JP2013531992A - Fatty acid supplementation to improve alcohol productivity - Google Patents

Abstract

  Fatty acids derived from biomass in steps during the fermentation process can be added to the fermentation medium containing the recombinant microorganisms that produce the product alcohol. At least one of microbial growth rate and fermentable carbon consumption in the presence of fatty acids is higher than microbial growth rate and fermentable carbon consumption in the absence of fatty acids. Addition of fatty acids can increase glucose consumption, improve microbial biomass production (cell growth / density) and growth rate, thereby shortening production time and increasing productivity of fermentation process .

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 is directed to achieving improved alcohol productivity, wherein the production of fermented alcohols such as butanol, in particular, the fermentative growth of recombinant microorganisms, is performed in the presence of fatty acids derived from biomass in a step during the fermentation process. It relates to the alcohol fermentation process.

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

  The production of alcohols such as butanol using fermentation by microorganisms is one environmentally friendly production method. Microorganisms such as yeast are used for the production of alcohol products, in which naturally produced pyruvate is used as the starting substrate in their biosynthetic pathway. Butanol can be produced biologically as a by-product of yeast fermentation, but its yield can usually be very low. To enhance the production of desired products, such as butanol, yeast can alter endogenous biosynthetic pathways or express enzymes that introduce new pathways and / or alter metabolite flow Has been modified by preventing the expression of endogenous enzymes. Introduced pathways using cellular pyruvate as a substrate include, for example, pathways for producing butanol isomers. The destruction of pyruvate decarboxylase has been used to increase pyruvate availability for pathways that produce desired products such as butanol. Further described are recombinant microbial production hosts that express the 1-butanol biosynthetic pathway (Patent Document 1), the 2-butanol biosynthetic pathway (Patent Documents 2 and 3), and the isobutanol biosynthetic pathway (Patent Document 4). ing.

  For example, Saccharomyces cerevisiae yeast can be metabolically engineered by disruption mutations in two major pyruvate decarboxylase (PDC) genes. These genes, commonly referred to as PDC1 and PDC5, produce enzymes that are directly involved in ethanol production, and disruption of these genes adversely affects growth. The knockout of pyruvate decarboxylase and alcohol dehydrogenase (ADH) alters the biosynthetic pathway to produce fewer fatty acids. Fatty acids are required for cell growth because they are required for cell wall formation. The importance of fatty acids for cell growth is demonstrated, for example, in Non-Patent Document 1, which describes the effect of the antibiotic cerulenin, a known inhibitor of fatty acid synthesis, on cell proliferation. Yes. Cerrenin is S. cerevisiae. Added to S. cerevisiae medium, resulting in growth inhibition, but growth was restored when oleic acid was added with certain saturated fatty acids (especially myristic acid, palmitic acid or pentadecanoic acid) .

  Glucose metabolism in yeast generally follows a pathway that converts glucose to pyruvate and pyruvate to acetyl-CoA, leading to the cell population. Correspondingly, there may be a conversion of pyruvate to acetaldehyde, acetaldehyde to ethanol, or acetaldehyde to acetyl-CoA, and acetyl-CoA to fatty acid synthesis. For recombinant microorganisms, one PDC deletion reduces maximum growth, but to a much lesser extent. If only one PDC gene is destroyed, the other PDC genes are sufficiently active to allow carbon to flow to acetaldehyde and then to ethanol and acetate. However, if a butanol product is desired, ethanol production reduces the butanol product yield relative to the substrate. The PDC gene is involved in converting pyruvate to acetaldehyde, and double mutations in PDC1 and PDC5 prevent the production of acetaldehyde and alter the pathway to fatty acid biosynthesis, thereby inhibiting cell growth .

US Patent Application Publication No. 2008 / 0182308A1 US Patent Application Publication No. 2007 / 0259410A1 US Patent Application Publication No. 2007/0292927 US Patent Application Publication No. 2007/0092957

Otoguro, et al. , J .; Biochem. 89: 523-529, 1981

  Accordingly, there remains a need for methods for producing fermented alcohols using recombinant microorganisms that can improve microbial growth rate and / or biomass production despite the reduction or loss of fatty acid biosynthesis by the microorganism. As will become apparent from the following description of the embodiments, the present invention provides further related advantages.

  The invention includes (a) providing a fermentation broth comprising a recombinant microorganism that produces product alcohol from a fermentable carbon source, wherein the recombinant microorganism comprises a reduction or elimination of pyruvate decarboxylase activity; (B) contacting the fermentation broth with a fermentable carbon source, whereby the recombinant microorganism consumes the fermentable carbon source and produces product alcohol; (c) derived from biomass in a step during the fermentation process; A step of contacting a fermentation broth with a fatty acid, wherein (i) the growth rate of the recombinant microorganism in the presence of the fatty acid and (ii) at least one of fermentable carbon consumption is the growth rate of the recombinant microorganism when no fatty acid is present And / or a process comprising higher than fermentable carbon consumption. In a further embodiment, steps (b) and (c) are performed substantially simultaneously. In one embodiment, the fatty acid is selected from oleic acid, palmitic acid, myristic acid, and mixtures thereof, and in another embodiment, the biomass is corn kernels, corn cobs, corn hulls, corn Ingredients from crop residues such as foliage, grass, wheat, rye, wheat straw, barley, barley straw, grass, rice straw, switchgrass, waste paper, sugarcane pomace, sorghum, sugarcane, soybeans, cereals Derived from cellulosic materials, lignocellulosic materials, trees, branches, roots, leaves, wood chips, sawdust, shrubs and shrubs, vegetables, fruits, flowers, animal manure, and mixtures thereof. In a further embodiment, the fermentable carbon source is derived from biomass. In one embodiment, the product alcohol is butanol, and in another embodiment, the fermentation broth further comprises ethanol. In another embodiment, the recombinant microorganism has one or more pyruvate decarboxylase (PDC) gene deletions.

  The present invention is a method for producing a product alcohol comprising: (a) providing a biomass comprising a fermentable carbon source and an oil; and (b) converting at least a portion of the oil into a fatty acid. Forming a biomass containing fatty acid; and (c) contacting the biomass with a fermentation broth containing a recombinant microorganism capable of producing product alcohol from a fermentable carbon source, wherein the recombinant microorganism is pyrubin. (D) contacting the fatty acid with the fermentation broth, wherein (i) the growth rate and (ii) fermentable carbon consumption of the recombinant microorganism in the presence of the fatty acid; Also relates to a method comprising a growth rate of recombinant microorganisms in the absence of fatty acids and / or a step higher than fermentable carbon consumption. In a further embodiment, the step (b) of converting at least a portion of the oil into fatty acids comprises contacting the oil with one or more substances capable of hydrolyzing a portion of the oil into fatty acids. . In one embodiment, the one or more substances include one or more enzymes, and in another embodiment, the one or more enzymes include a lipase enzyme. In further embodiments, prior to step (c), one or more enzymes may be inactivated after at least a portion of the oil has been hydrolyzed. In one embodiment, one or more of steps (b), (c), and (d) are performed in the fermentor, and in another embodiment, steps (b), (c), and (d) ) At about the same time. In one embodiment, the method further comprises separating the oil from the biomass prior to step (b) of converting at least a portion of the oil into fatty acids. In another embodiment, the method liquefies biomass to produce liquefied biomass, wherein the liquefied biomass includes an oligosaccharide; and the oligosaccharide can be converted into a fermentable sugar. The method further comprises contacting the saccharification enzyme with the liquefied biomass to form saccharified biomass, and step (c) includes contacting the saccharified biomass with a fermentation broth containing a recombinant microorganism. In one embodiment, the fatty acid is selected from oleic acid, palmitic acid, myristic acid, and mixtures thereof, and in another embodiment, the biomass is corn kernels, corn cobs, corn hulls, corn Ingredients from crop residues such as foliage, grass, wheat, rye, wheat straw, barley, barley straw, grass, rice straw, switchgrass, waste paper, sugarcane pomace, sorghum, sugarcane, soybeans, cereals Derived from cellulosic materials, lignocellulosic materials, trees, branches, roots, leaves, wood chips, sawdust, shrubs and shrubs, vegetables, fruits, flowers, animal manure, and mixtures thereof. In one embodiment, the method further comprises fermenting the fermentable carbon source to produce a product alcohol. In one embodiment, the product alcohol is butanol, and in another embodiment, the fermentation broth further comprises ethanol. In another embodiment, the recombinant microorganism has one or more pyruvate decarboxylase (PDC) gene deletions.

  Another method of the invention is a method for producing a product alcohol: (a) providing a raw material; (b) liquefying the raw material to produce a raw slurry; c) separating the raw slurry to produce a product comprising (i) an aqueous layer comprising a fermentable carbon source, (ii) an oil layer, and (iii) a solid layer; (d) obtaining oil from the oil layer; Converting at least a portion of the oil into fatty acids; and (e) the aqueous layer of (c) comprises a fermentation broth comprising a recombinant microorganism capable of producing product alcohol from a fermentable carbon source Supplying to the fermentor, wherein the recombinant microorganism comprises a decrease or disappearance of pyruvate decarboxylase activity; and (f) fermenting a fermentable carbon source in the aqueous layer to produce a product alcohol. And (g) fermenting broth with fatty acid A step of contacting, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism in the presence of fatty acid is a growth rate and / or fermentable carbon of the recombinant microorganism in the absence of fatty acid Including a step higher than consumption. In a further embodiment, the step (d) of converting at least a portion of the oil into fatty acids comprises contacting the oil with one or more substances capable of hydrolyzing a portion of the oil into fatty acids. . In one embodiment, the one or more substances include one or more enzymes, and in another embodiment, the one or more enzymes include a lipase enzyme. In one embodiment, the fatty acid is selected from oleic acid, palmitic acid, myristic acid, and mixtures thereof, and in another embodiment, the biomass is corn kernels, corn cobs, corn hulls, corn Ingredients from crop residues such as foliage, grass, wheat, rye, wheat straw, barley, barley straw, grass, rice straw, switchgrass, waste paper, sugarcane pomace, sorghum, sugarcane, soybeans, cereals Derived from cellulosic materials, lignocellulosic materials, trees, branches, roots, leaves, wood chips, sawdust, shrubs and shrubs, vegetables, fruits, flowers, animal manure, and mixtures thereof. In one embodiment, the product alcohol is butanol. In another embodiment, the recombinant microorganism has one or more pyruvate decarboxylase (PDC) gene deletions.

  The invention also relates to a composition comprising a recombinant microorganism comprising a reduction or elimination of pyruvate decarboxylase activity and a fatty acid.

  Fatty acids derived from biomass (eg, oleic acid, palmitic acid, and mixtures thereof) in steps during the fermentation process can be added to the fermentation medium containing recombinant microorganisms that produce product alcohol. The microorganism can be a yeast or other alcohol producing microorganism. The microorganism may also have one or more PDC gene deletions and / or have reduced or eliminated pyruvate decarboxylase activity. The addition of fatty acids can increase glucose consumption and improve microbial biomass production (cell growth) and growth rate. By increasing the growth rate, the production time can be shortened, thereby increasing the productivity of the alcohol fermentation process.

  In certain embodiments, a method for producing product alcohol in a fermentation process comprises (a) providing a fermentation broth comprising a recombinant microorganism that produces product alcohol from a fermentable carbon source; and (b) a fermentation broth. Contacting the fermentable carbon source, whereby the microorganism consumes the fermentable carbon source and produces a product alcohol; (c) fatty acid and fermentation broth derived from biomass in a step during the fermentation process; A step of contacting, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the microorganism in the presence of a fatty acid is greater than the growth rate and / or fermentable carbon consumption of the microorganism in the absence of the fatty acid. High process.

  In certain embodiments, the fatty acid is free fatty acid (FFA). In certain embodiments, the fatty acid comprises oleic acid. In certain embodiments, the fatty acid comprises a saturated fatty acid. In certain embodiments, the fatty acid comprises palmitic acid. In certain embodiments, the fatty acid comprises myristic acid.

  In certain embodiments, the product alcohol is butanol.

  In certain embodiments, the fermentable carbon source is derived from biomass. In certain embodiments, the biomass comprises corn and the fatty acid is corn oil fatty acid.

  In certain embodiments, the fermentation broth further comprises ethanol. In certain embodiments, the method further comprises contacting the fermentation broth with ethanol.

  In certain embodiments, the step of contacting the fermentation broth with a fatty acid contacts the triglyceride from the biomass with one or more enzymes capable of hydrolyzing the triglyceride to free fatty acids, thereby freeing the triglycerides. Hydrolyzing into fatty acids; contacting the fermentation broth with free fatty acids, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the microorganism in the presence of free fatty acids is: Higher steps than in the absence of free fatty acids. In certain embodiments, the one or more enzymes comprises a lipase enzyme.

  In certain embodiments, the recombinant microorganism has a single pyruvate decarboxylase (PDC) gene deletion. In certain embodiments, the recombinant microorganism has two PDC gene deletions. In certain embodiments, the recombinant microorganism has reduced or eliminated pyruvate decarboxylase activity.

  In certain embodiments, the concentration of fatty acid in the fermentation broth is about 0.8 g / L or less.

  In certain embodiments, a method for producing product alcohol from fermentation of biomass comprises the steps of: (a) providing an aqueous biomass feed stream comprising water, a fermentable carbon source, and a predetermined amount of oil; Both the fermentable carbon source and the oil are derived from said biomass; (b) hydrolyzing at least a portion of the oil into free fatty acids to form a biomass feed stream comprising free fatty acids; c) contacting the fermentation medium with the biomass feedstock stream in the fermentor, wherein the fermentation medium comprises recombinant microorganisms that produce product alcohol; and (d) a fermentable carbon source in the fermentor. Fermenting to produce the product alcohol, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the microorganism in the presence of free fatty acid is free fatty acid And a higher step growth rate and / or fermentable carbon consumption of microorganisms in the absence.

  In certain embodiments, the step (b) of hydrolyzing at least a portion of the oil to free fatty acids comprises a composition comprising one or more enzymes capable of hydrolyzing a portion of the oil to free fatty acids; A step of contacting the oil. In certain embodiments, the method further comprises inactivating one or more enzymes after at least a portion of the oil has been hydrolyzed prior to step (c).

  In certain embodiments, the aqueous biomass feed stream is a liquefied mash formed from crushed, unfractionated cereals. In certain embodiments, the ground, unfractionated cereal is corn and the oil is corn oil.

  In certain embodiments, a method of producing a product alcohol comprising: (a) providing a biomass comprising an oil comprising glucose and a predetermined amount of triglycerides; (b) converting the triglycerides to free fatty acids. Contacting the oil with a composition comprising one or more substances capable of converting, thereby converting at least some of the triglycerides in the oil to free fatty acids; (c) converting glucose to product alcohol Contacting the biomass with a fermentation broth containing microorganisms capable of producing, thereby producing a product alcohol; (d) contacting the free fatty acid with the fermentation broth, in the presence of the free fatty acid. Growth rate of microorganisms when at least one of (i) growth rate and (ii) glucose consumption in the absence of free fatty acids And / or and a higher step than glucose consumption.

  In certain embodiments, the method further comprises the step of separating the biomass-derived (a) oil prior to the step (b) of contacting the oil with one or more substances.

  In certain embodiments, the step (b) of contacting the oil with a composition comprising one or more substances comprises contacting the oil with one or more catalysts capable of hydrolyzing triglycerides to free fatty acids. Including.

  In certain embodiments, the step (b) of contacting the oil with a composition comprising one or more substances comprises contacting the oil with one or more reactants or solvents capable of chemically reacting triglycerides. And a step of obtaining a reaction product containing free fatty acids.

  In certain embodiments, a method for producing butanol comprises: (a) providing a biomass comprising starch and oil, wherein the oil comprises a predetermined amount of glycerides; and (b) liquefying the biomass. A step of producing liquefied biomass, wherein the liquefied biomass includes an oligosaccharide hydrolyzed from starch; (c) the biomass of step (a) or the liquefied biomass of step (b), Contacting with a composition comprising one or more enzymes capable of being converted to free fatty acids, whereby at least some of the glycerides in the oil are converted to free fatty acids; (d) Contacting the liquefied biomass with a saccharification enzyme capable of converting an oligosaccharide into a fermentable sugar containing monomeric glucose; (e) fermenting the liquefied biomass Contacting sugar with a recombinant microorganism capable of converting sugar to butanol, thereby producing butanol; (f) contacting free fatty acid with the recombinant microorganism in the presence of free fatty acid; A step in which at least one of (i) growth rate and (ii) glucose consumption of the recombinant microorganism is higher than the growth rate and / or glucose consumption of the recombinant microorganism in the absence of free fatty acids.

  In one embodiment, the fermentation process for producing product alcohol from a raw material comprises: (a) liquefying the raw material to produce a raw slurry; (b) centrifuging the raw slurry, and (i) glucose An aqueous layer comprising: (ii) an oil layer comprising glycerides; and (iii) producing a centrifuged product comprising a solid layer; (c) hydrolyzing at least a portion of the glycerides to free fatty acids; (D) supplying the aqueous layer of (b) to a fermentor containing a fermentation broth containing a recombinant microorganism capable of producing product alcohol from glucose; (e) fermenting the glucose in the aqueous layer; Producing a product alcohol; and (f) contacting the fermentation broth with free fatty acid, wherein (i) the growth rate and (ii) of the microorganism in the presence of the free fatty acid. At least one of the courses consumption, and a higher growth rate and / or glucose consumption of microorganisms in the absence of free fatty acid process.

  In certain embodiments, the method for producing product alcohol from a feedstock further comprises supplying glyceride to the fermentor prior to hydrolyzing the glyceride.

  In certain embodiments, the fermentation process comprises (a) providing a fermentation broth comprising a recombinant microorganism that produces product alcohol from a fermentable carbon source, a fermentable carbon source, product alcohol, and oil derived from biomass. Wherein the oil comprises a glyceride; (b) contacting the fermentation broth with a first extractant to form a two-phase mixture comprising an aqueous phase and an organic phase, wherein the product alcohol and oil Separating in the organic phase to form a product alcohol-containing organic phase; (c) separating the product alcohol-containing organic phase from the aqueous phase; and (d) removing the product alcohol from the organic phase. Separating to produce a lean organic phase; (e) a composition comprising one or more catalysts capable of hydrolyzing the dilute organic phase into glycerides into free fatty acids; Contacting to produce a second extractant comprising at least a portion of the first extractant and free fatty acids; (f) contacting the fermentation broth with the second extractant of step (e). Repeating step (b), wherein at least one of (i) growth rate and (ii) fermentable carbon consumption in the presence of free fatty acids is the growth rate of the microorganisms in the absence of free fatty acids and And / or higher than fermentable carbon consumption.

  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 liquefied biomass is contacted with one or more substances for lipid hydrolysis and fed to a fermentor. 1 schematically illustrates an exemplary method and system of the present invention in which liquefied and saccharified biomass is contacted with one or more substances for lipid hydrolysis and fed to a fermentor. Undissolved solids and lipids are removed from the liquefied biomass prior to fermentation, the removed lipids are hydrolyzed to free fatty acids using one or more substances, and the free fatty acids are fed to the fermentor. 1 schematically illustrates an exemplary method and system of the present invention. Fig. 4 schematically illustrates an exemplary method and system of the present invention in which lipids derived from natural oils are hydrolyzed to free fatty acids using one or more substances, and the free fatty acids are fed to the fermentor. Fig. 4 schematically illustrates an exemplary method and system of the present invention where biomass lipids present in the extractant exiting the fermentor are converted to free fatty acids, which are fed to the fermentor. FIG. 2 is a graph showing the effect of the presence of fatty acids in the fermentor on glucose consumption for butanologen strain NGCI-047. FIG. 4 is a graph showing the effect of the presence of fatty acids in the fermentor on glucose consumption for butanologen strain NGCI-049. It is a graph which shows the influence which the presence of the fatty acid in a fermenter has on glucose consumption about butanologen strain NYLA84.

  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.

  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, sludge from papermaking, garden 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. Low ammonia pretreatment is disclosed in US Patent Application Publication No. 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 production” as used herein refers to microbial biomass production (ie, cell biomass production or cell growth), such as during culturing prior to microbial fermentation or during microbial fermentative growth.

  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, sugar cane, barley, cellulosic materials, lignocellulosic materials, or mixtures thereof.

As used herein, “fermentation broth” refers to alcohol, water, and dioxide by water, sugars, dissolved solids, optionally microorganisms that produce alcohol, product alcohol, and microorganisms present. It means a mixture of all other components of the material to be retained in the fermentation vessel where the product alcohol is produced by reaction of a saccharide to a carbon (CO _2). Optionally, the terms “fermentation medium” and “fermented mixture” as used herein can be used interchangeably with “fermentation broth”.

  A “fermentable carbon source” or “fermentable carbon substrate” as used herein is metabolized (or “consumed” by the microorganisms disclosed herein to produce fermented alcohol. ") Means a carbon source capable of. 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. As used herein, the term “consumed” means that a compound, for example an organic compound such as glucose, is broken down by the action of an enzyme derived from the cell, thereby increasing the energy that can be used by the cell. Includes generated processes.

  A “fermentable sugar” as used herein is one species that can be metabolized (or “consumed”) by the microorganisms disclosed herein to produce fermented alcohol. It refers to the above sugars.

  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.

“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 specifically refers to butanol isomers 1-butanol (1-BuOH), 2-butanol (2-BuOH), tert-butanol, and / or isobutanol (iBuOH or i -BuOH or I-BUOH, also known as 2-methyl-1-propanol), refers 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.

  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.

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 “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.

  “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

  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 is for producing a product alcohol (eg, fermented alcohol) in which an alcohol-producing microorganism in a fermentor is contacted with a fatty acid derived from a natural oil, such as a biomass lipid, in a step during the fermentation process. Provide a method. This fatty acid supplementation during microbial fermentation growth can increase microbial fermentable carbon consumption, growth rate, and biomass production, particularly for recombinant microorganisms with reduced or eliminated pyruvate decarboxylase activity.

  In certain embodiments, glycerides in the oil can be chemically converted to fatty acids, which are contacted with a fermentation broth containing recombinant microorganisms that produce product alcohol from a fermentable carbon source. In other embodiments, glycerides in the oil can be hydrolyzed to fatty acids by catalysis (eg, enzymatically), which is contacted with a fermentation broth containing recombinant microorganisms. In certain embodiments, fatty acids can be obtained from hydrolysis of lipids found in biomass that provides a fermentable carbon source for fermentation. The product alcohol can also be removed from the fermentation broth using fatty acids as ISPR extractants.

  The fatty acid can be saturated, monounsaturated, polyunsaturated, and mixtures thereof. For example, an oil having a natural fatty acid composition comprising a mixture of palmitic acid and oleic acid (eg, corn oil) can be hydrolyzed to produce a mixture of free oleic acid and free palmitic acid, It can be contacted with the fermentation broth in the fermenter. 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.

  The growth rate and / or fermentable carbon consumption of microorganisms in the presence of fatty acids is higher than the growth rate and fermentable carbon consumption of microorganisms in the absence of fatty acids. Correspondingly, supplementation with fatty acids according to the method of the present invention results in increased cell concentration and increased alcohol production than would be obtained without such supplementation of fatty acids. In certain embodiments, the microorganism can be a butanol-producing microorganism or other microorganism that normally requires supplementation of a 2-carbon substrate, such as ethanol, to survive and grow. In such embodiments, supplementation with fatty acids according to the methods of the invention may allow such 2-carbon dependent microorganisms to survive and grow in the absence of ethanol supplementation. In certain embodiments, the microorganism may be free of acetyl-CoA production from pyruvate. In certain embodiments, the microorganism is metabolically modified such that a disruption mutation in one or more pyruvate decarboxylase (PDC) genes alters the pathway to fatty acid biosynthesis. In certain embodiments, the microorganism is metabolically altered and the pathway to fatty acid biosynthesis is altered by disruptive mutations in two PDC genes, such as genes PDC1 and PDC5. Thus, the method of the present invention can achieve improved alcohol productivity by providing an optimal environment for the fermentation growth of recombinant microorganisms.

  The invention will now be described with reference to the figures. FIG. 1 shows an exemplary process flow diagram for the production of fermented alcohol 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 starch that supplies a fermentable carbon source (for example, a fermentable sugar such as glucose), but is not limited to the following: rye, wheat, corn, sugar cane, barley, cellulosic material, It can be a biomass, such as a lignocellulosic material, or a mixture thereof, or it can be derived from biomass. In certain 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, feedstock 12 can be corn, such as dry-milled, unfractionated corn kernels, and the undissolved solid can include germs, 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. 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. In certain embodiments, the fatty acid or mixture thereof comprises an unsaturated fatty acid. 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.

  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 sugar, oil 26, and undissolved solids derived from the biomass forming the raw material 12. In certain embodiments, the oil is in an amount from about 0% to at least about 2% by weight of the composition of the fermentation broth. In certain embodiments, the oil is in an amount of at least about 0.5% by weight of the feedstock. 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.

  One or more materials 42 may be added to the raw slurry 16. Substance 42 can hydrolyze glycerides in oil 26 to free fatty acids (FFA) 28. 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). Therefore, after inoculating the material 42 into the raw slurry 16, at least a part of the glycerides in the oil 26 is hydrolyzed to the FFA 28 to obtain the raw slurry 18 having the FFA 28.

  In certain embodiments, the one or more materials 42 are one or more catalysts 42 that are capable of catalytically hydrolyzing glycerides in oil 26 to free fatty acids 28 (FFA). Therefore, after the catalyst 42 is introduced into the raw slurry 16, at least a part of the glycerides in the oil 26 is hydrolyzed to the FFA 28, and the raw slurry 18 having the FFA 28 and the catalyst 42 is obtained.

  The acid / oil composition obtained from hydrolyzed oil 26 is typically at least about 17% by weight FFA. In certain embodiments, the acid / oil composition obtained from hydrolyzed oil 26 comprises at least about 20 wt% FFA, at least about 25 wt% FFA, at least about 30 wt% FFA, at least about 35 wt%. FFA, at least about 40% by weight FFA, at least about 45% by weight FFA, at least about 50% by weight FFA, at least about 55% by weight FFA, at least about 60% by weight FFA, at least about 65% by weight FFA, At least about 70 wt% FFA, at least about 75 wt% FFA, at least about 80 wt% FFA, at least about 85 wt% FFA, at least about 90 wt% FFA, at least about 95 wt% FFA, or at least About 99% by weight of FFA.

  Alternatively, in certain embodiments, the substance 42 may instead constitute one or more reactants or solvents that can chemically react the oil 26 with the FFA 28 for contact with the recombinant microorganism 32. Further described in co-pending and co-assigned US Provisional Patent Application No. 61 / 368,436, filed Jul. 28, 2010, which is hereby incorporated by reference in its entirety. Thus, for example, corn oil fatty acids can be synthesized from corn oil as oil 26 by base hydrolysis using NaOH and water as material 42. Also, Roe, et al., Which is incorporated herein by reference in its entirety. (Am. Oil Chem. Soc. 29: 18-22, 1952), for example, corn oil triglyceride as oil 26 is reacted with an aqueous ammonium hydroxide solution as reactant 42, Fatty acids (and fatty amides) are obtained. For purposes of discussion herein with reference to the illustrated embodiment, one or more of the substances 42 as substances 42 for hydrolyzing biomass lipids into FFAs 28 that are supplemented during fermentative growth of recombinant microorganisms 32. Often described as constituting a catalyst. However, the exemplary methods and systems described herein can be modified such that substance 42 is a reactant and / or solvent capable of chemically converting biomass lipids to FFA 28. I want you to understand.

  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, Aureobasidium pullulans, Bacillus pumilus, Bacillus pumilus Allothermophilus (Bacillis streamothermophilus), Bacillus subtilis, Brochothrix thermosolagata (Candida cylindolacea), Candida cylindracea ), Candida Antarctic lipase A, Candida antarctica lipase B, Candida ernobiii, Candida deformans (Candida deforman) s), Chromobacter viscosum, Coprinus cinerius, Fusarium oxysporum, Fusarium solius, fusarium solius・ Kumorum (Fusarium roseum culmorum), Geotricum penicillatum (Hanenula anomala), Humicola brevispola (Humicola brevisporum) revis 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 Thermomyces lanuginosus, Aspergillus sp. Lipase, Aspergillus niger lipase, Candida antid sp.) lipase, Penicillium roqueforti lipase, Penicillium camemberti lipase, Mucor javanicus lipase, Burkholderia ur k oldia cepacia lipase, Alcaligenes sp. lipase, Candida rugosa lipase, Candida parapsilosis lipase, Candida deforma ri gen Derived lipases A and B, Neurospora crassa lipase, Nectria haematococca lipase, Fusarium heterosporum lipase, Rhizopus le les ar) 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 enzyme 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 heimescem , Porcine pancreas, Candida cylindracea, Rhizopus nibeus (Rhizopus niveus), Candida antarctica, Rhizopus arrizus or Aspergillus lipase.

  In some embodiments, the catalyst 42 can be deactivated after at least a portion of the glycerides have been hydrolyzed. The catalyst 42 can be deactivated using any method known in the art. For example, in certain embodiments, the catalyst 42 is heated and / or by adjusting the pH of the reaction mass to a pH at which the catalyst 42 is irreversibly deactivated, and / or the catalytic activity. It can be deactivated by adding chemical or biochemical species that can be selectively deactivated. As shown, for example, in the embodiment of FIG. 1, heat q is added to the raw slurry 18, thereby deactivating the catalyst 42. The application of heat q can be added to the raw slurry 18 before the raw slurry 18 is supplied to the fermenter 30. Next, the heat-treated raw material slurry 18 (along with the inert catalyst 42) is introduced into the fermenter 30 together with the microorganisms 32 to be included in the fermentation broth held in the fermenter 30. Alternatively, the raw slurry 18 can be supplied to the fermenter 30 and added with heat q in the fermenter prior to inoculation of the microorganisms 32 with the fermenter. For example, in certain embodiments, heat q is used to bring the raw slurry 18 to at least about 75 ° C. for at least about 5 minutes, at least about 75 ° C. for at least about 10 minutes, at least about 75 ° C. for at least about 15 minutes, at least about At least about 80 ° C for at least 5 minutes, at least about 80 ° C for at least about 10 minutes, at least about 80 ° C for at least about 15 minutes, at least about 85 ° C for at least about 5 minutes, at least about 85 ° C for at least about 10 minutes, or at least The catalyst deactivation treatment can be performed by heating to a temperature of at least about 85 ° C. for about 15 minutes. In some embodiments, the raw slurry 18 is suitable for fermentation after heat q is added and before introduction into the fermentor 30 (or before fermentor inoculation if heat q is applied to the fermentor). Cool to the correct temperature. For example, in certain embodiments, the temperature of the raw slurry 18 is about 30 ° C. prior to contact with the fermentation broth.

  Deactivation of catalyst 42 is preferred when it is desirable to prevent catalyst 42 from esterifying alcohol with fatty acid 28 in the fermentor. Copending, co-assigned US Provisional Patent Application No. 61 / 368,429, filed July 28, 2010, all incorporated herein by reference in its entirety; 2010 In addition to US Provisional Patent Application No. 61 / 379,546 filed on September 2, 2011; and US Provisional Patent Application No. 61 / 440,034 filed on February 7, 2011 As described, in certain embodiments, the production of alcohol esters by esterification of the product alcohol in a fermentation medium using an organic acid (eg, fatty acid) and a catalyst (eg, lipase) is desirable. For example, in butanol production, the active catalyst 42 in the fermentor (introduced via slurry 18) catalyzes the esterification of butanol with fatty acid 28 (introduced via slurry 18) to produce fatty acid butyl. Esters (FABE) can be formed in situ. In such embodiments where an alcohol ester of a fatty acid is desired, the methods described herein can be modified to exclude deactivation of the catalyst 42 prior to contacting the fermentation broth containing the product alcohol. Accordingly, with reference to the exemplary process flow diagrams of FIGS. 1-5, these alternative embodiments provide for catalyst 42 to esterify product alcohol with fatty acid 28 in fermentor 30. This can be accomplished by excluding the application of heat q to the containing process stream. Further, in some embodiments, the exemplary process flow diagrams of FIGS. 1-5 require chemical conversion of oil 26 to FFA 28 using one or more reactants or solvents as material 42 instead of catalyst 42. If not, it can be modified to exclude heat q.

  The fermenter 30 is configured to ferment the slurry 18 to produce a product alcohol such as butanol. In particular, the microorganism 32 metabolizes the fermentable sugar in the slurry 18 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 may include less than about 20 g / L monomer glucose, more preferably less than about 10 g / L or less than about 5 g / L monomer glucose. . 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 18 is subjected to a saccharification process to break down complex sugars (eg, oligosaccharides) in slurry 18 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.

  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 is used as the microorganism 32 for butanol production, the microorganism 32 is supplemented with a 2-carbon substrate (eg, ethanol) to survive and grow. You may need. Thus, in certain embodiments, ethanol 33 can be supplied to the fermenter 30.

  However, the method of the present invention in which free fatty acids (eg, FFA28) are 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. Surprisingly, I can do it. Furthermore, in certain embodiments, the method of the present invention allows an alcohol (eg, butanol) production rate that is not supplemented with ethanol to be comparable to a 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 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 and the disadvantages associated with storing large amounts of ethanol 33 and supplying ethanol to the fermentor during butanol fermentation may be reduced or eliminated. Is possible. 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. In accordance with the methods described herein, fatty acids are introduced into the fermentor 30 as FFA 28 and hydrolyzed from the feedstock 26 of the slurry 16 or hydrolyzed from natural oils such as biomass lipids during steps in the fermentation process. Can be done. Fatty acids can also be introduced into the fermentor as ISPR extractant 29.

In the fermenter 30, alcohol is produced by the microorganisms 32. In situ product removal (ISPR) can be used to remove product alcohol from the fermentation broth. In certain embodiments, ISPR includes liquid-liquid extraction. 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, the method comprising extracting a fermentation broth with a water-immiscible extractant. Contacting 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 ₁₂ -C ₂₂ fatty aldehydes, and obtain an organic extraction agent selected from the group consisting of mixtures thereof, which is in contact with the fermentation broth to form a two-phase mixture comprising an aqueous phase and an organic phase. 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 obtain an organic extraction agent selected from the group consisting of mixtures thereof, which is in contact with the fermentation broth to form a two-phase mixture comprising an aqueous phase and an organic phase. Free fatty acid 28 from slurry 18 can also serve as an ISPR extractant 28. For example, when the free fatty acid 28 is corn oil fatty acid (COFA), the ISPR extractant 28 is COFA. ISPR extractant (FFA) 28 contacts the fermentation broth and forms a two-phase mixture comprising an aqueous phase 34 and an organic phase. Product alcohol present in the fermentation broth is preferentially separated into the organic phase to form an alcohol-containing organic phase 36. In certain embodiments, the fermentor 30 receives one or more additional ISPR extractants 29 that form a two-phase mixture comprising an aqueous phase and an organic phase, with the product alcohol separated into the organic phase. One or more inlets.

  The biphasic mixture can be removed from the fermenter 30 as stream 39 and introduced into the vessel 35, in which the alcohol-containing organic phase 36 is separated from the aqueous phase 34. The alcohol-containing organic phase 36 may be formed using methods known in the art including, but not limited to, siphoning, decantation, inhalation, centrifugation, use of gravity settling, membrane assisted phase separation, and the like. Separated from the aqueous phase 34 of the phase stream 39. All or a portion of the aqueous phase 34 is recycled into the fermentor 30 as a fermentation medium (shown) or is discarded and replaced with fresh medium, or any remaining product alcohol is removed. Can be processed and then recycled to the fermenter 30. The alcohol-containing organic phase 36 is then processed in a separator 50 to recover product alcohol 54, and the resulting alcohol lean extractant 27 is then used for further extraction of product alcohol. Usually combined with fresh FFA 28 and / or fresh extractant 29 from slurry 18 can be returned to fermenter 30 for reuse. Alternatively, fresh FFA 28 and / or extractant 29 (from slurry 18) can be continuously added to the fermentor instead of ISPR extractant removed in the two-phase mixed stream 39.

  In certain embodiments, the optional additional 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. , An organic extractant from the outside, such as methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal, and mixtures thereof. In certain embodiments, ISPR extractant 29 can be a carboxylic acid or free fatty acid, in certain embodiments, the carboxylic acid or free fatty acid is 4 to 28 carbons, in other embodiments 4 to 22 carbons, 8-22 carbon in other embodiments, 10-28 carbon in other embodiments, 7-22 carbon in other embodiments, 12-22 carbon in other embodiments, other In embodiments, it has a chain of 4-18 carbons, in other embodiments 12-22 carbons, and in still other embodiments 12-18 carbons. In certain embodiments, the ISPR extractant 29 comprises the following fatty acids: azelaic acid, capric acid, caprylic acid, castor oil, coconut oil (ie, lauric acid, myristic acid, palmitic acid, caprylic acid, capric acid, stearic acid, Natural combinations of fatty acids including caproic acid, arachidic acid, oleic acid, and linoleic acid), dimer acid, isostearic acid, lauric acid, linseed oil, myristic acid, oleic acid, olive oil, palm oil, palmitic One or more of acids, palm kernel oil, peanut oil, pelargonic acid, ricinoleic acid, sebacic acid, soybean oil, stearic acid, tall oil, tallow, # 12 hydroxystearic acid, or any seed oil. In certain embodiments, ISPR extractant 29 is one or more of diacids, such as azelaic acid and sebacic acid. Thus, in certain embodiments, ISPR extractant 29 can be a mixture of two or more different free fatty acids. In certain embodiments, ISPR extractant 29 can be a fatty acid derived from chemical or enzymatic hydrolysis of glycerides derived from natural oils. For example, in certain embodiments, ISPR extractant 29 can be a free fatty acid 28 'obtained by enzymatic hydrolysis of natural oils such as biomass lipids described below with reference to the embodiment of FIG. In certain embodiments, ISPR extractant 29 is described, for example, in co-pending and co-pending US Provisional Patent Application No. 61 / 368,436, filed July 28, 2010. From the group consisting of fatty acids, fatty alcohols, fatty amides, fatty acid methyl esters, fatty acid lower alcohol esters, fatty acid glycol esters, hydroxylated triglycerides, and mixtures thereof, obtained from chemical conversion of natural oils such as biomass lipids It can be the fatty acid extractant selected. 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. In certain embodiments, the additional ISPR extractant 29 comprises COFA.

  In situ extraction fermentation may be performed in the fermenter 30 in batch mode or continuous mode. In situ extraction fermentation, 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, for example, the ISPR extractant can be contacted with the fermentation medium at a time before the butanol concentration reaches a level that would be toxic to microorganisms. After contacting the fermentation medium with the ISPR extractant, the butanol product separates into the extractant, reducing the concentration in the aqueous phase containing the microorganisms, thereby reducing the product microorganisms to an inhibitory butanol product. Exposure is reduced.

  The volume of ISPR extractant used depends on several factors including the volume of the fermentation medium, the size of the fermentor, the partition coefficient of the extractant for the butanol product, and the selected fermentation mode, as described below. It depends on. The volume of the extractant can be about 3% to about 60% of the fermenter use volume. Depending on the efficiency of extraction, the aqueous phase titer of butanol in the fermentation medium can be, for example, from about 1 g / L to about 85 g / L, from about 10 g / L to about 40 g / L, from about 10 g / L to about 20 g / L. From about 15 g / L to about 50 g / L, or from about 20 g / L to about 60 g / L. 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. Without being bound by theory, a higher butanol titer uses an extractive fermentation process to remove toxic butanol products from the fermentation medium, thereby keeping levels below levels that are toxic to microorganisms. It is thought that it is obtained from this.

  In batch mode of in situ extraction fermentation, a predetermined volume of organic extractant is added to the fermentor and the extractant is not removed during the process. This mode requires a larger volume of organic extractant to minimize the concentration of inhibitory butanol product in the fermentation medium. As a result, the volume of the fermentation medium is smaller and the amount of product produced is less than that obtained using the continuous mode. For example, the volume of extractant in batch mode may be from 20% to about 60% of the fermenter usage volume in one embodiment and from about 30% to about 60% in another embodiment.

  Gas stripping (not shown) can be used simultaneously with the ISPR extractant to remove product alcohol from the fermentation medium.

  In the embodiment of FIG. 1, the product alcohol is extracted in situ from the fermentation broth and separation of the two-phase mixture 39 occurs in a separate tank 35. In some embodiments, the separation of the two-phase mixture 39 can be performed in a fermentor, as shown in the embodiments of FIGS. 2 and 3, described below, in which the alcohol-containing organic phase stream 36 is fermented. Exit directly from tank 30. The aqueous phase stream 34 can also exit the fermenter 30 directly and can be treated to remove any remaining product alcohol, reused, or discarded and replaced with fresh fermentation medium. Extraction of the product alcohol with an organic extractant can be performed with or without removal of microorganisms from the fermentation broth. Microorganisms can 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.

  In a continuous mode of in-situ extraction fermentation, the volume of the extractant is about 3% to about 50% of the use volume of the fermenter in one embodiment, and about 3% to about 30% in another embodiment. In another embodiment, from 3% to about 20%; in another embodiment, from 3% to about 10%. Since the product is continuously removed from the reactor, a smaller volume of extractant is required, allowing the use of a larger volume of fermentation medium.

  As an alternative to in situ extraction fermentation, product alcohol can be extracted from the fermentation broth downstream of fermentor 30. In such a case, the fermentation broth is removed from the fermenter 30 and introduced into the vessel 35 for contact with the ISPR extractant to obtain a two-phase mixture 39 in the vessel 35, which is then organic Separated into phase 36 and aqueous phase 34. Alternatively, the ISPR extractant can be added to the fermentation broth in a separate tank (not shown) before being introduced into the tank 35.

  As a non-limiting data-proven example, referring to the embodiment of FIG. 1, ground whole corn (as raw material 12), which 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. Optionally, the liquefied and lipase treated mash 18 can be heated to deactivate the lipase 42 prior to fermentation. Mash 18 can be cooled to about 30 ° C. (eg, using a heat exchanger) and filled into 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 is much less than the amount of corn oil (eg, about 1.2 wt% corn oil) that may be present in the fermentation broth using liquefied mash that has not been treated with lipase 42. May be contained. In particular, lipase 42 treatment can convert corn oil lipid 26 (triglyceride (TG)) to COFA (and some diglyceride (DG) or monoglyceride (MG)) as FFA 28, which is in contact with the fermentation broth. To do. The growth rate and / or glucose consumption of microorganisms in the fermentation broth in the presence of COFA can be higher than the growth rate and fermentable carbon consumption of microorganisms in the absence of fatty acids. For example, as described below in the Examples, FIGS. 6-8 show increased glucose consumption by butanol producing microorganisms in the presence of supplemented fatty acids.

  In certain embodiments, the system and method of FIG. 1 is such that feed slurry 16 (with oil 26) and catalyst 42 are introduced and contacted in fermentor 30 to produce slurry 18 (with FFA 28). Can be changed. In that case, the fermenter temperature can be raised and the catalyst 42 can be deactivated by heat. At that time, the fermenter temperature can be lowered and the microorganism 32 can be inoculated into the fermentor, thereby fermenting the sugar of the slurry 18 to produce product alcohol.

  As should be apparent to those skilled in the art, in one embodiment, the system and method of FIG. 1 allows a simultaneous saccharification and fermentation (SSF) in fermentor 30 to be separated into separate saccharification tanks 60 ( (See FIG. 2). Thus, the slurry 18 can be saccharified before or during fermentation in the SSF process. It should also be clear that the catalyst 42 for the hydrolysis of the feedstock 26 can be introduced before, after or simultaneously with the saccharifying enzyme 38. Thus, in certain embodiments, the conversion of enzyme 38 and catalyst 42 may be gradual (eg, catalyst 42, then enzyme 38, or vice versa) or at about the same time (ie, a person or machine once 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.

  For example, as shown in the embodiment of FIG. 2, the system and method of FIG. 1 allows the simultaneous saccharification and fermentation (SSF) in the fermenter 30 to be replaced with a separate saccharification tank 60 before the fermentor 30. Can be changed. FIG. 2 includes a separate saccharification tank 60 that receives the enzyme 38 and is substantially identical to FIG. 1 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, oil 26, and undissolved solids derived from the feed. 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 certain embodiments, the catalyst 42 can be added with the saccharifying enzyme 38 to produce glucose and simultaneously hydrolyze the oil lipid 26 to the free fatty acid 28. The conversion of enzyme 38 and catalyst 42 can be gradual (eg, catalyst 42, then enzyme 38, or vice versa) or simultaneous. Alternatively, in some embodiments, slurry 62 can be introduced into the fermentor and catalyst 42 is added directly to fermentor 30.

  In the embodiment of FIG. 2, heat q is added to the feed slurry 64, thereby deactivating the catalyst 42, and then the heat treated slurry 64 metabolizes monosaccharides to produce product alcohols (eg, It is introduced into the fermenter 30 together with the alcohol-producing microorganism 32 that produces butanol). Alternatively, the slurry 64 can be fed to the fermentor 30 and heat q can be applied in the fermentor prior to inoculation with the microorganisms 32.

  As described above with reference to FIG. 1, the free fatty acid 28 can also serve as an ISPR extractant for preferentially separating the product alcohol from the aqueous phase. In certain embodiments, one or more additional ISPR extractants 29 may also be introduced into the fermentor 30. Separation of the two-phase mixture takes place in the fermentor 30, whereby the alcohol-containing organic phase stream 36 and the aqueous phase stream 34 exit directly from the fermentor 30. Alternatively, the separation of the biphasic mixture can be performed in a separate vessel 35 as provided in the embodiment of FIG. 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 yet another embodiment, the oil 26 derived from the feedstock 12 is either prior to liquefaction or being liquefied so that the feedstock slurry 18 having the FFA 28 can exit the liquefaction tank 10 directly and be fed to the fermenter 30. Can be hydrolyzed to FFA28 by a catalyst. For example, the raw material 12 having the oil 26 can be supplied to the liquefaction tank 10 along with a catalyst 42 for hydrolyzing at least a part of the glycerides in the oil 26 into the FFA 28. An enzyme 14 (eg, α-amylase) that hydrolyzes starch in the raw material 12 can also be introduced into the vessel 10 to produce a liquefied raw material. The addition of enzyme 14 and catalyst 42 can be stepwise or simultaneous. For example, after at least a portion of the oil 26 has been hydrolyzed, the catalyst 42 can be introduced and then the enzyme 14 can be introduced. Alternatively, enzyme 14 can be introduced and then catalyst 42 can be introduced. The liquefaction process may include application of heat q. In such embodiments, the catalyst 42 can be introduced before or during liquefaction so that the oil 26 can be hydrolyzed if the process temperature is lower than the temperature at which the catalyst 42 is deactivated. Thereafter, the application of heat q can serve two purposes: liquefaction and deactivation of the catalyst 42 if deactivation is desired.

  In certain embodiments, including any of the embodiments described above with respect to FIGS. 1-3, undissolved solids can be removed from the raw slurry before being introduced into the fermentor 30. For example, as shown in the embodiment of FIG. 4, 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 certain embodiments, separator 20 may include a filter press, vacuum filtration, or centrifuge to separate undissolved solids from feed slurry 16. Optionally, in certain embodiments, the separator 20 can also be configured to remove some or substantially all of the oil 26 present in the raw slurry 16. In such embodiments, separator 20 removes oil from an aqueous feed stream including, but not limited to, siphoning, inhalation, decantation, centrifugation, use of gravity settling, membrane assisted phase separation, and the like. It can be any suitable separator known in the art for removal. The remaining raw material containing sugar and water is discharged to the fermenter 30 as a water stream 22.

  In certain embodiments, separator 20 removes oil 26 but does not remove undissolved solids. Therefore, the water stream 22 supplied to the fermenter 30 contains undissolved solids. 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 26). A tricanter centrifuge 20 that stirs or rotates the raw slurry 16 to produce a centrifuge product comprising: A method and system for removing undissolved solids from a raw slurry 16 by centrifugation is described in a co-pending, co-pending application filed Jun. 18, 2010, which is hereby incorporated by reference in its entirety. It is described in detail in US Provisional Patent Application No. 61 / 356,290 owned by a human.

  In either case, as shown in FIG. 3, the catalyst 42 can be contacted with the removed oil 26 to produce a flow of FFA 28 containing the catalyst 42. Heat q can then be added to the FFA 28 stream, thereby deactivating the catalyst 42. Next, a stream of FFA 28 and inert catalyst 42 can be introduced into fermentor 30 along with stream 22 and microorganism 32. Alternatively, the FFA 28 and the active catalyst 42 can be supplied from the tank 40 to the fermentor 30, after which the active catalyst 42 can be added with heat q and inactivated in the fermentor prior to inoculation with the microorganisms 32.

  The FFA 28 can act as an ISPR extractant 28 and forms a two-phase mixture in the fermentor 30. The product alcohol produced by the SSF separates into the organic phase 36 constituted by the FFA 28. In certain embodiments, one or more additional ISPR extractants 29 may also be introduced into the fermentor 30. Thus, the oil 26 (eg, derived from the raw material) can be hydrolyzed to FFA 28 by the catalyst, thereby reducing the rate of lipid accumulation in the ISPR extractant, while also producing the ISPR extractant. The organic phase 36 can be separated from the aqueous phase 34 of the two-phase mixture 39 in the tank 35. In certain embodiments, the separation of the biphasic mixture 39 can be performed in a fermentor, as shown in the embodiments described in FIGS. 2 and 3, in which the alcohol-containing organic phase stream 36 is Get out of fermenter 30 directly. The organic phase 36 can be introduced into a separator 50 for recovery of the product alcohol 54 and optional reuse of the recovered extractant 27, as shown in FIG. The remaining process operations of the embodiment of FIG. 3 are the same as FIG. 1 and will not be described again in detail.

  When the wet cake 24 is removed via the centrifuge 20, in certain embodiments, if the feed is corn, some of the oil from the feed 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 20 can be dried by any suitable known method to form a solubilized dry cereal distiller (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 some 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 raw material 16 by centrifugation is described in a co-pending, commonly assigned, filed June 18, 2010, which is incorporated herein by reference in its entirety. Are described in detail in US Provisional Patent Application No. 61 / 356,290.

  In still other embodiments (not shown), saccharification is performed in a separate saccharification tank 60 (see FIG. 2) located between the separator 20 and the liquefaction tank 10, as should be apparent to those skilled in the art. ).

  For example, in yet another embodiment shown in the embodiment of FIG. 4, natural oil 26 ′ is fed to a tank 40 that is also fed with catalyst 42, so that at least a portion of the glycerides in oil 26 ′ are hydrolyzed. Decomposed to form FFA 28 '. Thereafter, the catalyst 42 can be deactivated, such as by application of heat q. Next, the product stream from vessel 40 containing FFA 28 'and inert catalyst 42 is fed to feed 26, and in some embodiments, undissolved solids are separated into separator 20 (see, eg, the embodiment of FIG. 3). Is introduced into the fermenter 30 together with the aqueous feed stream 22 removed in advance. Saccharification enzymes 38 and microorganisms 32 are also introduced into the fermentor 30, whereby product alcohol is produced by the SSF.

  Alternatively, oil 26 'and catalyst 42 can be fed directly to fermenter 30, in which oil 26' is hydrolyzed to FFA 28 'without using vessel 40. Thereafter, the active catalyst 42 can be added with heat q and deactivated in the fermentor prior to inoculation with the microorganisms 32. Alternatively, the FFA 28 'and the active catalyst 42 can be fed from the tank 40 to the fermentor 30, and the active catalyst 42 can then be added with heat q and deactivated in the fermentor prior to inoculation with the microorganism 32. In such an embodiment, rather than the stream 22 from which the oil 26 has been removed, the raw slurry 16 containing the oil 26 can be fed into the fermentor 30 and contacted with the active catalyst 42. Thus, the active catalyst 42 can be used to hydrolyze the oil 26 to the FFA 28, thereby leading to loss of extractant and / or extraction over time that can be attributed to the presence of oil in the fermentor. A decrease in the distribution coefficient of the agent can be suppressed.

  In certain embodiments, the system and method of FIG. 4 allows the simultaneous saccharification and fermentation in the fermentor 30 to be replaced with a separate saccharification tank 60 before the fermentor 30, as should be apparent to those skilled in the art. Can be changed.

  In certain embodiments, the natural oil 26 'is tallow, corn oil, canola oil, capric / caprylic triglyceride, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard, linseed oil, cow leg oil, juicy oil. , Palm oil, peanut oil, rapeseed oil, rice oil, safflower oil, soybean oil, sunflower oil, kiri oil, jatropha oil, vegetable oil blends, and mixtures thereof. 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 feedstock 12 (shown in FIG. 4 as stream 22) 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 possible combination of different biomass sources of oil 26 'relative to feedstock 12 can be used, as should be apparent to those skilled in the art. The remaining process operations of the embodiment of FIG. 4 are the same as FIG. 1 and will not be described again in detail.

  In certain embodiments of the present invention, biomass oil present in feed 12 may be converted to FFA 28 in a later step of alcohol fermentation. FFA 28 can then be introduced into the fermentor and contacted with the fermentation broth to achieve an improved growth rate and / or fermentable carbon consumption of the alcohol producing microorganism. The FFA 28 can also act as an ISPR extractant 28. For example, in the embodiment of FIG. 5, the raw material 12 is liquefied, and the raw material slurry 16 containing the oil 26 derived from the raw material is produced. The raw slurry 16 may also include undissolved solids derived from the raw material. Alternatively, undissolved solids can be separated from slurry 16 by a separator such as a centrifuge (not shown). The raw material slurry 16 containing the oil 26 is directly introduced into a fermenter 30 containing a fermentation broth containing saccharifying enzymes 38 and microorganisms 32. Product alcohol is produced by the SSF in the fermenter 30. Alternatively, in certain embodiments, the process can be modified to include a separate saccharification tank, as described in connection with FIG.

  ISPR extractant 29 is introduced into fermentor 30 to form a two-phase mixture, and product alcohol is removed by separation into the organic phase of ISPR extractant 29. Oil 26 also separates into the organic phase. Separation of the two-phase mixture takes place in the fermenter 30, whereby the alcohol-containing organic phase stream 36 and the aqueous phase stream 34 exit directly from the fermenter 30. Alternatively, the separation of the biphasic mixture can be performed in a separate tank 35 as provided in the embodiment of FIG. The organic phase stream 36 containing the oil 26 is introduced into the separator 50, and the product alcohol 54 is recovered from the extractant 29. The resulting alcohol dilute extractant 27 contains the recovered extractant 29 and oil 26. Extractant 27 is contacted with catalyst 42, thereby hydrolyzing at least a portion of the glycerides in oil 26 to form FFA 28. Next, heat q can be added to the extractant 27 containing the FFA 28 to deactivate the catalyst 42 before being returned to the fermenter 30 for reuse. Such recycled extractant stream 27 can be a separate stream or a stream combined with a fresh, make-up extractant stream 29. Next, the subsequent withdrawal of the alcohol-containing organic phase 36 from the fermenter 30 includes FFA 28 and ISPR extractant 29 (fresh extraction) in addition to the product alcohol and additional oil 26 from the newly introduced feed slurry 16. Agent 29 and reclaimed extractant 27). The organic phase 36 is then treated to recover the product alcohol and, as described above, contacted with the catalyst 42 for further hydrolysis of the oil 26 and then returned to the fermenter 30. Can be reused. In certain embodiments, the use of supplemental ISPR extractant 29 is phased out as the fermentation process is run over time because the process itself can produce FFA 28 as a supplemental ISPR extractant for extracting product alcohol. can do. Thus, ISPR extractant may be a stream of extractant 27 that is recycled with FFA 28.

  Accordingly, FIGS. 1-5 show various non-limiting embodiments of methods and systems including fermentation processes and FFAs 28 produced from hydrolysis of biomass-derived oil 26, and contact with microorganisms during fermentation growth. FFA 28 'produced from the catalytic hydrolysis of natural oils 26', such as plant derived oils, that can be used to increase microbial growth rate and / or fermentable carbon consumption more in the presence of free fatty acids. It becomes high and alcohol productivity can be improved.

  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, supplementation with fatty acids according to the present invention may be used prior to fermentation, ie, 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. Thus, according to certain embodiments, the seed medium can be contacted with FFA 28, whereby an improved growth rate and microbial biomass production can be achieved, and the pre-fermentation time associated with the seed culture stage of the alcoholic fermentation process. Can be shortened. Therefore, in order to improve the overall process efficiency without departing from the present invention, fatty acid supplementation according to the present invention is used at various stages during the alcohol fermentation process, for example, before culturing and during fermentation. It should be clear to get.

  In certain embodiments, including any of the above-described embodiments described with reference to FIGS. 1-5, the fermentation broth in fermentor 30 is via a biosynthetic pathway from at least one fermentable carbon source to butanol. At least one recombinant microorganism 32 that has been genetically modified (ie, genetically engineered) to produce butanol. 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; oligosaccharides such as lactose, maltose, or sucrose; polysaccharides such as starch or cellulose; or mixtures thereof, and whey permeate, corn Examples include unrefined mixtures from renewable raw materials such as steep liquor, sugar beet molasses, and 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 modified to use glucose, fructose, and sucrose, or these substrates, and C5 saccharides. A mixture with C5 sugars such as xylose and / or arabinose for fresh yeast. 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 growth of cultures and promotion of enzymatic pathways including dihydroxy acid dehydratase (DHAD). Must contain cofactors, buffers and other ingredients.

  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, or Saccharomyces. In one embodiment, the recombinant microorganism may be selected from the group consisting of Escherichia coli, Lactobacillus plantarum, and Saccharomyces cerevisiae. 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). The species of Crabtree positive yeast are not limited to the following, but include Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe Saccharomyces mikatae, Saccharomyces paradoxus, Zygosaccharomyces rouxii, and Candida glabra gata rata). For example, the production of butanol using fermentation by microorganisms, as well as microorganisms that produce butanol are known and are disclosed, for example, in U.S. Patent Application Publication No. 2009/0305370, which is hereby incorporated by reference. Yes. In certain embodiments, the microorganism comprises a butanol biosynthetic pathway. Suitable isobutanol biosynthetic pathways are known in the art (see, eg, US Patent Application Publication No. 2007/0092957, incorporated herein by reference). 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.

  Specific strain structures, including those used in the examples, are 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 (described in this specification and US Provisional Patent Application No. 61 / 246,709) genomic DNA as a template prepared using kit (Qiagen, Valencia, CA). 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: 144) 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 the gpd2 :: loxP-URA3-loxP cassette (SEQ ID NO: 145) 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).

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-114117-11D (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.

  The following non-limiting examples further illustrate the present invention. The following examples include COFA as ISPR extractant obtained from enzymatic hydrolysis of corn and corn lipids as feedstock, but without departing from the present invention, other biomass sources can be used as feedstock and biomass oil enzymes. It should be understood that it can be used for hydrolysis.

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 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 g / L dextrose 3.0 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 (No. 291920) without amino acids.

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 300 mL of 0.2 uM filter sterilized Cognis, 90-95% oleyl alcohol 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.

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.

Example 15
The experimental identifier was GNLOR432A. NGCI-047 (butanediol producing strain) was grown in 25 mL medium in a 250 mL flask from a frozen container to an OD of about 1. The pre-seed medium was transferred to a 2 L flask and grown to an OD of 1.7-1.8. The media for both flasks was as follows:
3.0 g / L dextrose 3.0 g / L absolute ethanol 6.7 g / L Difco Yeast Nitrogen Base (No. 291920) without amino acids
1.4 g / L Yeast Dropout Mix (Sigma Y2001)
10 mL / L of 1% w / v L-leucine stock solution 2 mL / L of 1% w / v L-tryptophan stock solution.

A 1 L Applikon fermentor was inoculated with a 60 mL seed flask. The fermentor contained 700 mL of the following sterile medium:
20.0 g / L dextrose 8.0 mL / L absolute ethanol 6.7 g / L Difco Yeast Nitrogen Base (No. 291920) without amino acids
2.8 g / L Yeast Dropout Mix (Sigma Y2001)
20 mL / L 1% w / v L-leucine stock solution 4 mL / L 1% w / v L-tryptophan stock solution 0.5 mL Sigma 204 Antifoam
1: 1: Tween 80: 0.8 mL / L of 1% w / v Ergestol solution in ethanol.

  The remaining glucose was kept in excess using a 50% w / w glucose solution. The dissolved oxygen concentration in the fermenter was controlled to 30% by stirring control. The pH was controlled at pH = 5.5. The fermentor was sparged with 0.3 slpm sterilized domestic water. The temperature was controlled at 30 ° C.

Example 16
The experimental identifier was GNLOR434A. This example was the same as Example 15 except for the addition of 3 g oleic acid and 3 g palmitic acid prior to inoculation. NGCI-047 (butanediol producing strain) was the biocatalyst.

  FIG. 6 shows that there were more grams per liter of glucose consumed in the fermentor that received the fatty acids. The square represents the fermentor that received oleic acid and palmitic acid. The round shape represents a fermentor that did not accept any additional fatty acids.

Example 17
The experimental identifier was GNLOR435A. This example was the same as Example 15 except that NGCI-049 (an isobutanol producing strain) was inoculated.

Example 18
The experimental identifier was GNLOR437A. This example was the same as Example 16 except that NGCI-049 (an isobutanol producing strain) was inoculated.

  FIG. 7 shows that there were more grams per liter of glucose consumed in the fermentor that received the fatty acids. The square represents the fermentor that received oleic acid and palmitic acid. The round shape represents a fermentor that did not accept any additional fatty acids.

Example 19
The experiment identifier was 090420_3212. This example was performed in the same manner as Example 15 except that it was inoculated with butanologen NYLA84 (an isobutanol producing strain). This fermentation was performed in a 1 L Sartorius fermentor.

Example 20
The experimental identifier was 2009Y047. This example was carried out in the same manner as Example 16 except that it was inoculated with butanologen NYLA84 (an isobutanol producing strain). This fermentation was carried out in a 1 L Sartorius fermentation.

  FIG. 8 shows that there were more grams per liter of glucose consumed in the fermentor that received the fatty acids. The square represents the fermentor that received oleic acid and palmitic acid. The round shape represents a fermentor that did not accept any additional fatty acids. The result of Examples 15-20 is shown in Table 4, and Table 4 shows the presence or absence of addition of a fatty acid, the maximum optical density, and the glucose (g / L) consumed.

Example 21
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 COFA is first produced by lipase-catalyzed hydrolysis of corn oil. The results are shown in Table 5.

Example 22
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 6.

Example 23
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 7.

Example 24
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 8.

Example 25
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 9.

Example 26
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% 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 10.

Example 27
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 active lipase is slightly less than the final total gram of i-BuOH present in the fermentation broth containing inactive lipase. Less so that the overall production of i-BuOH (as a combination of free i-BuOH and COFA isobutyl ester (FABE)) is more active than that obtained in the presence of heat-inactivated lipase. Much in the presence of lipase. The results are shown in Tables 11 and 12.

  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 (32)

(A) providing a fermentation broth comprising a recombinant microorganism that produces product alcohol from a fermentable carbon source, wherein the recombinant microorganism comprises a reduction or elimination of pyruvate decarboxylase activity;
(B) contacting the fermentation broth with a fermentable carbon source, whereby the recombinant microorganism consumes the fermentable carbon source and produces the product alcohol;
(C) contacting the fermentation broth with a fatty acid derived from biomass in a step in the fermentation process, wherein (i) the growth rate and (ii) fermentability of the recombinant microorganism in the presence of the fatty acid A process wherein at least one of the carbon consumption is higher than the growth rate and / or the fermentable carbon consumption of the recombinant microorganism in the absence of the fatty acid.   The method of claim 1, wherein the fatty acid is selected from oleic acid, palmitic acid, myristic acid, and mixtures thereof.   Biomass is crop residue such as corn kernel, corn cobs, corn peel, corn stover, grass, wheat, rye, wheat straw, barley, barley straw, pasture, 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, vegetables, fruits, 2. The method of claim 1, wherein the method is derived from flowers, animal manure, and mixtures thereof.   The method of claim 1, wherein the product alcohol is butanol.   The method of claim 1, wherein steps (b) and (c) are performed substantially simultaneously.   The method of claim 1, wherein the fermentable carbon source is derived from the biomass.   The method of claim 1, wherein the fermentation broth further comprises ethanol.   2. The method of claim 1, wherein the recombinant microorganism has one or more pyruvate decarboxylase (PDC) gene deletions. A method for producing a product alcohol comprising:
(A) providing a biomass comprising a fermentable carbon source and oil;
(B) converting at least a portion of the oil into fatty acids to form biomass containing the fatty acids;
(C) contacting the biomass with a fermentation broth containing a recombinant microorganism capable of producing a product alcohol from a fermentable carbon source, wherein the recombinant microorganism reduces or eliminates pyruvate decarboxylase activity. Including a process;
(D) contacting the fatty acid with the fermentation broth, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism in the presence of the fatty acid is free of the fatty acid. The growth rate of said recombinant microorganism and / or a step higher than said fermentable carbon consumption.   The step (b) of converting at least a part of the oil into a fatty acid comprises contacting the oil with one or more substances capable of hydrolyzing a part of the oil into a fatty acid. 10. The method of claim 9, comprising.   11. The method of claim 10, wherein the one or more substances comprise one or more enzymes.   12. The method of claim 11, wherein the one or more enzymes comprises a lipase enzyme.   12. The method of claim 11, further comprising inactivating the one or more enzymes after at least a portion of the oil has been hydrolyzed prior to step (c).   The method of claim 9, wherein one or more of steps (b), (c), and (d) are performed in the fermentor.   The method of claim 9, wherein one or more of steps (b), (c), and (d) are performed substantially simultaneously.   Biomass is crop residue such as corn kernel, corn cobs, corn peel, corn stover, grass, wheat, rye, wheat straw, barley, barley straw, pasture, 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, vegetables, fruits, 10. The method of claim 9, wherein the method is derived from flowers, animal manure, and mixtures thereof.   10. The method of claim 9, wherein the fatty acid is selected from oleic acid, palmitic acid, myristic acid, and mixtures thereof.   The method of claim 9, further comprising fermenting the fermentable carbon source to produce a product alcohol.   The method of claim 18, wherein the product alcohol is butanol.   The method of claim 9, wherein the fermentation broth further comprises ethanol.   10. The method of claim 9, wherein the recombinant microorganism has one or more pyruvate decarboxylase (PDC) gene deletions.   The method of claim 9, further comprising separating the oil from the biomass prior to the step (b) of converting at least a portion of the oil to fatty acids. Liquefying the biomass to produce liquefied biomass, wherein the liquefied biomass contains oligosaccharides;
Further comprising the step of contacting the liquefied biomass with a saccharification enzyme capable of converting oligosaccharides into fermentable sugars to form saccharified biomass,
The method of claim 9, wherein step (c) comprises contacting the saccharified biomass with the fermentation broth comprising recombinant microorganisms. A method for producing a product alcohol 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 (i) an aqueous layer containing a fermentable carbon source, (ii) an oil layer, and (iii) a solid layer;
(D) obtaining oil from the oil layer and converting at least a portion of the oil into fatty acids;
(E) supplying the aqueous layer of (c) to a fermentor containing a fermentation broth containing a recombinant microorganism capable of producing a product alcohol from a fermentable carbon source, wherein the recombinant microorganism comprises: Comprising reducing or eliminating pyruvate decarboxylase activity;
(F) fermenting the fermentable carbon source of the aqueous layer to produce the product alcohol;
(G) contacting the fermentation broth with the fatty acid, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism in the presence of the fatty acid is free of the fatty acid The growth rate of said recombinant microorganism and / or a step higher than said fermentable carbon consumption.   The step (d) of converting at least a part of the oil into a fatty acid comprises contacting the oil with one or more substances capable of hydrolyzing a part of the oil into a fatty acid. 25. The method of claim 24, comprising:   26. The method of claim 25, wherein the one or more substances comprise one or more enzymes.   27. The method of claim 26, wherein the one or more enzymes comprise a lipase enzyme.   Ingredients are crop residues such as corn kernels, corn cobs, corn peel, corn stover, grass, wheat, rye, wheat straw, barley, barley straw, pasture, 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, vegetables, fruits, 25. The method of claim 24, comprising one or more fermentable sugars derived from flowers, animal manure, and mixtures thereof.   25. The method of claim 24, wherein the fatty acid is selected from oleic acid, palmitic acid, myristic acid, and mixtures thereof.   The method of claim 18, wherein the product alcohol is butanol.   25. The method of claim 24, wherein the recombinant microorganism has one or more pyruvate decarboxylase (PDC) gene deletions.   A composition comprising a recombinant microorganism comprising reduced or eliminated pyruvate decarboxylase activity and a fatty acid.

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