ABUC PCR cassette using Frozen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, Calif.). The transformation mixture was plated at 30 ° C. in uracil-deficient synthetic complete medium supplemented with 1% ethanol. Gentra (R) Puregene (R) Yeast / Bact. Transformants with a fra2 knockout were screened by PCR with primers oBP602 (SEQ ID NO: 160) and oBP603 (SEQ ID NO: 161) using genomic DNA prepared using the kit (Qiagen, Valencia, CA). Correct transformants are grown in YPE (yeast extract, peptone, 1% ethanol) and plated in synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30 ° C. The isolates that lost the URA3 marker were selected. Gentra (R) Puregene (R) Yeast / Bact. Deletion and marker removal were confirmed by PCR with primers oBP602 (SEQ ID NO: 160) and oBP603 (SEQ ID NO: 161) using genomic DNA prepared using the kit (Qiagen, Valencia, CA). Negative PCR results with primers specific for the deleted coding sequence of FRA2, oBP605 (SEQ ID NO: 162) and oBP606 (SEQ ID NO: 163) demonstrated the absence of the FRA2 gene from the isolate. The correct isolate was obtained as strain CEN. PK 113-7D MATa ura3Δ :: loxP his3Δ pdc6Δ pdc1Δ :: P [PDC1] -DHAD | ilvD_Sm-PDC1t pdc5Δ :: P [PDC5] -ADH | sadB_Ax-PDC5f: P1135 Expressed as:

ADH1 deletion and kivD Ll (y) integration The ADH1 gene was deleted and replaced with a kivD coding region from Lactococcus lactis codon-optimized for expression in Saccharomyces cerevisiae. ADH1 deletion-kivD_Ll (y) incorporation, as described in US Provisional Patent Application No. 61 / 356,379, filed Jun. 18, 2010, which is incorporated herein by reference. The scarless cassette for was first cloned into the plasmid pUC19-URA3MCS. The vector is based on pUC19 and is located at the multiple cloning site (MCS) Saccharomyces cerevisiae CEN. It contains the sequence of the URA3 gene derived from PK 113-7D. pUC19 contains the pMB1 replicon and a gene encoding β-lactamase for replication and selection in Escherichia coli. In addition to the coding sequence for URA3, upstream (250 bp) and downstream (150 bp) sequences of this gene are present for expression of the URA3 gene in yeast.

A kivD coding region derived from Lactococcus lactis codon-optimized for expression in Saccharomyces cerevisiae, a primer oBP562 (SEQ ID NO: 164) containing a PmeI restriction site, and 5 'of ADH1 fragment B PLH468 (US Provisional Patent Application No. 61 / 246,709 filed on Sep. 29, 2009) using primer oBP563 (SEQ ID NO: 165) containing a 5 ′ tail having homology to the ends as a template. Amplification). Using the primer oBP564 (SEQ ID NO: 166) containing a 5 'tail having homology to the 3' end of kivD_Ll (y) and the primer oBP565 (SEQ ID NO: 167) containing the FseI restriction site Amplified from genomic DNA prepared as follows. The PCR product was purified using a PCR Purification kit (Qiagen, Valencia, CA). kivD_Ll (y) -ADH1 fragment B By overlapping PCR by mixing PCR products and amplifying with primers oBP562 (SEQ ID NO: 164) and oBP565 (SEQ ID NO: 167), kivD_Ll (y) -ADH1 fragment B Was generated. The resulting PCR product was digested with PmeI and FseI and, after digestion with an appropriate enzyme, ligated into the corresponding site of pUC19-URA3MCS using T4 DNA ligase. ADH1 fragment A was amplified from genomic DNA using primer oBP505 (SEQ ID NO: 168) containing a SacI restriction site and primer oBP506 (SEQ ID NO: 169) containing an AscI restriction site. The ADH1 fragment A PCR product was digested with SacI and AscI and ligated into the corresponding sites of the plasmid containing kivD_Ll (y) -ADH1 fragment B using T4 DNA ligase. ADH1 fragment C was amplified from genomic DNA using primer oBP507 (SEQ ID NO: 170) containing a PacI restriction site and primer oBP508 (SEQ ID NO: 171) containing a SalI restriction site. The ADH1 fragment C PCR product was digested with PacI and SalI and ligated into the corresponding sites of the plasmid containing the ADH1 fragment A-kivD_Ll (y) -ADH1 fragment B using T4 DNA ligase. The hybrid promoter UAS (PGK1) -P _FBA1 is _transformed into the vector pRS316-UAS (PGK1) -P _{Amplified from FBA1-} GUS (SEQ ID NO: 172). The UAS (PGK1) -P _FBA1 PCR product was digested with AscI and PmeI I and ligated to the corresponding site of the plasmid containing the kivD_Ll (y) -ADH1 fragment ABC using T4 DNA ligase. The entire integration cassette was amplified from the resulting plasmid using primers oBP505 (SEQ ID NO: 168) and oBP508 (SEQ ID NO: 171) and purified using the PCR Purification kit (Qiagen, Valencia, CA).

PNY1505 competent cells were generated and transformed with the ADH1-kivD_Ll (y) PCR cassette created above using the Frozen-EZ Yeast Transformation II ™ kit (Zymo Research Corporation, Irvine, Calif.). The transformation mixture was plated in uracil deficient synthetic complete medium supplemented with 1% ethanol at 30 ° C. Transformants were grown in YPE (1% ethanol) and plated at 30 ° C. in synthetic complete medium containing 5-fluoro-orotic acid (0.1%) to isolate isolates that lost the URA3 marker Selected. Gentra (R) Puregene (R) Yeast / Bact. Using genomic DNA prepared using the kit (Qiagen, Valencia, CA), external primers oBP495 (SEQ ID NO: 175) and oBP496 (SEQ ID NO: 176) and kivD_Ll (y) specific primer oBP562 (SEQ ID NO: 164) and ADH1 deletion and kivD_Ll (y) integration were confirmed by PCR with external primer oBP496 (SEQ ID NO: 176). The correct isolate was obtained as strain CEN. PK
113-7D MATa ura3Δ :: loxP his3Δ pdc6Δ pdc1Δ :: P [PDC1] -DHAD | ilvD_Sm-PDC1tpdc5Δ :: P [PDC5] -ADH | sadB_Ax-PDC5t gP22: h: P1P ] -KivD_Ll (y) -ADH1t, expressed as PNY1507 (BP1201). PNY1507 was transformed with isobutanol pathway plasmids pYZ090 (SEQ ID NO: 1) and pBP915 (described below).

Preparation of pRS316-UAS (PGK1) -FBA1p-GUS vector To clone the cassette UAS (PGK1) -FBA1p (SEQ ID NO: 177), first, the 602 bp FBA1 promoter (FBA1p) was used as a primer T-FBA1 (SalI) ( SEQ ID NO: 178) and B-FBA1 (SpeI) (SEQ ID NO: 179) were used to obtain CEN. After PCR amplification from PK genomic DNA and removal of the PGK1p promoter plasmid by SalI / SpeI digestion, cloning into the SalI and SpeI sites in plasmid pWS358-PGK1p-GUS (SEQ ID NO: 180) yields pWS358-FBA1p-GUS It was. The pWS358-PGK1p-GUS plasmid was generated by inserting the PGK1p and β-glucuronidase gene (GUS) DNA fragments into the multiple cloning site of pWS358 derived from the pRS423 vector (Christianson, et al., Gene 110: 119). -122, 1992). Second, the resulting pWS358-FBA1p-GUS plasmid was digested with SalI and SacI, the DNA fragment containing the FBA1p promoter, the GUS gene, and the FBAt terminator were gel purified and cloned into the SalI / SacI sites in pRS316. PRS316-FBA1p-GUS was generated. Third, an upstream activation sequence (UAS) located at positions −519 to −402 upstream of the 3-phosphoglycerate kinase (PGK1) open reading frame, ie a 118 bp DNA fragment containing UAS (PGK1) Using TU / PGK1 (KpnI) (SEQ ID NO: 181) and B-U / PGK1 (SalI) (SEQ ID NO: 182), CEN. PCR amplification was performed from the genomic DNA of PK. The PCR product was digested with KpnI and SalI and cloned into the KpnI / SalI site in pRS316-FBA1p-GUS to generate pRS316-UAS (PGK1) -FBA1p-GUS.

Construction of Integration Vector pUC19-kan :: pdc1 :: FBA-alsS :: TRX1 The 1.7 kb BbvCI / PacI fragment was transformed into pRS426 :: GPD :: alsS :: CYC (US Patent Application Publication No. 2007/0092957). ) To pRS426 :: FBA :: ILV5 :: CYC (US 2007/0092957, pre-digested with BbvCI / PacI to release the ILV5 gene). FBA-alsS-CYCt cassette was prepared. The ligation reaction was transformed into E. coli TOP10 cells and transformants were screened by PCR using primers N98SeqF1 (SEQ ID NO: 183) and N99SeqR2 (SEQ ID NO: 184). The FBA-alsS-CYCt cassette was inserted into the pUC19-URA3 :: ilvD-TRX1 (incorporated herein by reference, June 2010) at the AflII site (using a Klenow fragment to create a ligation compatible end). Isolated from the vector using BglII and NotI for cloning into clone “B”) described in US Provisional Patent Application No. 61 / 356,379, filed on the 18th. Transformants containing the alsS cassette in both orientations in the vector were obtained, using primers N98SeqF4 (SEQ ID NO: 185) and N1111 (SEQ ID NO: 186) for structure “A” and N98SeqF4 for structure “B”. (SEQ ID NO: 185) and N1110 (SEQ ID NO: 187) were used to confirm by PCR. Next, the geneticin selectable form of the “A” structural vector removes the URA3 gene (1.2 kb NotI / NaeI fragment) and the geneticin cassette described above (incorporated herein by reference). (SEQ ID NO: 655) described in US Provisional Patent Application No. 61 / 356,379 filed Jun. 18, 2010). The Klenow fragment was used to make all ends compatible with ligation, the transformants were screened by PCR, and using primers BK468 (SEQ ID NO: 188) and N160SeqF5 (SEQ ID NO: 189) with the previous URA3 marker Clones with the same orientation of geneticin resistance gene were selected. The resulting clone was called pUC19-kan :: pdc1 :: FBA-alsS :: TRX1 (clone A) (SEQ ID NO: 190).

  The pUC19-kan :: pdc1 :: FBA-alsS integration vector was linearized with PmeI and transformed into PNY1507 (above). PmeI cleaves the vector within the cloned pdc1-TRX1 integration region, thereby resulting in targeted integration at that position (Rothstein, Methods Enzymol. 194: 281-301, 1991). Transformants were selected in YPE plus 50 μg / ml G418. Patched transformants were screened by PCR for integration events using primers N160SeqF5 (SEQ ID NO: 189) and oBP512 (SEQ ID NO: 47). Two transformants were tested indirectly for the function of acetolactate synthase by assessing the ability of the strain to make isobutanol. For this purpose, additional isobutanol pathway genes were fed into an E. coli-yeast shuttle vector (pYZ090ΔalsS and pBP915 described below). One clone, strain MATa ura3Δ :: loxP his3Δ pdc6Δ pdc1Δ :: P [PDC1] -DHAD | ilvD_Sm-PDC1t-pUC19-loxP-kanMX-loxP-P [FBA1] -ALS | alcS_Bt5-C5 ] -ADH | sadB_Ax-PDC5t gpd2Δ :: loxP fra2Δ adh1Δ :: UAS (PGK1) P [FBA1] -kivD_Ll (y) -ADH1t was expressed as PNY2204. PNY2205 is PNY2204 transformed with pYZ090ΔalsS and pBP915 plasmid.

Isobutanol pathway plasmid (pYZ090ΔalsS and pBP915)
pYZ090 (SEQ ID NO: 1) was digested with SpeI and NotI to remove most of the CUP1 promoter and all of the alsS coding sequence and CYC terminator. Next, the vector was treated with the Klenow fragment, then self-gated and transformed into E. coli Stbl3 cells to select for ampicillin resistance. Removal of the DNA region was confirmed for two independent clones by DNA sequencing across the ligation junction by PCR with primer N191 (SEQ ID NO: 191). The resulting plasmid was called pYZ090ΔalsS (SEQ ID NO: 192).

  By deleting the kivD gene and 957 base pairs of the TDH3 promoter upstream of kivD, pBP915 was transformed into pLH468 (SEQ ID NO: 2; US Provisional Patent Application No. 61 / 246,709 filed September 29, 2009). Prepared from the description). pLH468 was digested with SwaI and the large fragment (12896 bp) was purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, Calif.). The isolated fragment of DNA was self-gated with T4 DNA ligase and used to transform electrocompetent TOP10 Escherichia coli (Invitrogen, Carlsbad, CA). Plasmids from the transformants were isolated and examined for appropriate deletions by restriction analysis using SwaI restriction enzyme. Isolates were also sequenced across the deletion site using primers oBP556 (SEQ ID NO: 193) and oBP561 (SEQ ID NO: 194). A clone with the appropriate deletion was designated pBP915 (pLH468ΔkivD) (SEQ ID NO: 195).

Creation of strains NYLA74, NYLA83, and NYLA84 Insertion inactivation of the endogenous PDC1 and PDC6 genes of S. cerevisiae. The PDC1, PDC5, and PDC6 genes encode the three major isozymes of pyruvate decarboxylase described below:

Construction of pRS425 :: GPM-sadB DNA fragment (SEQ ID NO: 70) encoding butanol dehydrogenase derived from Achromobacter xylosoxidans (disclosed in US Patent Application Publication No. 2009/0269823) Cloned). The coding region of this gene called sadB for secondary alcohol dehydrogenase (SEQ ID NO: 69) is recommended for gram-negative bacteria using forward and reverse primers N473 and N469 (SEQ ID NOs: 74 and 75), respectively. Prepared using the Gentra® Puregene® kit (Qiagen, Valencia, Calif.) According to Amplified from A. xylosoxidans genomic DNA using standard conditions. The PCR product was cloned into pCR®4 BLUNT (Invitrogen®, Carlsbad, Calif.) With TOPO®-Blunt to generate pCR4Blunt :: sadB, which was transformed into E. coli (E E. coli) transformed into Mach-1 cells. The plasmid was then isolated from 4 clones and the sequence was confirmed.

  The sadB coding region was PCR amplified from pCR4Blunt :: sadB. The PCR primer contained an additional 5 'sequence that could overlap with the yeast GPM1 promoter and ADH1 terminator (N583 and N584 provided as SEQ ID NOs 76 and 77). The PCR product was then cloned using the “gap repair” method in Saccharomyces cerevisiae (Ma, et al., Gene 58: 201-216, 1987) as follows. . Yeast-E. Coli shuttle vector pRS425 containing the GPM1 promoter (SEQ ID NO: 72), the kivD coding region (SEQ ID NO: 71) from Lactococcus lactis, and the ADH1 terminator (SEQ ID NO: 73): : GPM :: kivD :: ADH (described in Example 17 of US 2007/0092957 A1) was digested with BbvCI and PacI restriction enzymes to release the kivD coding region. About 1 μg of the remaining vector fragment was combined with 1 μg of sadB PCR product along with S. cerevisiae. Transformation into S. cerevisiae strain BY4741. Transformants were selected on leucine-deficient synthetic complete medium. Appropriate recombination events generating pRS425 :: GPM-sadB were confirmed by PCR using primers N142 and N459 (SEQ ID NOs: 108 and 109).

Generation of pdc6 :: PGPM1-sadB integration cassette and deletion of PDC6:
pdc6 :: PGPM1-sadB-ADH1t-URA3r integration cassette is converted to pRS425 :: GPM-sadB (SEQ ID NO: 78) GPM-sadB-ADHt segment (SEQ ID NO: 79) to URA3r gene derived from pUC19-URA3r Made by bonding. pUC19-URA3r (SEQ ID NO: 80) contains a URA3 marker derived from pRS426 (ATCC No. 77107) flanked by 75 bp homologous repeats, allowing homologous recombination and removal of the URA3 marker in vivo. . The two DNA segments were divided into Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) And primers 114117-11A-114117A-11D (SEQ ID NOs: 81, 82, 83, and 84), and 114117-13A. And 114117-13B (SEQ ID NOs: 85 and 86), as well as SOE PCR using pRS425 :: GPM-sadB and pUC19-URA3r plasmid DNA as templates (Horton, et al., Gene 77: 61-68, 1989). Combined).

  External primers for SOE PCR (114117-13A and 114117-13B) included approximately 50 bp of 5 'and 3' regions that were homologous to the upstream and downstream regions of the PDC6 promoter and terminator, respectively. By using standard gene recombination techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), a completed cassette PCR fragment was obtained as BY4700 (ATCC No. 60086). ) And the transformants were maintained on uracil-deficient synthetic complete medium supplemented with 2% glucose at 30 ° C. Transformants were screened by PCR using primers 112590-34G and 112590-34H (SEQ ID NOs: 87 and 88), and 112590-34F and 112590-49E (SEQ ID NOs: 89 and 90) to detect the absence of the PDC6 coding region. Integration at the PDC6 locus with loss was confirmed. According to standard protocols, the URA3r marker was reused by plating in synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. Colony removal from 5-FOA plates was patched onto SD-URA medium to confirm the absence of growth, confirming marker removal. The resulting identified strain has the genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t.

Generation of pdc1 :: PPDC1-ilvD integration cassette and deletion of PDC1:
Template pLH468 and pUC19-URA3r plasmid DNA with Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) And primers 114117-27A-114117-27D (SEQ ID NOs: 111, 112, 113, and 114) The ilvD-FBA1t segment (SEQ ID NO: 91) derived from pLH468 (SEQ ID NO: 2) was transformed into pUC19-URA3r by SOE PCR (described by Horton, et al., Gene 77: 61-68, 1989) used as Pdc1 :: PPDC1-ilvD-FBA1t-URA3r integration cassette was made by binding to the URA3r gene derived from.

  External primers for SOE PCR (114117-27A and 114117-27D) contained approximately 50 bp of 5 'and 3' regions that were homologous to regions downstream of the PDC1 promoter and downstream of the PDC1 coding sequence. Using a standard gene recombination technique (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), the completed cassette PCR fragment was obtained as BY4700 pdc6: PG1-PGc6: PG SadB-ADH1t was transformed and the transformants were maintained on uracil-deficient synthetic complete medium supplemented with 2% glucose at 30 ° C. Transformants were screened by PCR using primers 114117-36D and 135 (SEQ ID NOs: 92 and 93), and primers 112590-49E and 112590-30F (SEQ ID NOs: 90 and 94) to delete the PDC1 coding sequence. Integration at the PDC1 locus with According to standard protocols, the URA3r marker was reused by plating in synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. The removal of the marker was confirmed by patching colonies from the 5-FOA plate onto SD-URA medium to confirm no growth. The obtained identified strain “NYLA67” has the genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t.

HIS3 Deletion To delete the endogenous HIS3 coding region, the his3 :: URA3r2 cassette was PCR amplified from URA3r2 template DNA (SEQ ID NO: 95). URA3r2 contains a URA3 marker derived from pRS426 (ATCC No. 77107) flanked by a 500 bp homologous repeat that allows homologous recombination and removal of the URA3 marker in vivo. PCR is performed using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) And primers 114117-45A and 114117-45B (SEQ ID NOs: 96 and 97) to produce an approximately 2.3 kb PCR product. did. The HIS3 portion of each primer was derived from the 5 ′ region upstream of the HIS3 promoter and the 3 ′ region downstream of the coding region, such that incorporation of the URA3r2 marker resulted in replacement of the HIS3 coding region. Using standard genetic recombination techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), the PCR product is transformed into NYLA67 and transformed. Were selected on uracil-deficient synthetic complete medium supplemented with 2% glucose at 30 ° C. Transformants were screened to confirm correct integration by replica plating transformants in complete histidine-deficient synthetic medium supplemented with 2% glucose at 30 ° C. The URA3r marker was reused by plating in synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. according to standard protocols. The removal of the marker was confirmed by patching colonies from the 5-FOA plate onto SD-URA medium to confirm no growth. The resulting identified strain called NYLA73 has the genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t Δhis3.

Generation of pdc5 :: kanMX integration cassette and deletion of PDC5:
The pdc5 :: kanMX4 cassette was transformed into strain YLR134W chromosomal DNA using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) and primers PDC5 :: KanMXF and PDC5 :: KanMXR (SEQ ID NO: 98 and 99). PCR amplification from (ATCC No. 4034091) produced a PCR product of approximately 2.2 kb. The PDC5 portion of each primer was derived from the 5 ′ region upstream of the PDC5 promoter and the 3 ′ region downstream of the coding region, such that incorporation of the kanMX4 marker resulted in substitution of the PDC5 coding region. Using standard genetic recombination techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), the PCR product is transformed into NYLA73 and transformed. Were selected on YP medium supplemented with 1% ethanol and geneticin (200 μg / mL) at 30 ° C. Transformants were screened by PCR to confirm correct integration at the PDC locus by replacement of the PDC5 coding region with primers PDC5kofor and N175 (SEQ ID NOs: 100 and 101). The exact transformant identified has the genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1tΔhis3 pdc5 :: kanMX4. The strain was called NYLA74.

  Plasmid vectors pRS423 :: CUP1-alsS + FBA-budA and pRS426 :: FBA-budC + GPM-sadB were transformed into NYLA74 to generate a butanediol-producing strain (NGCI-047).

  Plasmid vectors pLH475-Z4B8 (SEQ ID NO: 140) and pLH468 were transformed into NYLA74 to generate an isobutanol producing strain (NGCI-049).

Deletion of HXK2 (Hexokinase II):
hxk2 :: URA3r cassette was PCR amplified from URA3r2 template (above) using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) and primers 384 and 385 (SEQ ID NO: 102 and 103), An approximately 2.3 kb PCR product was generated. The HXK2 portion of each primer was derived from the 5 ′ region upstream of the HXK2 promoter and the 3 ′ region downstream of the coding region so that incorporation of the URA3r2 marker resulted in replacement of the HXK2 coding region. Using standard genetic recombination techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), the PCR product is transformed into NYLA73 and transformed. Were selected on uracil-deficient synthetic complete medium supplemented with 2% glucose at 30 ° C. Transformants were screened by PCR to confirm correct integration at the HXK2 locus by replacement of the HXK2 coding region with primers N869 and N871 (SEQ ID NOs: 104 and 105). The URA3r2 marker was reused by plating in synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. according to standard protocols. Colonies from 5-FOA plates are patched onto SD-URA medium to confirm the absence of growth, and by PCR, using primers N946 and N947 (SEQ ID NOs 106 and 107) to identify the correct marker. By confirming the removal, the removal of the marker was confirmed. The resulting identified strain, referred to as NYLA83, has the genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t Δhis3 Δhxk2.

Generation of pdc5 :: kanMX integration cassette and deletion of PDC5:
The pdc5 :: kanMX4 cassette was PCR amplified as described above. As described above, the PCR fragment was transformed into NYLA83, and transformants were selected and screened. The identified correct transformant, referred to as NYLA84, has the genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1tΔhis3 Δhxk2 pdc5 :: kanMX4.

  Using standard genetic recombination techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), plasmid vectors pLH468 and pLH532 were transformed into strain NYLA84 (BY4p: p4dp: pc1p1p: p4dp: p1pd :: PPDC1-ilvD-FBA1t Δhis3 Δhxk2 pdc5 :: kanMX4), and the resulting “butanologen NYLA84” was synthesized on histidine and uracil-deficient synthetic complete medium supplemented with 1% ethanol at 30 ° C. Maintained.

Expression vector pLH468
The pLH468 plasmid (SEQ ID NO: 2) was generated for expression of DHAD, KivD, and HADH in yeast, which is described in US Patent Application Publication No. 2009/030363, which is incorporated herein by reference. Have been described. For the expression of DHAD, S. Coding region of ilvD gene derived from S. cerevisiae FBA1 promoter (nt 2109-3105), followed by FBA1 terminator (nt 4858-5857) (Streptococcus mutans) (nt position 3313- 4849) for the expression of ADH; Chimera with Codon-Optimized Horse Liver Alcohol Dehydrogenase Coding Region (nt 6286-7413) Expressed from S. cerevisiae GPM1 Promoter (nt 7425-8181), followed by ADH1 Terminator (nt 5962-6277) Codon-optimized kivD gene derived from TDH3 promoter (nt 10896-11918) followed by TDH3 terminator (nt 8237-9235) for expression of the gene; and KivD PLH486 was prepared to contain a chimeric gene having the coding region (nt 9249-10895).

  The coding regions for Lactococcus lactis ketoisovalerate decarboxylase (KivD) and horse liver alcohol dehydrogenase (HADH) (SEQ ID NOs: 71 and 118, respectively) for expression in Saccharomyces cerevisiae Based on codons optimized for DNA 2.0, Inc. (Menlo Park, CA) and provided in plasmids pKivDy-DNA2.0 and pHadhy-DNA2.0. The encoded proteins are SEQ ID NOs: 117 and 119, respectively. Individual expression vectors for KivD and HADH were created. To construct pLH467 (pRS426 :: PTDH3-kivDy-TDH3t), the vector pNY8 (SEQ ID NO: 121; pRS426.GPD-ald-GPDt, also referred to herein as US Pat. Appl. Publ. No. 2008). Digested with AscI and SfiI enzymes, thereby cleaving the GPD promoter and the ald coding region. To add an AscI site at the 5 ′ end and a SpeI site at the 3 ′ end using 5 ′ primer OT1068 and 3 ′ primer OT1067 (SEQ ID NO: 123 and 124), a TDH3 promoter fragment derived from pNY8 (SEQ ID NO: 122 ) Was PCR amplified. The AscI / SfiI digested pNY8 vector fragment was ligated with the AscI and SpeI digested TDH3 promoter PCR product, and the SpeI-SfiI fragment containing the codon optimized kivD coding region was obtained from the vector pKivD-DNA2.0. Isolated. The vector pLH467 (pRS426 :: PTDH3-kivDy-TDH3t) was generated by three ligations. pLH467 was confirmed by restriction mapping and sequencing.

  pLH435 (pRS425 :: PGPM1-Hadhy-ADH1t) is the vector pRS425 :: GPM-sadB described in Example 3 of US Provisional Patent Application No. 61 / 058,970, incorporated herein by reference. (SEQ ID NO: 78). pRS425 :: GPM-sadB is a coding region derived from the butanol dehydrogenase of the GPM1 promoter (SEQ ID NO: 72), Achromobacter xylosoxidans (sadB; DNA SEQ ID NO: 69; protein SEQ ID NO: 70: US patent) And the pRS425 vector (ATCC No. 77106) having a chimeric gene containing the ADH1 terminator (SEQ ID NO: 73). pRS425 :: GPMp-sadB contains BbvI and PacI sites at the 5 'and 3' ends of the sadB coding region, respectively. By site-directed mutagenesis using primers OT1074 and OT1075 (SEQ ID NOs: 126 and 127), a NheI site was added to the 5 ′ end of the sadB coding region to generate the vector pRS425-GPPMp-sadB-NheI; It was confirmed by sequencing. pRS425 :: PGPM1-sadB-NheI is digested with NheI and PacI to exclude the sadB coding region and ligate with the NheI-PacI fragment containing the codon-optimized HADH coding region from vector pHadhy-DNA2.0. PLH435 was generated.

  To combine the KivD and HADH expression cassettes in a single vector, the yeast vector pRS411 (ATCC No. 87474) was digested with SacI and NotI and pLH435 containing the PGPM1-Hadhy-ADH1t cassette in three ligation reactions. Was ligated with the SacI-SalI fragment derived from pLH467 containing the PTDH3-kivDy-TDH3t cassette together with the SalI-NotI fragment derived from. This generated the vector pRS411 :: PTDH3-kivDy-PGPM1-Hadhy (pLH441), which was confirmed by restriction mapping.

  To generate a co-expression vector for all three genes in the lower isobutanol pathway: ilvD, kivDy, and Hadhy, described in US 2010/0081154 as a source of the IlvD gene. PRS423 FBA ilvD (Strep) (SEQ ID NO: 128) was used. This shuttle vector contains an F1 origin of replication (nt 1423-1879) for maintenance in E. coli and a 2 micron origin (nt 8082-9426) for replication in yeast. The vector has an FBA1 promoter (nt 2111-3108; SEQ ID NO: 120) and an FBA terminator (nt 4861-5860; SEQ ID NO: 129). In addition, the vector has a His marker (nt 504-1163) for selection in yeast and an ampicillin resistance marker (nt 7092-7949) for selection in E. coli. The ilvD coding region (nt 3116-4828; SEQ ID NO: 115; protein SEQ ID NO: 116) derived from Streptococcus mutans UA159 (ATCC No. 700610) is between the FBA promoter and the FBA terminator and is expressed Form a chimeric gene for In addition, there is a lumio tag fused to the ilvD coding region (nt 4829-4849).

  The first step is to use pRS423 FBA ilvD (Strep) (pRS423-FBA (SpeI) -IlvD (mutans streptococci ( Streptococcus mutans)) — also called Lumio) was linearized to obtain a vector having a total length of 9,482 bp. The second step was to isolate the kivDy-hADHy cassette from pLH441 using SacI and KpnI (the KpnI site was blunted using T4 DNA polymerase) to obtain a 6,063 bp fragment. . This fragment was ligated with a 9,482 bp vector fragment derived from pRS423-FBA (SpeI) -IlvD (Streptococcus mutans) -Lumio. This generated the vector pLH468 (pRS423 :: PFBA1-ilvD (Strep) Lumio-FBA1t-PTDH3-kivDy-TDH3t-PGPM1-hadhy-ADH1t), which was confirmed by restriction mapping and sequencing.

Generation of pLH532 The pLH532 plasmid (SEQ ID NO: 130) was generated for the expression of ALS and KARI in yeast. pLH532 consists of the following chimeric genes: 1) CUP1 promoter (SEQ ID NO: 139), acetolactate synthase coding region derived from Bacillus subtilis (AlsS; SEQ ID NO: 137; protein SEQ ID NO: 138) and CYC1 terminator 2 (sequence) No. 133); 2) ILV5 promoter (SEQ ID NO: 134), Pf5. The IlvC coding region (SEQ ID NO: 132) and the ILV5 terminator (SEQ ID NO: 135); and 3) the FBA1 promoter (SEQ ID NO: 136), S. A pHR81 vector (ATCC No. 87541) containing the S. cerevisiae KARI coding region (ILV5; SEQ ID NO: 131); and the CYC1 terminator.

  Pf5. The IlvC coding region is a sequence encoding KARI derived from Pseudomonas fluorescens described in US Patent Application Publication No. 2009/0163376, which is incorporated herein by reference.

  Pf5. The IlvC coding region was derived from DNA 2.0, Inc., based on codons optimized for expression in Saccharomyces cerevisiae. (Menlo Park, CA; SEQ ID NO: 132).

Generation of pYZ090 pYZ090 (SEQ ID NO: 1) is based on the pHR81 (ATCC No. 87541) backbone and for expression of ALS, the yeast CUP1 promoter (nt 2-449), followed by the CYC1 terminator (nt 2181-2430) A chimeric gene with the coding region of the alsS gene from Bacillus subtilis (nt positions 457 to 2172) expressed from, and for expression of KARI, the yeast ILV5 promoter (2433 to 3626), followed by ILV5 Created to contain a chimeric gene having the coding region (nt 3634-4656) of the ilvC gene derived from Lactococcus lactis expressed from a terminator (nt 46682-5304) It is.

Generation of pYZ067 pYZ067 is a murine expressed from the yeast FBA1 promoter (nt 1161-2250) followed by an FBA terminator (nt 4005-4317) for expression of the following chimeric gene: 1) dihydroxy acid dehydratase (DHAD). The coding region of the ilvD gene from S. mutans UA159 (nt positions 2260-3971), 2) for expression of alcohol dehydrogenase, the yeast GPM promoter (nt 5819-6575), followed by the ADH1 terminator ( nt 4356-4671), the coding region for horse liver ADH (nt 4680-5807), and 3) the expression of ketoisovalerate decarboxylase for the yeast TDH3 promoter (nt 8830-9493), followed by the coding region (nt 7175-8821) of the KivD gene derived from Lactococcus lactis expressed from the TDH3 terminator (nt 5682-7161).

pRS423 :: CUP1-alsS + FBA-budA and pRS426 :: FBA-budC + GPM-sadB and pLH475-Z4B8 are created It is described in US Patent Application Publication No. 2009/0305363, which is incorporated herein by reference.

Creation of Saccharomyces cerevisiae strain PNY2242 Strain PNY2242 was created from PNY1507 (above) in several steps. First, a chimeric gene containing an FBA1 promoter, an alsS coding region, and a CYC1 terminator was integrated into chromosome XII upstream of the TRX1 gene. The sequence of the modified locus is shown as SEQ ID NO: 196. Next, two copies of the gene encoding horse liver alcohol dehydrogenase were integrated into chromosomes VII and XVI. In chromosome VII, a chimeric gene containing the PDC1 promoter, hADH coding region, and ADH1 terminator was placed in the fra2Δ locus (the original deletion of FRA2 has been described above). The sequence of the modified locus is shown as SEQ ID NO: 197. In chromosome XVI, a chimeric gene containing the PDC5 promoter, the hADH coding region, and the ADH1 terminator was integrated into the region originally occupied by the long term repeat element YPRCdelta15. The sequence of the modified locus is shown as SEQ ID NO: 198. Next, the native genes YMR226c and ALD6 were deleted. The disappearance of YMR226c was a scarless deletion of only the coding region. The sequence of the modified locus is shown as SEQ ID NO: 199. The upstream sequence of the ALD6 coding region plus 700 bp is deleted using CRE-lox mediated marker removal (as described above), whereby the resulting locus contains one loxP site. The sequence of the modified locus is shown as SEQ ID NO: 200. Finally, the plasmid was introduced into the strain for expression of KARI (pLH702, SEQ ID NO: 201) and DHAD (pYZ067DkivDDhADH, SEQ ID NO: 202), resulting in strain PNY2242.

  When the recombinant microorganism produces isobutanol, in certain embodiments, the microorganism exhibits a higher specific productivity. Furthermore, the volume velocity was improved by about 50%.

  While not wishing to be bound by theory, it is believed that the process described herein provides an extractive fermentation process with improved product alcohol yield. As mentioned above, alcohol production using fermentation by microorganisms can be inefficient due to the alcohol toxicity threshold of microorganisms. In certain embodiments, the methods herein provide a means to convert the product alcohol into a material that is less toxic to microorganisms. For example, the product alcohol can be contacted with the carboxylic acid in the presence of a catalyst that esterifies the alcohol with the carboxylic acid, thereby producing an alcohol ester that is less toxic to microorganisms. Furthermore, the production of alcohol esters from the product alcohol results in a lower concentration of product alcohol in the fermentation medium. When the concentration of product alcohol is reduced, the product alcohol yield is improved because the toxic effects of product alcohol on microorganisms are minimized.

  The carboxylic acid can act as an extractant and the alcohol ester can be separated into the extractant. However, the partition coefficient of the extractant can be reduced by lipid contamination. In order to suppress the reduction of the extractant's partition coefficient, the lipid present in the fermentation medium can be converted to extractant and then the lipid concentration can be minimized. In certain embodiments, the methods herein provide a means to convert lipids present in the feedstock or biomass into extractants by catalytic hydrolysis of the lipids to carboxylic acids. The carboxylic acid produced by this hydrolysis can act as an extractant or can be esterified with the product alcohol to form an alcohol ester. Thus, the methods described herein provide means for maintaining the partition coefficient of the extractant (eg, lipid hydrolysis) as well as means for minimizing the toxic effects of the product alcohol (eg, product alcohol). Esterification).

  Carboxylic acids can be supplied to the fermentor or derived from the lipid (eg, biomass) supplied to the fermentor by hydrolysis. The amount of carboxylic acid needs to be sufficient to form a biphasic mixture comprising an organic phase and an aqueous phase. That is, the appropriate concentration of carboxylic acid (ie, extractant) is contacted with the fermentation broth to form a two-phase mixture. The alcohol esters that are formed in the fermentation broth preferentially separate into the organic phase because these esters are formed at concentrations that exceed the equilibrium concentration of the aqueous phase. The alcohol ester-containing organic phase can be separated from the fermentation broth, the product alcohol can be recovered from the organic phase, and the extractant can be recycled to the fermenter.

  Moreover, while various embodiments of the invention have been described above, it should be understood that they have been presented by way of example only and not limitation. It will be apparent to those skilled in the relevant art that various modifications can be made in the form and details of the invention without departing from the spirit and scope of the invention. Accordingly, the breadth and scope of the present invention is not limited by any of the above-described exemplary embodiments, but is defined only in accordance with the following claims and their equivalents.

  All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and each individual publication, patent or patent application is incorporated by reference. To the same extent as specifically and individually indicated herein by reference.

  The following non-limiting examples further illustrate the present invention. The following examples include corn as the raw material and COFA as the carboxylic acid, but other biomass sources can be used as the raw material without departing from the invention, and acids other than COFA serve as the carboxylic acid. Please understand that you get. In addition, the following examples include the production of butanol and butyl esters, but other alcohols, including ethanol, and alcohol esters can be produced without departing from the invention.

As used herein, the meanings of the abbreviations used were as follows: “g” means gram, “kg” means kilogram, “L” means liter, “ML” means milliliters, “μL” means microliters, “mL / L” means milliliters / liter, “mL / minute” means milliliters / minute, and “DI” means depleted. Means ionized, “uM” means micrometer, “nm” means nanometer, “w / v” means weight / volume, “OD” means optical density “OD ₆₀₀ ” means optical density at a wavelength of ₆₀₀ nM, “dcw” means dry cell weight, “rpm” means number of revolutions per minute, “° C.” means degree Celsius. , "° C / min" means Celsius temperature / min, “slpm” means standard liters per minute, “ppm” means parts per million, “pdc” means pyruvate decarboxylase enzyme, followed by the enzyme number.

General Method Seed Flask Growth (Seed Flash Growth)
Saccharomyces cerevisiae strains modified to produce isobutanol from carbohydrate sources, dpc1 deficient, pdc5 deficient, and pdc6 deficient, were seeded in seed flasks from frozen medium. Growing to .55-1.1 g / L dcw (OD ₆₀₀ 1.3-2.6-Thermo Helios α Thermo Fisher Scientific Inc., Waltham, Massachusetts). The medium was grown at 23-26 ° C. in an incubator rotating at 300 rpm. The frozen medium was pre-stored at -80 ° C. The composition of the first seed flask medium was as follows:
3.0-5.0 g / L dextrose 3.0-3.5 g / L absolute ethanol 3.7 g / L ForMedium ™ Synthetic Complete Amino Acid (Kaiser) Drop-Out: No HIS, No URA ( Reference number DSCK162CK)
6.7 g / L Difco Yeast Nitrogen Base (No. 291920) without amino acids.

8-12 milliliters from the first seed flask medium was transferred to a 2 L flask and grown at 30 ° C. in an incubator rotating at 300 rpm. The second seed flask has 220 mL of the following medium:
30.0 g / L dextrose 5.0 g / L absolute ethanol 3.7 g / L ForMedium ™ Synthetic Complete Amino Acid (Kaiser) Drop-Out: no HIS, no URA (reference number DSCK162CK)
6.7 g / L Difco Yeast Nitrogen Base without amino acid (No. 291920)
0.2 M MES buffer titrated to pH 5.5-6.0.

The medium was grown at 0.55-1.1 g / L dcw (OD ₆₀₀ 1.3-2.6). An additional 30 mL solution containing 200 g / L peptone and 100 g / L yeast extract was added to the cell concentrate. Next, an additional 250-300 mL of 0.2 uM filter sterilized HD OCENOL® 90/95 oleyl alcohol (Cognis, Monheim, DE) was added to the flask. Medium was continued to grow to> 4 g / L dcw (OD ₆₀₀ > 10) before being harvested and added to the fermentation.

Fermentation Preparation Initial Fermenter Preparation A glass-coated 2 L fermenter (Sartorius AG, Goettingen, Germany) was charged with domestic water to a liquefied weight of 66%. A pH probe (Hamilton Easyferm Plus K8, part number: 238627, Hamilton Bondadz AG, Bondaduz, Switzerland) was calibrated by the Sartorius DCU-3 Control Tower Calibration menu. Zero calibration was performed at pH = 7. Span calibration was performed at pH = 4. The probe was then placed into the fermentor through a stainless steel head plate. A dissolved oxygen probe (pO ₂ probe) was also placed in the fermentor through the head plate. Tubes used to deliver nutrients, seed medium, extraction solvent, and base were attached to the head plate and foiled at the ends. The entire fermentor was placed in a Steris (Steris Corporation, Mentor, Ohio) autoclave and sterilized in a liquid cycle for 30 minutes.

  The fermenter was removed from the autoclave and placed on the load cell. The jacket water supply and return piping were connected to domestic water and clean drain, respectively. A condenser for cooling the incoming and outgoing water lines was connected to a 6 L recirculation temperature bath operating at 7 ° C. A vent line for transferring gas from the fermentor was connected to a transfer line connected to a Thermo mass spectrometer (Prima dB, Thermo Fisher Scientific Inc., Waltham, Massachusetts). The sparger line was connected to the gas supply line. Tubes for adding nutrients, extraction solvent, seed medium, and base were plumbed through the pump or secured.

  The fermenter temperature was controlled at 55 ° C. using a thermocouple and a domestic water circulation loop. Wet corn nuts (# 2 yellow dent) are ground using a hammer mill equipped with a 1.0 mm sieve, and the resulting ground whole corn nuts are then 30% (dry corn solid weight) was added to the fermentor at a charge of liquefied reaction mass.

Lipase treatment before liquefaction The lipase enzyme stock solution was added to the fermentor so that the final lipase concentration was 10 ppm. The fermentor was held at 55 ° C., 300 rpm, and 0.3 slpm N ₂ overlay for over 6 hours. After the lipase treatment was completed, liquefaction was performed as described later (liquefaction).

While applying household _{N 2} liquefied sterilized 0.3slpm by sparger, while mixing fermentor 300～1200Rpm, the α- amylase was added to the fermentor to its specifications as expected. The temperature set point was changed from 55 ° C to 85 ° C. If the temperature exceeded 80 ° C, the liquefaction cooking time was started and the liquefaction cycle was held above 80 ° C for 90-120 minutes. After the liquefaction cycle was completed, the fermentor temperature set point was set to a fermentation temperature of 30 ° C. N ₂ was redirected from the sparger to the headspace to prevent foaming without adding a chemical defoamer.

Lipase treatment after liquefaction After the liquefaction cycle was completed (liquefaction), the fermenter temperature was set to 55 ° C instead of 30 ° C. The pH was manually controlled to pH = 5.8 by adding an acid or base bolus as needed. The lipase enzyme stock solution was added to the fermentor so that the final lipase concentration was 10 ppm. The fermentor was held at 55 ° C., 300 rpm, and 0.3 slpm N ₂ bilayer for over 6 hours. After the lipase treatment was completed, the fermenter temperature was set to 30 ° C.

Inactivation treatment by heat of lipase (heat sterilization treatment method)
The fermenter temperature was held above 80 ° C. for longer than 15 minutes to deactivate the lipase. After the heat deactivation treatment was completed, the fermenter temperature was set to 30 ° C.

Nutritional addition before inoculation Ethanol (6.36 mL / L, volume after inoculation, 200 proof, anhydrous) was added to the fermentor just prior to inoculation. Thiamine was added to a final concentration of 20 mg / L, and 100 mg / L nicotinic acid was also added immediately before inoculation.

Addition of oleyl alcohol or corn oil fatty acid before inoculation Immediately after inoculation, 1 L / L (volume after inoculation) of oleyl alcohol or corn oil fatty acid was added.

Fermentation Fermenter Inoculation The fermenter pO ₂ probe was calibrated to zero while N ₂ was added to the fermentor. The fermenter pO ₂ probe was calibrated to its span using a sterile air sparge at 300 rpm. The fermenter was inoculated after the second seed flask exceeded 4 g / L dcw. The shake flask was removed from the incubator / shaker for 5 minutes to allow phase separation between the oleyl alcohol phase and the aqueous phase. The aqueous phase (110 mL) was transferred to a sterile inoculation bottle. The inoculum was injected into the fermentor through a peristaltic pump.

Fermenter operating conditions The fermentor was operated at 30 ° C for the entire growth and production phase. The pH was lowered from a pH of 5.7-5.9 to a control set point of 5.2 without any acid added. For the remainder of the growth and production phase, ammonium hydroxide was used to control the pH to pH = 5.2. For the remainder of the growth and production phase, sterile air was added to the fermentor at 0.3 slpm through a sparger. With the DCU-3 Control Box PID control loop, using only agitation control, set the stirrer to a minimum of 300 rpm, set to a maximum of 2000 rpm, and set pO ₂ to be controlled to 3.0% did. Glucose was supplied by simultaneous saccharification and fermentation of liquefied corn mash by adding a-amylase (glucoamylase). As long as starch was available for saccharification, the glucose was kept in excess (1-50 g / L).

Analysis of Analyzed Gas Treated air was analyzed with a Thermo Prima (Thermo Fisher Scientific Inc., Waltham, Massachusetts) mass spectrometer. This was the same treated air that was sterilized and added to each fermentor. The offgas of each fermentor was analyzed with the same mass spectrometer. The Thermo Primer dB has a function of executing a calibration check at 6:00 am every Monday. Calibration checks were scheduled by Gas Works v1.0 (Thermo Fisher Scientific Inc., Waltham, Massachusetts) software associated with the mass spectrometer. The gas to be calibrated was as follows:

  Carbon dioxide was checked at 5% and 15% during the calibration cycle using other known bottled gases. Oxygen was checked at 15% using other known bottled gases. Based on the analysis of offgas in each fermentor, the amount of stripped isobutanol, consumed oxygen, and carbon dioxide inhaled into the offgas, the mass fraction analysis of the mass spectrometer and the gas flow into the fermentor Measurement was performed using a (mass flow controller). The gas rate per hour was calculated and then integrated during the fermentation.

Biomass Measurement A 0.08% Trypan Blue solution was prepared from a 1: 5 dilution of 0.4% Trypan Blue in NaCl (VWR BDH8721-0) with 1 × PBS. A 1.0 mL sample was withdrawn from the fermentor, placed in a 1.5 mL Eppendorf centrifuge tube, and centrifuged at 14,000 rpm for 5 minutes in an Eppendorf, 5415. After centrifugation, the upper solvent layer was removed using an m200 Variable Channel BioHit pipette with a 20-200 μL BioHit pipette tip. Care was taken not to remove the layer between the solvent and the aqueous layer. After removing the solvent layer, the sample was resuspended using Vortex-Genie® set at 2700 rpm.

  A series of dilutions was required to prepare the ideal concentration for the hemacytometer count. If the OD was 10, a 1:20 dilution could be made to obtain an OD of 0.5 that would allow an ideal amount (20-30) of cells to be counted per square. Multiple dilutions with cut 100-1000 μL BioHit pipette tips were required to reduce dilution inaccuracies due to corn solids. In order to prevent the tip from becoming clogged and widen the opening, about 1 cm was cut from the tip. For a final dilution of 1:20, an initial 1: 1 dilution of the fermentation sample and 0.9% NaCl solution was prepared. Next, after producing a 1: 1 dilution of the previous solution (ie, the initial 1: 1 dilution) and a 0.9% NaCl solution (second dilution), the second dilution And 1: 5 dilutions of Trypan Blue solution. Samples were vortexed between each dilution and the cut tip was rinsed into 0.9% NaCl and Trypan Blue solution.

  The coverslip was carefully placed on top of a haemocytometer (Hauser Scientific Bright-Line 1492). An aliquot (10 μL) was withdrawn from the final Trypan Blue dilution and injected into the hemacytometer using an m20 Variable Channel BioHit pipette with a 2-20 μL BioHit pipette tip. The hemacytometer was placed on a Zeis Axioskop 40 microscope with a 40 × magnification. The center quadrant was divided into 25 squares, then the four corners and the center square in both chambers were counted and recorded. After counting both chambers, take the average value, multiply by the dilution factor (20), then multiply by the square number 25 in the middle quarter in the hemacytometer, then count the 4 minutes The volume was divided by 0.0001 mL, which is the volume of 1. The sum of this calculation is the number of cells per mL.

LC analysis of fermentation products in the aqueous phase Samples were refrigerated until ready for processing. Samples were removed from refrigeration and allowed to reach room temperature (approximately 1 hour). Approximately 300 μL of sample is transferred to a 0.2 um centrifuge filter (Nanosep® MF modified nylon centrifuge filter) using an m1000 Variable Channel BioHit pipette with a 100-1000 μL BioHit pipette tip. And then centrifuged for 5 min at 14,000 rpm using an Eppendorf, 5415C. Approximately 200 μL of the filtered sample was transferred to a 1.8 autosampler container equipped with a 250 μL glass container insert with polymer feet. The vessel was capped using a screw cap with a PTFE septum and the sample was vortexed using a Vortex-Genie® set at 2700 rpm.

  The samples were then run on an Agilent 1200 series LC equipped with a binary isocratic pump, vacuum degasser, heated column compartment, sampler cooling system, UV DAD detector and RI detector. The column used was an Aminex HPX-87H, 300 × 7.8 with a Bio-Rad Cation H refill, 30 × 4.6 guard column. The column temperature was 40 ° C. for 40 minutes at a flow rate of 0.6 mL / min using a mobile phase of 0.01 N sulfuric acid. The results are shown in Table 1.

  Samples were refrigerated until ready for processing. Samples were removed from refrigeration and allowed to reach room temperature (approximately 1 hour). Approximately 150 μL of sample was transferred to a 1.8 autosampler container with a 250 μL glass container insert with polymer feet using an m1000 Variable Channel BioHit pipette with a 100-1000 μL BioHit pipette tip. The container was capped using a screw cap with a PTFE septum.

  The samples were then run on an Agilent 7890A GC equipped with a 7683B injector and a G2614A autosampler. The column was an HP-InnoWax column (30 m × 0.32 mm ID (ID), 0.25 μm film). The carrier gas is helium at a flow rate of 1.5 mL / min measured at 45 ° C. using a constant top pressure; the injector split is 1:50 at 225 ° C .; the oven temperature is 1.5 For a run time of 45 ° C. per minute for 0 minute at 10 ° C./minute, 45 ° C. to 160 ° C., then 29 minutes, it was 14 minutes at 35 ° C./minute for 230 minutes. Flame ionization detection was used at 260 ° C. with 40 mL / min helium makeup gas. The results are shown in Table 2.

  Samples analyzed for fatty acid butyl esters were run on an Agilent 6890 GC equipped with a 7683B injector and a G2614A autosampler. The column was an HP-DB-FFAP column (15 meter x 0.53 mm ID), 1-micron film thickness column (30 m x 0.32 mm ID, 0.25 μm film). The carrier gas is helium at a flow rate of 3.7 mL / min measured at 45 ° C. using a constant top pressure; the injector split is 1:50 at 225 ° C .; the oven temperature is 2.0 100 ° C. per minute, 100 ° C. to 250 ° C. at 10 ° C./min, then for 26 minutes run time was 9 minutes 250 ° C. Flame ionization detection was performed using helium makeup gas at 40 mL / min. Used at <RTIgt; C. </ RTI> The following GC standard (Nu-Chek Prep; Elysian, MN) was used to produce the fatty acid isobutyl ester product: isobutyrate palmitate. , Iso stearic acid - was confirmed attributes of butyl - butyl, iso oleate - butyl, iso linoleic acid - butyl, iso-linolenic acid - butyl, arachidonic acid iso.

  Examples 1-14 illustrate various fermentation conditions that can be used in the claimed method. As an example, there was fermentation that performed lipase treatment before liquefaction and fermentation that performed lipase treatment after liquefaction. In other examples, the fermentation was subjected to a heat deactivation treatment. After fermentation, the effective isobutanol titer (effective isopotency), ie the total grams of isobutanol produced per liter of aqueous volume, was measured. The results are shown in Table 3.

Example 1-(Control)
The experimental identifier 2010Y014 included the following: seed flask growth method, initial fermentor preparation method, liquefaction method, nutrient addition prior to inoculation method, fermenter inoculation method, fermenter operating condition method, and All of the analysis methods. Oleyl alcohol was added to a single batch for 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 2
The experiment identifier 2010Y015 included: seed flask growth method, initial fermentor preparation method, liquefaction method, lipase treatment method after liquefaction, nutrient addition before inoculation method, fermenter inoculation method, fermentation All tank operating condition methods and analysis methods. Oleyl alcohol was added to a single batch for 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 3
The experimental identifier 2010Y016 included: seed flask growth method, initial fermenter preparation method, liquefaction method, lipase treatment method after liquefaction, nutrient addition prior to inoculation method excluding ethanol, fermentation All of the inoculation method of the tank, the operating condition method of the fermenter, and the analysis method. Oleyl alcohol was added to a single batch for 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 4
The experiment identifier 2010Y017 included: seed flask growth method, initial fermentor preparation method, liquefaction method, heat sterilization method after liquefaction, nutrient addition prior to inoculation method excluding ethanol, Fermenter inoculation method, fermenter operating condition method, and analysis method all. Oleyl alcohol was added to a single batch for 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 5
The experimental identifier 2010Y018 included: seed flask growth method, initial fermentor preparation method, liquefaction method, lipase treatment method after liquefaction except that 7.2 ppm lipase was added after liquefaction, Heat sterilization treatment method after liquefaction, nutrient addition before inoculation method, inoculation method of fermenter, operating condition method of fermenter, and analysis method. Oleyl alcohol was added to a single batch for 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 6 (Control)
The experimental identifier 2010Y019 included: seed flask growth method, initial fermenter preparation method, liquefaction method, heat sterilization treatment method after liquefaction, nutrient addition before inoculation method, fermenter inoculation method, All fermenter operating condition methods and analytical methods. Oleyl alcohol was added to a single batch for 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 7-(Control)
The experiment identifier 2010Y021 included: seed flask growth method, initial fermentor preparation method, lipase treatment method before liquefaction, liquefaction method, heat sterilization treatment during liquefaction, nutrition addition before inoculation method, Fermenter inoculation method, fermenter operating condition method, and analysis method all. Oleyl alcohol was added to a single batch for 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 8
The experimental identifier 2010Y022 included: seed flask growth method, initial fermenter preparation method, liquefaction method, nutrient addition prior to inoculation method, fermenter inoculation method, fermenter operating condition method, and All of the analysis methods. Oleyl alcohol was added to a single batch for 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 9
The experiment identifier 2010Y023 included: seed flask growth method, initial fermentor preparation method, liquefaction method, lipase treatment method after liquefaction, no heat sterilization treatment, nutrition addition before inoculation method, fermenter Inoculation method, fermenter operating condition method, and analysis method. Corn oil fatty acids made from crude corn oil were added to a single batch for 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 10
The experiment identifier 2010Y024 included: except seed flask growth method, initial fermentor preparation method, lipase treatment method prior to liquefaction, liquefaction method, heat sterilization during liquefaction, no ethanol added Nutritional addition before the inoculation method, fermenter inoculation method, fermenter operating condition method, and analysis method all. Oleyl alcohol was added to a single batch for 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 11
The experimental identifier 2010Y029 included: seed flask growth method, initial fermentor preparation method, lipase treatment method before liquefaction, liquefaction method, heat sterilization treatment during liquefaction, nutrition addition before inoculation method, Fermenter inoculation method, fermenter operating condition method, and analysis method all. Corn oil fatty acids made from crude corn oil were added to a single batch for 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 12
The experiment identifier 2010Y030 included: Seed flask growth method, initial fermentor preparation method, lipase treatment method prior to liquefaction, liquefaction method, heat sterilization during liquefaction, no ethanol added All of the nutrient additions before the inoculation method, fermenter inoculation method, fermenter operating condition method, and analysis method. Corn oil fatty acids made from crude corn oil were added to a single batch for 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 13-(Control)
The experimental identifier 2010Y031 included the following: seed flask growth method, initial fermentor preparation method, liquefaction method, lipase treatment method after liquefaction, no heat sterilization treatment, no inoculation except that no ethanol was added Nutritional addition before method, fermenter inoculation method, fermenter operating condition method, and analysis method all. Corn oil fatty acids made from crude corn oil were added to a single batch for 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 14
Experiment identifier 2010Y032 included: seed flask growth method, initial fermentor preparation method, liquefaction method, lipase treatment method after liquefaction, no heat sterilization treatment, nutrient addition before inoculation method, fermentor Inoculation method, fermenter operating condition method, and analysis method. Corn oil fatty acids made from crude corn oil were added to a single batch for 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

  Examples 15 and 16 show a comparison of fermentation and isobutanol production with and without lipase treatment after liquefaction. The results are shown in Tables 4 and 5.

Example 15
The experiment identifier 2010Y026 included: seed flask growth method, initial fermenter preparation method, liquefaction method, lipase treatment method after liquefaction, nutrient addition method before inoculation, fermenter inoculation method, fermentation All tank operating condition methods and analysis methods. Corn oil fatty acids made from crude corn oil were added to a single batch for 0.1-1.0 hours after inoculation. Corn oil fatty acid extraction solvent was added in a volume equal to the volume of broth. The butanologen was PNY2205. Since most of the glucose made from corn mash was deleted, 274 g of 50% w / w sterilized glucose solution was added in a fermentation time of 46 hours to 61 hours.

Example 16
The experimental identifier 2010Y027 included: seed flask growth method, initial fermentor preparation method, liquefaction method, nutrient addition method before inoculation, fermenter inoculation method, fermenter operating condition method, and All of the analysis methods. HD OCENOL® 90/95 (oleyl alcohol, CAS number 143-28-2, Cognis, Monheim, DE) was added to a single batch 0.1-1.0 hours after inoculation. The oleyl alcohol extraction solvent was added in a volume equal to the broth volume. Butanologen was PNY2205.

  Examples 17-22 show a comparison of the effect of fresh extractant on recycled extractant on fermentation and isobutanol production. The results are shown in Table 6. In these examples, 2 L and 10 L fermentations were prepared as described below.

10L pre-seed flask growth Saccharomyces cerevisiae strain (above strain PNY2242), which is dpc1-deficient, pdc5-deficient, and pdc6-deficient, modified to produce isobutanol from a carbohydrate source, 0.6-0.7 g / L dcw (OD ₆₀₀ 1.5-2.5-Thermo Helios α Thermo Fisher Scientific in seed flasks from frozen medium (10 mL synthetic medium in 125 mL vented flask) Inc., Waltham, Massachusetts). The medium was grown at 29-31 ° C. in an incubator rotating at 260 rpm. The frozen medium was pre-stored at -80 ° C. The composition of the synthetic seed flask medium was as follows:
10.0 g / L dextrose 3.5 mL / L absolute ethanol 3.7 g / L ForMedium ™ Synthetic Complete Amino Acid (Kaiser) Drop-Out: no HIS, no URA (reference number DSCK162CK)
6.7 g / L Difco Yeast Nitrogen Base without amino acid (No. 291920)
1: 1: Tween 80: 1% ergosterol in ethanol.

  Two milliliters from the first seed flask medium were transferred to 25 mL in a 250 mL vented flask and grown at 29-31 ° C. in an incubator rotating at 260 rpm. The second seed flask uses the same synthetic medium as used above.

The medium was grown to 0.6-0.7 g / L dcw (OD ₆₀₀ 1.0-3.0). Next, 8 mL of this second flask medium was added to three flasks (2 L flask, vented flask, baffled flask) containing 200 mL of synthetic medium. The medium was grown in an incubator at 29-31 ° C. for 18-24 hours. The three seed flasks use the same synthetic media used for the first two seed flasks. These three flasks (600 mL flask broth) are used to inoculate a breeding tank with a final aqueous volume of 6 L.

10L breeding tank liquefaction 10L of B. Prepared to use the Braun BioStat C fermentor. An in-line pH probe was placed in the fermentation apparatus. Zero calibration was performed at pH = 7. Span calibration was performed at pH = 4. The probe was then placed in the fermentor through the side port. A dissolved oxygen probe (pO ₂ probe) was also placed in the fermentor through the side port. Tubes used to deliver nutrients, seed medium, extraction solvent, and base were attached to the head plate and foiled at the ends. The valves for intake and collection were sterilized with low pressure steam and a steam trap for over 20 minutes above 121 ° C.

  The fermenter temperature was controlled at 30 ° C. using a thermocouple and a household water circulation loop. Wet corn nuts (# 2 yellow dent) are ground using a hammer mill equipped with a 1.0 mm sieve, and the resulting ground whole corn nuts are then 20% (dry corn solid weight) of the liquefied reaction mass was added to the fermentor at an input rate. Difco Yeast Extract was added to the fermentor at a total batch weight of 0.5% w / w.

While applying sterile household _{N 2} at 1~2slpm by sparger, while mixing fermentor 300～1500Rpm, the α- amylase was added to the fermentor to its specifications as expected. The temperature set point was changed from 55 ° C. to 95 ° C. with a step change of 5 ° C. with a 5-15 minute hold at each stage to ensure thorough mixing. If the temperature exceeded 90 ° C, the liquefaction cooking time was started and the liquefaction cycle was held above 90 ° C for 60 minutes. After the liquefaction cycle was completed, the fermentor temperature set point was set to a fermentation temperature of 30 ° C. N ₂ was redirected from the sparger to the headspace to prevent foaming without adding a chemical defoamer.

10 L breeding tank operation The fermenter pO ₂ probe was calibrated to zero while N ₂ was added to the fermentor. The fermenter pO ₂ probe was calibrated to its span using a sterile air sparge at 400 rpm. The fermentor was inoculated from the final stage of the pre-seed flask growth process. Three shake flasks were removed from the incubator / shaker and added to the sterilized bath. The contents of the sterilized tank were added to the 5.3-5.5 L liquefied mash made during the breeding tank liquefaction process.

The fermentation temperature was controlled at 29-31 ° C. The stirring speed was fixed at 400 rpm. Air was sparged for the entire fermentation at 2.0 slpm. Using _NH 4 OH and PID control loop for the fermentor was controlled to pH 5.4 to 5.5. A back pressure of 0.3-0.5 bar, controlled by a PID loop that controls the back pressure control valve, was set in the fermentor.

  At 16-20 hours after inoculation, glucoamylase (1.8 mL Distilase® L-400, Genencor, Palo Alto, Calif.) Was added to initiate simultaneous saccharification and fermentation, and glucose from the dissolved starch Was released. Also 5.5 L of HD OCENOL® 90/95 (oleyl alcohol, Cognis, Monheim, DE) was added to the fermenter. At 34-36 hours, the stirrer speed was reduced to 100 rpm. After 10 minutes, the agitator was turned off and the air flow to the fermentor was changed from sparge mode to overlay mode.

10 L Production Tank Liquefaction 10 L production tank liquefaction was performed as described above. The fermenter temperature was controlled at 30 ° C. using a thermocouple and a household water circulation loop. Wet corn nuts (# 2 yellow dent) are ground using a hammer mill equipped with a 1.0 mm sieve, and the resulting ground whole corn nuts are then converted into 25- 25 35% (dry corn solid weight) was added to the fermentor at a charge of liquefied reaction mass. 75 mL of 100-fold vitamin solution (100 × Vitamin Solution) (2 g / L thiamine and 10 g / L nicotinic acid) was added to the fermentor. α-Amylase was added to the fermentor as described above. Moreover, after the fermenter was returned to 30 ° C., 6 to 7 mL / L of absolute ethanol was added to the fermenter.

10 L Production Tank Operation The fermenter pO ₂ probe was calibrated to zero while N ₂ was added to the fermentor. The fermenter pO ₂ probe was calibrated to its span using a sterile air sparge at 400 rpm. The fermenter was inoculated from the breeding tank. After 36 hours of growth time in the breeding tank, aseptic transfer was performed from the breeding tank and fermentation agitation was turned off for more than 10 minutes. This allowed considerable separation of oleyl alcohol and aqueous phase. Aseptic transfer was performed from a collection valve (located at the bottom of the fermenter) in the breeding tank. Approximately 10% v / v was added to the production tank, based on the final non-solvent volume of the tank after transfer.

The fermentation temperature was controlled at 29-31 ° C. The stirring speed was fixed at 400 rpm. Air was sparged for the entire fermentation at 2.0 slpm. The pH was controlled between 5.2 and 5.3 using NH ₄ OH and PID control loop for the fermentor. A back pressure of 0.3-0.5 bar, controlled by a PID loop that controls the back pressure control valve, was set in the fermentor.

  Just prior to inoculation, 25-35% v / v Cognis Emery® 610 soy fatty acid was aseptically added to the fermentor. After the 10 L breeding tank operation method was completed, the fermenter was inoculated with 10% v / v fermentation broth. Immediately after inoculation, glucoamylase (Distilase® L-400) was added to release glucose from the starch. Additional glucoamylase additions were made as needed to maintain an excess of glucose. Immediately after inoculation, lipase (Novozymes Lipolase® 100 L) was added to the fermenter at 4-15 ppm.

2 L Preseed Flask Growth A 2 L preseed flask growth was prepared using a Saccharomyces cerevisiae strain (strain PNY2242 above) and seeded flask from frozen medium (in a 125 mL aerated flask, 10 mL In synthetic medium) to 0.6-0.7 g / L dcw (OD ₆₀₀ 1.5-2.5-Thermo Helios α Thermo Fisher Scientific Inc., Waltham, Massachusetts). The medium was grown at 29-31 ° C. in an incubator rotating at 260 rpm. The frozen medium was pre-stored at -80 ° C. The composition of the synthetic seed flask medium was as follows:
10.0 g / L dextrose 3.5 mL / L absolute ethanol 3.7 g / L ForMedium Synthetic Complete Amino Acid (Kaiser) Drop-Out: no HIS, no URA (reference number DSCK162CK)
6.7 g / L Difco Yeast Nitrogen Base (No. 291920) without amino acids
1: 1: Tween 80: 1% ergosterol in ethanol.

  Two milliliters from the first seed flask medium were transferred to 25 mL in a 250 mL vented flask and grown at 29-31 ° C. in an incubator rotating at 260 rpm. The second seed flask uses the same synthetic medium as used above.

The medium was grown to 0.6-0.7 g / L dcw (OD ₆₀₀ 1.0-3.0). Next, 4 mL of this second flask medium was added to 100 mL of corn mash centrifuge in a 2 L flask. The medium was grown in an incubator at 29-31 ° C. for 18-24 hours. Next, 500 mL of HD OCENOL® 90/95 (oleyl alcohol, Cognis, Monheim, DE) was added to the flask. The flask was grown for 6-8 hours. Next, 2 mL of 1.2 g Distilase® L-400, (Genencor, Palo Alto, Calif.) In 80 mL of deionized water is added to the centrifuge and the dissolved starch in the centrifuge is added. Glucose was released. The medium was allowed to grow for 18-24 hours. The final biomass concentration was 6-12 g / L dcw.

A corn mash centrifuge was made by liquefying corn with the following formulation:
1150 g tap water 340.5 g 1 mm sieved ground corn 13.5 g yeast extract (Difco number 9102333, low dusting)
27 g peptone 4.1 g urea 40.5 mg nicotinic acid 40.5 mg thiamine.

  The material was then centrifuged for 30 minutes in a Sorval RC5C centrifuge. The supernatant was separated from the solid pellet. The supernatant was heated in a Steris autoclave for a 5 minute liquid cycle. This is defined as the centrifuge.

2L Fermentation Preparation 2L Initial Fermenter Preparation A 2L initial fermentor preparation was prepared as described above. The fermenter temperature was controlled at 55 ° C. using a thermocouple and a household water circulation loop. Wet corn nuts (# 2 yellow dent) are ground using a hammer mill equipped with a 1.0 mm sieve, and the resulting ground whole corn nuts are then converted into 25- 25 30% (dry corn solid weight) was added to the fermentor at a charge of liquefied reaction mass. In addition, liquefaction was performed as described above. While applying sterile household _{N 2} at 0.3slpm by sparger, while mixing fermentor 300～1200Rpm, the α- amylase was added to the fermentor to its specifications as expected.

2L addition before inoculation The following nutrients were added to the fermentor on a volume basis after inoculation before inoculation, after liquefaction:
30 mg / L nicotinic acid 30 mg / L thiamine 1: 1: Tween 80: 1 mL / L 1% ergosterol w / v solution in ethanol 6.3 mL / L ethanol 2 g / L urea.

Fermentor pO ₂ probe was calibrated to zero while ₂ L fermentor inoculation N ₂ was added to the fermentor. The fermenter pO ₂ probe was calibrated to its span using a sterile air sparge at 300 rpm. The fermentor was inoculated from the final stage of the pre-seed flask growth process. The shake flask was removed from the incubator / shaker and centrifuged for 30 minutes. The liquid (oleyl alcohol and aqueous supernatant) was discarded and the cell pellet was resuspended in pre-seed flask growth medium (synthetic medium). 100 mL of the aqueous phase was transferred to a sterile inoculation bottle. The inoculum was injected into the fermentor through a peristaltic pump.

2 L lipase addition after inoculation Lipolase® solution (100 L stock solution) was prepared at an enzyme concentration of 1.2-1.4 mg / mL. After inoculating the fermentor to the desired parts per million concentration based on the non-solvent volume, the solution was added to the fermentation. The addition time was less than 1 hour after inoculating the fermenter.

2 L Soybean Oil Fatty Acid Addition Either unused Cognis Emery (R) 610 soy fatty acid or recycled Cognis Emery (R) 610 soy fatty acid containing 0-30 weight percent fatty acid butyl ester in the fermentor 0.1 to 0.5 L / L (volume after inoculation) was added.

2 L Fermenter Operating Conditions The fermentor was operated at 30 ° C. for the entire growth and production stage. The pH was lowered from a pH of 5.7-5.9 to a control set point of 5.25 without any acid added. For the remainder of the growth and production phase, ammonium hydroxide was used to control the pH to pH = 5.2. For the remainder of the growth and production phase, sterile air was added to the fermentor at 0.2-0.3 slpm through a sparger. pO ₂ was not controlled. The stirrer was set at a constant rpm of 300 rpm. The agitation shaft had two Rushton impellers below the liquid level and one inclined impeller above the liquid level. Glucose was supplied by simultaneous saccharification and fermentation of liquefied corn mash by adding glucoamylase. As long as starch was available for saccharification, the glucose was kept in excess (1-50 g / L).

  A 5-20 mL sample was withdrawn from the fermentor, placed in a centrifuge tube, and centrifuged for cell mass measurement using the procedure described above. Furthermore, analysis methods such as gas analysis and LC analysis of the fermentation product in the aqueous phase and GC analysis of the fermentation product in the solvent phase were performed as described above.

  The fermentation conditions of Examples 17-22 are shown below, and a summary of the results (unused soybean oil fatty acids including fatty acid butyl esters and recycled soy fatty acids) is shown in Table 6.

Example 17
The experimental identifier GLNOR1050 included: 10 L pre-seed flask growth, 10 L breeding tank liquefaction, 10 L breeding tank operation, 10 L production tank liquefaction, 10 ppm Lipolase® 100 L (Genencor) fermenter 10 L production tank operation, extractant: unused Cognis Emery® 610 soybean fatty acid (unused soybean oil fatty acid). Liquid solvent and non-solvent materials were separated in a Sorval RC-12 centrifuge and all analytical methods.

Example 18
The experimental identifier GNLOR1051 included: 10L pre-seed flask growth, 10L breeding tank liquefaction, 10L breeding tank operation, 10L production tank liquefaction, 4ppm Lipolase® 100L (Genencor) fermenter 10 L production tank operation, extractant: unused Cognis Emery® 610 soy fatty acid (unused soybean oil fatty acid). Liquid solvent and non-solvent materials were separated in a Sorval RC-12 centrifuge and all analytical methods.

Example 19
The identifier 2011Y029 included: 2L pre-seed flask growth, 2L fermentation preparation, 2L liquefaction, 2L addition before inoculation, 2L fermentor inoculation, 2L after inoculation at 10ppm final concentration Lipase addition, 2 L recycled soybean oil fatty acid addition (recycled Cognis Emery® 610 soybean fatty acid and fatty acid butyl ester from Example 56A-50% v / v solvent loading), 2 L Fermenter operating conditions and all analytical methods.

Example 20
The identifier 2011Y030 included: 2L pre-seed flask growth, 2L fermentation preparation, 2L liquefaction, 2L addition before inoculation, 2L fermentor inoculation, 2L after inoculation at 10ppm final concentration Lipase addition, 0.4 L / L (volume after inoculation) of unused Cognis Emery® 610 soy fatty acid containing 20-30% fatty acid butyl ester, 2 L fermentor operating conditions, and all Analysis method.

Example 21
Identifier 2011Y031 included: 2L pre-seed flask growth, 2L fermentation preparation, 2L liquefaction, 2L addition before inoculation, 2L fermentor inoculation, 2L after inoculation at 10ppm final concentration Lipase addition, 2 L recycled soybean oil fatty acid addition (recycled Cognis Emery® 610 soybean fatty acid and fatty acid butyl ester from Example 56B-10% v / v solvent charge), 2 L Fermenter operating conditions and all analytical methods.

Example 22
The identifier 2011Y032 included: 2L pre-seed flask growth, 2L fermentation preparation, 2L liquefaction, 2L addition before inoculation, 2L fermentor inoculation, 2L after inoculation at 10ppm final concentration Lipase addition, 0.4 L / L (volume after inoculation) of unused Cognis Emery® 610 soy fatty acid, 2 L fermentor operating conditions, and all analytical methods.

Example 23
The following examples illustrate the production of isobutanol by fermentation using sucrose as the fermentable carbon source.

Production of biomass Inoculum: A seed medium was prepared to initiate the growth of isobutanologen. The composition of the seed medium was as follows: ammonium sulfate, 5 g / L; potassium dihydrogen phosphate, 3 g / L; magnesium sulfate heptahydrate, 0.5 g / L; ethanol, 3.2 g / L; yeast Extract (BBL), 5 g / L; glucose, 10 g / L; MES buffer, 150 mmol / L; biotin, 50 μg / L; and 15 g EDTA, 4.5 g zinc sulfate heptahydrate in 1 L water 0.8 g anhydrous manganese chloride, 0.3 g cobalt chloride hexahydrate, 0.3 g copper sulfate pentahydrate, 0.4 g anhydrous disodium molybdate, 4.5 g calcium chloride dihydrate 3 g iron sulfate heptahydrate, 1 g boric acid, 0.1 g potassium iodide trace element solution, 1 mL / L. The pH was adjusted to 5.5 and then the media filter was sterilized with a 0.22μ sterile filter device.

Preparation of 10 L Fermentor for Biomass Production One vial of isobutanologen PNY2205 is aseptic to 15 mL seed medium in a 125 mL vented flask for overnight growth at 30 ° C. and shaking at 260 rpm Moved. The medium is aseptically transferred to 500 mL of the same medium in a 2 L baffled vented flask for overnight growth at 30 ° C. and shaking at 260 rpm, and is prepared when the medium reaches OD ₆₀₀ 7 And transferred to a 10 L Sartorius C fermenter (Sartorius AG, Goettingen, Germany).

  A 10 L Sartorius C fermentor containing 6 L initial volume of growth medium was prepared. The composition and preparation of the growth medium was as follows: prior to sterilization, ammonium sulfate, 1 g / L; potassium dihydrogen phosphate, 5 g / L; magnesium sulfate heptahydrate, 2 g / L; yeast extract ( Amberex ™ 695), 2 g / L; Antifoam Sigma 204, 0.5 mL / L; biotin, 100 μg / L; and 1 mL / L trace element solution (prepared in 1 L water: 15 g EDTA, 4. 5 g zinc sulfate heptahydrate, 0.8 g anhydrous manganese chloride, 0.3 g cobalt chloride hexahydrate, 0.3 g copper sulfate pentahydrate, 0.4 g anhydrous sodium disodium molybdate, 4 g 0.5 g calcium chloride dihydrate, 3 g iron sulfate heptahydrate, 1 g boric acid, 0.1 g potassium iodide). After steam sterilization at 121 ° C. in place, the tank was cooled and 60 g of raw medium was added. The raw medium was prepared as follows: sucrose, 50% solution, 2.97 L; biotin, 1.4 mg; 34 mL trace element solution; titrated to pH 7.5 using 5N sodium hydroxide and steam sterilized Cooled after sterilization and added 320 mL of 20% (w / v) filter sterilized solution of ethanol and yeast extract (Amberex ™ 695). Here, the initial sugar concentration in the 10 L fermentor was 3.7 g / L sucrose, 0.8 g / L glucose, and 0.8 g / L fructose.

The fermentation is controlled at pH 5.5 (by addition of ammonium hydroxide), air flow at 30 ° C., 2.0 standard liters / minute, oxygen dissolved at 30% by stirring control, and back pressure of 0.5 bar. did. After inoculation, sugar was consumed until the residual glucose measurement was below 0.1 g / L, then the feeding program was started; this was done with an elapsed fermentation time of 11 hours. The program was set to maintain the sucrose limit until an OD ₆₀₀ of 20 (approximately 8 g / L dry cell weight) was achieved with a programmed growth rate of 0.1 / hour. The actual measured growth rate for this experiment was 0.18 / hour. After 20 hours fermentation time, the target OD ₆₀₀ was achieved.

  Once the goal was achieved, the medium was collected aseptically and centrifuged in a Sorvall RC12BP centrifuge. The resulting pellet was resuspended with the isobutanol production medium described below to a final volume of 300 mL. This medium was used as an inoculum for an isobutanol producing fermentation apparatus.

Production of isobutanol Production Fermenter Preparation: Two 1 liter glass Applikon (Applikon, Inc, Holland) fermentors equipped with a Sartorius BioStat B Plus Twin control unit (Sartorius AG, Goettingen, Germany) used. The fermenter was prepared with 1 L of deionized water and sterilized by autoclaving at 121 ° C. for 30 minutes. When the fermenter had cooled, water was removed aseptically and a volume of filter sterilized production medium as shown in Table 7 was added. The composition of the production medium was as follows: yeast nitrogen base without amino acids (Difco), 6.7 g / L; yeast synthetic dropout medium supplement without histidine, leucine, tryptophan, and uracil (Sigma) 2.70 g / L; tryptophan, 1.6 mg / L; leucine, 8 mg / L; ethanol, 2.8 g / L; Antifoam Sigma 204, 0.2 mL / L; sucrose, 25 g / L. The lipase solution sterilized by filtration immediately before inoculation was as shown in Table 7. A lipase solution was prepared by diluting Lipolase® L100 (Sigma) in 10 mM potassium phosphate buffer at pH 7 to a final concentration of 1.25 mg protein / mL. Solutions were prepared and stored at 5 ° C. for 1 day before being added to the fermenter.

  The fermentor was controlled at 3% dissolved oxygen by pH 5.2 (by addition of 20% potassium hydroxide), air flow at 30 ° C., 0.2 standard liters / minute, and stirring control.

Each fermentor was inoculated with 40 mL of concentrated biomass to an initial OD _{600 of} 20-25 (about 8-10 g / L dry cell weight). Addition of 4 mL of filter sterilized vitamin solution (in water, thiamine-HCl, 1 mg / mL; nicotinic acid, 1 mg / mL), similar to the volume of filter sterilized soybean oil fatty acid (SOFA) shown in Table 7. Added at the time of inoculation. Samples (5-10 mL) were withdrawn every 2-3 hours and assayed for glucose and sucrose by YSI Select Biochemistry Analyzer (YSI, Inc., Yellow Springs, Ohio). As sucrose was consumed, 50% sucrose (w / w) raw material was added to maintain a concentration of 5-30 g / L. The aqueous and organic phases of the sample were separated and analyzed by the HPLC method described above by Agilent 1100 HPLC. A Shodex® Sugar SH1011 column was used with 0.01 N sulfuric acid mobile phase for analysis of organic acids and alcohols. A BioRad Aminex® HPX-87N column was used with 0.01 M Na ₂ HPO ₄ (pH 8) mobile phase for analysis of sucrose, glucose, and fructose.

  Each of the fermenters to which lipase was added had lower concentrations of isobutanol in the aqueous phase and free isobutanol in the solvent phase. The aqueous and solvent phase concentrations of isobutanol are shown in FIG. Also, the addition of more lipase at the same solvent loading resulted in a lower aqueous titer of isobutanol and lower free isobutanol in the solvent, and more isobutanol as FABE.

  The medium containing lipase gave higher isobutanol potency than the control fermenter without lipase. FIG. 7 shows the effective titer of isobutanol. In this example, the effective titer was calculated based on the initial measured weight of broth in the fermenter after inoculation and the initial measured weight of solvent introduced into the fermenter. Throughout the fermentation, the solvent density was assumed to be 0.88 g / mL and the aqueous broth density was assumed to be 1.00 g / mL. The addition of more lipase at a lower solvent loading resulted in a higher effective titer of isobutanol (D vs. C) but not as much as an increase in the relative volume of the solvent (C vs. B).

  As shown in FIG. 8, the sugar consumed, calculated in glucose equivalents, is higher in the fermenter with lipase added. The glucose equivalent consumed is calculated from the measured sugar remaining fed to the fermentor, counting each mole of sucrose as 2 moles of glucose and each mole of fructose as 1 mole of glucose, then , Converted to grams by the molecular weight of glucose. The concentration of glucose equivalent consumed is also calculated based on the initial volume of fermentation broth after inoculation.

Example 24
Lipase treatment of liquefied corn mash for simultaneous saccharification fermentation using in-situ product removal using oleyl alcohol Broth and oleyl alcohol taken from fermentation performed as described above in Examples 1, 2, and 3 Samples of E. G. Bligh and W.W. J. et al. % By weight of lipid (fatty acid methyl ester, derivatized as FAME) and free fatty acid according to the method described by Dyer (Canadian Journal of Biochemistry and Physiology, 37: 911-17, 1959, ref. 1) % By weight (derivatized as FFA, fatty acid methyl ester, FAME). The liquefied corn mash prepared for each of the three fermentations was also Lipolase® 100 L (Novozymes) (10 ppm Lipolase (kg) per kg of liquefied reaction mass containing 30 wt% ground corn nuts. After treatment with (trademark) total soluble protein (BCA protein analysis, Sigma Aldrich)), it was analyzed for wt% lipid and wt% FFA. The fermentation described in Examples 2 and 3 containing liquefied corn mash treated with lipase (without heat inactivation of lipase) without adding lipase to the liquefied corn mash of Example 1 (control) Was the same except that it was not added to the fermentation described in Example 3.

  The% of FFA of lipase-treated liquefied corn mash prepared for fermentation performed as described in Examples 2 and 3 was 31% of that without lipase treatment (Example 1). In comparison, they were 88% and 89%, respectively. At 70 hours (end of run (EOR)), the concentration of FFA in the OA phase of the fermentation performed as described in Examples 2 and 3 (containing active lipase) was 14% and 20%, respectively. The corresponding increase in lipids (measured as corn oil fatty acid methyl ester derivatives) was found by GC / MS to be due to lipase-catalyzed esterification of COFA by OA, in which liquefied corn mash Lipase-catalyzed hydrolysis of corn oil first produced COFA; the formation of oleyl palmitate, oleyl stearate, and oleyl oleate was confirmed by GC / MS, and the fourth ester was tentatively designated as oleyl linoleate Identified. The results of FFA and lipid analysis are shown in Table 8.

Example 25
Thermal deactivation of lipase in lipase-treated liquefied corn mash to limit the production of oleyl alcohol esters of corn oil free fatty acids Tap water (918.4 g) is added to the coated 2 L resin kettle, Next, 474.6 g wet weight (417.6 g dry weight) of ground whole corn corn (1.0 mm sieve in a hammer mill) was added with stirring. The mixture was heated to 55 ° C. with stirring at 300 rpm and the pH was adjusted to 5.8 using 2N sulfuric acid. To the mixture is added 14.0 g of an aqueous solution containing 0.672 g of Spezyme®-FRED L (Genencor®, Palo Alto, Calif.), And the temperature of the mixture is stirred at 600 rpm and pH 5. 8 and raised to 85 ° C. After 120 minutes at 85 ° C., the mixture was cooled to 50 ° C., and a 45.0 mL aliquot of the resulting liquefied corn mash was transferred to a 50 mL polypropylene centrifuge tube and stored frozen at −80 ° C.

  In the first reaction, 50 g of liquefied corn mash prepared as described above was mixed with 10 ppm of Lipolase® 100 L (Novozymes) for 6 hours at 55 ° C. and 1 hour of lipase at 85 ° C. The mixture was cooled to 30 ° C. without quenching. In the second reaction, 50 g of liquefied corn mash is mixed with 10 ppm of Lipolase® for 6 hours at 55 ° C., then heated to 85 ° C. for 1 hour (lipase deactivation), then 30 Cooled to ° C. In the third reaction, 50 g of liquefied corn mash without lipase was mixed at 55 ° C. for 6 hours, without heating at 85 ° C. for 1 hour, the mixture was cooled to 30 ° C. and 38 g of oleyl alcohol was added. The resulting mixture was stirred at 30 ° C. for 73 hours. In the fourth reaction, 50 g of liquefied corn mash without lipase was mixed at 55 ° C. for 6 hours, then heated to 85 ° C. over 1 hour and then cooled to 30 ° C. Each of the four reaction mixtures was taken at the 6 hour time point, then 38 g oleyl alcohol was added and the resulting mixture was stirred at 30 ° C. and taken at the 25 hour and 73 hour time points. According to the method described by reference 1, samples (both liquefied mash and oleyl alcohol (OA)) were prepared by weight% lipid (fatty acid methyl ester, derivatized as FAME) and weight% free fatty acid. (FFA, fatty acid methyl ester, derivatized as FAME) was analyzed.

  % Of FFA in the OA phase of the second reaction carried out with thermal inactivation of the lipase prior to OA addition, if the lipase in the liquefied corn mash treated with lipase was not inactivated by heat (First reaction) was 99% at 25 hours and 95% at 73 hours compared to only 40% FFA and 21% FFA at 25 and 73 hours. No significant change in% FFA was observed in the two control reactions where lipase was not added. The results are shown in Table 9.

Example 26
Thermal inactivation of lipase in lipase-treated liquefied corn mash for simultaneous saccharification fermentation using in situ product removal with oleyl alcohol. Three fermentations were described above in Examples 4, 5, and 6. Performed as follows. In the liquefied corn mash of Examples 4 and 6, lipase was not added before fermentation, and lipase treatment of liquefied corn mash in fermentation described in Example 5 (7.2 ppm of Lipolase® total soluble protein was used) Immediately after the inactivation, a heat inactivation treatment (to completely inactivate the lipase) was performed, and thereafter, nutrient addition and fermentation before inoculation were performed. The% of FFA in liquefied corn mash prepared without lipase treatment for the fermentation performed as described in Examples 4 and 6 compared to 89% with lipase treatment (Example 5). And 31% and 34%, respectively. During the fermentations listed in Table 10, the concentration of FFA in the OA phase did not decrease in any of the three fermentations, including those containing lipase deactivated by heat. The% of FFA in the OA phase of the fermentation performed according to Example 5 (with heat inactivation of lipase prior to fermentation) is the remaining two fermentations in which the liquefied corn mash was not treated with lipase (Examples) 95% at 70 hours (end of execution (EOR)) compared to only 33% FFA for 4 and 6). The results are shown in Table 10.

Example 27
Milled whole corn lipase treatment prior to liquefaction Tap water (1377.6 g) is added to each of the two coated 2 L resin kettles and then 711.9 g wet weight (625 .8 g dry weight) of ground whole corn (1.0 mm sieve in a hammer mill) was added to each kettle with stirring. Each mixture was heated to 55 ° C. with stirring at 300 rpm, and the pH was adjusted to 5.8 using 2N sulfuric acid. To each mixture was added 21.0 g of an aqueous solution containing 1.008 g of Spezyme®-FRED L (Genencor®, Palo Alto, Calif.). Next, 10.5 mL of an aqueous solution of Lipolase® 100 L solution (21 mg total soluble protein, 10 ppm lipase final concentration) is added to one mixture, and Lipolase® 100 L solution is added to the second mixture. A 1.05 mL aqueous solution of (2.1 mg total soluble protein, 1.0 ppm lipase final concentration) was added. Samples were withdrawn from each reaction mixture at 55 ° C for 1 hour, 2 hours, 4 hours and 6 hours, then the temperature of the mixture was raised to 85 ° C at pH 5.8 with stirring at 600 rpm. A sample was taken when the temperature first reached 85 ° C. After 120 hours at 85 ° C., a sample is taken, the mixture is cooled to 50 ° C., and the final sample of the resulting liquefied corn mash is transferred to a 50 mL polypropylene centrifuge tube; all samples are at −80 ° C. Stored frozen.

  In two separate reactions, a 50 g sample of liquefied corn mash treated with 10 ppm lipase or a 55 g sample of liquefied corn mash treated with 1.0 ppm lipase, prepared as described above, was prepared using oleyl alcohol. (OA) (38 g) at 30 ° C. for 20 hours, and then the liquefied mash and OA in each reaction mixture are separated by centrifugation and separated according to the method described by reference 1 Were analyzed for weight percent lipid (fatty acid methyl ester, derivatized as FAME) and free fatty acid weight percent (FFA, fatty acid methyl ester, derivatized as FAME). The% FFA in the OA phase of the liquefied mash / OA mixture prepared using the thermal inactivation of 10 ppm lipase during liquefaction uses the thermal inactivation of 1.0 ppm lipase during liquefaction. Compared to only 62% FFA in the OA phase of the liquefied mash / OA mixture prepared in the past, it was 98% in 20 hours. The results are shown in Table 11.

Example 28
Screening of lipase for the treatment of ground whole corn before liquefaction Tap water (67.9 g) and ground whole corn (35.1 g wet weight, pH 5.8) 7 reaction mixtures containing those milled with a 1.0 mm sieve using a hammer mill) were stirred at 55 ° C. in a stoppered flask. A 3 mL sample (t = 0 hour) is removed from each flask and the sample is immediately frozen with dry ice, then the following lipase: Lipolase® 100 L, Lipex® 100 L About 1 mg of total soluble protein (final concentration of 10 ppm in the reaction mixture) of one of Lipoclean® 2000T, Lipozyme® CALB L, Novozyme® CALA L, and Palatase 20000L 0.5 mL of 10 mM sodium phosphate buffer (pH 7.0) was added to one of each flask; no lipase was added to the seventh flask. The resulting mixture was stirred at 55 ° C. in a stoppered flask and 3 mL samples were withdrawn from each reaction mixture at 1 h, 2 h, 4 h and 6 h and are described by reference 1. According to method, immediately on dry ice until analyzed for weight percent of lipid (fatty acid methyl ester, derivatized as FAME) and free fatty acid weight percent (FFA, fatty acid methyl ester, derivatized as FAME) Upon freezing, the percent free fatty acid content was calculated for the total concentration of lipid and free fatty acid combined and measured for each sample. The results are shown in Table 12.

Example 29
Milled whole corn lipase treatment prior to simultaneous saccharification fermentation using in-situ product removal with oleyl alcohol. Three fermentations are performed as described above in Examples 7, 8, and 10. did. In fermentations performed as described in Examples 7 and 10, lipase (10 ppm of Lipolase® total soluble protein) is added to the ground corn suspension and 6 ° C. at 55 ° C. prior to liquefaction. Heated for hours to produce liquefied corn mash containing lipase inactivated by heat. No lipase was added to the ground corn suspension used to prepare the liquefied corn mash for fermentation described in Example 8, but this suspension was subjected to 55 ° C. prior to liquefaction. The same heating step in was performed. The% of FFA in the lipase-treated liquefied corn mash prepared for fermentation performed as described in Examples 7 and 10 is 41% of the case without lipase treatment (Example 8). And 83% and 86%, respectively. During fermentation, the concentration of FFA did not decrease in any of the fermentations, including those containing lipase inactivated by heat. The percentage of FFA in the OA phase of the fermentation carried out according to Examples 7 and 10 (with thermal inactivation of lipase prior to fermentation) is that lipase of ground whole corn fruit prior to liquefaction. 97% each at 70 hours (end of run (EOR)) compared to only 49% FFA for the fermentation carried out according to Example 8 which was not treated with. The results are shown in Table 13.

Example 30
Lipase treatment of ground whole corn nuts or liquefied corn mash for simultaneous saccharification and fermentation using in situ product removal with corn oil fatty acids (COFA). Performed as described above for 12, 13, and 14. In fermentations performed as described in Examples 9, 13, and 14, lipase (10 ppm of Lipolase® total soluble protein) was added after liquefaction and lipase was not deactivated by heat. Fermentations performed as described in Examples 9 and 14 were supplemented with 5 g / L ethanol prior to inoculation, while fermentations performed as described in Example 13 had ethanol. It was not added. Fermentations performed as described in Examples 11 and 12 were performed using lipase during liquefaction using the addition of 10 ppm Lipolase® total soluble protein to a suspension of ground corn prior to liquefaction. Was deactivated by heat. For fermentations performed as described in Example 11, 5 g / L ethanol was added prior to inoculation, whereas for fermentations performed as described in Example 12, ethanol was added. I couldn't. The final total grams of isobutanol (i-BuOH) present in the COFA phase of the fermentation containing the active lipase is the final i-BuOH present in the COFA phase of the fermentation containing the inactive lipase. Much more than total grams. The final total gram of isobutanol (i-BuOH) present in the fermentation broth containing the active lipase is i-BuOH (present as FABE) present in the fermentation broth containing the inactive lipase (aqueous phase). Slightly less than the final total gram of i-BuOH), so that the overall production of i-BuOH (as a combination of free i-BuOH and COFA isobutyl ester (FABE)) is thermally deactivated Considerably more in the presence of active lipase compared to that obtained in the presence of lipase. The results are shown in Tables 14 and 15.

Example 31
Formation of iso-butyl COFA ester by phospholipase catalyzed reaction of isobutanol and corn oil fatty acid (COFA) Aqueous 2- (N-morpholino) ethanesulfonic acid buffer (0.20 M, pH 5.3), isobutanol (2-methyl) -1-propanol), phospholipase (phospholipase A; Sigma Aldrich, L3295-250) and a corn oil fatty acid prepared reaction mixture (Table 16) from corn oil was stirred at 30 ° C. and the samples were It was withdrawn from the reaction mixture, immediately centrifuged, the aqueous and organic layers separated and analyzed for isobutanol (i-BuOH) and isobutyl ester of corn oil fatty acid (i-BuO-COFA) (Table 17).

Example 32
Dependence of isobutyl-COFA ester concentration on aqueous / COFA ratio for lipase catalyzed reaction Aqueous 2- (N-morpholino) ethanesulfonic acid buffer (0.20 M, pH 5.2), isobutanol (2-methyl-1-propanol) ), Lipase (Lipolase® 100 L; Novozymes) and a corn oil fatty acid (Table 18) prepared from corn oil, the reaction mixture is stirred at 30 ° C., and the sample is added to each reaction mixture at a given time. Extracted from and immediately centrifuged, the aqueous and organic layers were separated and analyzed for isobutanol (i-BuOH) and isobutyl ester of corn oil fatty acid (i-BuO-COFA) (Table 19).

Example 33
Dependence of butyl-COFA ester concentration on esterified alcohol in lipase catalyzed reaction Aqueous 2- (N-morpholino) ethanesulfonic acid buffer (0.20 M, pH 5.2), isobutanol (2-methyl-1-propanol) Or a reaction mixture containing corn oil fatty acid (Table 20) prepared from n-butanol, lipase (Lipolase® 100L; Novozymes) and corn oil is stirred at 30 ° C. and the sample is allowed to react for each reaction at a given time. Extract from the mixture, immediately centrifuge, separate the aqueous and organic layers, isobutanol (i-BuOH) or n-butanol (n-BuOH) and isobutyl- or butyl ester of corn oil fatty acid (BuO-COFA) ) (Table 21).

Example 34
Formation of iso-butyl oleate by lipase-catalyzed reaction of isobutanol and oleic acid Aqueous 2- (N-morpholino) ethanesulfonic acid buffer (0.20 M, pH 5.2), isobutanol (2-methyl-1-propanol) ), A reaction mixture containing lipase (0 ppm or 10 ppm Lipolase® 100 L; Novozymes) and oleic acid (Alfa Aesar) (Table 22) is stirred at 30 ° C., and samples are added to each reaction mixture at predetermined times. Extracted from and immediately centrifuged, the aqueous and organic layers were separated and analyzed for isobutanol (i-BuOH) and iso-butyl oleate (i-BuO-oleate) (Table 23).

Example 35
Comparison of formation of iso-butyl oleate by lipase catalyzed reaction of isobutanol and oleic acid Aqueous 2- (N-morpholino) ethanesulfonic acid buffer (MES, 0.20 M, pH 5.2), isobutanol (2-methyl) -1-propanol), oleic acid (Alfa Aesar), and Liporose (registered trademark) 100 L, Lipex (registered trademark) 100 L, Lipozyme (registered trademark) CALB L, Novozyme (registered trademark) CALA L, Palatase (manufactured by Novozymes) Lipase derived from (registered trademark) (10 ppm), or a fluorescent bacterium (Pseudomonas fluorescens), Pseudomonas cepacia, Muco, manufactured by Sigma Aldrich Mucor miehei, porcine pancreas, Candida cylindracea, Rhizopus niveus, Candida antarctica, iRus p The reaction mixture containing lipase (10 ppm) derived from Aspergillus (10 ppm) (Table 24) is stirred at 30 ° C., samples are withdrawn from each reaction mixture at predetermined time points, immediately centrifuged, and the aqueous and organic layers are separated. Separated and analyzed for isobutanol (i-BuOH) and iso-butyl oleate (i-BuO-oleate) (Table 25).

Formation of Ethyl-COFA Ester by Lipase-Catalyzed Reaction of Ethanol and Corn Oil Fatty Acid (COFA) Aqueous 2- (N-morpholino) ethanesulfonic acid buffer (0.20M, pH 5.5), ethanol, lipase (Lipolase®) ) A reaction mixture containing corn oil fatty acids (Table 26) prepared from 100 L or Lipozyme® CALB L; Novozymes) and corn oil is stirred at 30 ° C. and samples are stirred from each reaction mixture at predetermined times. The mixture was immediately extracted and centrifuged, and the aqueous layer and the organic layer were separated and analyzed for ethanol and ethyl ester of corn oil fatty acid (EtO-COFA) (Table 27).

Example 37
Formation of ethyl-COFA ester by lipase catalyzed reaction of ethanol and corn oil fatty acid (COFA) during yeast fermentation Wild type yeast strain CEN. PK113-7D is grown overnight in medium containing yeast nitrogen base without amino acids (6.7 g / L), dextrose (25 g / L), and MES buffer (0.1 M at pH 5.5). It was. The overnight culture was diluted in fresh medium so that the optical density obtained at 600 nm was 0.1. The diluted medium was aliquoted into six 250 mL closed cap shake flasks, 25 mL per flask. Four of the media contain two lipase enzyme stock solutions (Lipozyme® CALB L or Lipolase® 100 L of 10 mM phosphate buffer so that the final lipase concentration in the media is 10 ppm. One of the solutions (2 mg protein per mL of pH 7.0). Corn oil fatty acid (COFA) was added to the three aqueous media (no enzyme, CALB L, or Lipolase® 100 L) in a flask at a 1: 1 volume ratio. One flask had no replenishment. The medium was grown for 23 hours in a shaking incubator temperature controlled at 30 ° C. at a shaking speed of 250 rpm. Samples for cell mass measurement were phase separated in a 15 mL conical bottom tube. The optical density of the sample at 600 nm was measured at a 20-fold dilution in saline. Samples for chromatographic analysis (5 mL aqueous or 10 mL media / COFA emulsion) were immediately centrifuged at 4000 rpm for 5 minutes in a TX-400 swing bucket rotor in a 15 mL conical bottom tube. For aqueous samples, a 0.22 μm spin filter was used prior to analysis. The aqueous sample was analyzed on a Shodex SH1011 column equipped with a SH-G guard column using 0.01 M sulfuric acid mobile phase at 50 ° C. and a flow rate of 0.5 mL per minute. The detection of saccharides and alcohol was based on refractive index and absorption at 210 nm and was quantified using a standard curve. Samples were taken from aqueous medium (no addition of COFA) or medium / COFA emulsion and analyzed as described in the previous example for the ethyl ester of COFA. The results are shown in Tables 28 and 29.

Example 38
Formation of methyl-COFA ester by lipase-catalyzed reaction of methanol and corn oil fatty acid (COFA) Aqueous 2- (N-morpholino) ethanesulfonic acid buffer (0.20 M, pH 5.5), methanol, lipase (Lipolase®) ) 100 L (Novozymes), Lipozyme® CALB L (Novozymes), Rhizopus arrizus lipase (Sigma Aldrich), and Candida cylind algae (Candida cereal oil) Stir reaction mixtures containing corn oil fatty acids (Table 30) at 30 ° C. and stir samples from each reaction mixture at predetermined times. Reluctant extraction, immediately centrifuged and the aqueous and organic layers were separated and analyzed for ethyl ester of ethanol and corn oil fatty acids (EtO-COFA) (Table 31).

Example 39
Formation of 1-propyl-COFA ester by lipase catalyzed reaction of 1-propanol and corn oil fatty acid (COFA) Aqueous 2- (N-morpholino) ethanesulfonic acid buffer (0.20M, pH 5.5), 1-propanol, Lipase (R) 100 L (Novozymes), Lipozyme (R) CALB L (Novozymes), Rhizopus arrizus lipase (Sigma Aldrich) and Candida gir Cid And a reaction mixture (Table 32) containing corn oil fatty acid prepared from corn oil is stirred at 30 ° C. Extraction with stirring from the mixture, immediately centrifuged and the aqueous and organic layers were separated and analyzed for 1-propyl ester of 1-propanol and corn oil fatty acids (PrO-COFA) (Table 33).

Example 40
Formation of 1-pentyl-COFA ester by lipase catalyzed reaction of 1-pentanol and corn oil fatty acid (COFA) Aqueous 2- (N-morpholino) ethanesulfonic acid buffer (0.20M, pH 5.5), 1-pen Tanol, lipase (Lipolase® 100L (Novozymes), Lipozyme® CALB L (Novozymes), Rhizopus arrizus lipase (Sigma Aldrich), Candida lind ) And a reaction mixture containing corn oil fatty acids prepared from corn oil (Table 34) is stirred at 30 ° C. and the samples are reacted at a given time. The mixture was withdrawn with stirring and immediately centrifuged to separate the aqueous and organic layers and analyzed for 1-pentanol and 1-pentyl ester of corn oil fatty acid (PenO-COFA) (Table 35).

Example 41
Formation of 2-methyl-1-butyl-COFA ester by lipase catalyzed reaction of 2-methyl-1-butanol and corn oil fatty acid (COFA) Aqueous 2- (N-morpholino) ethanesulfonic acid buffer (0.20 M, pH 5) .5), 2-methyl-1-butanol, lipase (Lipolase® 100 L (Novozymes), Lipozyme® CALB L (Novozymes), Rhizopus arrizus lipase (Sigma Aldiz, SigmaAld) A reaction mixture (Table 36) containing corn oil fatty acids prepared from Candida cylindracea lipase (Sigma Aldrich) and corn oil at 30 ° C. Stir and remove samples from each reaction mixture with stirring at a given time, immediately centrifuge, separate the aqueous and organic layers, and remove 2-methyl-1-butanol and corn oil fatty acid 2-methyl- 1-Butyl ester (MeBO-COFA) (Table 37) was analyzed.

Example 42
Formation of isopropyl-COFA ester by lipase-catalyzed reaction of isopropanol and corn oil fatty acid (COFA) Aqueous 2- (N-morpholino) ethanesulfonic acid buffer (0.20 M, pH 5.5), isopropanol (2-propanol), lipase (Lipolase® 100 L (Novozymes), Lipozyme® CALB L (Novozymes), Rhizopus arrhizus lipase (Sigma Aldrich) and Candida kilind a cirdida cirida girder A reaction mixture (Table 38) containing corn oil fatty acids prepared from corn oil was stirred at 30 ° C. and tested. Is extracted from each reaction mixture with stirring at a given time, immediately centrifuged, and the aqueous and organic layers are separated, isopropanol and isopropyl ester of corn oil fatty acid (i-PrO-COFA) (Table 39) analyzed.

Example 43
Comparison of partition coefficients for isobutanol between water and extractant. An aqueous solution of isobutanol (30 g / L) is mixed with corn oil fatty acid (COFA), or oleic acid or corn oil triglyceride and their measured The distribution coefficient was reported in the table in comparison with the distribution coefficient measured for oleyl alcohol. The results are shown in Table 40.

Example 44
Corn Oil Fatty Acid Generation A 750 g crude corn oil (non-food grade, ethanol) was added to a 5 liter (5 L) round bottom flask equipped with a mechanical stirrer, thermocouple, heating mantle, condenser and nitrogen tea. 2112 g of water and 285 g of 50% sodium hydroxide solution (charged from the fermentation facility) were charged. The mixture was heated to 90 ° C. and held for 2 hours, during which time the mixture became one high viscosity, emulsion-like single phase. At the end of this time, TLC shows no corn oil remaining in the mixture. The mixture was then cooled to 74 ° C. and 900 g of 25% sulfuric acid was added to acidify the mixture. The mixture was then cooled to 50 ° C. and the aqueous layer was drained. The oil layer was washed twice with 1500 mL of 40 ° C. water and then once with 1 liter of saturated brine. It was dried over magnesium sulfate and filtered through celite. Yield was 610 g of a clear red oil. Titration of free fatty acids by AOCS method Ca 5a-40 shows a 95% fatty acid content expressed as oleic acid. The sample was silanized by reacting 104 mg with 100 uL N-methyl-N- (trimethylsilyl) trifluoroacetamide in 1 mL dry pyridine. Gas chromatography-mass spectrometry (GCMS) of the silanized product shows the presence of TMS derivatives of 16: 0, 18: 2, 18: 1, 18: 0, and 20: 0 acids.

Example 45
Chemical synthesis of FABE The 3 L flask was equipped with a mechanical stirrer, thermocouple, nitrogen inlet, heating mantle and condenser. The flask was charged with COFA (595 g) (prepared as in Example 44), isobutanol (595 g), and sulfuric acid (12 g). The mixture was refluxed for 1.5 hours, at which time the condenser was removed and replaced with a still head. Distillate was collected over 3 hours at an initial head temperature of 90 ° C and a final head temperature of 105 ° C. The mixture was then cooled to room temperature and 500 mL of deionized water was added. The layers were separated and the organic layer was washed 5 times with 500 mL deionized water. It was then washed once with 500 mL of 10% calcium chloride solution and then 6 times with 500 mL of deionized water. The oil was then dried over magnesium sulfate and filtered through a celite bed, yielding 601 g of a clear red oil. GC analysis shows the presence of 0.36% by weight of isobutanol. GC / MS analysis indicates the presence of isobutyl palmitate, isobutyl stearate, isobutyl oleate, isobutyl linoleate, and isobutyl linolenate.

Example 46
Recovery of butanol using inorganic acid catalyst A 1 liter round bottom flask equipped with mechanical stirring and a 12 inch column equipped with a Raschig ring with a still head and nitrogen inlet on top was used. The flask was charged with 254 g FABE synthesized as in Example 45, 255 g COFA, 100 mL water, and 5 g sulfuric acid and heated to a pot temperature of 93 ° C. The head temperature was equilibrated at 89.7 ° C. The first fraction was collected at a reflux ratio that maintained a head temperature of 89-94 ° C.

  The reaction was cooled and allowed to stand at room temperature for 3 days. GC analysis of the pot shows a total of 1 g isobutanol in the pot. Distillation was resumed and three or more fractions were collected, each 25 mL. After collecting fraction # 2, 100 mL of water was added to the pot. Four fractions were collected and analyzed and the results are shown in Table 41.

  GC analysis was performed using a Hewlett Packard 6890 GC using a 30 m FFAP column. Samples were dissolved in isopropanol and 1-pentanol was added as an internal standard. Standard curves were generated for isobutanol, isobutyl palmitate, isobutyl stearate, isobutyl oleate, isobutyl linoleate, isobutyl linolenate, isobutyl arachidate, palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid. The FABE content was reported as the sum of butyl esters and the COFA content was reported as the sum of fatty acids.

Example 47
Recovery of butanol using organic acid catalyst A 1 liter three-necked round bottom flask equipped with a magnetic stirrer, thermocouple, dropping funnel and still head was used. The flask was charged with 100 g FABE synthesized as in Example 45, 100 g COFA, 5 g p-toluenesulfonic acid, and 25 mL water. Initial pot isobutanol analysis indicates that 1.1 g of isobutanol is present (contaminant in FABE). The pot was heated to 125 ° C. When the pot reached 116 ° C., the head temperature was 96 ° C. and 125 mL of water was added over 2.5 hours. Six fractions were collected over the time the water was added and they were analyzed by GC as in Example 46. The results are shown in Table 42.

  The butanol analysis of the remaining still pot shows that 0.9 g of free isobutanol is present. The initial COFA: FABE mixture analyzed was 45 wt% FABE. The final pot analyzed was 32 wt% FABE.

Example 48
Hydrolysis of FABE with Water at High Temperature A 1 liter autoclave was charged with FABE synthesized as in Example 45, 300 mL and 300 mL of water. The autoclave was sealed and purged with nitrogen. Stirring was started and then heated to 250 ° C. for 45 minutes and after reaching temperature, samples were removed every hour. Samples were analyzed by GC as in Example 46. The oil phase sample showed a composition according to the time shown in Table 43.

Example 49
Hydrolysis of FABE with Dilute Acid at High Temperature A 1 liter autoclave was charged with FABE synthesized as in Example 45, 450 g of a 75/25 mixture of COFA and 150 g of 2% sulfuric acid. The autoclave was sealed and purged with nitrogen. Stirring was started and then it was heated to 225 ° C. over 45 minutes and after reaching temperature, samples were removed every hour. Samples were analyzed by GC as in Example 46. The oil phase sample showed a composition according to the time shown in Table 44.

Example 50
Hydrolysis of FABE with sulfuric acid in solvent at 100 ° C. A solution of 5 g FABE synthesized as in Example 45, 5 g 25% sulfuric acid and 60 g diethylene glycol dimethyl ether was prepared. 10 g of solution was added to each of the five vials, which were then sealed. All vials were heated to 100 ° C. and one vial was removed from the heater and analyzed every hour. The resulting composition was measured by GC (as described in Example 46) and reported in Table 45.

Example 51
Hydrolysis of FABE by Reactive Distillation A 12 liter flask was equipped with an insulated 2 "x 30" column with a raw material inlet and a still head on top. The column was randomly packed with 1 liter of Pro-pak® (316 SS 0.16 inch) still packing and 500 g of Amberlyst® 36 solid acid catalyst (Dow). The flask was charged with 6 liters of water and boiled. The heat was controlled to have a water distillation rate of about 1.8 mL / min. FABE synthesized as described in Example 45 was added to the top of the column at a rate of 2 g / min. Feeding continued for a total of 60 minutes. Distillation continued for another 30 minutes. A total of 194 g of distillate was collected and contained 2.1 g of isobutanol. Based on the amount of FABE fed, this represents a 9% conversion of FABE to butanol.

Example 52
Hydrolysis of FABE with Backflow Steam The apparatus described in Example 50 is heated with a heat tape (heat) wrapped around a distillation column.
changed with the addition of tape). The temperature of the upper half of the tower was adjusted to 115 ° C and the temperature of the lower half of the tower was adjusted to 104 ° C. The pot was boiled and the pot heat was adjusted until the water distilled at a rate of 1.5-2 mL / min. With continued distillation, FABE (346 g) synthesized as described in Example 45 was fed to the top of the packed column over 3 hours. After the feeding period, distillation continued for another 90 minutes. A total of 486 g of distillate was collected and contained 30.1 g of isobutanol. This represents a 39% conversion of FABE to isobutanol.

Example 53
Hydrolysis catalyzed by water-insoluble organic acids 150 g FABE in a 1 liter three-necked round bottom flask equipped with an oil bath, mechanical stirrer, nitrogen inlet, subsurface water inlet, and still head , 50 g of water, and 5 g of dodecylbenzenesulfonic acid were added. The oil bath was heated to 95-100 ° C. and a slow nitrogen sweep was initiated. Distillate fractions were collected every 30 minutes for a total of 5 hours. After 3 hours, water was fed into the still pot at a rate of 15 mL / hour. The distillate fraction was analyzed for isobutanol content by the GC method described in Example 46 and the results are shown in Table 46. Approximately 44% of the isobutanol contained in FABE was collected over 5 hours.

Example 54
Hydrolysis catalyzed by solid acid catalyst 150 g FABE and 50 g dry in a 1 liter three-neck round bottom flask equipped with oil bath, mechanical stirrer, subsurface nitrogen inlet, subsurface water inlet, and still head Amberlyst 15 solid acid catalyst was charged. The flask was heated to 110 ° C. with an oil bath and water was added via a syringe pump at a rate of 15 mL / hr. Distillate fractions were collected every 30 minutes for a total of 5 hours. The fraction was analyzed for isobutanol content by the GC method described in Example 46 and the results are shown in Table 47. Approximately 44% of the theoretical amount of isobutanol contained in the FABE was collected over 5 hours.

Example 55
Hydrolysis catalyzed by water-soluble organic acid catalyst 1 liter flask equipped with mechanical stirrer, subsurface nitrogen inlet, subsurface water inlet, and still head is charged with 200 g FABE and 10 g p-toluenesulfonic acid did. The flask was stirred and heated to 110 ° C. with an oil bath, during which time water was added via a syringe pump at a rate of 20 mL / hour. Distillate fractions were collected every 30 minutes for a total of 3 hours. The fraction was analyzed for isobutanol content by the GC method described in Example 46 and the results are shown in Table 48. Approximately 30% of the theoretical amount of isobutanol contained in FABE was collected over 5 hours.

Example 56
Hydrolysis of the solvent phase from fermentation Solvent phase 1
The solvent phase from the fermentation shown in Example 17 was analyzed by the GC method shown in Example 46 and the results are shown in Table 49. The analysis shows mainly FABE and fatty acids with a small amount of material having a retention time consistent with FAEE. Analysis of butyl ester and acid alone shows a ratio of 62% FABE and 39% fatty acid.

  The solvent phase (1.25 liters, 1090 g) and 1.25 liters of water were charged to a 1 gallon autoclave. The autoclave was sealed, heated to 250 ° C. and held at that temperature for 4 hours. Next, the autoclave was cooled and opened to obtain an emulsion. The mixture was filtered through a celite bed and the layers were separated. The organic layer was washed 3 times with 1 liter of water. The sample was then heated to 50 ° C. and purged with nitrogen for 6 hours. GC analysis shows the absence of i-BuOH and a ratio of 33% FABE and 67% fatty acids. An amber oil (993.9 g) was obtained. A detailed composition analysis of the original solvent phase from fermentation Example 17 and the solvent phase after hydrolysis is shown in Table 50.

B. Solvent phase 2
The solvent phase from the fermentation shown in Example 18 was analyzed by the GC method shown in Example 46 and the results are shown in Table 49. The analysis shows mainly FABE and fatty acids with a small amount of material having a retention time consistent with FAEE. Analysis of butyl ester and acid alone shows a ratio of 45% FABE and 55% fatty acid.

  Solvent (1.25 liters, 1100 g) and 1.25 liters of water were charged to a 1 gallon autoclave. The autoclave was sealed, heated to 250 ° C. and held at that temperature for 4 hours. Next, the autoclave was cooled and opened to obtain an emulsion. The mixture was filtered through a celite bed and the layers were separated. The organic layer was washed 3 times with 1 liter of water. The sample was then heated to 50 ° C. and purged with nitrogen for 6 hours. GC analysis shows the absence of i-BuOH and a ratio of 28% FABE and 72% fatty acids. An amber oil (720.5 g) was obtained.

Example 57
Product Alcohol Recovery-Lipase Catalyzed Hydrolysis FABE was synthesized from corn oil fatty acids as described in Example 44. Novozyme 435 (Novo 435, Candida Antarctic lipase B, immobilized on acrylic resin) was purchased from Sigma Aldrich (St. Louis, Mo). Candida Antarctic lipase B was purchased from Novozymes (Franklinton, NC). t-BuOH, acetone, ethanol, methanol, and glycerol were all purchased from Sigma Aldrich (St. Louis, Mo). For gas chromatography (GC) analysis, the gas chromatograph used was a Hewlett Packard 5890 Series II GC chromatogram, using methyl pentadecanoate as an internal standard.

A. Atmospheric pressure, 40 ° C
To a mixture of 2 mL FABE and 5 mL water was added 40 mg Novozyme 435 and the reaction mixture was placed in a 20 mL vial and incubated at 40 ° C. on a rotary shaker (300 rpm). The reaction mixture was analyzed using GC during the 24 hour reaction to produce the following% conversion profiles shown in Table 51:

B. Atmospheric pressure, 40 ° C., 65 ° C. and 80 ° C., no organic solvent Together with Part A of this example, these data show how the equilibrium changes with temperature.

  To a mixture of 1 mL FABE and 2 mL water was added 20 mg Novozyme 435 and the reaction mixture was rotated in a 6 mL septum-capped vial at 40 ° C. for 45 hours. Analysis of the reaction mixture using GC showed a 18.2% conversion of FABE at equilibrium.

  To a mixture of 1 g FABE and 2 mL water was added 20 mg Novozyme 435 and the reaction mixture was spun for 42 hours at 65 ° C. in a 6 mL septum capped vial. Analysis of the reaction mixture using GC showed 19.8% conversion of FABE at equilibrium.

  To a mixture of 1 g FABE and 2 mL water, 20 mg Novozyme 435 was added and the reaction mixture was rotated in a 6 mL septum cap vial at 80 ° C. for 42 hours. Analysis of the reaction mixture using GC showed 21.4% conversion of FABE at equilibrium.

C. Example showing the effect of organic solvent (t-BuOH) on equilibrium Three reaction mixtures containing 0.25 mL FABE, 0.75 mL t-BuOH, and 0.1-0.3 mL water were 20 mg Novozyme 435 was added and the mixture was allowed to rotate at 40 ° C. overnight in 6 mL septum capped vials when they reached equilibrium. Analysis of the reaction mixture using GC after 24 hours of reaction gave 77-82% FABE conversion shown in Table 52. Replacing t-BuOH with 3-Me-3-pentanol under similar reaction conditions resulted in 70-80% FABE hydrolysis yield.

D. Acetone as solvent To three reaction mixtures containing 0.25 mL FABE, 0.75 mL acetone, and 0.1-0.3 mL water, 20 mg Novozyme 435 was added and the mixture was added to a 6 mL septum cap. In a vial with vials, they were allowed to rotate overnight at 40 ° C. when they reached equilibrium. Analysis of the reaction mixture using GC after 24 hours of reaction showed 71-78% FABE conversion as shown in Table 53.

E. Example showing the effect of removing i-BuOH during hydrolysis on FABE conversion-nitrogen purge at atmospheric pressure A 25 mL round bottom flask is charged with 2 mL FABE, 5 mL water, and 40 mg Novozyme 435. did. The reaction mixture was heated to 95 ° C. and i-BuOH formed during the reaction was removed by bubbling nitrogen through the reaction mixture. A sample was taken from the mixture during the reaction and the organic phase was analyzed using GC. A conversion of 94% was obtained after 6 hours as shown in Table 54:

F. Example showing the effect of removing i-BuOH during hydrolysis on conversion-Vacuum distillation A 25 mL round bottom flask was charged with 3 mL FABE, 7.5 mL water, and 60 mg Novozyme 435. The flask was attached to a vacuum distillation apparatus and the pressure was set to 91 mm Hg. The reaction mixture was then heated to 74 ° C. and i-BuOH formed during the reaction was removed by distillation. A sample was taken from the mixture during the reaction and the organic phase was analyzed using GC. As shown in Table 55, a 91% conversion was obtained after 10 hours.

G. Example showing the effect of removing i-BuOH on hydrolysis on conversion-Vacuum distillation example-Variable FABE / COFA starting ratio: 23% FABE: 77% COFA v / v
A 25 mL round bottom flask was charged with 0.69 mL FABE, 2.31 mL COFA, 7.5 mL water, and 60 mg Novozyme 435. The flask was attached to a vacuum distillation apparatus and the pressure was set to 91 mm Hg. The reaction mixture was then heated to 74 ° C. and i-BuOH formed during the reaction was removed by distillation. A sample was taken from the mixture during the reaction and the organic phase was analyzed using GC. As shown in Table 56, 98% conversion was obtained after 10 hours.

H. Example showing the effect of removing i-BuOH on hydrolysis on conversion-Vacuum distillation example-Variable FABE / COFA starting ratio: 70% FABE: 30% COFA v / v
A 25 mL round bottom flask was charged with 2.1 mL FABE, 0.9 mL COFA, 7.5 mL water, and 60 mg Novozyme 435. The flask was attached to a vacuum distillation apparatus and the pressure was set to 91 mm Hg. The reaction mixture was then heated to 74 ° C. and i-BuOH formed during the reaction was removed by distillation. A sample was taken from the mixture during the reaction and the organic phase was analyzed using GC. As shown in Table 57, 96% conversion was obtained after 10 hours:

I. Example showing free Cal B enzyme during FABE hydrolysis under vacuum distillation conditions Two round bottom flasks were charged with 3 mL (2.7 g) FABE and 7.5 mL H ₂ O, respectively. To one mixture, 5.9 mg of Candida Antarctic lipase B was added and to the other 0.59 mg of enzyme was added. The reaction flasks were separately connected to a distillation apparatus and subjected to a pressure of 91 mm Hg. The reaction mixture was heated to 65-68 ° C. A sample was taken from the reaction mixture over 10 hours and analyzed using gas chromatography. The final FABE conversion was 96 and 78%, respectively. Experiments show that when the amount of enzyme concentration is reduced by a factor of 10, the rate and conversion are reduced by a third and by 18%, respectively. The results are shown in Table 58.

Example 58
Product Alcohol Recovery-Transesterification FABE was synthesized from corn oil fatty acids as described in Example 44; Novozyme 435 (Candida Antarctica lipase B, immobilized on acrylic resin Were purchased from Sigma Aldrich (St. Louis, Mo.). Candida Antarctic lipase B was purchased from Novozymes (Franklinton, NC). t-BuOH, acetone, ethanol, methanol, and glycerol were all purchased from Sigma Aldrich (St. Louis, Mo). For GC analysis, the gas chromatograph used was a Hewlett Packard 5890 Series II GC chromatogram, using methyl pentadecanoate as an internal standard.

A. Lipase testing-FABE to FAME
Reagents used are: t-BuOH (Aldrich); MeOH (Aldrich); Novozyme 435 (Aldrich); PS30 (Burkholderia cepacia), Amano Enzymes, Inc, IL, Inc .; ) 100T (Thermomyces lanuginosa), immobilized on silica, Novozymes, Franklinton, NC; Lipolase® 100L (Thermomyces lanuginosla, Thermomyces lanuginosla Registered trademark) TLIM (Immobilized Thermo Imoss lanuginosa (Thermyces lanuginosa), Novozymes, Franklinton, NC; Lipoclean® 2000T (an immobilized mixture of lipases; Novozymes, Franklinton, NC); NZL-103 Lipase, Novozymes, Franklinton, NC).

  To a 6 mL vial was added 500 mg FABE (1.48 mmol), 400 μL t-BuOH, 60 μL MeOH (1.48 mmol), 3 μL water, and 2.5 mg lipase (see Table 57). The resulting mixture was placed in an incubator / shaker and left at 40 ° C. overnight. GC analysis of the reaction mixture showed 9-56% conversion. The results are shown in Table 59.

B. FABE to FAME Transformation-Optimization of Methanol Amount In a 6 mL vial, 500 mg FABE (1.48 mmol), 400 μL t-BuOH, 60-240 μL MeOH (1.48-5.92 mmol), 3 μL Of water, and 2.5 mg Novozyme 435 were added. The resulting mixture was placed in an incubator / shaker and left at 40 ° C. overnight. GC analysis of the reaction mixture showed 53-73% conversion as shown in Table 60.

C. Enzyme quantity optimization-FABE to FAME
To a 6 mL vial, 500 mg FABE (1.48 mmol), 400 μL t-BuOH, 132 μL MeOH (3.26 mmol), and 5-25 mg Novozyme 435 were added. The resulting mixture was placed in an incubator / shaker and left at 40 ° C. overnight. GC analysis of the reaction mixture showed 76-79% conversion as shown in Table 61.

D. Minimizing the amount of solvent (t-BuOH)-FABE to FAME
To a 6 mL vial, 500 mg FABE (1.48 mmol), 0-300 μL t-BuOH, 132 μL MeOH (3.26 mmol), and 10 mg Novozyme 435 were added. The resulting mixture was placed in an incubator / shaker and left at 40 ° C. overnight. GC analysis of the reaction mixture showed 30-81% conversion as shown in Table 62.

E. Minimizing the amount of solvent (3-Me-3-pentanol)-FABE to FAME
To a 6 mL vial, 500 mg FABE (1.48 mmol), 0-300 μL 3-Me-3-pentanol, 132 μL MeOH (3.26 mmol), and 10 mg Novozyme 435 were added. The resulting mixture was placed in an incubator / shaker and left at 40 ° C. overnight. GC analysis of the reaction mixture showed 30-78% conversion as shown in Table 63.

F. Conversion of FABE to FAME without solvent To a mixture of FABE (500 mg, 1.48 mmol) and methanol (0.13 μL, 3.25 mmol) was added 40 mg Novozyme 435 and the reaction mixture was stirred at 40 ° C. overnight. did. The mixture was then filtered and analyzed by GC, showing 76% conversion.

G. Enzyme recycling-from FABE to FAME
To a 6 mL vial was added 500 mg FABE (1.48 mmol), 400 μL t-BuOH, 132 μL MeOH (3.26 mmol), and 10 mg Novozyme 435. The resulting mixture was placed in an incubator / shaker and left at 40 ° C. overnight. The reaction mixture was then filtered and analyzed for conversion using GC and the filter cake containing the immobilized enzyme was used for another conversion from FABE to FAME. This process was repeated 10 times (Table 64). Experiments show that it is possible to reuse the enzyme up to 10 times without reducing the conversion rate in an overnight reaction.

H. Conversion of FABE to FAEE To a 6 mL septum capped vial was added 0.8 mL FABE (2.08 mmol) and 0.2 mL EtOH (3.43 mmol) to form a single phase. No enzyme was added or 20 mg Novozyme 435 was added to the vial. The vials were then incubated for 17 hours at 25 ° C. and 40 ° C. in a culture shaker (300 rpm), after which the solution was analyzed by gas chromatography to determine the content and FABE to FAEE shown in Table 65. A percent conversion was obtained.

Example 59
Glycolysis of FABE To a 6 mL vial with a septum cap, add 0.75 mL t-BuOH, 0.25 mL FABE, 0.1 mL (0.126 g) glycerol + 4 μL H ₂ O, 20 mg of enzyme, respectively. A single phase was formed. The reaction was cultured with various lipases at 40 ° C. in a rotary shaker at 300 rpm. After 20 hours, the sample was analyzed by gas chromatography to obtain the contents shown in Table 66. Comparison of the COFA and FABE content indicates that the product is a mixture of i-BuOH and COFA and acylglycerol (approximately 64% acylglycerol / 36% COFA on a molar basis).

A. Dependence on water and glycerol / FABE In a 6 mL septum capped vial, either 0.75 mL t-BuOH, 0.25 mL FABE, 0.1 or 0.2 g glycerol + either Amano PS-30 or Novozyme 435 20 mg of each was added to form a single phase. No water was added. The reaction was incubated at 40 ° C. on a rotary shaker at 300 rpm. After 20 hours, the sample was analyzed by gas chromatography to obtain the contents shown in Table 67. Comparison of the COFA and FABE content indicates that the product is i-BuOH and predominantly acylglycerol (mostly monoglycerides). Compared to the previous example, the percent of product in the form of acylglycerol increases with no added water and with increasing glycerol / FABE (1.6 glycerol / FABE, mol / mol, mol basis) About 91% acylglycerol / 9% COFA and 3.2 glycerol / FABE, in the case of mol / mol, about 95% acylglycerol / 5% COFA on a molar basis). In the absence of added water, the enzyme activity of Amano PS-30 is lost. However, Novozyme 435 with water (about 3% w / w) in the acrylic resin to be immobilized is still active.

B. Dependence on enzyme concentration In a 6 mL septum capped vial, 0.75 mL t-BuOH, 0.25 mL FABE, 0.2 g glycerol (glycerol / FABE 3.2 / 1, mol / mol) +2 or 20 mg Novozyme. 435 (Novo 435) was added. No water was added. The reaction was incubated at 40 ° C. on a rotary shaker at 300 rpm and the reaction was followed as a function of time by gas chromatography. The yield is shown in Table 68. About 97% of the FABE reacted on a molar basis was converted to acylglycerol (mostly monoglycerides).

The rate of the reaction is linearly related to the enzyme concentration, with t _1/2 of 2 mg and 20 mg Novozyme 435 being 208 minutes and 20 minutes, respectively. However, the reaction reaches approximately the same yield of FABE conversion after 24 hours.

  These last reactions were repeated using 0.75 mL 3-methyl-3-pentanol instead of 0.75 mL t-BuOH. The degree of FABE hydrolysis obtained after 24 hours was the same for both solvents. The advantage of 3-methyl-3-pentanol is that, with a boiling point of 122 ° C., i-BuOH can be distilled off first in pure form (boiling point 108 ° C.). The 3-methyl-3-pentanol is then distilled off and reused in the hydrolysis reaction and remains in the retentate acylglycerol, COFA, and glycerol and is returned to the fermentation tank for re-use for FABE production. Can be reused. Tertiary alcohols have the advantage that they act alone as solvents and do not react with fatty acids to form fatty acid alkyl esters in the presence of CALB.

C. Glycolysis of FABE in the absence of organic cosolvent (FABE to COFA + acylglycerol)-Dependence on glycerol concentration 1 g FABE mixed with 2 mL 50, 70, 90 and 100% (w / w) glycerol And placed in a vial sealed with a 6 mL septum in the presence of 20 mg of Lipobond (Sprin Technologies, Trieste, Italy). The vial was rotated at 62 ° C. for 24 hours (tabled end-over-end). As the concentration of glycerol in the aqueous phase increases, the percentage of product in the form of acylglycerol (Table 69, mostly monoglycerides) increases. However, the degree of FABE conversion does not show a dependence on glycerol concentration.

Example 60
Conversion of COFA to FAEE and monoacylglycerol The following examples show that COFA can be esterified with EtOH or glycerol in high yield under mild conditions using immobilized enzyme.

  Novozyme 435 (Candida Antarctic lipase B, immobilized on acrylic resin) was purchased from Sigma Aldrich (St. Louis, Mo). Acetone, t-BuOH, ethanol, methanol, and glycerol were all purchased from Sigma Aldrich (St. Louis, Mo). For GC analysis, the gas chromatograph used was a Hewlett Packard 5890 Series II GC chromatogram, using methyl pentadecanoate as an internal standard.

A. Conversion of COFA to FAEE + i-BuOH using ethanol Corn oil fatty acid (COFA, 0.25 g) was dissolved in 2.0 mL EtOH to form a single phase. 20 mg of Candida Antarctica Lipase B (CALB) (Novozyme 435) immobilized on acrylic resin is added (containing 1.7 mg of enzyme) and the suspension is sealed with a septum cap In a 6 mL glass vial, the cells were cultured at 40 ° C. for 24 hours on a rotary shaker (300 rpm). When the reaction was actually completed, 98% COFA was converted to FAEE (fatty acid ethyl ester). GC analysis after 24 hours showed 98% conversion of COFA to fatty acid ethyl ester as shown in Table 70.

B. Conversion of COFA to monoacylglyceride (MAG) + i-BuOH using glycerol Corn oil fatty acid (COFA, 0.25 g) and 0.325 g glycerol were dissolved in 2.0 mL acetone. There was a large upper phase in which most of the ingredients were dissolved and a small remaining glycerol-containing phase. 20 mg of Candida Antarctica Lipase B (CALB) (Novozyme 435) immobilized on acrylic resin is added (containing 1.7 mg of enzyme) and the suspension is sealed with a septum cap In a 6 mL glass vial, the cells were cultured at 40 ° C. for 24 hours on a rotary shaker (300 rpm). The upper phase GC showed that 87% of the COFA was converted to acylglycerides (mostly expected to be monoacylglycerides). The results are shown in Table 71.

Example 61
Conversion of COFA to FAME The following example demonstrates that COFA can be esterified with MeOH, EtOH, and glycerol in high yield under mild conditions using immobilized lipase.

A. Conversion of COFA to FAME without solvent To a 6 mL vial was added 500 mg COFA (1.48 mmol), 132 μL MeOH (3.26 mmol), and 10 mg Novozyme 435. The resulting mixture was placed in an incubator / shaker and left at 40 ° C. overnight. GC analysis of the reaction mixture showed 95% conversion.

B. Time course measurement of reaction from COFA to FAME To a 6 mL vial was added 500 mg COFA (1.48 mmol), 132 μL MeOH (3.26 mmol), and 10 mg Novozyme 435. Samples were cultured in an incubator / shaker at 40 ° C., time points during the reaction were taken and analyzed using GC. The results are shown in Table 72.

C. Addition of more MeOH to the COFA → FAME reaction In a 6 mL vial, 500 mg of COFA (1.48 mmol), 180, 240, 300, or 1320 μL of MeOH (4.44, 5.92, 7.41, and 14.82 mmol), and 10 mg Novozyme 435 were added. The resulting mixture was placed in an incubator / shaker and left at 40 ° C. overnight. GC analysis of the reaction mixture showed 96-97% conversion. The results are shown in Table 73.

Example 62
This example showed the removal of solids from the distillation waste and extraction with a desolvator to recover fatty acids, esters, and triglycerides from the solids. During the fermentation, the solid is separated from the total distillation effluent and fed to the desolventizer where the solid is contacted with 1.1 ton / hour of steam. Table 74 shows the flow rate of the total distillation waste liquid wet cake (extractor raw material), solvent, extractor miscella, and extractor discharged solids. The unit of the values in the table is short ton / hour.

  The solid leaving the desolvator is fed to the dryer. The vapor exiting the desolvator contains 0.55 ton / hour hexane and 1.102 ton / hour water. This stream is condensed and fed to the decanter. The water-enriched phase exiting the decanter contains about 360 ppm of hexane. This stream is fed to a distillation column where hexane is removed from the water-enriched stream. The hexane enriched stream exiting the top of the distillation column is condensed and fed to the decanter. The organic enriched stream leaving the decanter is fed to a distillation column. Steam (11.02 tons / hour) is fed to the bottom of the distillation column. The composition of the top and bottom products of this tower are shown in Table 75. The unit of the values in the table is ton / hour.

Example 63
Preparation of solid extraction hydrous isobutanol In a 100 mL volumetric flask, 65 g of anhydrous reagent grade isobutanol (supplied by Aldrich) is combined with 10 g of distilled water and shaken until a clear, colorless, homogeneous phase is obtained. That ’s it. An additional 10 g of distilled water was added to the volumetric flask and shaken again, resulting in two viscous, colorless and transparent liquid layers. The top layer is considered to be hydrous isobutanol, usually containing 20% by weight water, and the bottom layer is mainly water containing usually 8% by weight dissolved alcohol.

Extraction with sieving filtration and displacement washing The fermentation was completed with the recycled fatty acid (Example 19). An 185 g portion representative of the resulting heterogeneous mixture is removed and placed in a sealed 80MESH sieve dish supported in a Nalgene® plastic filter funnel using a slight vacuum (water column—20 inches) on the bottom. Threaded for a minute. The filtrate was separated into 90.5 g of a reddish brown oil phase and 50.9 g of a turbid aqueous phase containing dispersed particulates but no sedimentation of particulate matter. The wet cake remained on the sieve plate. A 1.5 g sample of this unwashed wet cake was removed and allowed to air dry. Hydrous isobutanol (23 g) was withdrawn from the upper layer in the volumetric flask and passed through the wet cake for 5 minutes while maintaining a low vacuum on the underside of the sieve dish until no further droplets were collected. The total filtrate mass of 18 g consisted of a small amount of an immiscible bottom turbid aqueous layer and a yellow clear hydrous isobutanol layer. The wet cake was removed from the sieve dish and a total mass of 38.4 g was removed. A sample of 1.5 g of this washed wet cake was removed and allowed to air dry. Analysis of the unwashed solid dry sample showed that it contained 53.35 wt% total fat on a triglyceride basis, and analysis of the washed solid dry sample showed 15.9 wt% on a triglyceride basis. Of total fat.

Extraction with Centrifugation and Reslurry Wash The fermentation was completed with the recycled fatty acid (Example 19). A 225 g portion representative of the resulting heterogeneous mixture was removed and centrifuged at 10,000 rpm for 10 minutes using a Beckman Coulter Allegra 64R machine. The clear reddish brown oil phase totaling 67.2 g was removed by decanting. The remaining material was centrifuged again and 95.1 g of cloudy aqueous filtrate was removed by decanting. A 1.5 g sample of the wet solid was removed and air dried, 56.5 g was collected and transferred to a 400 mL beaker. Hydrous isobutanol (20 g) extracted from the upper layer in the volumetric flask was added to the beaker to repulp the wet solid and agitation was carried out for 5 minutes. An additional 32 g of hydroisobutanol was added along with 32 g of the centrifuge and the solid was stirred for 5 minutes in the aqueous suspension below the quiescent organic layer. The mixture is then centrifuged at 10,000 rpm for 10 minutes to remove the clear yellow hydrous isobutanol layer by decanting, and a 1.5 g sample of the washed wet solid is isolated and dried again to dry. Centrifuged. Analysis of a dry sample of unwashed wet solids revealed 21.6% by weight total fat on a triglyceride basis, and analysis of a dry sample of washed wet solids found 4.04 on a triglyceride basis. It was found to contain weight percent total fat.

Example 64
Removal of corn oil by removing undissolved solids About 1000 g of liquefied corn mash was prepared in a 1 L glass coated resin kettle. The kettle was equipped with mechanical agitation, temperature control, and pH control. The following protocol was used: ground corn was mixed with tap water (26 wt% corn on a dry basis) and the slurry was heated to 55 ° C. with agitation to either NaOH or H ₂ SO _4. Use to adjust the pH to 5.8, add α-amylase (0.02 wt% based on dry corn), continue to heat to 85 ° C, adjust the pH to 5.8, and adjust the pH to 5.8 While maintaining, the temperature was maintained at 85 ° C. for 2 hours and cooled to 25 ° C. The corn used was a whole grain yellow corn (3335) from Pioneer. Corn was ground on a hammer mill using a 1 mm sieve. The moisture content of ground corn was about 11.7% by weight, and the starch content of ground corn was about 71.4% by weight on a dry corn basis. The α-amylase enzyme was Liquizyme® SC DS made by Novozymes (Franklinton, NC). The total amount of ingredients used was: 294.5 g ground corn (11.7% moisture), 705.5 g tap water, and 0.059 g Liquizyme® SC DS. H ₂ O (4.3 g) was added to dilute the enzyme and a total of 2.3 g of 20% NaOH solution was added to control the pH. About 952 g of mash was recovered. Note that there was a loss due to mash sticking to the walls of the kettle and CF bottle.

  The liquefied corn mash was centrifuged at 5000 rpm (7260 g's) for 30 minutes at 40 ° C. to remove undissolved solids from the oligosaccharide aqueous solution. Removing solids by centrifugation also removes free corn oil as a separate organic liquid layer on top of the aqueous phase. About 1.5 g of corn oil was recovered from the organic layer suspended above the aqueous phase. It was found by hexane extraction that the ground corn used to produce the liquefied mash contained about 3.5 wt% corn oil on a dry corn basis. This corresponds to about 9 g of corn oil fed to the liquefaction process, including ground corn.

  About 1 g of corn oil was recovered from the organic layer suspended above the aqueous phase. When about 617 g of liquefied starch solution was recovered, about 334 g of wet cake remained. The wet cake contained most of the undissolved solid that was in the liquefied mash. The liquefied starch solution contained about 0.2% by weight of undissolved solids. The wet cake contained about 21% by weight undissolved solids. The wet cake was washed with 1000 g of tap water to remove the oligosaccharide remaining in the cake. This was done by mixing the cake with water to form a slurry. The slurry was then centrifuged under the same conditions used to centrifuge the original mash to recover the washed solid. Removal of the washed solids by centrifugation also removed some additional free corn oil as a separate organic liquid layer at the top of the aqueous phase. Corn oil was recovered from the organic layer suspended above the aqueous phase.

  The wet solid was washed twice more with 1000 g of tap water each time to remove substantially all of the liquefied starch. The final washed solid was dried in a vacuum oven overnight at 80 ° C. and approximately 20 inches of mercury vacuum. The amount of corn oil remaining in the dry solid (possibly remaining in the germ) was measured by hexane extraction. It was determined that a 3.60 g sample of a relatively dry solid (about 2 wt% moisture) contained 0.22 g of corn oil. This result corresponds to 0.0624 g corn oil per g dry solid. This was the case for the washed solid, which meant no residual oligosaccharide in the wet solid. After the liquefied corn mash was centrifuged to separate the layer of free corn oil and the aqueous solution of oligosaccharides from the wet cake, there remained about 334 g of wet cake containing about 21 wt% undissolved solids. I understood. This corresponds to a wet cake containing about 70.1 g of undissolved solids. At 0.0624 grams of corn oil per gram of dry solids, the solids in the wet cake should contain about 4.4 grams of corn oil.

Example 65
Lipid Analysis Lipid analysis was performed by converting various fatty acid-containing compound species to fatty acid methyl esters (“FAME”) by transesterification. Glycerides and phospholipids were transesterified with sodium methoxide in methanol. Glycerides, phospholipids, and free fatty acids were transesterified using acetyl chloride in methanol. The resulting FAME was equipped with a 30-m × 0.25 mm (inner diameter) OMEGAWAX ™ (Supelco, Sigma Aldrich, St. Louis, MO) column after dilution in toluene / hexane (2: 3). Analysis was by gas chromatography using an Agilent 7890 GC. The oven temperature was increased from 160 ° C. to 200 ° C. at 5 ° C./min and then from 200 ° C. to 250 ° C. (held for 10 minutes) at 10 ° C./min. The FAME peaks recorded by GC analysis were identified by their retention times when compared to the retention times of known methyl esters (ME), and the FAME peak area was determined by a known amount of internal standard (C15: 0 Quantification by comparison with the area of triglycerides, taken by the transesterification procedure with the sample). Thus, an approximate amount (mg) of any fatty acid FAME (“mg FAME”) is calculated according to the following formula: (area of FAME peak for a given fatty acid / area of FAME peak at 15: 0) ^* (internal Standard C15: 0 FAME mg). The FAME result can then be corrected to the corresponding fatty acid mg by dividing by an appropriate molecular weight conversion factor of 1.052. All internal standards and reference standards are from Nu-Chek Prep, Inc. Obtained from.

  The fatty acid result obtained from the sample transesterified with sodium methoxide in methanol is converted to the corresponding triglyceride level by multiplying by a molecular weight conversion factor of 1.045. Triglycerides generally account for about 80-90% of the glycerides in the samples examined for this example, with the remainder being diglycerides. Monoglyceride and phospholipid contents are generally negligible. The total fatty acid results obtained for the sample transesterified with acetyl chloride in methanol are corrected for glyceride content by subtracting the fatty acid determined for the same sample using the sodium methoxide procedure. The result is the free fatty acid content of the sample.

  The distribution of glyceride content (monoglyceride, diglyceride, triglyceride, and phospholipid) was determined using thin layer chromatography. A solution of oil dissolved in 6: 1 chloroform / methanol was hung near the bottom of a glass plate pre-coated with silica gel. The spot is then chromatographed on a plate using a 70: 30: 1 hexane / diethyl ether / acetic acid solvent system. Next, isolated spots corresponding to monoglycerides, diglycerides, triglycerides, and phospholipids are detected by staining the plate with iodine vapor. The spot is then dropped from the plate and transesterified with acetyl chloride in a methanol procedure and analyzed by gas chromatography. The ratio of the total peak area of each spot to the total peak area of all spots is the distribution of the various glycerides.

Example 66
This example demonstrates the recovery of by-products from mash. Maize corn oil under the conditions described in Example 64 except that a tricanter centrifuge (Flottweg Z23-4 bowl diameter, 230 mm, length to diameter ratio 4: 1) was used under these conditions. Separated from:
・ Bowl speed: 5000rpm
・ Differential speed: 10rpm
・ Supply speed: 3 gallons / minute ・ Phase separator disk: 138 mm
・ Impeller setting: 144 mm.

  The isolated corn oil has 81% triglycerides, 6% free fatty acids, 4% diglycerides, and a total of 5% phospholipids as measured by the method described in Example 65 and thin layer chromatography. Had monoglycerides.

  The solid separated from the mash under the above conditions has a moisture content of 58% as measured by weight loss upon drying and 1.2% as measured by the method described in Example 65. It had triglycerides and 0.27% free fatty acids.

  The composition of the solids separated from the total distillation effluent in Table 78, the oil extracted during the evaporator stage, the by-product extractant and the condensed distillation solubles (CDS) is shown in Table 76. The calculation was made on the assumption of Table 77 (separation in a tricanter centrifuge). The values in Table 75 were obtained from an Aspen Plus® model (Aspen Technology, Inc., Burlington, Mass.). This model assumes that no corn oil has been extracted from the mash. It is estimated that the protein content on a dry basis of cells, lysed solids and suspended solids is about 50%, 22% and 35.5%, respectively. The composition of the byproduct extractant is estimated to be 70.7% fatty acid and 29.3% fatty acid isobutyl ester on a dry basis.

  While various embodiments of the invention have been described above, it should be understood that they have been presented by way of example only and not limitation. It will be apparent to those skilled in the relevant art that various modifications can be made in the form and details of the invention without departing from the spirit and scope of the invention. Accordingly, the breadth and scope of the present invention is not limited by any of the above-described exemplary embodiments, but is defined only in accordance with the following claims and their equivalents.

  All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and each individual publication, patent or patent application is incorporated by reference. To the same extent as specifically and individually indicated herein by reference.

Claims (197)

A method for producing a butyl ester comprising:
Contacting butanol produced in the fermentation process with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with the butanol to form a butyl ester of the carboxylic acid; Because;
A method comprising the step of the carboxylic acid in the fermentation process being present at a concentration sufficient to produce a biphasic mixture.   The method of claim 1, wherein the production of butanol and the production of butyl ester occur simultaneously or sequentially.   The method of claim 1, wherein the fermentation process feedstock comprises one or more fermentable sugars.   The raw material of the fermentation process is corn kernels, corn cobs, corn hulls, corn stover and other crop residues, grass, wheat, rye, wheat straw, barley, barley straw, grass, rice straw , Switchgrass, waste paper, sugarcane pomace, sorghum, sugarcane, soy, ingredients obtained from grinding cereals, cellulosic materials, lignocellulosic materials, trees, branches, roots, leaves, wood chips, sawdust, shrubs and shrubs The method of claim 1, comprising one or more fermentable sugars derived from plants, fruits, flowers, animal manure, and mixtures thereof.   The method of claim 1, further comprising providing a natural oil and converting at least a portion of the natural oil to a carboxylic acid by contacting the oil with one or more enzymes. Method.   The method of claim 1, wherein the carboxylic acid comprises a fatty acid.   The method of claim 1, wherein the carboxylic acid comprises 12 to 22 carbons.   The method of claim 1, wherein the carboxylic acid is a mixture of carboxylic acids.   The method of claim 1, wherein the butyl ester of the carboxylic acid is a butyl ester of a fatty acid.   The method of claim 1, wherein the catalyst is an enzyme capable of esterifying the carboxylic acid with the butanol to form a butyl ester of the carboxylic acid.   The method according to claim 10, wherein the enzyme is esterase, lipase, phospholipase, or lysophospholipase. A method for producing butanol and butyl esters from raw materials comprising:
(A) providing a raw material;
(B) liquefying the raw material to produce liquefied biomass containing oligosaccharides;
(C) separating the raw slurry to produce a water stream containing an oligosaccharide, an oil stream, and a product containing solids;
(D) adding the water stream to a fermentor containing a fermentation broth;
(E) saccharifying the oligosaccharide of the water stream;
(F) fermenting the product of the oligosaccharide saccharification present in the water stream to produce butanol, simultaneously, butanol is esterified with at least one carboxylic acid, and butanol together with the butanol Contacting with at least one catalyst capable of forming a butyl ester of the carboxylic acid, wherein the carboxylic acid is present in a concentration sufficient to form a biphasic mixture; Including:
A method in which steps (e) and (f) are optionally performed at the same time.   The method of claim 12, further comprising obtaining oil from the oil stream and converting at least a portion of the oil to carboxylic acid.   The raw slurry is decanter bowl centrifugal separation, tricanter centrifugal separation, disk stack centrifugal separation, filtration centrifugal separation, decanter centrifugal separation, filtration, vacuum filtration, belt filter, pressure filtration, sieving filtration, sieving separation, lattice, porous lattice 13. The method of claim 12, wherein the method is separated by flotation, hydroclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof.   The method of claim 12, wherein the carboxylic acid comprises a fatty acid.   The method of claim 12, wherein the carboxylic acid comprises 12 to 22 carbons.   13. The method of claim 12, further comprising adding the oil to the fermentor prior to the step of converting at least a portion of the oil to carboxylic acid.   13. The method of claim 12, further comprising adding additional carboxylic acid to the fermentor.   19. The method of claim 18, wherein after adding the additional carboxylic acid, the oil is converted to a carboxylic acid.   19. The method of claim 18, wherein the carboxylic acid is corn oil fatty acid, soybean oil fatty acid, or a mixture of corn oil fatty acid and soybean oil fatty acid.   13. The method of claim 12, wherein the oil obtained from the oil stream comprises glycerides and the one or more catalysts hydrolyze the glycerides into fatty acids.   13. The method of claim 12, wherein the butyl ester of carboxylic acid is a butyl ester of fatty acid.   13. The method of claim 12, wherein the catalyst is an enzyme capable of esterifying the carboxylic acid with the butanol to form a butyl ester of the carboxylic acid.   24. The method of claim 23, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.   The method of claim 12, further comprising washing the solid with a solvent.   26. The method of claim 25, wherein the solvent is selected from petroleum distillates such as hexane, isobutanol, isohexane, ethanol, petroleum ether, or mixtures thereof.   27. The method of claim 26, wherein the solid is processed to form an animal feed product.   13. The method of claim 12, wherein the solid is treated to form an animal feed product.   The animal feed product comprises one or more crude proteins, crude fat, triglyceride, fatty acid, fatty acid isobutyl ester, lysine, neutral detergent fiber (NDF), and acidic detergent fiber (ADF). 28. The method according to 28.   30. The method of claim 29, wherein the animal feed product further comprises one or more vitamins, minerals, flavors, or colorants.   The animal feed product comprises 20-35 wt% crude protein, 1-20 wt% crude fat, 0-5 wt% triglyceride, 4-10 wt% fatty acid, and 2-6 wt% fatty acid isobutyl ester. 30. The method of claim 29, comprising:   Whether the step of separating the solid from the raw slurry increases the efficiency of butanol production by increasing the liquid-liquid mass transfer coefficient of the butanol from the fermentation broth to the extractant; Increase the efficiency of the butanol production by increasing the extraction efficiency; increase the efficiency of the butanol production by increasing the speed of phase separation between the fermentation broth and the extractant; recovery and recycling of the extractant 13. The method of claim 12, wherein the efficiency of butanol production is increased by increasing utilization; or the efficiency of butanol production is increased by decreasing the flow rate of the extractant. A method for producing butanol and butyl esters from raw materials comprising:
(A) providing a raw material;
(B) liquefying the raw material to produce liquefied biomass containing oligosaccharides;
(C) separating the raw slurry to produce a stream comprising oligosaccharides and oil, and a solid;
(D) adding the stream to a fermentor containing a fermentation broth;
(E) saccharifying the oligosaccharides of the stream;
(F) fermenting the product of the oligosaccharide saccharification present in the stream to produce butanol and at the same time esterifying the carboxylic acid with at least one carboxylic acid and butanol. Contacting with at least one catalyst capable of forming a butyl ester of the carboxylic acid, wherein the carboxylic acid is present in a concentration sufficient to form a biphasic mixture. ;
A method in which steps (e) and (f) are optionally performed at the same time.   34. The method of claim 33, further comprising converting at least a portion of the oil to carboxylic acid.   The raw slurry is decanter bowl centrifugal separation, tricanter centrifugal separation, disk stack centrifugal separation, filtration centrifugal separation, decanter centrifugal separation, filtration, vacuum filtration, belt filter, pressure filtration, sieving filtration, sieving separation, lattice, porous lattice 34. The method of claim 33, separated by flotation, hydroclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof.   34. The method of claim 33, wherein the carboxylic acid comprises a fatty acid.   34. The method of claim 33, wherein the carboxylic acid comprises 12-22 carbons.   34. The method of claim 33, further comprising adding oil to the fermentor.   34. The method of claim 33, further comprising adding additional carboxylic acid to the fermentor.   34. The method of claim 33, wherein after the step of adding the additional carboxylic acid, the oil is converted to a carboxylic acid.   34. The method of claim 33, wherein the carboxylic acid is corn oil fatty acid, soybean oil fatty acid, or a mixture of corn oil fatty acid and soybean oil fatty acid.   34. The method of claim 33, wherein the oil comprises glycerides and the one or more catalysts hydrolyze the glycerides into fatty acids.   34. The method of claim 33, wherein the butyl ester of a carboxylic acid is a butyl ester of a fatty acid.   34. The method of claim 33, wherein the catalyst is an enzyme capable of esterifying the carboxylic acid with the butanol to form a butyl ester of the carboxylic acid.   45. The method of claim 44, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.   34. The method of claim 33, further comprising washing the solid with a solvent.   47. The method of claim 46, wherein the solvent is selected from petroleum distillates such as hexane, isobutanol, isohexane, ethanol, petroleum ether, or mixtures thereof.   49. The method of claim 46, wherein the solid is processed to form an animal feed product.   34. The method of claim 33, wherein the solid is treated to form an animal feed product.   The animal feed product comprises one or more crude proteins, crude fat, triglyceride, fatty acid, fatty acid isobutyl ester, lysine, neutral detergent fiber (NDF), and acidic detergent fiber (ADF). 49. The method according to 49.   51. The method of claim 50, wherein the animal feed product further comprises one or more vitamins, minerals, flavors, or colorants.   The animal feed product comprises 20-35 wt% crude protein, 1-20 wt% crude fat, 0-5 wt% triglyceride, 4-10 wt% fatty acid, and 2-6 wt% fatty acid isobutyl ester. 51. The method of claim 50, comprising:   Whether the step of separating the solid from the raw slurry increases the efficiency of butanol production by increasing the liquid-liquid mass transfer coefficient of the butanol from the fermentation broth to the extractant; Increase the efficiency of the butanol production by increasing the extraction efficiency; increase the efficiency of the butanol production by increasing the speed of phase separation between the fermentation broth and the extractant; recovery and recycling of the extractant 34. The method of claim 33, wherein the efficiency of the butanol production is increased by increasing utilization; or the efficiency of the butanol production is increased by decreasing the flow rate of the extractant. A method for producing butanol comprising:
(A) at least one catalyst capable of esterifying butanol produced in the fermentation process with at least one carboxylic acid and the butanol together with the butanol to form a butyl ester of the carboxylic acid; The contacting step;
The carboxylic acid in the fermentation process is present in a concentration sufficient to produce a biphasic mixture comprising an aqueous phase and a butyl ester-containing organic phase;
(B) separating the butyl ester-containing organic phase from the aqueous phase;
(C) recovering butanol from the butyl ester.   55. The method of claim 54, wherein recovering butanol from the butyl ester comprises hydrolyzing the ester to a carboxylic acid and butanol.   56. The method of claim 55, wherein the butyl ester is hydrolyzed in the presence of a hydrolysis catalyst.   56. The method of claim 55, wherein the butyl ester is hydrolyzed in the presence of water and the hydrolysis catalyst comprises an acid catalyst, an organic acid, an inorganic acid, a water soluble acid, or a water insoluble acid.   57. The method of claim 56, wherein the hydrolysis catalyst comprises an enzyme capable of hydrolyzing the butyl ester to form a carboxylic acid and butanol.   59. The method of claim 58, wherein the enzyme is esterase, lipase, phospholipase, or lysophospholipase.   60. The method of claim 59, wherein the enzymatic reaction conditions favor enzymatic hydrolysis over esterification.   61. The method of claim 60, wherein the enzymatic reaction conditions include a cosolvent.   62. The method of claim 61, wherein fatty acid butyl ester, fatty acid, isobutanol, and water are soluble in the co-solvent and free fatty acids do not react with the co-solvent.   63. The method of claim 62, wherein the co-solvent is selected from acetone, tert-butanol, 2-Me-2-butanol, 2-Me-2-pentanol, and 3-Me-3-pentanol.   61. The method of claim 60, wherein the enzymatic reaction conditions include final product removal.   65. The method of claim 64, wherein the final product is isobutanol or a fatty acid.   66. The method of claim 65, wherein the isobutanol is removed by vacuum distillation, pervaporation, permselective filtration, or gas sparging.   68. The method of claim 66, wherein the fatty acid is removed by precipitation, permselective filtration, or electrophoresis.   56. The method of claim 55, wherein the hydrolysis reaction occurs in a reaction vessel.   55. The method of claim 54, wherein recovering butanol from the butyl ester comprises transesterifying the butyl ester into butanol and a fatty acid alkyl ester or acylglyceride.   70. The method of claim 69, wherein the fatty acid alkyl ester comprises a fatty acid methyl ester, a fatty acid ethyl ester, or a fatty acid propyl ester.   55. The method of claim 54, further comprising providing a natural oil and converting at least a portion of the natural oil to a carboxylic acid by contacting the oil with one or more enzymes. Method.   72. The method of claim 71, wherein the enzyme is an enzyme capable of hydrolyzing or transesterifying the butyl ester to form butanol.   73. The method of claim 72, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.   55. The method of claim 54, wherein the carboxylic acid comprises a fatty acid.   55. The method of claim 54, wherein the carboxylic acid has a carbon chain length in the range of 12-22 carbons.   55. The method of claim 54, wherein at least about 10% of butanol is recovered from the butyl ester.   55. The method of claim 54, wherein at least about 50% of butanol is recovered from the butyl ester.   55. The method of claim 54, wherein at least about 90% of butanol is recovered from the butyl ester.   55. The method of claim 54, wherein carboxylic acid is recovered from the butyl ester. Removing butanol from the fermentation apparatus as an extractant stream;
55. The method of claim 54, further comprising adding the extractant stream to two or more distillation columns.   81. The method of claim 80, wherein the distillation column is an overpressure distillation column with a steam heated reboiler.   81. The method of claim 80, further comprising recovering water and solvent from the distillation column; and recycling the water and solvent.   81. The method of claim 80, further comprising recovering heat from the distillation process; and reusing the heat to evaporate water. A method for producing butanol from a raw material comprising:
(A) providing a raw material;
(B) liquefying the raw material to produce a raw material slurry;
(C) separating the raw slurry to produce a product comprising a water stream, an oil stream, and a solid;
(D) adding the water stream to a fermentor containing a fermentation broth;
(E) saccharifying said water stream;
(F) fermenting the saccharified water stream to produce butanol and simultaneously esterifying the butanol with at least one carboxylic acid and the butanol together with the butanol to form a butyl ester of the carboxylic acid; Contacting with at least one catalyst capable of: wherein the carboxylic acid is present in a concentration sufficient to form a biphasic mixture;
(G) separating the butyl ester-containing organic phase from the aqueous phase;
(H) recovering butanol from the butyl ester;
A method in which steps (e) and (f) are optionally performed at the same time.   85. The method of claim 84, further comprising obtaining oil from the oil stream and converting at least a portion of the oil to carboxylic acid.   85. The method of claim 84, wherein the raw slurry is separated by centrifugation, filtration, or decantation.   85. The method of claim 84, wherein the carboxylic acid comprises a fatty acid.   85. The method of claim 84, wherein the carboxylic acid has a carbon chain length in the range of 12-22 carbons.   85. The method of claim 84, further comprising adding the oil to the fermentor prior to the step of converting at least a portion of the oil to carboxylic acid.   85. The method of claim 84, further comprising adding additional carboxylic acid to the fermentor.   94. The method of claim 90, wherein after the step of adding the additional carboxylic acid, the oil is converted to a carboxylic acid.   85. The method of claim 84, wherein the carboxylic acid is corn oil fatty acid, soybean oil fatty acid, or a mixture of corn oil fatty acid and soybean oil fatty acid.   85. The method of claim 84, wherein the oil obtained from the oil stream comprises glycerides and the one or more catalysts hydrolyze the glycerides into fatty acids.   85. The method of claim 84, wherein the butyl ester of a carboxylic acid is a butyl ester of a fatty acid.   85. The method of claim 84, wherein the catalyst is an enzyme capable of esterifying the carboxylic acid with the butanol to form a butyl ester of the carboxylic acid.   96. The method of claim 95, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.   85. The method of claim 84, wherein the solid is processed to form an animal feed product.   85. The method of claim 84, wherein recovering butanol from the butyl ester comprises hydrolyzing the ester to a carboxylic acid and butanol.   99. The method of claim 98, wherein the butyl ester is hydrolyzed in the presence of a hydrolysis catalyst.   99. The method of claim 99, wherein the butyl ester is hydrolyzed in the presence of water and the hydrolysis catalyst comprises an acid catalyst, an organic acid, an inorganic acid, a water soluble acid, or a water insoluble acid.   100. The method of claim 99, wherein the hydrolysis catalyst comprises an enzyme capable of hydrolyzing the butyl ester to form carboxylic acid and butanol.   102. The method of claim 101, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.   99. The method of claim 98, wherein the hydrolysis reaction occurs in a reaction vessel.   85. The method of claim 84, wherein recovering butanol from the butyl ester comprises transesterifying the butyl ester into butanol and a fatty acid alkyl ester or acylglyceride.   105. The method of claim 104, wherein the fatty acid alkyl ester comprises a fatty acid methyl ester, a fatty acid ethyl ester, or a fatty acid propyl ester.   85. The method of claim 84, further comprising providing a natural oil and converting at least a portion of the natural oil to a carboxylic acid by contacting the oil with one or more enzymes. Method.   107. The method of claim 106, wherein the enzyme is an enzyme capable of hydrolyzing or transesterifying the butyl ester to form butanol.   108. The method of claim 107, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. A method for producing butanol and butyl esters from raw materials comprising:
(A) providing a raw material;
(B) liquefying the raw material to produce liquefied biomass containing oligosaccharides;
(C) separating the raw slurry to produce a stream containing oligosaccharides and oil, and solids;
(D) adding the water stream to a fermentor containing a fermentation broth;
(E) saccharifying the oligosaccharide of the water stream;
(F) fermenting the product of the oligosaccharide saccharification present in the water stream to produce butanol, simultaneously, butanol is esterified with at least one carboxylic acid, and butanol together with the butanol Contacting with at least one catalyst capable of forming a butyl ester of the carboxylic acid, wherein the carboxylic acid is present in a concentration sufficient to form a biphasic mixture; ;
(G) separating the butyl ester-containing organic phase from the aqueous phase;
(H) recovering butanol from the butyl ester;
A method in which steps (e) and (f) are optionally performed at the same time.   110. The method of claim 109, further comprising converting at least a portion of the oil to carboxylic acid. The raw slurry is separated by centrifugation, filtration, or decantation;
110. The method of claim 109.   110. The method of claim 109, wherein the carboxylic acid comprises a fatty acid.   110. The method of claim 109, wherein the carboxylic acid comprises 12-22 carbons.   110. The method of claim 109, further comprising adding oil to the fermentor.   110. The method of claim 109, further comprising adding additional carboxylic acid to the fermentor.   118. The method of claim 115, wherein after the step of adding the additional carboxylic acid, the oil is converted to a carboxylic acid.   110. The method of claim 109, wherein the carboxylic acid is corn oil fatty acid or soybean oil fatty acid.   110. The method of claim 109, wherein the carboxylic acid is a mixture of corn oil fatty acid and soybean oil fatty acid.   110. The method of claim 109, wherein the oil comprises glycerides and the one or more catalysts hydrolyze the glycerides into fatty acids.   110. The method of claim 109, wherein the butyl ester of a carboxylic acid is a butyl ester of a fatty acid.   110. The method of claim 109, wherein the catalyst is an enzyme capable of esterifying the carboxylic acid with the butanol to form a butyl ester of the carboxylic acid.   122. The method of claim 121, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.   110. The method of claim 109, wherein the solid is treated to form an animal feed product.   110. The method of claim 109, wherein recovering butanol from the butyl ester comprises hydrolyzing the ester to a carboxylic acid and butanol.   125. The method of claim 124, wherein the butyl ester is hydrolyzed in the presence of a hydrolysis catalyst.   126. The method of claim 125, wherein the butyl ester is hydrolyzed in the presence of water and the hydrolysis catalyst comprises an acid catalyst, an organic acid, a water soluble acid, or a water insoluble acid.   126. The method of claim 125, wherein the hydrolysis catalyst comprises an enzyme capable of hydrolyzing the butyl ester to form a carboxylic acid and butanol.   128. The method of claim 127, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.   129. The method of claim 124, wherein the hydrolysis reaction occurs in a reaction vessel.   110. The method of claim 109, wherein recovering butanol from the butyl ester comprises transesterifying the butyl ester to butanol and other fatty acid alkyl esters or acylglycerides.   131. The method of claim 130, wherein the other fatty acid alkyl ester comprises a fatty acid methyl ester, a fatty acid ethyl ester, or a fatty acid propyl ester.   110. The method of claim 109, further comprising providing a natural oil and converting at least a portion of the natural oil to a carboxylic acid by contacting the oil with one or more enzymes. Method.   135. The method of claim 132, wherein the enzyme is an enzyme capable of hydrolyzing or transesterifying the butyl ester to form butanol.   134. The method of claim 133, wherein the enzyme is esterase, lipase, phospholipase, or lysophospholipase. A method for producing an alcohol ester comprising:
Contacting the alcohol produced in the fermentation process with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with the alcohol to form an alcohol ester of the carboxylic acid. Because;
A method comprising the step of the carboxylic acid in the fermentation process being present at a concentration sufficient to produce a biphasic mixture.   138. The method of claim 135, wherein the production of the alcohol and the production of an alcohol ester are performed simultaneously or sequentially.   138. The method of claim 135, wherein the fermentation process feedstock comprises one or more fermentable sugars.   The raw material of the fermentation process is corn kernels, corn cobs, corn hulls, corn stover and other crop residues, grass, wheat, rye, wheat straw, barley, barley straw, grass, rice straw , Switchgrass, waste paper, sugarcane pomace, sorghum, sugarcane, soy, ingredients obtained from grinding cereals, cellulosic materials, lignocellulosic materials, trees, branches, roots, leaves, wood chips, sawdust, shrubs and shrubs 138. The method of claim 135, comprising one or more fermentable sugars derived from food, vegetables, fruits, flowers, animal manure, and mixtures thereof.   140. The method of claim 135, further comprising providing a natural oil and converting at least a portion of the natural oil to a carboxylic acid by contacting the oil with one or more enzymes. Method.   138. The method of claim 135, wherein the carboxylic acid comprises a fatty acid.   138. The method of claim 135, wherein the carboxylic acid has a carbon chain length in the range of 12-22 carbons.   138. The method of claim 135, wherein the alcohol ester of the carboxylic acid is an alcohol ester of a fatty acid.   138. The method of claim 135, wherein the catalyst is an enzyme capable of esterifying the carboxylic acid with the butanol to form a butyl ester of the carboxylic acid.   145. The method of claim 143, wherein the enzyme is esterase, lipase, phospholipase, or lysophospholipase. Wherein the alcohol _is an alkyl alcohol of C 2 -C _8, The method of claim 135. Alkyl alcohols of the _C 2 -C ₈ is ethanol, propanol, butanol, pentanol or 2-methyl-1-butanol, The method of claim 145. A method for producing alcohol comprising:
(A) at least one catalyst capable of esterifying the alcohol produced in the fermentation process with at least one carboxylic acid and the carboxylic acid together with the alcohol to form an alcohol ester of the carboxylic acid; Contacting; wherein the carboxylic acid in the fermentation process is present in a concentration sufficient to produce a two-phase mixture comprising an aqueous phase and an alcohol ester-containing organic phase;
(B) separating the alcohol ester-containing organic phase from the aqueous phase;
(C) recovering alcohol from the alcohol ester.   148. The method of claim 147, wherein recovering alcohol from the alcohol ester comprises hydrolyzing the ester to a carboxylic acid and an alcohol.   149. The method of claim 148, wherein the alcohol ester is hydrolyzed in the presence of a hydrolysis catalyst.   149. The method of claim 148, wherein the alcohol ester is hydrolyzed in the presence of water and the hydrolysis catalyst comprises an acid catalyst, an organic acid, a water soluble acid, or a water insoluble acid.   149. The method of claim 149, wherein the hydrolysis catalyst comprises an enzyme capable of hydrolyzing the butyl ester to form a carboxylic acid and butanol.   152. The method of claim 151, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.   149. The method of claim 148, wherein the hydrolysis reaction occurs in a reaction vessel.   148. The method of claim 147, wherein recovering the alcohol from the alcohol ester comprises transesterifying the alcohol ester into an alcohol and a fatty acid alkyl ester or acylglyceride.   155. The method of claim 154, wherein the fatty acid alkyl ester comprises a fatty acid methyl ester, a fatty acid ethyl ester, or a fatty acid propyl ester.   148. The method of claim 147, further comprising providing a natural oil and converting at least a portion of the natural oil to a carboxylic acid by contacting the oil with one or more enzymes. Method.   157. The method of claim 156, wherein the enzyme is an enzyme capable of hydrolyzing or transesterifying the alcohol ester to form an alcohol.   158. The method of claim 157, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.   148. The method of claim 147, wherein the carboxylic acid comprises a fatty acid.   148. The method of claim 147, wherein the carboxylic acid has a carbon chain length in the range of 12-22 carbons. Wherein the alcohol _is an alkyl alcohol of C 2 -C _8, The method of claim 147. Alkyl alcohols of the _C 2 -C ₈ is ethanol, propanol, butanol, pentanol or 2-methyl-1-butanol, The method of claim 161.   149. The method of claim 148, wherein carboxylic acid is recovered from the butyl ester. (A) a recombinant microorganism capable of producing a product alcohol;
(B) a fermentable carbon substrate;
(C) a catalyst capable of transesterifying fatty acids together with the product alcohols into fatty acid alcohol esters extracellularly;
(D) A fermentation broth comprising a fatty acid alcohol ester produced during fermentation.   165. The fermentation broth of claim 164, further comprising one or more of acyl glycerides, fatty acids, product alcohols, or oleic acid.   165. The fermentation broth further comprising a catalyst, wherein the catalyst esterifies a fatty acid with an alcohol to a fatty acid alcohol ester and hydrolyzes a triglyceride to a free fatty acid.   173. The fermentation broth of claim 166, wherein the catalyst is one or more lipase enzymes.   168. The fermentation broth of claim 167, further comprising a saccharifying enzyme capable of converting oligosaccharides into fermentable sugars.   169. The fermentation broth of claim 168, wherein the saccharifying enzyme comprises glucoamylase.   170. The fermentation broth of claim 169, wherein the fermentable sugar comprises monomeric glucose.   166. The fermentation broth of claim 164, wherein the recombinant microorganism is capable of producing butanol.   166. The fermentation broth of claim 164, wherein the fatty acid alcohol ester is a fatty acid butyl ester.   165. The fermentation broth of claim 164, further comprising isobutanol. A recombinant yeast cell capable of producing isobutanol, including an isobutanol biosynthetic pathway, wherein the isobutanol biosynthetic pathway comprises the following substrate-product conversion:
i) pyruvate to acetolactate;
ii) 2,3-dihydroxyisovaleric acid from acetolactate;
iii) 2,3-dihydroxyisovaleric acid to α-ketoisovaleric acid;
iv) comprising α-ketoisovaleric acid to isobutyraldehyde; and v) at least one enzyme catalyzing each of isobutyraldehyde to isobutanol;
A recombinant yeast cell wherein at least one of said enzyme of iii) or said enzyme of v) is encoded by a heterologous polynucleotide integrated into a chromosome.   175. The recombinant yeast cell of claim 174, wherein the yeast cell is substantially free of pyruvate decarboxylase activity.   175. The recombinant yeast cell of claim 175, comprising a deletion of the pdc1, pdc5, and pdc6 genes.   177. The recombinant yeast cell of claim 176, wherein the yeast cell is substantially free of an enzyme having NAD-dependent glycerol-3-phosphate dehydrogenase activity.   179. The recombinant yeast cell of claim 177, comprising a deletion of gpd2.   179. The recombinant yeast cell of claim 178, wherein both the enzyme of iii) and the enzyme of iv) are encoded by heterologous polynucleotides integrated into a chromosome.   179. The recombinant yeast cell of claim 179, wherein said isobutanol biosynthetic pathway comprises at least one heterologous polynucleotide encoding an enzyme for each of said substrate-product conversions.   181. The recombinant yeast cell of claim 180, further comprising a plasmid having at least 80% identity to each of said coding regions of pYZ090 or a plasmid having at least 80% identity to each of said coding regions of pLH468.   181. The recombinant yeast cell of claim 180, comprising the plasmid of SEQ ID NO: 1, the plasmid of SEQ ID NO: 2, or both. (A) a fermentable carbon substrate;
(B) a catalyst capable of esterifying free fatty acids together with alcohols into fatty acid alkyl esters, and optionally hydrolyzing glycerides into free fatty acids;
(C) with alcohol;
(D) free fatty acids;
(E) A fermentation composition comprising a fatty acid alcohol ester formed in situ from esterification of the free fatty acid with the alcohol using the catalyst.   184. The composition of claim 183, further comprising an oil comprising glycerides.   185. The composition of claim 184, wherein the oil, the free fatty acid, and the fermentable carbon substrate are derived from biomass.   186. The composition of claim 185, wherein the oil and the fermentable carbon substrate are derived from the same biomass source or different biomass sources.   186. The composition of claim 185, wherein the biomass source of the oil is soy or corn oil and the biomass source of the fermentable carbon substrate is corn.   188. The composition of claim 183, wherein (d) the free fatty acid is corn oil fatty acid.   184. The composition of claim 183, wherein (d) the free fatty acid is formed from hydrolysis of at least a portion of the glyceride in the oil using the catalyst.   184. The composition of claim 183, further comprising at least one of a diglyceride and a monoglyceride formed from partial hydrolysis of a portion of the glyceride in the oil using the catalyst.   184. The composition of claim 183, further comprising glycerol.   184. The composition of claim 183, further comprising an undissolved solid derived from the biomass source of the fermentable carbon substrate.   193. The composition of claim 192, comprising less than about 25% by weight of the undissolved solid.   184. The composition of claim 183, further comprising a saccharifying enzyme capable of converting starch into fermentable sugar, wherein the alcohol is butanol.   110. A composition comprising butanol produced by the method of claim 1, 12, 33, 54, 84, and 109.   196. The composition of claim 195, used as a fuel additive, raw chemical, reagent, solvent, disinfectant, or food grade extractant.   110. Use of butanol produced by the method of claim 1, 12, 33, 54, 84, and 109 as a fuel additive, raw chemical, reagent, solvent, disinfectant, or food grade extractant.

Images