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WO2010059616A2 - Biocatalyseurs et procédés pour la conversion d'hydrolysats d'hémicellulose en produits à base biologique - Google Patents

Biocatalyseurs et procédés pour la conversion d'hydrolysats d'hémicellulose en produits à base biologique Download PDF

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WO2010059616A2
WO2010059616A2 PCT/US2009/064773 US2009064773W WO2010059616A2 WO 2010059616 A2 WO2010059616 A2 WO 2010059616A2 US 2009064773 W US2009064773 W US 2009064773W WO 2010059616 A2 WO2010059616 A2 WO 2010059616A2
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gene encoding
inactivation
asburiae
strain
megax
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PCT/US2009/064773
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WO2010059616A3 (fr
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James F. Preston
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University Of Florida Research Foundation, Inc.
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Priority to US13/122,985 priority Critical patent/US8993287B2/en
Publication of WO2010059616A2 publication Critical patent/WO2010059616A2/fr
Publication of WO2010059616A3 publication Critical patent/WO2010059616A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/54—Acetic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • yeast and bacterial biocatalysts has been applied to the commercial production of ethanol as an alternative fuel from starch and sucrose derived from commodity crops, e.g. corn and sugarcane (Dien, B. S., M. A. Cotta, and T. W. Jeffries. 2003. Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 63:258-266).
  • lignocellulosic resources including forest and agricultural residues, have become targets for bioconversion cellulose and hemicellulose to fermentable sugars (Aden, A., M. Ruth, K. Ibsen, J.
  • Cellulose consists of long chains of beta glucosidic residues linked through the 1,4 positions. These linkages cause the cellulose to have a high crystallinity and thus a low accessibility to enzymes or acid catalysts.
  • Hemicellulose is an amorphous hetero-polymer which is easily hydrolyzed.
  • Lignin an aromatic three-dimensional polymer, is interspersed among the cellulose and hemicellulose within the plant fiber cell.
  • Previously reported processes for hydrolysing cellulose include biological and non- biological means of depolymerization.
  • the biological methods involve the use a cellulase enzyme.
  • the oldest and best known non-biological method of producing sugars from cellulose is the use of acid hydrolysis.
  • the acid most commonly used in this process is sulfuric acid.
  • sulfuric acid hydrolysis can be categorized as either dilute acid hydrolysis or concentrated acid hydrolysis.
  • the dilute acid processes generally involve the use of 0.5% to 15% sulfuric acid to hydrolyze the cellulosic material.
  • temperatures ranging from 90°-600° C, and pressure up to 800 psi are necessary to effect the hydrolysis.
  • the sugars degrade to form furfural and other undesirable by-products.
  • the resulting glucose yields are generally low, less than 50%. Accordingly, the dilute acid processes have not been successful in obtaining sugars from cellulosic material in high yields at low cost.
  • the fermentation of the sugars produced by dilute acid hydrolysis presents additional problems.
  • the hydrolysis of cellulose and hemicellulose results in the production of pentose sugars for fermentation (Y. Y.
  • Microbial strategies for the depolymerization of ghicuronoxylan leads to biotechnological applications of endoxylanases, p. 191 -210, Applications of Enzymes to Lignocellulosics. American Chemical Society, Washington D. C). Resistance of the a- 1,2 glucuronosyl linkages to dilute acid hydrolysis results in the release of methylglucuronoxylose (MeGAX), which is not fermented by bacterial biocatalysts currently used to convert hemicellulose to ethanol, e.g. E.coli KOI l .
  • MeGAX methylglucuronoxylose
  • the frequency of MeGAX substitutions on the xylose residues of methylglucuronoxylan ranges from less than one in ten in crop residues to one in six to seven in hardwoods, e.g. sweetgum, and as much as 21% of the carbohydrate may reside in this unfermentable fraction following dilute acid pretreatment (Maria E. Rodriguez, Alfredo Martinez, Lonnie Ingram, Keelnatham T Shamugam and James F Preston. 2001. Properties of the hemicellulose fractions of lignocellulosic biomass affecting bacterial ethanol production. ASM National Meeting, 2001.).
  • the invention relates to processes and biocatalysts for producing ethanol and other useful products from biomass and/or other materials.
  • Initial processing of lignocellulosic biomass frequently yields methylglucuronoxylose (MeGAX) and related products which are resistant to further processing by common biocatalysts.
  • MeGAX methylglucuronoxylose
  • Strains of Enterobacter asburiae are shown to be useful in bioprocessing of MeGAX and other materials into useful bioproducts such as ethanol, acetate, lactate, and many others. Genetic engineering may be used to enhance production of desired bioproducts.
  • FIGURE 1 Scheme for the release of xylose and MeGAX by dilute acid hydrolysis of sweetgum xylan.
  • FIGURES 2A-2B Aerobic growth, substrate utilization, and formation of products from acid hydrolysates of MeGAX 11 by A) E. asburiae JDR-I and B) E. c ⁇ li B. Xylose (diamonds), MeGAX (squares), and acetic acid (triangles) were determined in media by HPLC. Growth was determined by measuring turbidity as OD 6OO (open circles).
  • FIGURES 3A-3C Aerobic growth of E. asburiae JDR-I on different combinations of sugar substrates. Concentrations of substrates and acetic acid as a product were determined by HPLC. Growth was determined as turbidity (OD 6 O 0 )- A) Growth on glucose (7.5 mM) and xylose (7.5 mM). Concentrations of glucose (closed circles), xylose (diamonds) and acetic acid (triangles); OD 6 oo (open circles); B) Growth on glucuronic acid (10 mM) and xylose
  • FIGURES 4A-4D Pathway determination for the metabolism of xylose and glucose by E. asburiae JDR-I.
  • FIGURES 5A-5D Fermentation time course for different strains in media containing 0.5% sweetgum xylan hydrolysate.
  • Figure 5A depicts E. asburiae JDR-I in minimal medium
  • Figure 5B depicts E. asburiae Ll in minimal medium
  • Figure 5C depicts E. asburiae JDR-I in LB
  • Figure 5D depicts E. asburiae Ll in LB.
  • Substrates and fe ⁇ nentation products xylose (closed diamonds ⁇ ), MeGAX (closed squares ⁇ ), acetic acid (open triangles ⁇ ), ethanol (open squares ⁇ ), lactic acid (open diamonds 0).
  • FIGURE 6 Diagram to illustrate deletion of als and pflB genes modifying mixed-acid fermentation of E. asburiae JDR-I into a homolactate production pathway in E. asburiae Ll. Deletion of pathways is indicated in the figure as symbol X.
  • FIGURE 7 HPLC profiles of fermentation media of E. asburiae JDR-I, E. coli KOl 1 and E. asburiae El (pLOI555) in 0.5% sweetgum xylan hydrolysate with 0.1 M MOPS buffer after 48 hours of fermentation. (The unlabeled peaks with retention times of 11 min and 21 min were for salts and buffers.)
  • FIGURES 8A-8D Fermentation time course for different strains in media of buffered sweetgum xylan hydrolysate.
  • Figure 8 A depicts E. asburiae JDR-I
  • Figure 8B depicts E. coli KOI l
  • Figure 8C depicts E.
  • the present invention provides novel microorganisms that are capable of fermenting by-products of acid hydrolysis of renewable biomass materials.
  • the fermentation of MeGAX sugars produced from acid hydrolysis of biomass materials involves the use of bacteria, namely Enterobacler asbiiriae. Because Mc ⁇ X is not fermented by bacterial biocatalysts currently used to convert biomass materials into useful bioproducts, the presence of MeGAX retards the overall production rate and yield in a fermentation process.
  • Enterobacter asbiiriae has been found to ferment MeGAX very well, thereby assisting in providing higher bioproduct yield over other known fermenting methods following acid hydrolysis.
  • Enterobacter asbvriae strain JDR-I is applied to by-products following dilute acid hydrolysis of biomass materials to produce high yields and concentrations of cthanol or other bioproducts.
  • thermochemical and bioconversion processes involving the use of the microorganisms or enzymes derived therefrom may be used for processing lignocellulosics to MeGAX, hexoses (e.g.
  • biocatalysts of the invention are particularly suited to facile bioprocessing of MeGAX-containing materials derived from biomass, the biocatalysts are by no means limited to bioprocessing of MeGAX-containing materials or materials derived from biomass.
  • the biocatalysts of the invention may be used to convert a wide variety of different substrates into useful products regardless of the source of the substrates.
  • the substrate comprises a monsaccharide, disaccharide, trisaccharide, or oligosaccharide (wherein the oligosaccharide contains 4, 5, 6, 7, 8, or more simple sugars).
  • the substrate comprises a monosaccharide selected from xylose, glucose, mannose, galactose, arabinose, fructose, or rhamnose.
  • the substrate comprises glucuronic acid (or its conjugate base) and/or MeGAX.
  • the substrate comprises an aldopentose, a ketopentose, an aldohexose, or a ketopentose.
  • the substrate comprises a sugar acid or a sugar alcohol.
  • the subject invention provides microorganisms useful in the production of ethanol, lactate, and other resources from recyclable photosynthetic resources.
  • a means for fermenting aldobiuronate methyl glucuronoxylose is provided.
  • MeGAX is fe ⁇ nented following dilute acid hydrolysis of hemicellulose containing materials, thereby providing an inexpensive, effective, and improved bioproduct production rate than that observed with previous methods for fermenting acid-treated hemicellulose materials.
  • Enterobacter asburiae strain is used following dilute acid treatment of materials containing hemicellulose for the large-scale bioconversion of MeGAX along with hexoses and pentoses to valuable resources such as ethanol, acetate, lactate, and other bioproducts.
  • Enterobacter asburiae strain either alone or in combination with other bacteria useful in the breakdown of sugars following dilute acid hydrolysis of hemicellulose containing materials, the subject invention provides improved rates and yields of ethanol and other bioproducts.
  • One aspect of the present invention is therefore related to a process for fermenting MeGAX to produce improved ethanol yields from biomass materials following acid hydrolysis comprising the steps of:
  • the substrate is inoculated with other strains of bacteria such as E. coli KOl 1 or other ethanogenic strains of bacteria in addition to Enterobacter asburiae.
  • a species of the genus was isolated from a soil sample and maintained on an agar plate. This specific strain was biologically pure and is identified as namely Enterobacter asburiae strain JDR-I (NRRL B-S0074).
  • Biomass materials that are applied to the process described herein are any known materials containing hemicellulose.
  • biomass materials that can be used as described herein include, but are not limited to, materials comprising: sweetgum wood as representative of forest energy crops, wood preprocessed for cellulose production, rice straw, wood primings, wood, wood waste, newspaper and/or other paper products, plant materials and/or tree cuttings obtained from, for example, miscanthus, switchgrass, elephant grass, energy cane, hemp, corn, Eucalyptus spp., poplar (including, for example, yellow poplar or tulip tree (Lirodendron tulipifera) or cottonwood), willow, sorghum, sugarcane, sugarcane bagasse, corn stalks, corn stover, wheat straw and/or various combinations thereof.
  • the culture medium used for fermentation in the present process can be any known culturing composition with suitable nitrogen sources, mineral supplements, vitamins, and carbon sources.
  • the culture medium comprises MeGAX.
  • Carbon sources may include D-glucose, D-xylose, D-xylobiose, D-xylotriose, D-mannose, L- arabinose, D-galactose, glucuronate and various combinations of such carbon sources.
  • oxygen tension for the fermentation process may vary widely and the oxygen tension can be either microaerophilic for batch fermentation, or the inoculated substrate may be sparged with a small amount of air in continuous fermentation techniques. Moreover, anaerobic fermentation may also be used. The technique will depend on the initial cell density, the substrate concentration, and the incubation condition of the inoculum.
  • the pH of the fermentation medium can range from a pH of about 5.0-7.0. Other embodiments provide for the fermentation of MeGAX and/or other carbon sources at a pH greater than, or equal to, 5.0.
  • the temperature of the fermentation process of the present invention can also vary considerably (from about 28°C to about 37°C). In various embodiments, the temperature can range from about 28 0 C to about 35° C, 28 0 C to about 33°C or be maintained around about 3O 0 C.
  • Additional embodiments relate to Enterobacter asburiae strains genetically modified to facilitate production of xylitol. Genetic modifications suitable for this purpose are set forth in U.S. Pat. App. 11/523,403, published as US-2007-0072280-A1, the disclosures of which are incorporated herein by reference in their entirety.
  • the genetically modified Enterobacter asburiae strains may contain, for example, one or more genetic modifications selected from the group consisting of:
  • the inactivated gene is a native gene or is an exogenous gene previously introduced into the Enterobacter asburiae strain. Additional embodiments relate to Enterobacter asburiae strains genetically modified to facilitate production of lactic acid (D(-)-lactic acid and/or L(+)-lactic acid). Genetic modifications suitable for this purpose are set forth in U.S. Pat. 7,098,009 and U.S. Pat. App. 11/501,137 (published as US-2007-0037265-A1), the disclosures of which are incorporated herein by reference in their entirety.
  • the genetically modified Enterobacter asburiae strains may contain, for example, one or more genetic modifications selected from the group consisting of:
  • L-lactate dehydrogenase and D-lactate dehydogenase are independently native to Enterobacter asbu ⁇ ae or exogenous. It is understood, for example, that when L(+)- lactate production is desired, and the native lactate dehydrogenase is D-lactate dehydrogenase then the native lactate dehydrogenase may be inactivated and replaced with an exogenous L- lactate dehydrogenase, and so on. It is thus understood that the strains may be engineered to produce D-lactate, L-lactate, or a mixture of the two.
  • the inactivated genes are native gene(s) and/or are exogenous gene(s) previously introduced into the Enterobacter asburiae strain.
  • Additional embodiments relate to Enterobacter asburiae strains genetically modified to facilitate production of ethanol. Genetic modifications suitable for this purpose are set forth in U.S. Pat. 7,098,009 and U.S. Pat. 5,000,000, the disclosures of which are incorporated herein by reference in their entirety.
  • the genetically modified Enterobacter asburiae strains may contain, for example, one or more genetic modifications selected from the group consisting of:
  • a gene encoding pyruvate decarboxylase is supplied.
  • a gene encoding pyruvate decarboxylase and a gene encoding alcohol dehydrogenase are supplied, and preferably the two genes are Z.
  • the inactivated genes are native gene(s) and/or are exogenous gene(s) previously introduced into the Enterobacter asburiae strain. Additional embodiments relate to Enterobacter asburiae strains genetically modified to facilitate production of succinate and/or malate. Genetic modifications suitable for this purpose are set forth in PCT/US2008/057439 (published as WO2008/1 15958A3) and U.S. Pat. App. 61/166,093, the disclosures of which are incorporated herein by reference in their entirety.
  • the genetically modified Enterohacter asburiae strains may contain, for example, one or more genetic modifications selected from the group consisting of:
  • the PEP carboxykinase gene may be native to
  • Enterobacter asburiae may be an exogenous gene.
  • the PEP carboxykinase gene is from Escherichia coli.
  • the inactivated genes are native gene(s) and/or are exogenous gene(s) previously introduced into the Enterobacter asburiae strain.
  • the genetically modified Enterobacter asburiae strains may contain, for example, one or more further genetic modifications selected from the group consisting of:
  • Examples of various combinations of the above referenced genetic modifications include, and are not limited to:: d only, e only, f only, g only, h only , i only, j only, k only, 1 only, m only, d.e, d.f, d.g, d.h, d.i, d.j, d.k, d.l, d.m, e.f, e.g, e.h, e.i, e.j, e.k, e.l, e.m, f.g, f.h, f.i, f.j, f.k, f.l, f.m, g.h, g.i, g.j, g.k, g.l, g.m, hi, h.j, h.k, h.l, h.m, i.j, i.k, i.l, i.m, j
  • g.j.l e. g.j.m, e. g.k.l, e.g.k.m, e.g.l.m, e.h.i.j, e.h.i.k, e.h.i.1, e.h.i.m, e.h.j.k, e.h.j.1, e.h.j.m, e.h.k.l, e.h.k.m, e.h.i.m, e.i.j.k, e.i.j .1, e.i.j.m, e.i.k.l, e.i.k.m, e.i.l.m, e.j.k.l, e.j.k.m, e.k.l.m, f.g.h.i, f.g.h.j, f.g.h.k, f.g.h.k, f.g.
  • a gene encoding formate transporter may also be inactivated.
  • the inactivated genes are native gene(s) and/or are
  • Additional embodiments relate to Enterobacter asburiae strains genetically modified to facilitate production of alanine. Genetic modifications suitable for this purpose are set forth in PCT/US2008/058410 (published as WO2008/119009A2), the disclosures of which are incorporated herein by reference in their entirety.
  • the genetically modified Enterobacter asburiae strains may contain, for example, one or more genetic modifications selected from the group consisting of:
  • Combinations of these modifications suitable to the invention include: a, b, c, d, e, f, g, h, a.b, a.c, a.d, a.e, a.f, a.g, a.h, b.c, b.d, b.e, b.f, b.g, b.h, c.d, c.e, c.f, eg, c.h, d.e, d.f, d.g, d.h, e.f, e.g, e.h, f.g, f.h, g.h, a.b.c, a.b.d, a.b.e, a.b.f, a.b.g, a.b.h, a.c.d, a.c.e, a.c.f, a.c.g, a.c.h, a.
  • incorporation and/or overexpression of a gene encoding alanine dehydrogenase is a present in the genetically modified Enterobacter asburiae strain intended for the production of alanine.
  • the gene encoding alanine dehydrogenase is from Geobacillus stearothermophilus or from another thermophilic microorganism.
  • the inactivated genes are native gene(s) and/or are exogenous gene(s) previously introduced into the Enterobacter asburiae strain.
  • Additional embodiments relate to Enterobacter asburiae strains geneticalfy modified to enhance their capacity to utilize lignocellulose. Genetic modifications suitable for this purpose are set forth in PCT/US2008/058410 (published as WO2008/119009A2); in Ingram et al., Appl Environ Microbiol 67(1): 6-14 (2001); and in Ingram et ah, Appl Environ Microbiol 63(12): 4633-4637 (1997); the disclosures of which are incorporated herein by reference in their entirety.
  • the genetically modified Enterobacter asburiae strains may contain, for example, one or more genetic modifications selected from the group consisting of: (a) incorporation and/or overexpression of a gene encoding cellobiose utilizing enzyme;
  • the gene encoding cellobiose utilizing enzyme and/or the gene encoding phospho- ⁇ -glucosidase are genes from Klebsiella, and preferably are Klebsiella oxytoca casAB.
  • the gene encoding an endoglucanase or cellulase is a gene from Erwinia, and preferably is Erwinia chrysanthemi celY or Erwinia chrysanthemi celZ.
  • the genes are integrated such that transcription is via a promoter native to Enterobacter generally or to Enterobacter asburiae specifically.
  • Additional embodiments relate to Enterobacter asburiae strains genetically modified to facilitate production of acetate and/or pyruvate. Genetic modifications suitable for this purpose are set forth in U.S. Pat. App.10/703,812, the disclosure of which is incorporated herein by reference in its entirety.
  • the genetically modified Enterobacter asburiae strains may contain, for example, one or more genetic modifications selected from the group consisting of:
  • any strain containing any of these combinations of modifications may be further modified to inactivate a gene encoding formate transporter, for example focA.
  • the inactivation of the gene encoding (FiFo)H + -ATP synthase preserves the hydrolytic activity of Fl-ATPase in the cytoplasm while disrupting oxidative phosphorylation.
  • the gene encoding (Fi Fo)H -ATP synthase is atpF or atpH or both.
  • the gene encoding lactate dehydrogenase is ldhA.
  • the gene encoding pyruvate formate lyase is pflB.
  • the gene encoding fumarate reductase is one or more of the component genes of frdABCD, for example frdBC or frdCD.
  • the gene encoding alcohol/aldehyde dehydrogenase is adhE.
  • the gene encoding 2-ketoglutarate dehydrogenase is sucA.
  • Enterobact ⁇ r asburiae strains may contain, for example, one or more further genetic modifications selected from the group consisting of;
  • the gene encoding acetate kinase is ackA.
  • the gene encoding pyruvate oxidase is poxB.
  • Additional embodiments relate to Enterobacter asburiae strains genetically modified to facilitate production of propanediols. Genetic modifications suitable for this purpose are set forth in U.S. Pat. 7,098,009, the disclosure of which is incorporated herein by reference in its entirety.
  • the genetically modified Enterobacter asburiae strains may contain, for example, one or more genetic modifications selected from the group consisting of:
  • E. coli host cell W 1485 harboring plasmids pDT20 and pAH42 can be used as sources of nucleic acids that encode glycerol-3-phosphate dehydrogenase (G3PDH), glycerol-3-phosphatase (G3P phosphatase), glycerol dehydratase (dhaB), and 1 ,3-propanediol oxidoreductase (dhaT).
  • G3PDH glycerol-3-phosphate dehydrogenase
  • G3P phosphatase glycerol-3-phosphatase
  • dhaB glycerol dehydratase
  • dhaT 1 ,3-propanediol oxidoreductase
  • cerevisiae YPH500 (deposited as ATCC 74392 under the terms of the Budapest Treaty) harboring plasmids pMCKlO, pMCK17, pMCK30 and pMCK35 containing genes encoding glycerol-3-phosphate dehydrogenase (G3PDH), glycerol-3- phosphatase (G3P phosphatase), glycerol dehydratase (dhaB), and 1,3 -propanediol oxidoreductase (dhaT) can be used as a source of nucleic acid(s) that encode the enzymes.
  • G3PDH glycerol-3-phosphate dehydrogenase
  • G3P phosphatase glycerol-3- phosphatase
  • dhaB glycerol dehydratase
  • dhaT 1,3 -propanediol oxidoreductase
  • E. coli DH5a containing pKPl which has about 35kb insert of a Klebsiella genome which contains glycerol dehydratase, protein X and proteins 1, 2 and 3 (deposited with the ATCC under the terms of the Budapest Treaty and designated ATCC 69789); E. coli DH5a cells containing pKP4 comprising a portion of the Klebsiella genome encoding diol dehydratase enzyme, including protein X was deposited with the ATCC under the terms of the Budapest Treaty and was designated ATCC 69790.
  • Preferred enzymes for the production of 1 ,2-propanediol are aldose reductase, glycerol dehydrogenase, or both.
  • the gene encoding aldose reductase is the gene for rat lens aldose reductase.
  • the gene encoding glycerol dehydrogenase is the E. coli gene that encodes glycerol dehydrogenase.
  • Aldose reductase sequences are highly conserved, thus the source of the aldose reductase gene is not critical to the present invention. The source of the glycerol dehydrogenase gene also is not critical.
  • L A process for fermenting MeGAX comprising:
  • biomass materials comprise sweetgum.
  • a process for fermenting MeGAX comprising:
  • a process for fermenting a substrate comprising:
  • the isolated E. asburiae strain of embodiments 15-16 wherein said strain comprises one or more genetic modifications selected from the group consisting of: incorporation and/or over expression of a gene encoding CRP*; incorporation and/or overexpression of a gene encoding xylose reductase; incorporation and/or overexpression of a gene encoding xylitol dehydrogenase; and inactivation of a gene encoding xylulokinase.
  • the isolated E. comprises one or more genetic modifications selected from the group consisting of: insertion and/or overexpression of a gene encoding pyruvate decarboxylase; insertion and/or overexpression of a gene encoding alcohol dehydrogenase; inactivation of a gene encoding lactate dehydrogenase; in
  • asburiae strain of embodiments 15-19 wherein said strain comprises one or more genetic modifications selected from the group consisting of: overexpression of a gene encoding PEP carboxykinase; inactivation of a gene encoding pyruvate formate lyase; and inactivation of a PEP-dependent phosphotransferase system gene.
  • biomass comprises sweetgum, wood preprocessed for cellulose production, rice straw, wood prunings, wood, wood waste, newspaper, paper products, plant materials and/or tree cuttings, miscanthus, switchgrass, elephant grass, energy cane, hemp, corn, Eucalyptus spp., poplar, yellow poplar, cottonwood, willow, sorghum, sugarcane, sugarcane bagasse, corn stalks, corn stover, wheat straw and combinations thereof.
  • Sweetgum methylglucuronoxylan (MeGAX n ) was prepared from sweetgum stem wood ⁇ Liquidambar styraciflua) as previously described and characterized by 13 C-NMR
  • Dilute acid hydrolysates of methylglucuronoxylan were prepared by hydrolysis with 0.1 N H 2 SO 4 (4 g methylglucuronoxylan in 400 ml 0.1 N H 2 SO 4 ) at 121 0 C for 60 min, followed by neutralization with BaCO 3 .
  • Anion exchange resin Bio-Rad AG2- X8 in the acetate form was used to adsorb the charged aldouronates; the uncharged xylose and xylooligosaccharides, mainly small amounts of xylobiose, were eluted with water. The aldouronates were then eluted with 20 % (v/v) acetic acid.
  • aldouronates were separated on a 2.5 cm x 160 cm BioGel P-2 column (BioRad, Hercules, CA) with 50 mM formic acid as the eluent.
  • the formic acid was removed from the purified sugar sample fractions by lyophilization.
  • MeGAX and MeGAX 2 were identified by thin layer chromatography (TLC) analysis using MeGAX and MeGAX 2 standards structurally defined by 13 C and 1 H-NMR spectrometry (Zuobi-Hasona et al. ASM National Meeting (2001)). Xylobiose and xylotriose were obtained and purified from MeGAX n digested with Paenihacillus sp.
  • strain JDR-2 XynAi catalytic domain CD
  • a recombinant GHlO endoxylanase XynAi CD overexpressed in E. coli St. John et al. Appl Environ Microbiol 72: 1496-1506 (2006).
  • the substrate containing 30 mg/ml MeGAX n was prepared with 10 mM sodium phosphate buffer, pH 6.5. Digestions were initiated by the addition of 3.5 U of XynAi CD into 50 ml substrate and incubated with rocking at 30 0 C for 24 h. An additional 1 U was added after 24 h and incubation was continued for 40 h.
  • Minimal medium containing the substrates described above was prepared upon mixing sterile substrate solutions (2x concentration) with the same volume of a 2x solution of Zucker and Hankin mineral salts (ZH salts) at pH 7.4 (Zucker & Hankin J Bacteriol 104:13- 18 (1970)). Neutralized MeGAX n acid hydrolysate (0.5% w/v) was also added to ZH salts directly as a growth substrate. Where indicated, some media preparations were supplemented with 0.1% yeast extract (YE medium).
  • E. asburiae JDR-I was isolated from discs of sweetgum stem wood ⁇ Liquidambar styraciflua) buried, soon after cutting, about one inch below the soil surface in a sweetgum stand for approximately three weeks.
  • Discs were suspended in 50 ml sterile deionized water and sonicated in a 125 Watt Branson Ultrasonic Cleaner water bath for 10 min. The sonicate was inoculated into 0.2% (w/v) MeGAX YE medium and incubated at 37 0 C. Cultures were streaked on MeGAX minimal medium agar plates. Isolated colonies were passed several times between MeGAX broths and agars until pure. Exponential phase cultures growing on 0.2 % MeGAX minimal media were cryostored in 25% sterile glycerol at -7O 0 C.
  • the purified isolate was submitted to MIDI Labs (world wide web site: midilabs.com) for partial 16s rRNA sequencing and FAME analysis.
  • BBL EnterotubeTM II Becton, Dickinson and Company, USA
  • inoculation was also used to identify the isolate based upon metabolic capability using the standard protocol.
  • Differential Interference Contrast (DIC) micrographs of E. asburiae JDR-I growing in MeGAX minimal medium at exponential phase were obtained with a Zeiss DIC microscope at 40xl5-fold magnification. Negative stain electron micrographs were obtained with a Zeiss EMlOA electron microscope.
  • Samples were delivered with a 710B WISP automated injector and chromatography controlled with a Waters 610 solvent delivery system at flow rate of 0.5 ml/min. Products were detected by differential refractometry with a Waters 2410 RI detector. Data analysis was performed with Waters Millennium Software. To determine and quantify methanol, unfiltered supernatants from fermentation cultures were also analyzed by gas chromatography (6890N Network GC system, Agilent Technologies), using isopropanol as an internal standard. This detection method was used since diffusion during HPLC precluded quantitative detection of methanol by differential refractometry.
  • NMR spectra were obtained using a VXR300 NMR spectrometer (NMR facility of the Department of Chemistry, University of Florida) operating in the Fourier transform mode as follows: 75.46 MHz; excitation pulse width, 7.0 s; spectral width, 16502; 256 acquisitions.
  • Acetone (30 ⁇ l) containing 13 C at natural abundance in 700 ⁇ l sample was used as an internal reference of 31.07 ppm for the 13 C methyl carbon (Kardosova et al. Carbohydr Res 308:99-105 (1998)).
  • Individual carbon atoms for fermentation products were identified by shift assignments and quantified by comparison with standards ( 13 C at natural abundance) of known concentrations.
  • anaerobic growth was performed in 50 ml minimal medium containing either 0.26% glucose, 0.36% xylose, 0.35% glucuronate and 0.2% MeGAX as sole carbon sources with the fermentation conditions described above. After 24 hours of growth and complete utilization of the carbon source, cells were harvested by centrifugation and the resulting pellets were washed twice with deionized water. The pellets were dried to constant weight in a Sargent vacuum dryer at 60 0 C for up to 36 hours. The culture supernants were analyzed by HPLC to determine substrate consumption. The molar cell dry weight yield was calculated as cell dry weight (gram) divided by consumed substrate (mole).
  • a biocode of 32061 obtained from the Enterotube II (BBL) test, also corresponded to Enterobacter asburiae species. Based upon these three criteria, the isolate was identified within Enterobacter asburiae species and designated as Enterobacter asburiae strain JDR-I. The strain has been deposited with the Agriculture Research Service Patent Culture Collection of the USDA, Peoria, IL., under NRRL number NRRL B-S0074.
  • E. asburiae JDR-I appeared as short motile rods.
  • Negative stain electron microscopy revealed 3 ⁇ m x 1 ⁇ m cells with peritrichous flagella. These morphological characteristics were similar to those of other isolates of Enterobacter asburiae (Hoffman et al. Syst Appl Microbiol 28:196-205 (2005)).
  • colonies of E. asburiae JDR-I were morphologically indistinguishable from E. coli colonies.
  • E. asburiae JDR-I Utilization of acid hydrolysates of methylgluronoxylan by E. asburiae JDR-I
  • the unique ability of E. asburiae JDR-I to grow on the aldobiuronate MeGAX as the sole carbon source suggested a potential for the complete metabolism of the carbohydrates generated by the dilute acid pretreatment currently applied for the release and fermentation of xylose in hemicellulose fractions.
  • E. asburiae JDR-I was grown aerobically in minimal medium comprised of neutralized MeGAX n acid hydrolysate and Zucker and Hankin mineral salts. Based upon HPLC analysis of media samples taken at different stages of growth, E.
  • E. asburiae JDR-I utilized 17.5% more substrate (mass amount) than E. coli B, which was unable to utilize MeGAX (Fig. 2B).
  • both E. asburiae JDR-I and E. coli B formed acetic acid during exponential growth phase that was metabolized upon complete utilization of the carbon sources in the MeGAX n hydrolysates.
  • E. asburiae JDR-I was also able to grow in xylobiose and xylotriose minimal medium, which E. coli B could not utilize.
  • E. asburiae JDR-I was unable to utilize MeGAX 2 and MeGAX 3 (data not shown).
  • E. asburiae JDR-I was found to grow aerobically in minimal media containing different sole carbon sources, such as glucose, xylose, mannitol, maltose, rhamnose, mannose, glucuronate and glycerol. As noted above, it was able to quantitatively metabolize MeGAX, but was unable to utilize MeGAX 2 generated by acid hydrolysis, or MeGAX 3 generated by a GHlO endoxylanase.
  • E. asburiae JDR-I displayed a diauxic growth pattern typical of species of Enterobacteraceae (Fig. 3A). Glucose (8 niM) was consumed within approximately 8 hours, while xylose utilization began when glucose was almost entirely consumed and was depleted in 14 hours.
  • JDR-I as a biocatalyst for the production of biobased products, and define the processes involved in the metabolism of MeGAX.
  • E. asburiae JDR-I was able to ferment all major sugars constituting hemicellulose, including D-glucose, D-xylose, D-mannose, L- arabinose and D-galactose.
  • the major products from xylose and galactose fermentation were acetic acid and ethanol present in similar molar quantities. Acetic acid, ethanol and small amounts of lactic acid were produced from glucose, mannose and arabinose (Table 1).
  • Sweetgum methylglucuronoxylan (MeGAX n ) was prepared from sweetgum stem wood (Liquidambar styraciflu ⁇ ) as previously described and characterized by C 13 -NMR (Hurlbert and Preston 2001; Kardosova et al. 1998).
  • Dilute acid hydrolysates of methyglucuronoxylan were prepared by acid hydrolysis of 1% sweetgum xylan with 0.05 M HoSO 4 at 121 0 C for 60 min, followed by neutralization with BaCO 3 .
  • Total carbohydrate concentrations of substrate preparations were determined by the phenol-sulfuric acid assay (Dubois et al. 1956) with xylose as reference or by HPLC as previously described (Bi et al.
  • Fermentation media were supplemented with Zucker and Hankin mineral salts (ZH salts) at pH 7.4 (Zucker and Hankin 1970) or LB broth.
  • the media were buffered with 100 mM sodium phosphate buffer (pH 7.0) or 100 mM 3-(N-morpholino) propane sulfonic acid (MOPS) buffer (pH 7.0) when necessary.
  • Batch fermentations were carried out in medium saturated with nitrogen in tubes set in a Glas-Col minirotator at 60 rpm in a 30 0 C incubator. Fermentations were inoculated to an initial optical density at 600 nm of 0.8. Fermentation products were resolved on a Bio-Rad HPX-87H column with a Waters HPLC system or an Agilent HPLC system.
  • E. asburiae JDR-I pflB gene gene bank accession number: EU719655
  • a segment of the E. asburiae JDR-I als gene was amplified using degenerate primers designed from conserved sequences in homologous als genes found in Enterobacter sp. 638, Erwinia carotovora subsp. alroseptica SCRIl 043, Yersinia enterocolitica subsp. enterocolitica 8081 and Serratia proteamaculans 568.
  • Reactions were initiated by adding 4 Kunitz units (1 ⁇ mol/min) of either L-lactate dehydrogenase (rabbit muscle, 140 U/mg protein) or D-lactate dehydrogenase ⁇ Lactobacillus leichmanii, 232 U/mg protein) in 100 ⁇ l colorimetric reagent and 100 ⁇ l sample at room temperature. The reduction of iodonitrotetrazolium dye was measured at room temperature at 503 nm. Sodium salts of L and D-lactate (Sigma) were used as standards to define enantiomer specificity of the reaction.
  • the initial concentrations of substrates in the medium containing 0.5% sweetgum hemicellulose hydrolysate were determined by HPLC to be 20 mM xylose, 1.4 mM MeGAX and a small amount Of MeGAX 2 .
  • the major competing pathway to lactate production initiates from the pyruvate formate lyase catalyzed reaction, which produces formate and acetyl-CoA in the wild type strain E. asburiae JDR-I. Both acetate and ethanol are produced from acetyl-CoA.
  • the pj W gene of JDR-I was deleted to obtain strain E. asburiae El . Since 2,3-butanediol was also produced by E. asburiae El in the fermentation of glucose (Table 6), the als gene which encodes acetolactate synthase was deleted in E. asburiae El to eliminate 2,3-butanediol production (Moat et al. 2002).
  • the resulting strain E. asburiae Ll was a double mutant lacking pflB and als genes (Fig. 6).
  • E. asburiae El and Ll produced lactate as the predominant product in glucose, xylose and arabinose fermentations.
  • E. asburiae El produced 2.9 mM 2,3-butanediol in 0.8% glucose fermentation.
  • the Ll strain with an interrupted 2,3-butanediol-producing pathway produced no 2,3-butanediol and achieved a higher lactate yield (94.1% of the theoretical maximum).
  • the Ll strain also achieved higher lactate yield than El strain (Table 6).
  • the E. asburiae Ll fermented slowly in the xylan hydrolysate with ZH minimal salts.
  • MeGAX The utilization of MeGAX by the Ll strain was markedly enhanced with LB supplementation, while the original isolate, E. asburiae JDR-I, readily utilized MeGAX in both minimal (Fig. 5A) and LB supplemented (Fig. 5C) media during the mixed acid fermentation that produced acetate and lactate in nearly equal amounts (Table 6). Supplementation with LB doubled the rate of utilization of xylose and nearly trebled the production rate of lactate in the Ll strain (Table 7).
  • D-Lactate was produced by E. asburiae Ll
  • optical enantiomer(s) of lactate produced by E. asburiae Ll from the fermentation of xylan hydrolysates was determined by measuring the oxidation of lactate catalyzed by D- or L-lactate dehydrogenase with the reduction of iodonitrotetrazolium dye mediated via NADH formation as described in the Materials and Methods section.
  • a sample of medium containing 3.6 ⁇ mol lactate (determined by HPLC) of an E. asburiae Ll fermentation (72 h) of 0.5% xylan hydrolysate supplemented with LB resulted in an increase inA503 from 0 to 0.113 in 5 min when assayed with 4 units of D-lactate dehydrogenase.
  • Ampicillin 50 mg l ' 1
  • tetracycline (12.5 mg I " 1 )
  • kanamycin (20 mg F and 50 mg F
  • apramycin (20 mg F ]
  • chloramphenicol (10 mg F l and 40 mg F ') were added as needed.
  • Sweetgum methylglucuronoxylan (MeGAX n ) was prepared from sweetgum stem wood (Liquidambar styraciflua) as previously described and characterized by C 13 -NMR (Hurlbert and Preston J Bacteriol 183:2093-2100 (2001); Kardosova et al. Carbohydr Res 308:99-105 (1998)).
  • Dilute acid hydrolysates of methyglucuronoxylan were prepared by acid hydrolysis of 1% (w/v) sweetgum xylan with 0.1 N H 2 SO 4 at 121 0 C for 60 min. followed by neutralization with BaCO 3 .
  • Total carbohydrate concentrations of substrate preparations were determined by the phenol-sulfuric acid assay (Dubois et al. Anal Chern 28:350-356 (1956)) with xylose as a reference or by HPLC (Bi et al. Appl Envron Microbiol 75:395-404 (2009)).
  • Minimal media were supplemented with Zucker and Hankin mineral salts (ZH salts) at pH 7.4 (Zucker and Hankin J Bacteriol 104:13-18 (1970)). Growth media were buffered with 100 DiM sodium phosphate buffer (pH 7.0) or 100 niM 3-(N-morpholino) propane sulfonic acid (MOPS) buffer (pH 7.0) when necessary.
  • the cell dry weight was determined based on the OD 600 of the fermentation culture, which was 1.0 (0.5 Ig I "1 ) initially and did not appreciably change during the fermentation in 0.5% xylan hydrolysate.
  • E. asburiae JDR-I was grown with one of several antibiotics at different concentrations in LB and minimal media on agar plates or in liquid media to test its antibiotic resistance. Based upon its sensitivity to chloramphenicol and tetracycline respectively, plasmids pLOI555 ( cm ) and pLOI297(tet ), both containing the PET operon, were transformed into E. asburiae JDR-I or E. asburiae El by electroporation in a 100 ⁇ l cuvette under the condition of 1.8kV, 25 ⁇ F capacitance and 200 ⁇ resistance.
  • E.c ⁇ li The method for gene deletion in E.c ⁇ li was used as previously described (Jantama et al. Biotechnol Bioeng 99:1140-53 (2008); Zhang et al. Appl Microbiol Biolechnol 77:355-366 (2007)), with minor modifications applied to E. asburiae JDR-I.
  • the pflB gene in E. asburiae JDR-I was also selected as an integration site for the PET operon.
  • Several sets of primers were designed based on sequences of pflB orthologs in other Enterobacter spp. to amplify this gene fragment from E. asburiae JDR-I. Only one set derived from E.coli B was found to amplify the E.
  • E. asburiae JDR-I pflB gene fragment The amplified E. asburiae JDR-I DNA sequence and E.coli K12 pflB sequence were found to have 93% identity.
  • the plasmids constructed are listed in Table 8.
  • the partial sequence of the E. asburiae JDR-I pflB gene (gene bank accession number: EU719655) was determined within a DNA fragment amplified by PCR using specific primers based on the E. colipflB sequence.
  • the 3 kb cat-sacB cassette was obtained by digesting pLOI4162 with Smal and Sfol, and used in subsequent ligations.
  • asburiae JDR-I was cloned into pCR 4-TOPO vector (Invitrogen) to obtain a plasmid, pTOPOpfl.
  • This plasmid was diluted 500-fold and served as template for inside-out PCR amplification using the pfl inside-out primers.
  • the resulting 5.5 kb fragment containing the replicon was ligated to the blunt-end cat-sacB cassette from pLOI4162 to produce a new plasmid, pTOPO4162pfl.
  • This 5.5 kb fragment was also used to construct a second plasmid, pTOPODpfl, by phosphorylation and self- ligation.
  • Both pTOPO4162pfl and pTOPODpfl were then digested with Xmnl, diluted 500- fold and used as templates for amplification using the pfl primer set to produce linear DNA fragments for integration step 1 (pfl'-cat—sacB—pfl”) and step 2 (pfl -pfl”), respectively.
  • step 1 fragment E. asburiae JDR-I containing pLO13240
  • cells were incubated for 2 hr at 30 0 C.
  • the recombinant candidates were selected for chloramphenicol (20 mg 1 " x ) resistance in Luria broth plates after overnight incubation (15 h) at 39°C.
  • Colonies were patched on both kanamycin (50 mg I " 1 ) plates and chloramphenicol (40 mg r ') plates. Those colonies growing on chloramphenicol (40 mg F l ) plates but not on kanamycin (50 mg F 1 ) plates were subjected for PCR confirmation. The confirmed mutant colonies were transformed with pLOI3240, and prepared for electroporation with the step 2 fragment (pfl'-pfl"). After electroporation, cells were incubated at 30 0 C for 4 h and then transferred into a 250-ml flask containing 100 ml of LB minus NaCl with 10% sucrose.
  • One generation was defined as a 2-fold increase in culture turbidity.
  • Appropriate dilutions of cultures were plated on Luria agar with and without antibiotic; colonies formed were counted and calculated to obtain the ratio of cells retaining antibiotic resistance to total cells.
  • the supernatant was collected after 15 min centrifugation at 1.8k rpm (Eppendorf centrifuge 5414). The entire process was carried out at 4 0 C. Heat treatment for 15 min at 60 0 C was used to inactivate competing native enzymes of E. asburiae JDR-I which might affect quantitative measurements of PDC activities in transformants.
  • the enzyme activity assay of PDC was performed in the reaction mixture of 1.0 mM TPP (thiamine pyrophosphate), 1.0 mM MgCl 2 , 0.40 mM NADH, 20 mM sodium pyruvate and
  • the assay was started by adding 20 ⁇ l crude cell extract. Protein concentration of the crude extract was determined with BCA protein assay reagent kit (Pierce Chemical Co., Rockford, IL).
  • E. asburiae JDR-I performed a mixed-acid fermentation in low substrate concentration.
  • the wild type strain produced a wide range of products, including succinate, lactate, acetate, formate, 2,3- butanediol and ethanol (Table 9).
  • succinate and acetate were produced at low concentrations, approximately 1 mM.
  • Lactate was produced at approximately 10 mM, and the major products were formate, 2,3-butanediol and ethanol, each at approximately 40 mM. More acetate and less 2,3-butanediol were produced in xylose fermentation (Table 9).
  • Plasmids pLOI297 and pLOI555 were transformed into E. asburiae JDR-I for overexpression of pdc and adh genes. Both transformed strains were able to completely utilize 2.5% (w/v) glucose or 2% (w/v) xylose within 48 hours, with ethanol as the predominant fermentation product. The ethanol yields of glucose fermentation were 94.1% and 95.3% for E. asburiae JDR-I (pLOI297) and E. asburiae JDR-I (pLOI555), respectively (Table 9). E. asburiae JDR-I (pLOI555) was further tested in xylose fermentation, and the ethanol yield was even higher, greater than 98% of theoretical. There were also other fermentation products present at concentrations below 10 mM (Table 9).
  • E. asburiae El Fermentation characteristics of E. asburiae El (pLOI555) compared with E. coli KOIl and other E. ashuriae JDR-I derivatives Neither 2,3-butanediol nor lactic acid was produced in the hydrolysate fermentation by either E. asburiae JDR-I (pLO1297) or JDR-I (pLOI555).
  • E. asburiae JDR-I pLO1297)
  • JDR-I555 JDR-I555
  • the E. coli KOI l which was reported to be able to produce 0.54 gram ethanol per gram glucose (Ohta et al. Appl Environ Microbiol 57:893-900 (1991)), could only produce ethanol at 63% of the theoretical maximum in the sweetgum xylan hydrolysate medium, and accumulated a substantial amount (10.6 ⁇ 0.3 mM) of acetate (Fig. 7, Fig. 8C).
  • the sum of ethanol and acetate was 33.1 mM for E. coli KOl 1, and 40.2 mM for JDR-I (pLOI555), 39.9 mM for JDR-I (pLOI297) and 40.5 niM for El (pLO1555) (Table 10).
  • E. coli KOl 1 utilized less substrate in the hydrolysate than the 3 engineered E. asburiae strains and produced lower quantities of products as a result of the inability of £ coli KOl lto utilize MeGAX in the hydrolysate (Fig. 7, Fig. 8B).
  • the ethanol specific production rate of E. coli KOI l (0.074 ⁇ 0.006 g ethanol/g DCW/h) was much lower than E. asburiae El
  • the PDC enzyme activity produced as a result of expression of heterologous gene pdc in engineered E. asburiae strains (Table 12). Because of the relative thermal stability of PDC encoded by the pdc gene of Zymomonas mobilis, a heat treatment at 65 0 C for 15 minutes was used to inactivate competing native enzymes, e.g. activities associated with the pyruvate dehydrogenase complex, could affect measurements of PDC activity (Conway et al. J Bacterial 169:2591-2597 (1987); Ohta et al. Appl Environ Microbiol 57:2810-2815 (1991)).
  • the pLOI297 transformant was relatively unstable, with only 10.7% of transformed E. asburiae JDR-I cells retaining tetracycline resistance after cultivation for 72 generations without antibiotic selection pressure.
  • the pLOI555 transformant was quite stable, with 98.1% of pLOI555 transformed E. asburiae JDR-I cells retaining chloramphenicol resistance after growth for 72 generations in the absence of antibiotic (Table 13). Fermentation analysis of 10 descendent colonies retaining antibiotic resistance from strains carrying pLOI297 and pLO1555 was also performed to confirm that strains with retained antibiotic resistance also retained the homoethanolgenic phenotype. Discussion
  • E. asburiae JDR-I pCR4- TOPO plasmid with a small insertion was electroporated into the competent cells and the transformants were able to be selected on a kanamycin (50 mg F ) plate.
  • the transformed pCR4-TOPO plasmid in E. asburiae JDR-I was qualitatively determined by DNA gel electrophoresis to have a lower concentration than in E. coli ToplO host (data not shown). With these transformation systems, E. asburiae JDR-I (pLOI297) and E. asburiae
  • JDR-I (pLOI555), were able to produce ethanol at 94.1% and 95.3% of theoretical yield in glucose, but failed to achieve such high yield in the dilute acid hydrolysates of methylglucuronxylan.
  • the pflB gene was then deleted.
  • the convenient one-step gene inactivation method successfully applied to E. coli failed to knock out the pflB gene in E. asburiae JDR-I, requiring the development of a different protocol.
  • An alternative gene deletion method used PCR fragments with several hundred bases of homologous sequence at both ends instead of 40 bp used by the one-step method (Jantama et al. Biotechnol Bioeng 99:1140-53 (2008)). Recombinants were not selected on the plates containing levels of antibiotics used for selection of E.
  • kanamycin (20 mg F ) and chloramphenicol (10 mg F 1 ) to be used. This is likely the basis for growth of non-recombinant as well as recombinant colonies and required a second selection that was achieved by patching colonies onto kanamycin (50 mg F 1 ) and chloramphenicol (40 mg F ') plates.
  • kanamycin 50 mg F 1
  • chloramphenicol 40 mg F '
  • E. asburiae JDR-I recombinants 5 ⁇ g/ ⁇ l and cell concentrations of 10 10 cells/1 OO ⁇ l in electroporation transformation, usually 3 to 6 E. asburiae JDR-I recombinants could be obtained by this process.
  • the E. asburiae strain with a genomic pflB deletion was transformed with a plasmid, pLOI555, to obtain E. asburiae El (pLOI555), a strain capable of efficiently converting the xylose residues derived from methyglucuronoxylan to ethanol, achieving a yield at 99% of the theoretical maximum.
  • pLOI555 E. asburiae El
  • Plasmid stability is critical for biocatalysts engineered with genes conferring a desired metabolic potential confined within a plasmid, as consistent traits are required for long-term applications.
  • the plasmid pLOI297, containing colEl replicon was present in high copy numbers in E. coli strains, but was unstable in Klebsiella oxytoca M5A1.
  • pLOI555 derived from cryptic low-copy-number plasmids in E. coli B (ATCC 11303), however, was very stable in Klebsiella oxytoca M5A1 (Ohta et al. Appl Environ Microbiol 57:2810-2815 (1991)).
  • pLOI555 plasmids were found to be more stable than pLOI297 in E. asburiae JDR-I.
  • the relative stability of the plasmid in E, asburiae El recommend it for further development, perhaps through introduction of the pdc and adh genes into the chromosome as has been achieved for the successful development of E. coli KOl 1 and its derivatives as ethanologenic biocatalysts (Jarboc et al. Adv Biochem Eng Biotechnol ⁇ 08:237 -61 (2007)).
  • Y]vrsubstrate molar cell dry weight yields for different substrates, determined in triplicate with indicated standard deviations. Table 5. Bacterial strains and plasmids.
  • E. asburiae El 0.32 ⁇ 0.28 0.077 ⁇ 0.13 0.022+0.003 O.l l ⁇ O.Ol a)
  • q xylose is defined as consumed g xylose /g DCW(dry cell weight) /h:
  • q MeGAX is defined as consumed g MeGAX /g DCW(dry cell weight) /h;
  • q acetate is defined as produced g acetate /g DCWfdry cell weight) /h;
  • q ethanol is defined as produced g ethanol /g DCW(dry cell weight)/h.
  • Table 12 Specific activity of PDC in cell crude extract from E. asburiae JDR-I derived strains. Results were averages of 3 experiments.
  • E. asburiae El 0.53 ⁇ 0.10 a
  • One U is defined as that amount of the enzyme that catalyzes the conversion of 1 ⁇ mole of substrate per minute at room temperature.
  • Enterobacter as Enterobacter dissohens comb-nov and Enterobacter nimipressuralis comb-nov. J. Clin. Microbiol. 23:1114-1 120.
  • Enterobacter cloacae as E cloacae subspecies dissolvens comb, nov and emended description of Enterobacter asburiae and Enterobacter kobei. Syst. Appl. Microbiol.
  • An aldouronic acid-utilization operon in Paenibacillus sp. JDR encodes an alpha-glucuronidase with activity on aldouronic acids generated by acid and enzyme-mediated digestion of methyglucuronoxylan. ASM National Meeting, Atlanta, GA, 2005.
  • D-lactate dehydrogenase is a member of the D-isomer-specif ⁇ c 2- hydroxyacid dehydrogenase family — cloning, sequencing, and expression in Escherichia coli of the D-Lactate dehydrogenase gene of Lactobacillus plantarum. J Biol Chem 266:12588-12594.

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  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des procédés et des biocatalyseurs pour produire de l’éthanol et d’autres produits utiles à partir de biomasse et/ou d’autres matériaux. Le traitement initial de biomasse lignocellulosique produit fréquemment du méthylglucuronoxylose (MeGAX) et des produits apparentés qui sont résistants au traitement plus avant par des biocatalyseurs communs. Il est démontré que des souches d’Enterobacter asburiae sont utiles dans le biotraitement de MeGAX et d’autres matériaux en bioproduits utiles tels que l’éthanol, l’acétate, le lactate, et de nombreux autres. La modification génétique peut être utilisée pour augmenter la production de bioproduits souhaités.
PCT/US2009/064773 2008-11-18 2009-11-17 Biocatalyseurs et procédés pour la conversion d'hydrolysats d'hémicellulose en produits à base biologique WO2010059616A2 (fr)

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