WO2010059616A2 - Biocatalysts and methods for conversion of hemicellulose hydrolsates to biobased products - Google Patents

Biocatalysts and methods for conversion of hemicellulose hydrolsates to biobased products 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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WO2010059616A3 (en
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James F. Preston
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University Of Florida Research Foundation, Inc.
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Publication of WO2010059616A3 publication Critical patent/WO2010059616A3/en

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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.

Abstract

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. 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.

Description

DESCRIPTION
BIOCATALYSTS AND METHODS FOR CONVERSION OF HEMICELLULOSE
HYDROLYSATES TO BIOBASED PRODUCTS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application Serial No. 61/1 15,722, filed November 18, 2008, and Serial No. 61/229,536, filed July 29, 2009, the disclosures of which are hereby incorporated by reference in their entirety, including all figures, tables and amino acid or nucleic acid sequences.
GOVERNMENT SUPPORT
This invention was made with government support under grants awarded as follows: Department of Energy grant numbers USDOE G012026-161, DE FC36-99GO10476, and DE FC36-00GO10594. The government has certain rights in this invention.
BACKGROUND OF THE INVENTION
The need for alternatives to petroleum resources for production of fuels and chemicals has become a major quest, generated from economic incentives associated with limited and diminishing supply (Kheshgi, H. S., R. C. Prince, and G. Marland. 2000. The potential of biomass fuels in the context of global climate change: focus on transportation fuels. Annu. Rev. Energy Environ. 25:199-244). The connection between increasing carbon dioxide and global warming has directed this quest toward formation of fermentation products derived from resources renewable through photosynthesis (McMillan J.D. (1997) Bioethanol production: status and prospects. Renew Energy 10:295-302). The development of 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). To expand production of ethanol and chemical feedstocks from renewable resources that do not economically impact these commodities, 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. Jechura, K. Neeves, J. Sheehan, B. Wallace, L. Montague, A. Slayton, and J. Lukas. 2002. Lignocellulosic biomass to ethanol process design and economics utilizing co-current dilute acid prehydrolysis and enzymatic hydrolysis for corn stover. NREL/TP-510-32438. National Renewable Energy Laboratory, Golden, Colo. Available online at nrel.gov/docs/fy02osti/32438.pdf. Cellulose comprises the major part of all plant biomass. The source of all cellulose is the structural tissue of plants. It occurs in close association with hemicellulose and lignin, which together comprise the major components of plant fiber cells. 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. In general, 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. In addition, temperatures ranging from 90°-600° C, and pressure up to 800 psi are necessary to effect the hydrolysis. At high temperatures, 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. In addition to these difficulties, it has been recognized that 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. Lee Al, Prashant Iyer, R.W. Torget. 1999. Dilute- Acid Hydrolysis of Lignocellulosic Biomass. Advances in Biochemical Engineering/Biotechnology Volume 65 pp. 93-115). The predominant structural polymer in the hemicellulose fraction of hardwoods and crop residues is methylglucuronoxylan (MeGAXn), a β-1,4 linked xylan in which xylose residues are periodically substituted with a-l,2-linked 4-O-methyl-glucuronic acid (Preston, J. F., J. C. Hurlbert, J. D. Rice, A. Ragunathan, and F. J. St. John. 2003. 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 . 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.). As a result of the sometimes large yield of MeGAX following dilute acid processes, the sugar yield is low and fermentation is hampered in producing useful biofuels and chemical feedstocks from renewable photosynthetic resources. Thus, there is an urgent need for an economically viable, environmentally safe microorganism that can ferment MeGAX resulting from dilute acid hydrolysis of photosynthetic resources to produce useful biofuels (such as ethanol) and chemical feedstocks (such as acetate).
BRIEF SUMMARY OF THE INVENTION
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. 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.
BRIEF DESCRIPTION OF THE FIGURES 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 MeGAX11 by A) E. asburiae JDR-I and B) E. cυli B. Xylose (diamonds), MeGAX (squares), and acetic acid (triangles) were determined in media by HPLC. Growth was determined by measuring turbidity as OD6OO (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 (OD6O0)- A) Growth on glucose (7.5 mM) and xylose (7.5 mM). Concentrations of glucose (closed circles), xylose (diamonds) and acetic acid (triangles); OD6oo (open circles); B) Growth on glucuronic acid (10 mM) and xylose
(10.5 mM). Concentrations of glucuronic acid (open squares) and xylose (diamonds); OD6oo
(open circles) C) Growth on MeGAX (6.5 mM). Concentrations of MeGAX (squares): OD6Oo (open circles).
FIGURES 4A-4D: Pathway determination for the metabolism of xylose and glucose by E. asburiae JDR-I. Media from anaerobic cultures of E. asburiae JDR-I and E. coli B grown with xylose or glucose enriched with 13C in specific carbons were analyzed by 75.5
MHz 13C-NMR spectrometry. A) [2-13C]-xylose fermented by E. asburiae JDR-I; B) [2-13C]- xylose fermented by E. coli B; C) [l-bC]-glucose fermented by E. asburiae JDR-I; D) [6-
C]-glucose fermented 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 feπnentation products: xylose (closed diamonds ♦), MeGAX (closed squares ■), acetic acid (open triangles Δ), ethanol (open squares α), 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. asburiae JDR-I (pLO1555); and Figure 8D depicts E. asburiae El (pLOI555). Substrates and fermentation products: xylose (closed diamonds ♦), MeGAX (closed squares ■), acetic acid (open triangles Δ), formic acid (open circles o), cthanol (open squares D).
DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel microorganisms that are capable of fermenting by-products of acid hydrolysis of renewable biomass materials. According to the invention, the fermentation of MeGAX sugars produced from acid hydrolysis of biomass materials involves the use of bacteria, namely Enterobacler asbiiriae. Because McϋΛX 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.
There exist commercially available industrial microorganisms (e.g., E. coli KOI l) that will ferment sugar by-products of acid hydrolysis, but not MeGAX, and this occasions the need for supplementation of the fermentation process with a microorganism having the ability to ferment MeGAX in order to avoid low ethanol production rates and low ethanol yields, or low production rates and yields of other desirable bioproducts.
According to the subject invention, 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. In one embodiment of the invention, 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. In a related embodiment, 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. fructose, glucose, mannose, galactose) or pentoses (e.g. arabinose and mannose) in combination with Enterobacter asburiae to ferment acid hydrolysis by-products of biomass materials. Other embodiments provide for the utilization of Enterobacter asburiae strains in combination with other bacterial strains for the simultaneous saccharification and fermentation of hexoses and pentoses to targeted biobased products. Although the 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. As would be appreciated by one of skill in the art, 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. In one embodiment, the substrate comprises a monsaccharide, disaccharide, trisaccharide, or oligosaccharide (wherein the oligosaccharide contains 4, 5, 6, 7, 8, or more simple sugars). In one embodiment, the substrate comprises a monosaccharide selected from xylose, glucose, mannose, galactose, arabinose, fructose, or rhamnose. In one embodiment, the substrate comprises glucuronic acid (or its conjugate base) and/or MeGAX. In one embodiment the substrate comprises an aldopentose, a ketopentose, an aldohexose, or a ketopentose. In one embodiment, 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. According to the subject invention, a means for fermenting aldobiuronate methyl glucuronoxylose (MeGAX) is provided. In one embodiment, MeGAX is feπnented 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.
In one embodiment, 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. By using 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:
(a) foπning a substrate from biomass materials containing hemicellulose;
(b) subjecting the substrate to acid hydrolysis;
(c) selecting and isolating a strain of Enterobacter asburiae that has the ability to ferment MeGAX to ethanol; (d) inoculating the substrate with the selected strain of Enterobacter asburiae to ferment
MeGAX under conditions favorable for cell viability and conversion of MEGAX to ethanol; and (e) optionally, recovering ethanol produced through the fermentation process. In one embodiment of the method of the present invention, 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.
Additional advantages of this invention will become readily apparent from the ensuing description.
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. Examples of 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. In certain embodiments, 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.
Conditions suitable for cell viability and conversion of hydrolysates to ethanol and other bioproducts are well known to the skilled artisan. For example, 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. In certain embodiments, 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 280C to about 35° C, 280C to about 33°C or be maintained around about 3O0C.
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:
(a) incorporation and/or overexpression of a gene encoding CRP*; (b) incorporation and/or overexpression of a gene encoding xylose reductase;
(c) incorporation and/or overexpression of a gene encoding xylitol dehydrogenase; and
(d) inactivation of a gene encoding xylulokinase.
Combinations of these modifications suitable to the invention include: (a), (b), (c), (d), (a)&(b), (a)&(c), (a)&(d), (b)&(c), (b)&(d), (c)&(d), (a)&(b)&(c). (a)&(b)&(d), (a)&(c)&(d), (b)&(c)&(d), and (a)&(b)&(c)&(d). The genes encoding CRP*, xylose reductase, and xylitol dehydrogenase are independently native to Enterobacter asburiae or are exogenous, but preferably are exogenous. 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:
(a) incorporation and/or overexpression and/or inactivation of a gene encoding L-lactate dehydrogenase;
(b) incorporation and/or overexpression and/or inactivation of a gene encoding D -lactate dehydogenase;
(c) inactivation of a gene encoding fumarate reductase (frd);
(d) inactivation of a gene encoding alcohol/ aldehyde dehydrogenase (adh);
(e) inactivation of a gene encoding pyruvate formate lyase (pfl); (f) inactivation of a gene encoding acetate kinase (ack); and
(g) inactivation of a gene encoding methylglyoxal synthase (mgs). Combinations of these modifications suitable to the invention include: (a), (b), (c),
(d), (e), (f), (g), (a)&(b), (a)&(c), (a)&(d), (a)&(e), (a)&(f), (a)&(g), (b)&(c), (b)&(d), (b)&(e), (b)&(f), (b)&(g), (c)&(d), (c)&(e), (c)&(f), (c)&(g), (d)&(e), (d)&(f), (d)&(g),
(e)&(f), (e)&(g), (f)&(g), (a)&(b)&(c), (a)&(b)&(d), (a)&(b)&(e), (a)&(b)&(f), (a)&(b)&(g), (a)&(c)&(d), (a)&(c)&(e), (a)&(c)&(f), (a)&(c)&(g), (a)&(d)&(e), (a)&(d)&(f), (a)&(d)&(g), (a)&(e)&(f), (a)&(e)&(g), (a)&(f)&(g), (b)&(c)&(d), (b)&(c)&(e), (b)&(c)&(f), (b)&(c)&(g), (b)&(d)&(e), (b)&(d)&(f), (b)&(d)&(g), (b)&(e)&(f), (b)&(e)&(g), (b)&(f)&(g), (c)&(d)&(e), (c)&(d)&(f), (c)&(d)&(g), (c)&(e)&(f), (c)&(e)&(g), (c)&(f)&(g), (d)&(e)&(f), (d)&(e)&(g), (d)&(f)&(g), (e)&(f)&(g), (a)&(b)&(c)&(d), (a)&(b)&(c)&(e), (a)&(b)&(c)&(f), (a)&(b)&(c)&(g), (a)&(b)&(d)&(e), (a)&(b)&(d)&(f), (a)&(b)&(d)&(g), (a)&(b)&(e)&(f), (a)&(b)&(e)&(g), (a)&(b)&(f)&(g), (a)&(c)&(d)&(e), (a)&(c)&(d)&(f), (a)&(c)&(d)&(g), (a)&(c)&(e)&(f), (a)&(c)&(e)&(g), (a)&(c)&(f)&(g), (a)&(d)&(e)&(f), (a)&(d)&(e)&(g), (a)&(d)&(f)&(g), (a)&(e)&(f)&(g), (b)&(c)&(d)&(e), (b)&(c)&(d)&(f), (b)&(c)&(d)&(g), (b)&(c)&(e)&(f), (b)&(c)&(e)&(g), (b)&(c)&(f)&(g), (b)&(d)&(e)&(f), (b)&(d)&(e)&(g), (b)&(d)&(f)&(g), (b)&(e)&(f)&(g), (c)&(d)&(e)&(f), (c)&(d)&(e)&(g), (c)&(d)&(f)&(g), (c)&(e)&(f)&(g), (d)&(e)&(f)&(g), (a)&(b)&(c)&(d)&(e),
(a)&(b)&(c)&(d)&(f), (a)&(b)&(c)&(d)&(g), (a)&(b)&(c)&(e)&(f), (a)&(b)&(c)&(e)&(g), (a)&(b)&(c)&(f)&(g), (a)&(b)&(d)&(e)&(f), (a)&(b)&(d)&(e)&(g), (a)&(b)&(d)&(f)&(g), (a)&(b)&(e)&(f)&(g), (a)&(c)&(d)&(e)&(f), (a)&(c)&(d)&(e)&(g), (a)&(c)&(d)&(f)&(g), (a)&(c)&(e)&(f)&(g), (a)&(d)&(e)&(f)&(g), (b)&(c)&(d)&(e)&(f), (b)&(c)&(d)&(e)&(g), (b)&(c)&(d)&(f)&(g), (b)&(c)&(e)&(f)&(g), (b)&(d)&(e)&(f)&(g), (c)&(d)&(e)&(f)&(g), (a)&(b)&(c)&(d)&(e)&(f), (a)&(b)&(c)&(d)&(e)&(g), (a)&(b)&(c)&(d)&(f)&(g), (a)&(b)&(c)&(e)&(f)&(g), (a)&(b)&(d)&(e)&(f)&(g), (a)&(c)&(d)&(e)&(f)&(g),
(b)&(c)&(d)&(e)&(f)&(g), and (a)&(b)&(c)&(d)&(e)&(f)&(g).
The genes L-lactate dehydrogenase and D-lactate dehydogenase are independently native to Enterobacter asbuήae 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) insertion and/or overexpression of a gene encoding pyruvate decarboxylase;
(b) insertion and/or overexpression of a gene encoding alcohol dehydrogenase; (c) inactivation of a gene encoding lactate dehydrogenase;
(d) inactivation of a gene encoding phosphoenolpyruvate carboxylase;
(e) inactivation of a gene encoding acetate kinase; and
(f) inactivation of a gene encoding pyruvate formate lyase. Combinations of these modifications suitable to the invention include: (a), (b), (c), (d), (e), (f), (a)&(b), (a)&(c), (a)&(d), (a)&(e), (a)&(f), (b)&(c), (b)&(d), (b)&(e), (b)&(f),
(c)&(d), (c)&(e), (c)&(f), (d)&(e), (d)&(f), (e)&(f), (a)&(b)&(c), (a)&(b)&(d), (a)&(b)&(e), (a)&(b)&(f), (a)&(c)&(d), (a)&(c)&(e), (a)&(c)&(f), (a)&(d)&(e), (a)&(d)&(f), (a)&(e)&(f), (b)&(c)&(d), (b)&(c)&(e), (b)&(c)&(f), (b)&(d)&(e), (b)&(d)&(f), (b)&(e)&(f), (c)&(d)&(e), (c)&(d)&(f), (c)&(e)&(f), (d)&(e)&(f), (a)&(b)&(c)&(d), (a)&(b)&(c)&(e), (a)&(b)&(c)&(f), (a)&(b)&(d)&(e), (a)&(b)&(d)&(f), (a)&(b)&(e)&(f), (a)&(c)&(d)&(e), (a)&(c)&(d)&(f), (a)&(c)&(e)&(f), (a)&(d)&(e)&(f), (b)&(c)&(d)&(e), (b)&(c)&(d)&(f), (b)&(c)&(e)&(f), (b)&(d)&(e)&(f), (c)&(d)&(e)&(f), (a)&(b)&(c)&(d)&(e),
(a)&(b)&(c)&(d)&(f), (a)&(b)&(c)&(e)&(f), (a)&(b)&(d)&(e)&(f), (a)&(c)&(d)&(c)&(f), (b)&(c)&(d)&(e)&(f), and (a)&(b)&(c)&(d)&(e)&(f). Preferably, a gene encoding pyruvate decarboxylase is supplied. In one embodiment, a gene encoding pyruvate decarboxylase and a gene encoding alcohol dehydrogenase are supplied, and preferably the two genes are Z. mobilis genes such as those contained within the PET operon. 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:
(a) overexpression of a gene encoding PEP carboxykinase;
(b) inactivation of a gene encoding pyruvate formate lyase; and (c) inactivation of a PEP-dependent phosphotransferase system gene.
Combinations of these modifications suitable to the invention include: (a), (b), (c),
(a)&(b), (a)&(c), (b)&(c), and (a)&(b)&(c). The PEP carboxykinase gene may be native to
Enterobacter asburiae or may be an exogenous gene. In one embodiment, 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.
For any strain modified to contain a combination of overexpression of a PEP carboxykinase gene, inactivation of a pyruvate formate lyase gene, and/or inactivation of a PEP-dependent phosphotransferase system gene, as set forth immediately above, additional genetic modifications are also suitable to the invention. The genetically modified Enterobacter asburiae strains may contain, for example, one or more further genetic modifications selected from the group consisting of:
(d) inactivation of a gene encoding acetate kinase;
(e) inactivation of a gene encoding alcohol dehydrogenase;
(f) inactivation of a gene encoding aspartate aminotransferase; (g) inactivation of a gene encoding citrate lyase;
(h) inactivation of a gene encoding lactate dehydrogenase; (i) inactivation of a gene encoding methylglyoxal synthase; (j) inactivation of a gene encoding pyruvate oxidase; (k) inactivation of a gene encoding phosphate acetyltransferase; (1) inactivation of a gene encoding malic enzyme; and
(m) inactivation of a gene encoding threonine dehydratase.
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.k, j.L j.m. k.l, k.m, I.m, d.e.f d.e.g, d.e.h, d.e.i, d.e.j, d.e.k, d.e.l, d.e.m, d.f.g, d.f.h, d.f.i, d.f.j, d.f.k, d.f.l, d.f.m, d.g.h, d.g.i, d.g.j, d.g.k, d.g.l, d.g.m, d.h.i, d.h.j, d.h.k, d.h.i, d.h.m, d.i.j, d.i.k, d.i.l, d.i.m, d.j.k, d.j.l, d.j.m, d.k.l, d.k.m, d.l.m, e.f.g, e.f.h, e.f.i, e.f.j, e.f.k, e.f.l, e.f.m, e.g.h, e.g.i, e.g.j, e.g.k, e.g.l, e.g.m, e.h.i, e.h.j, e.h.k, e.h.l, e.h.m, e.i.j, e.i.k, e.i.l, c.i.m, e.j.k, e.j.l, e.j.m, e.k.l, e.k.m, e.l.m, f.g.h, f.g.i, f.g.j, f.g.k, f.g.l, f.g.m, f.h.i, f.h.j, f.h.k, f.h.l, f.h.m, f.i.j, f.i.k, f.i.l, f.i.m, f.j.k, f.j.l, f.j.m, f.k.l, f.k.m, f.l.m, g.h.i, g.h.j, g.h.k, g.h.l, g.h.m, g.i.j, g.i.k, g.i.l, g.i.m, g.j.k, g.j.l, g.j.m, g.k.l, g.k.m, g.l.m, h.i.j, h.i.k, h.i.l, h.i.m, h.j.k, h.j.l, h.j.m, h.k.l, h.k.m, h.l.m, i.j.k, i.j.l, i.j.m, i.k.l, i.k.m, i.l.m, j.k.l, j.k.m, j.l.m, k.l.m, d.e.f.g, d.e.f.h, d.e.f.i, d.e.f.j, d.e.f.k, d.e.f.i, d.e.f.m, d.e.g.h, d.e.g.i, d. e.g.j, d.e.g.k, d.e.g.l, d.e.g.m, d.e.h.i, d.e.h.j, d.e.h.k, d.e.h.i, d.e.h.m, d.e.i.j, d.e.i.k, d.e.i.l, d.e.i.m, d. e.j.k, d.e.j.l, d.e.j.m, d.e.k.l, d.e.k.m, d.e.l.m, d.f.g.h, d.f.g.i, d.f.g.j, d.f.g.k, d.f.g.l, d.f.g.m, d.f.h.i, d.f.h.j, d.f.h.k, d.f.h.l, d.f.h.m, d.f.i.j, d.f.i.k, d.f.i.l, d.f.i.m, d.f.j.k, d.f.j.l, d.f.j.m, d.f.k.l, d.f.k.m, d.f.l.m, d.g.h.i, d.g.h.j, d.g.h.k, d.g.h.i, d.g.h.m, d. g.i.j, d.g.i.k, d.g.i.l, d.g.i.m, d.g.j.k, d.g.j.l, d.g.j.m, d.g.k.l, d. g.k.m, d.g.l.m, d.h.i.j, d.h.i.k, d.h.i.l, d.h.i.m, d.h.j.k, d.h.j.l, d.h.j.ni, d.h.k.l, d.h.k.m, d.h.l.m, d. i.j.k, d.i.j.l, d.i.j.m, d.i.k.l, d.i.k.m, d.i.l.m, d.j.k.l, d.j.k.m, d.j.l.m, d.k.l.m, e.f.g.h, e.f.g.i, e.f.g.j, e.f.g.k, e.f.g.i, e. f.g.m, e.f.h.i, e.f.h.j, e.f.h.k, e.f.h.l, e.f.h.m, e. f.i.j, e. f.i.k, e. f.i.l, e.f.i.m, e. f.j.k, e.f.j.l, e.f.j.m, e.f.k.l, e.f.k.m, e.f.i.m, e.g.h.i, e.g.h.j, e. g.h.k, e.g.h.l, e. g.h.m, e.g.i.j, e.g.i.k, e.g.i.l, e.g.i.m, e.g.j.k, e. 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.j.l.m, e.k.l.m, f.g.h.i, f.g.h.j, f.g.h.k, f.g.h.l, f.g.h.m, f.g.i.j, f.g.i.k, f.g.i.l, f.g.i.m, f.g.j.k, f.g.j.1, f.g.j.m, f.g.k.l, f.g.k.m, f.g.l.m, f.h.i.j, f.h.i.k, f.h.i.l, f.h.i.m, f.h.j.k, f.h.j .1, f.h.j.m, f.h.k.l, f.h.k.m, f.h.i.m, f.i.j.k, f.i.j.1, f.i.j .m, f.i.k.l, f.i.k.m, f.i.l.m, f j.k.l, f.j.k.m, f.j.l.m, f.k.l.m, g.h.i.j, g.h.i.k, g.h.i.l, g.h.i.m, g.h.j.k, g.h.j.l, g.h.j.m, g.h.k.l, g.h.k.m, g.h.l.m, g.i.j.k, g.i.j.l, g.i.j.m, g.i.k.l, g.i.k.m, g.i.l.m, g.j.k.l, g.j.k.m, g.j.l.m, g.k.l.m, h.i.j.k, h.i.j.l, h.i.j.m, h.i.k.l, h. i.k.m, h.i.Lm, h.j.k.l, h.j.k.m, h.j.l.m, h.k.l.m, i.j.k.l, i.j.k.m, i.j.l.m, i.k.l.m, j. k.l.m, d.e.f.g.h, d.e.f.g.i, d.e.f.g.j, d.e.f.g.k, d.e.f.g.l, d.e.f.g.m, d.e.f.h.i, d. e.f.h.j, d.e. f.h.k, d.e.f.h.l, d.e.f.h.m, d.e.f.i.j, d.e.f.i.k, d.e.f.i.1, d. e.f.i.m, d.e. f.j.k, d.e.f.j.1, d.e.f.j.m, d.e.f.k.l, d.e.f.k.m, d.e.f.l.m, d.e.g.h.i, d.e.g.h.j, d.e.g.h.k, d.e.g.h.l, d.e.g.h.m, d.e. g.i.j, d.e.g.i.k, d. e.g.i.l, d.e.g.i.m, d.e.g.j.k, d.e.g.j.l, d.e.g.j.m, d.e. g.k.l, d. e.g.k.m, d.e.g.l.m, d.e.h.i.j, d.e.h.i.k, d. e.h.i.1, d.e.h.i.m, d.e.h.j.k, d.e.h.j.1, d.e.h.j.m, d.e.h.k.l, d.e.h.k.m, d.e.h.l.m, d. e.i.j.k, d.e.i.j.1, d.e.i.j.m, d.e.i.k.l, d.e. i.k.m, d.e.i.l.m, d.e.j.k.l, d.e.j.k.m, d. e.j.l.m, d.e.k.l.m, d.f.g.h.i, d. f.g.h.j, d.f.g.h.k, d.f.g.h.l, d. f.g.h.m, d. f.g.i.j, d.f.g.i.k, d.f.g.i.1, d.f.g.i.m, d.f.g.j.k, d.f.g.j.l, d.f.g.j.m, d.f.g.k.l, d.f.g.k.m, d.f.g.i.m, d.f.h.i.j, d.f.h.i.k, d.f.h.i.l, d.f.h.i.m, d.f.h.j.k, d.f.h.j.l, d.f.h.j.m, d.f.h.k.l, d.f.h.k.m, d.f.h.i.m, d.f.i.j.k, d.f.i.j.l, d.f.i.j.m, d.f.i.k.l, d.f.i.k.m, d.f.i.l.m, d.f.j.k.l, d.f.j.k.m, d. f.j.l.m, d. f.k.l.m, d.g.h.i.j, d. g.h.i.k, d.g.h.i.l, d.g.h.i.m, d.g.h.j.k, d.g.h.j .1, d.g.h.j.m, d.g.h.k.l, d.g.h.k.m, d.g.h.i.m, d.g.i.j.k, d.g.i.j.l, d.g.i.j.m, d.g.i.k.1, d.g.i.k.m, d.g.i.l.m, d.g.j.k.l, d.g.j.k.m, d.g.j.l.m, d.g.k.l.m, d.h.i.j.k, d.h.i.j.l, d.h.i.j.m, d.h.i.k.l, d.h.i.k.m, d.h.i.l.m, d.h.j.k.l, d.h.j.k.m, d.h.j.l.m, d.h.k.l.m, d.i.j.k.l, d.i.j.k.m, d.i.j.l.m, d.i.k.l.m, d.j.k.l.m, e.f.g.h.i, e.f.g.h.j, e.f.g.h.k, e.f.g.h.l, e.f.g.h.m, e.f.g.i.j, e.f.g.i.k, e.f.g.i.l, e.f.g.i.m, e.f.g.j.k, e.f.g.j.l, e.f.g.j.m, e.f.g.k.l, e.f.g.k.m, e.f.g.l.m, e.f.h.i.j, e.f.h.i.k, e.f.h.i.l, e.f.h.i.m, e.f.h.j.k, e.f.h.j.l, e.f.h.j.m, e.f.h.k.l, e.f.h.k.ra, e.f.h.l.m, e.f.i.j.k, e.f.i.j.l, e.f.i.j.m, e.f.i.k.l, e.f.i.k.m, e.f.i.Lm, e.f.j.k.l, e.f.j.k.m, e.f.j.l.m, e.f.k.l.m, e.g.h.i.j, e.g.h.i.k, e.g.h.i.l, e.g.h.i.m, e.g.h.j.k, e.g.h.j.l, e.g.h.j.m, e.g.h.k.l, e.g.h.k.m, e.g.h.l.m, e.g.i.j.k, e.g.i.j.l, e.g.i.j.m, e.g.i.k.l, e.g.i.k.m, e.g.i.l.m, e.g.j.k.l, e.g.j.k.m, e.g.j.l.m, e.g.k.l.m, e.h.i.j.k, e.h.i.j.l, e.h.i.j.m, e.h.i.k.l, e.h.i.k.m, e.h.i.l.m, e.h.j.k.l, e.h.j.k.m, e.h.j.l.m, e.h.k.l.m, e.i.j.k.l, e.i.j.k.m, e.i.j.l.m, e.i.k.l.m, e.j.k.l.m, f.g.h.i.j, f.g.h.i.k, f.g.h.i.l, f.g.h.i.m, f.g.h.j.k. f.g.h.j.l, f.g.h.j.m, f.g.h.k.l, f.g.h.k.m, f.g.h.l.m, f.g.i.j.k, f.g.i.j.l, f.g.i.j.m, f.g.i.k.l, f.g.i.k.m, f.g.i.l.m, f.g.j.k.l, f.g.j.k.m, f.g.j.l.m, f.g.k.l.m, f.h.i.j.k, f.h.i.j.l, f.h.i.j.m, f.h.i.k.l, f.h.i.k.m, f.h.i.l.m, f.h.j.k.l, f.h.j.k.m, f.h.j.l.m, f.h.k.l.m, f.i.j.k.l, f.i.j.k.m, f.i.j.l.m, f.i.k.l.m, f.j.k.l.m, g.h.i.j.k, g.h.i.j.l, g.h.i.j.m, g.h.i.k.l, g.h.i.k.m, g.h.i.l.m, g.h.j.k.l, g.h.j.k.ra, g.h.j.l.m, g.h.k.l.m, g.i.j.k.l, g.i.j.k.m, g.i.j.l.m, g.i.k.l.m, g.j.k.l.m, h.i.j.k.l, h.i.j.k.m, h.i.j.l.m, h.i.k.l.m, h.j.k.l.m, i.j.k.l.m, d.e.f.g.h.i, d.e.f.g.h.j, d.e.f.g.h.k, d.e.f.g.h.i, d.e.f.g.h.m, d.e.f.g.i.j, d.e.f.g.i.k, d.e.f.g.i.l, d.e.f.g.i.m, d.e.f.g.j.k, d.e.f.g.j.l, d.e.f.g.j.m, d.e.f.g.k.l, d. e.f.g.k.m, d.e.f.g.i.m, d.e.f.h.i.j, d. e.f.h.i.k, d.e.f.h.i.l, d.e.f.h.i.m, d.e.f.h.j.k, d.e.f.h.j.l, d.e.f.h.j.m, d.e.f.h.k.l, d.e.f.h.k.m, d.e.f.h.l.m, d.e.f.i.j.k, d. e.f.i.j.l, d.e.f.i.j.m, d.e.f.i.k.l, d.e.f.i.k.m, d.e.f.i.l.m, d.e.f.j.k.l, d.e.f.j.k.m, d.e.f.j.l.m, d. e.f.k.l.m, d.e.g.h.i.j, d.e.g.h.i.k, d.e.g.h.i.l, d.e.g.h.i.m, d.e.g.h.j.k, d.e.g.h.j.l, d.e.g.h.j.m, d.e.g.h.k.l, d.e.g.h.k.m, d.e.g.h.i.m, d.e.g.i.j.k, d.e.g.i.j.l, d.e.g.i.j.m, d.e.g.i.k.l, d.e.g.i.k.m, d.e.g.i.l.m, d.e.g.j.k.l, d.e.g.j.k.m, d.e.g.j.l.m, d. e.g.k.l.m, d. e.h.i.j.k, d.e.h.i.j.l, d.e.h.i.j.m, d.e.h.i.k.l, d.e.h.i.k.m, d.e.h.i.l.m, d.e.h.j.k.l, d.e.h.j.k.m, d.e.h.j.l.m, d.e.h.k.l.m, d.e.i.j.k.l, d.e.i.j.k.m, d. e.i.j.l.m, d. e.i.k.l.m, d.e.j.k.l.m, d.f.g.h.i.j, d. f.g.h.i.k, d. f.g.h.i.l, d.f.g.h.i.m, d.f.g.h.j.k, d. f.g.h.j.l, d.f.g.h.j.m, d. f.g.h.k.l, d.f.g.h.k.m, d.f.g.h.l.m, d.f.g.i.j.k, d. f.g.i.j.l, d.f.g.i.j.m, d.f.g.i.k.l, d.f.g.i.k.m, d.f.g.i.l.m, d.f.g.j.k.l, d.f.g.j.k.m, d. f.g.j.l.m, d.f.g.k.l.m, d.f.h.i.j.k, d.f.h.i.j.l, d.f.h.i.j.m, d.f.h.i.k.l, d.f.h.i.k.m, d.f.h.i.l.m, d.f.h.j.k.l, d.f.h.j.k.m, d.f.h.j.l.m, d.f.h.k.l.m, d.f.i.j.k.l, d.f.i.j.k.m, d.f.i.j.l.m, d.f.i.k.Lm, d.f.j.k.l.m, d.g.h.i.j.k, d.g.h.i.j.l, d.g.h.i.j.m, d.g.h.i.k.l, d. g.h.i.k.m, d.g.h.i.l.m, d.g.h.j.k.l, d.g.h.j.k.m, d.g.h.j.l.m, d.g.h.k.l.m, d.g.i.j.k.l, d.g.i.j.k.m, d.g.i.j.l.m, d.g.i.k.l.m, d.g.j.k.l.m, d.h.i.j.k.l, d.h.i.j.k.m, d.h.i.j.l.m, d.h.i.k.l.m, d.h.j.k.l.m, d.i.j.k.l.m, e.f.g.h.i.j, e.f.g.h.i.k, e.f.g.h.i.l, e.f.g.h.i.m, e.f.g.h.j.k, e. f.g.h.j.l, e.f.g.h.j.m, e.f.g.h.k.l, c.f.g.h.k.m, e.f.g.h.l.m, e.f.g.i.j.k, e.f.g.i.j .1, e. f.g.i.j.m, e. f.g.i.k.l, e.f.g.i.k.m, e.f.g.i.l.m, e. f.g.j.k.l, e.f.g.j.k.m, e.f.g.j.l.m, e.f.g.k.l.m, e.f.h.i.j.k, e.f.h.i.j.l, e.f.h.i.j.m, e.f.h.i.k.l, e.f.h.i.k.m, e.f.h.i.l.m, e.f.h.j.k.l, e.f.h.j.k.m, e.f.hj.l.m, e.f.h.k.l.m, e.f.i.j.k.l, e.f.i.j.k.m, e.f.i.j.l.m, e.f.i.k.l.m, e.f.j.k.l.m, e.g.h.i.j.k, e.g.h.i.j.l, e.g.h.i.j.m, e.g.h.i.k.l, e.g.h.i.k.m, e.g.h.i.l.m, e.g.h.j.k.l, e.g.h.j.k.m, e.g.h.j.l.m, e.g.h.k.l.m, e.g.i.j.k.l, e.g.i.j.k.m, e.g.i.j.l.m, e.g.i.k.l.m, e.g.j.k.l.m, e.h.i.j.k.l, e.h.i.j.k.m, e.h.i.j.l.m, e.h.i.k.l.m, c.h.j.k.l.m, e.i.j.k.l.m, f.g.h.i.j.k, f.g.h.i.j.l, f.g.h.i.j.m, f.g.h.i.k.l, f.g.h.i.k.m, f.g.h.i.l.m, f.g.h.j.k.l, f.g.h.j.k.m, f.g.h.j.l.m, f.g.h.k.l.m, f.g.i.j.k.l, f.g.i.j.k.m, f.g.i.j.l.m, f.g.i.k.l.m, f.g.j.k.l.m, f.h.i.j.k.l, f.h.i.j.k.m, f.h.i.j.l.m, f.h.i.k.l.m, f.h.j.k.l.m, f.i.j.k.l.m, g.h.i.j.k.l, g.h.i.j.k.m, g.h.i.j.l.m, g.h.i.k.l.m, g.h.j.k.l.m, g.i.j.k.l.m, h.i.j.k.l.m, d.e.f.g.h.i.j, d.e.f.g.h.i.k, d.e.f.g.h.i.l, d.e.f.g.h.i.m, d.e.f.g.h.j.k, d.e.f.g.h.j.l, d.e.f.g.h.j.m, d.e.f.g.h.k.l, d.e.f.g.h.k.m, d.e.f.g.h.i.m, d.e.f.g.i.j.k, d.e.f.g.i.j.l, d.e.f.g.i.j.m, d.e.f.g.i.k.l, d.e.f.g.i.k.m, d.e.f.g.i.l.m, d.e.f.g.j.k.l, d.e.f.g.j.k.m, d.e.f.g.j.l.m, d.e.f.g.k.l.m, d.e.f.h.i.j.k, d.e.f.h.i.j.l, d.e.f.h.i.j.m, d. e.f.h.i.k.l, d. e.f.h.i.k.m, d.e.f.h.i.l.m, d.e.f.h.j.k.l, d.e.f.h.j.k.m, d.e.f.h.j.l.m, d.e.f.h.k.l.m, d.e.f.i.j.k.l, d. e.f.i.j.k.m, d.e.f.i.j.l.m, d. e.f.i.k.l.m, d.e.f.j.k.l.m, d.e.g.h.i.j.k, d.e.g.h.i.j.l, d.e.g.h.i.j.m, d.e.g.h.i.k.l, d.e.g.h.i.k.m, d.e.g.h.i.l.m, d.e.g.h.j.k.l, d.e.g.h.j.k.m, d.e.g.h.j.l.m, d.e.g.h.k.l.m, d.e.g.i.j.k.l, d.e.g.i.j.k.rn, d.e.g.i.j.l.m, d.e.g.i.k.l.m, d.e.g.j.k.l.m, d. e.h.i.j.k.l, d.e.h.i.j.k.m, d.e.h.i.j.l.m, d.e.h.i.k.l.m, d.e.h.j.k.l.m, d.e.i.j.k.l.m, d.f.g.h.i.j.k, d. f.g.h.i.j.l, d.f.g.h.i.j.m, d.f.g.h.i.k.l, d. f.g.h.i.k.m, d. f.g.h.i.l.m, d.f.g.h.j.k.l, d.f.g.h.j.k.m, d. f.g.h.j.l.m, d.f.g.h.k.l.m, d.f.g.i.j.k.l, d.f.g.i.j.k.m, d.f.g.i.j.l.m, d.f.g.i.k.l.m, d.f.g.j.k.l.m, d.f.h.i.j.k.l, d.f.h.i.j.k.m, d.f.h.i.j.l.m, d.f.h.i.k.l.m, d.f.h.j.k.l.m, d.f.i.j.k.l.m, d. g.h.i.j.k.l, d.g.h.i.j.k.m, d.g.h.i.j.l.m, d.g.h.i.k.l.m, d. g.h.j.k.l.m, d.g.i.j.k.l.m, d.h.i.j.k.l.m, e.f.g.h.i.j.k, e.f.g.h.i.j.l, e.f.g.h.i.j.m, e. f.g.h.i.k.l, e. f.g.h.i.k.m, e.f.g.h.i.l.m, e.f.g.h.j.k.l, e.f.g.h.j.k.m, e.f.g.h.j.l.m, e.f.g.h.k.l.m, e.f.g.i.j.k.l, e.f.g.i.j.k.m, e.f.g.i.j.l.m, e.f.g.i.k.l.m, e. f.g.j.k.l.m, e.f.h.i.j.k.l, e.f.h.i.j.k.m, e.f.h.i.j.l.m, e. f.h.i.k.l.m, e.f.h.j.k.l.m, e.f.i.j.k.l.m, e.g.h.i.j.k.l, e.g.h.i.j.k.m, e.g.h.i.j.l.m, e.g.h.i.k.l.m, e.g.h.j.k.l.m, e.g.i.j.k.l.m, e.h.i.j.k.l.m, f.g.h.i.j.k.l, f.g.h.i.j.k.m, f.g.h.i.j.l.m, f.g.h.i.k.l.m, f.g.h.j.k.l.m, f.g.i.j.k.l.m, f.h.i.j.k.l.m, g.h.i.j.k.l.m, d.e.f.g.h.i.j .k, d.e.f.g.h.i.j .1, d.e.f.g.h.i.j .m, d.e.f.g.h.i.k.l, d.e.f.g.h.i.k.m, d.e.f.g.h.i.l.m, d.e.f.g.h.j.k.l, d. e.f.g.h.j.k.m, d.e.f.g.h.j.l.m, d.e.f.g.h.k.l.m, d.e. f.g.i.j.k.l, d. e.f.g.i.j.k.m, d.e. f.g.i.j.l.m, d. e.f.g.i.k.l.m, d.e.f.g.j.k.l.m, d.e.f.h.i.j.k.l, d.e.f.h.i.j.k.m, d.e. f.h.i.j.l.m, d.e.f.h.i.k.l.m, d.e.f.h.j.k.l.m, d.e. f.i.j.k.l.m, d.e.g.h.i.j.kl, d.e.g.h.i.j.k.m, d.e.g.h.i.j.l.m, d. e.g.h.i.k.l.m, d.e.g.h.j.k.l.m, d.e.g.i.j.k.l.m, d.e.h.i.j.k.l.m, d.f.g.h.i.j.k.l, d.f.g.h.i.j.k.m, d.f.g.h.i.j.l.m, d.f.g.h.i.k.l.m, d.f.g.h.j.k.l.m, d.f.g.i.j.k.l.m, d.f.h.i.j.k.l.m, d. g.h.i.j.k.l.m, e.f.g.h.i.j.k.l, e.f.g.h.i.j.k.m, e.f.g.h.i.j.l.m, e. f.g.h.i.k.l.m, e.f.g.h.j.k.l.m, e.f.g.i.j.k.l.m, e.f.h.i.j.k.l.m, e.g.h.i.j.k.l.m, f.g.h.i.j.k.l.m, d. e.f.g.h.i.j.k.l, d.e.f.g.h.i.j.k.m, d.e.f.g.h.i.j.l.m, d.e.f.g.h.i.k.l.m, d.e.f.g.h.j.k.l.m, d.e.f.g.i.j.k.l.m, d.e.f.h.i.j.k.l.m, d.e.g.h.i.j.k.l.m, d.f.g.h.i.j.k.l.m, e.f.g.h.i.j.k.l.m, and d.e.f.g.h.i.j.k.l.m, wherein commas separate the individual combinations. Optionally, a gene encoding formate transporter may also be inactivated. 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 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:
(a) incorporation and/or overexpression of a gene encoding alanine dehydrogenase;
(b) inactivation of a gene encoding alanine racemase; (c) inactivation of a gene encoding lactate dehydrogenase;
(d) inactivation of a gene encoding alcohol dehydrogenase;
(e) inactivation of a gene encoding fumarate reductase;
(f) inactivation of a gene encoding pyruvate formate lyase;
(g) inactivation of a gene encoding acetate kinase; and Qx) inactivation of a gene encoding methyl glyoxal synthase.
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.d.e, a.d.f, a.d.g, a.d.h, a.e.f, a.e.g, a.e.h, a.f.g, a.f.h, a.g.h, b.c.d, b.c.e, b.c.f, b.c.g, b.c.h, b.d.e, b.d.f, b.d.g, b.d.h, b.e.f, b.e.g, b.e.h, b.f.g, b.f.h, b.g.h, c.d.e, c.d.f, c.d.g, c.d.h, c.e.f, c.e.g, c.e.h, c.f.g, c.f.h, c.g.h, d.e.f, d.e.g, d.e.h, d.f.g, d.f.h, d.g.h, e.f.g, e.f.h, e.g.h, f.g.h, a.b. c.d, a.b.c.e, a.b. c.f, a.b. eg, a.b. c.h, a.b.d.e, a.b. d.f, a.b.d.g, a.b.d.h, a.b. e.f, a.b. e.g, a.b.e.h, a.b. f.g, a.b. f.h, a.b. g.h, a.c. d.e, ax.d.f, a.c.d.g, a.c. d.h, a.c.e.f, a.c.e.g, a.c.e.h, a.c.f.g, a.c.f.h, a.c.g.h, a.d.e.f, a.d.e.g, a.d. e.h, a.d.f.g, a.d.f.h, a.d.g.h, a.e.f.g, a.e.f.h, a.e.g.h, a.f.g.h, b. c.d.e, b. c.d.f, b.c.d.g, b. c.d.h, b. c.e.f, b. c.e.g, b. c.e.h, b.c. f.g, b.c. f.h, b. c.g.h, b.d. e.f, b. d.e.g, b.d.e.h, b.d. f.g, b.d.f.h, b. d.g.h, b. e.f.g, b. e.f.h, b. e.g.h, b. f.g.h, c.d.e.f, c.d.e.g, c.d.e.h, c.d.f.g, c.d. f.h, c.d.g.h, c.e.f.g, c.e.f.h, c.e.g.h, c.f.g.h, d. e.f.g, d.e.f.h, d.e.g.h, d.f.g.h, e. f.g.h, a.b. c.d.e, a.b. c.d.f, a.b. c.d.g, a.b. c.d.h, a.b. c.e.f, a.b. c.e.g, a.b. c.e.h, a.b. c.f.g, a.b. c.f.h, a.b. c.g.h, a.b.d.e.f, a.b.d.e.g, a.b.d.e.h, a.b.d.f.g, a.b.d.f.h, a.b.d.g.h, a.b.e.f.g, a.b.e.f.h, a.b.e.g.h, a.b.f.g.h, a.c.d.e.f, a.c.d.e.g, a.c.d.e.h, a.c.d.f.g, a.c.d.f.h, a.c.d.g.h, a.c.e.f.g, a.c.e.f.h, a.c.e.g.h, a.c.f.g.h, a.d.e.f.g, a.d.e.f.h, a.d.e.g.h, a.d.f.g.h, a.e.f.g.h, b.c.d.e.f, b.c.d.e.g, b.c.d.e.h, b.c.d.f.g, b.c.d.f.h, b.c.d.g.h, b.c.e.f.g, b.c.e.f.h, b.c.e.g.h, b.c.f.g.h, b.d.e.f.g, b.d.e.f.h, b.d.e.g.h, b.d.f.g.h, b.e.f.g.h, c.d.e.f.g, c.d.e.f.h, c.d.e.g.h, c.d.f.g.h, c.e.f.g.h, d.e.f.g.h, a.b.c.d.e.f, a.b.c.d.e.g, a.b.c.d.e.h, a.b.c.d.f.g, a.b.c.d.f.h, a.b.c.d.g.h, a.b.c.e.f.g, a.b.c.e.f.h, a.b.c.e.g.h, a.b.c.f.g.h, a.b.d.e.f.g, a.b.d.e.f.h, a.b.d.e.g.h, a.b.d.f.g.h, a.b.e.f.g.h, a.c.d.e.f.g, a.c.d.e.f.h, a.c.d.e.g.h, a.c.d.f.g.h, a.c.e.f.g.h, a.d.e.f.g.h, b. c.d.e.f.g, b. c.d.e.f.h, b. c.d.e.g.h, b. c.d.f.g.h, b. c.e.f.g.h, b. d.e.f.g.h, c.d.e.f.g.h, a.b. c.d.e.f.g, a.b. c.d.e.f.h, a.b. c.d.e.g.h, a.b. c.d.f.g.h, a.b. c.e.f.g.h, a.b.d.e.f.g.h, a.c. d.e.f.g.h, b. c.d.e.f.g.h, and a.b. c.d.e.f.g.h. Preferably 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. In one embodiment, 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;
(b) incorporation and/or overexpression of a gene encoding phospho-β-glucosidase; and
(c) incorporation and/or overexpression of a gene encoding an endoglucanase or cellulase.
Combinations of these modifications suitable to the invention include: a; b; c; a & b; a & c; b & c; and a & b & c. In one embodiment, the gene encoding cellobiose utilizing enzyme and/or the gene encoding phospho-β-glucosidase are genes from Klebsiella, and preferably are Klebsiella oxytoca casAB. In one embodiment the gene encoding an endoglucanase or cellulase is a gene from Erwinia, and preferably is Erwinia chrysanthemi celY or Erwinia chrysanthemi celZ. In one embodiment 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:
(a) inactivation of a gene encoding lactate dehydrogenase;
(b) inactivation of a gene encoding pyruvate foπnatelyase;
(c) inactivation of a gene encoding fumarate reductase; (d) inactivation of a gene encoding (FiF0)H" -ATP synthase;
(e) inactivation of a gene encoding alcohol/aldehyde dehydrogenase; and
(f) inactivation of a gene encoding 2-ketoglutarate dehydrogenase.
Combinations of these modifications suitable to the invention include: a; b; c; d; e; f; a & b; a & c; a & d: a & e; a & f; b & c; b & d; b & e; b & f; c & d; c & e; c & f; d & e; d & f; e&f;a&b&c;a&b&d;a&b&e;a&b&f;a&c&d;a&c&e;a&c&f;a&d& e;a&d&f;a&e&f;b&c&d;b&c&e;b&c&f;b&d&e;b&d&f;b&e&f;c &d&e;c&d&f;c&e&f;d&e&f;a&b&c&d;a&b&c&e;a&b&c&f;a&b &d&e;a&b&d&f;a&b&e&f;a&c&d&e;a&c&d&f;a&c&e&f;a&d& e&f;b&c&d&e;b&c&d&f;b&c&e&f;b&d&e&f;c&d&e&f;a&b&c &d&e;a&b&c&d&f;a&b&c&e&f;a&b&d&e&f;a&c&d&e&f;b&c & d & e & f ; and a&b&c&d&e&f. Any strain containing any of these combinations of modifications may be further modified to inactivate a gene encoding formate transporter, for example focA. In one embodiment, the inactivation of the gene encoding (FiFo)H+-ATP synthase preserves the hydrolytic activity of Fl-ATPase in the cytoplasm while disrupting oxidative phosphorylation. In one embodiment, the gene encoding (Fi Fo)H -ATP synthase is atpF or atpH or both. In one embodiment, the gene encoding lactate dehydrogenase is ldhA. hi one embodiment, the gene encoding pyruvate formate lyase is pflB. In one embodiment, the gene encoding fumarate reductase is one or more of the component genes of frdABCD, for example frdBC or frdCD. In one embodiment the gene encoding alcohol/aldehyde dehydrogenase is adhE. In one embodiment, the gene encoding 2-ketoglutarate dehydrogenase is sucA.
For any strain modified to contain any combination of inactivation of a gene encoding lactate dehydrogenase, inactivation of a gene encoding pyruvate formate lyase, inactivation of a gene encoding fumarate reductase, inactivation of a gene encoding (F]Fo)H+-ATP synthase, inactivation of a gene encoding alcohol/aldehyde dehydrogenase, and/or inactivation of a gene encoding 2-ketoglutarate dehydrogenase, as set forth immediately above (and optionally including inactivation of a gene encoding formate transporter), additional genetic modifications are also suitable to the invention, and may serve, for example, to increase amounts of pyruvate that can be harvested. The genetically modified
Enterobactβr asburiae strains may contain, for example, one or more further genetic modifications selected from the group consisting of;
(g) inactivation of a gene encoding acetate kinase; and (h) inactivation of a gene encoding pyruvate oxidase.
Combinations of these further modifications suitable to the invention include: g; h; and g & h. In one embodiment of the invention, the gene encoding acetate kinase is ackA. In one embodiment of the invention, 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:
(a) incorporation and/or overexpression of a gene encoding glycerol-3- phosphate dehydrogenase;
(b) incoiporation and/or overexpression of a gene encoding glycerol-3- phosphatase; (c) incorporation and/or overexpression of a gene encoding glycerol dehydratase;
(d) incorporation and/or overexpression of a gene encoding 1,3-propanediol oxidoreductase: (e) incorporation and/or overexpression of a gene encoding aldose reductase; and
(f) incorporation and/or overexpression of a gene encoding glycerol dehydrogenase. Combinations of these modifications suitable to the invention include: a; b; c; d; e; f; a & b; a & c; a & d; a & e; a & f; b & c; b & d; b & e; b & f; c & d; c & e; c & f; d & e; d & f; e&f;a&b&c;a&b&d;a&b&e;a&b&f;a&c&d;a&c&e;a&c&f;a&d& e;a&d&f;a&e&f;b&c&d;b&c&e;b&c&f;b&d&e;b&d&f;b&e&f;c &d&e;c&d&f;c&e&f;d&e&f;a&b&c&d;a&b&c&e;a&b&c&f;a&b &d&e;a&b&d&f;a&b&e&f;a&c&d&e;a&c&d&f;a&c&e&f;a&d& e&f;b&c&d&e;b&c&d&f;b&c&e&f;b&d&e&f;c&d&e&f;a&b&c &d&e;a&b&c&d&f;a&b&c&c&f;a&b&d&e&f;a&c&d&e&f;b&c & d & e & f ; and a&b&c&d&e&f. In one embodiment, E. coli host cell W 1485 harboring plasmids pDT20 and pAH42 (Accession Number ATCC 98188 and deposited in the ATCC under the terms of the Budapest Treaty) 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). In one embodiment, S. 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. Yet another source of readily available genetic material for the production of recombinant organisms capable of producing 1,3-propanediol is 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. In one embodiment, the gene encoding aldose reductase is the gene for rat lens aldose reductase. In one embodiment, 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.
Various aspects of the invention provide the following non-limiting embodiments: L A process for fermenting MeGAX comprising:
(a) forming a substrate from biomass materials;
(b) subjecting the substrate to acid hydrolysis;
(c) selecting and isolating a strain of Enterobacter asburiae that has the ability to ferment MeGAX; (d) inoculating the acid hydrolyzed substrate with the selected strain of Enterobacter asburiae to ferment MeGAX under conditions favorable for cell viability and conversion of MEGAX to a fermentation product; and (e) optionally, recovering said fermentation product.
2. The process of embodiment 1 , wherein the Enterobacter asburiae is the
Enterobacter asburiae strain JDR-I, El, or Ll .
3. The process of any preceding embodiment, wherein the biomass materials contain hemicellulose.
4. The process of any preceding embodiment, wherein the biomass materials comprise sweetgum.
5. The process of any preceding embodiment, wherein the acid hydrolysis is dilute acid hydrolysis.
6. A process for fermenting MeGAX comprising:
(a) selecting and/or isolating a strain of Enterobacter asburiae that has the ability to ferment MeGAX; (b) inoculating culture media comprising MeGAX with the selected strain of Enterobacter asburiae to ferment MeGAX under conditions favorable for cell viability and conversion of MEGAX to a fermentation product; and (e) optionally, recovering fermentation product from the substrate. 7. The process of embodiment 6, wherein the Enterobacter asburiae is the Enterobacier asburiae strain JDR-I, El, or Ll .
8. The process of any embodiments 6-7, wherein the culture media contains hemicellulose.
9. The process of embodiments 6-8, wherein the culture media comprises sweetgum or other biomass.
10. The process according to embodiments 6-9, wherein said fermentation product is acetate/acetic acid; ethanol; methanol; succinate/succinic acid; lactate/lactic acid; formate/formic acid; acetate/acetic acid; 2,3-butanediol; or combinations thereof.
11. A process for fermenting a substrate comprising:
(a) selecting and isolating a strain of Enterobacter asburiae that has the ability to ferment a substrate;
(b) inoculating culture media comprising said substrate with the selected strain of Enterobacter asburiae and fermenting said substrate under conditions favorable for cell viability and conversion of the substrate to a fermentation product; and
(c) optionally, recovering fermentation product from the substrate.
12. The process of embodiment 11, wherein the Enterobacter asburiae is the Enterobacter asburiae strain JDR-I, El. or Ll .
13. The process of embodiments 1 1 -12, wherein said fermentation product acetate/acetic acid; ethanol; methanol; succinate/succinic acid; lactate/lactic acid; formate/formic acid; acetate/acetic acid; 2,3-butanediol; or combinations thereof.
14. The process of embodiments 11-13, wherein said substrate is D-glucose, D- xylose, D-mannose, L-arabinose, D-galactose, glucuronate, or various combinations thereof. 15. An isolated strain of Enterobacter asburiae.
16. The isolated E. asburiae strain according to embodiment 15, wherein said strain is selected from the group consisting of JDR-I, El , and Ll .
17. 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.
18. The isolated E. asburiae strain of embodiments 15-17, wherein said strain comprises one or more genetic modifications selected from the group consisting of: incorporation and/or overexpression and/or inactivation of a gene encoding L- lactate dehydrogenase; incorporation and/or overexpression and/or inactivation of a gene encoding D- lactate dehydogenase; inactivation of a gene encoding fumarate reductase (frd); inactivation of a gene encoding alcohol/aldehyde dehydrogenase (adh); inactivation of a gene encoding pyruvate formate lyase (pfl); inactivation of a gene encoding acetate kinase (ack); and inactivation of a gene encoding methylglyoxal synthase (mgs).
19. The isolated E. asburiae strain of embodiments 15-18, wherein said strain 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; inactivation of a gene encoding phosphoenolpyruvate carboxylase; inactivation of a gene encoding acetate kinase; and inactivation of a gene encoding pyruvate formate lyase. 20. The isolated E. 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.
21. The isolated E. asburiae strain of embodiments 15-20, wherein said strain comprises one or more further genetic modifications selected from the group consisting of: inactivation of a gene encoding acetate kinase; inactivation of a gene encoding alcohol dehydrogenase; inactivation of a gene encoding aspartate aminotransferase; inactivation of a gene encoding citrate lyase; inactivation of a gene encoding lactate dehydrogenase; inactivation of a gene encoding methylglyoxal synthase: inactivation of a gene encoding pyruvate oxidase; inactivation of a gene encoding phosphate acetyltransferase; inactivation of a gene encoding malic enzyme; and inactivation of a gene encoding threonine dehydratase.
22. The isolated E. asburiae strain of embodiments 15-21, wherein said strain comprises one or more genetic modifications selected from the group consisting of: incorporation and/or overexpression of a gene encoding alanine dehydrogenase; inactivation of a gene encoding alanine racemase; inactivation of a gene encoding lactate dehydrogenase; inactivation of a gene encoding alcohol dehydrogenase: inactivation of a gene encoding fumarate reductase; inactivation of a gene encoding pyruvate formate lyase; inactivation of a gene encoding acetate kinase; and inactivation of a gene encoding methylglyoxal synthase. 23. The isolated E. asburiae strain of embodiments 15-22, wherein said strain comprises one or more genetic modifications selected from the group consisting of: incorporation and/or overcxpression of a gene encoding cellobiose utilizing enzyme; incorporation and/or overexpression of a gene encoding phospho-β- glucosidase; and incorporation and/or overexpression of a gene encoding an endoglucanase or cellulase.
24. The isolated E, asburiae strain of embodiments 15-23, wherein said strain comprises one or more genetic modifications selected from the group consisting of: inactivation of a gene encoding lactate dehydrogenase; inactivation of a gene encoding pyruvate formatelyase; inactivation of a gene encoding fumarate reductase; inactivation of a gene encoding (F]Fo)H+-ATP synthase; inactivation of a gene encoding alcohol/aldehyde dehydrogenase; and inactivation of a gene encoding 2-ketoglutarate dehydrogenase.
25. The isolated E. asburiae strain of embodiments 15-24, wherein said strain comprises one or more further genetic modifications selected from the group consisting of: inactivation of a gene encoding acetate kinase; and inactivation of a gene encoding pyruvate oxidase.
26. The isolated E. asburiae strain of embodiments 15-25, wherein said strain comprises one or more genetic modifications selected from the group consisting of: incorporation and/or overexpression of a gene encoding glycerol-3-phosphate dehydrogenase; incorporation and/or overexpression of a gene encoding glycerol-3- phosphatase; incorporation and/or overexpression of a gene encoding glycerol dehydratase; incorporation and/or overexpression of a gene encoding 1,3 -propanediol oxidoreductase; incorporation and/or overexpression of a gene encoding aldose reductase; and incorporation and/or overexprcssion of a gene encoding glycerol dehydrogenase.
27. The isolated E. asburiae strain of embodiments 15-26, wherein said strain comprises one or more genetic modifications selected from the group consisting of: inactivation of a gene encoding pyruvate formate lyase; and inactivation of a gene encoding acetolactate synthase.
28. The process according to embodiments 1-14, wherein said 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.
Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
EXAMPLE 1
STRAIN ISOLATION AND CHARACTERIZATION
Preparation of substrates and culture media
Sweetgum methylglucuronoxylan (MeGAXn) was prepared from sweetgum stem wood {Liquidambar styraciflua) as previously described and characterized by 13C-NMR
(Hurlbert & Preston J Bacterial 183:2093-2100 (2001); Kardosova et al. Carbohydr Res
308:99-105 (1998)). Dilute acid hydrolysates of methylglucuronoxylan were prepared by hydrolysis with 0.1 N H2SO4 (4 g methylglucuronoxylan in 400 ml 0.1 N H2SO4) at 1210C for 60 min, followed by neutralization with BaCO3. 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. After concentration under vacuum at 50 0C, 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 MeGAX2 were identified by thin layer chromatography (TLC) analysis using MeGAX and MeGAX2 standards structurally defined by 13C and 1H-NMR spectrometry (Zuobi-Hasona et al. ASM National Meeting (2001)). Xylobiose and xylotriose were obtained and purified from MeGAXn 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 MeGAXn 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 300C for 24 h. An additional 1 U was added after 24 h and incubation was continued for 40 h. Aldouronates, xylobiose, and xylotriose were separated with the P2 column and identified by TLC. Total carbohydrate concentrations related to substrate preparations were determined by the phenol- sulfuric acid assay (Dubois et al. Anal Chem 28:350-356 (1956)), with xylose as the reference. The conditions of acid hydrolysis generated mostly MeGAX and a small amount of MeGAX2 from MeGAXn, with no aldouronates larger than MeGAX2 detected. MeGAX3 was prepared from GHlO endoxylanase-catalyzed depolymerization of sweetgum MeGAXn and then purified by gel filtration on BioGel P4 (St. John et al. Appl Environ Microbiol 72:1496- 1506 (2006)). 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 MeGAXn 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).
Isolation and identification of E. asburiae JDR-I
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 370C. 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 -7O0C.
The purified isolate was submitted to MIDI Labs (world wide web site: midilabs.com) for partial 16s rRNA sequencing and FAME analysis. BBL Enterotube™ 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.
Substrate utilization and fermentation product analysis
Growth and substrate utilization analysis was performed in cultures aerated by shaking. For preparing inocula, cultures of E. coli B (ATCC 11303) and E. asburiae JDR-I from cryostored samples were directly streaked on Luria-Bertani (LB) agar plates. After overnight incubation at 37°C, isolated colonies were picked to inoculate liquid media specified for a particular experiment. Growth studies were performed at 37°C in 16 mm x 100 mm test tubes containing 6 ml medium. Optical densities of cultures were measured at 600 nm (ODgoo) with a Beckman DU500 series spectrophotometer. The relationship of cell density and OD6Oo was experimentally determined as CDW/L (g cell dry weight/L) = 0.490Deoo+0.02. Sample dilutions were made to obtain OD60O readings between 0.2 and 0.8 absorbance units which, corrected for dilution factors, provided turbidity values for growth studies. Individual 6 ml cultures for study were inoculated with 12 μl (0.2% volume) of overnight cultures and maintained at 37°C with constant shaking (Eberbach shaker set at "low"). Batch fermentations under anaerobic conditions at 37°C were conducted in 13 mm x
100 mm screw cap tubes containing 3.0 ml medium. Inocula (0.5% [v/v]) were from overnight aerobic cultures grown in the same medium. After inoculation, nitrogen gas was used to flush and saturate the sealed batch culture. The tubes were set in a Glas-Col minirotator at 60 rpm. For analysis of substrates and fermentation products, cells were removed by centrifugation and supernatants were passed through 0.22 um filters and subjected to HPLC analysis. Products were resolved on a Bio-Rad HPX-87H column with 0.01 N H2SO4 as the eluent at 650C. 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.
Determination of metabolic pathways by 13C-NMR The central metabolic pathways utilized by E. asburiae JDR-I during glucose and xylose fermentation were evaluated with 13C-NMR (Scott & Baxter Annu Rev Biophys Bioeng 10:151-174 (1981)). Cultures were grown in LB medium to mid-exponential phase at 37°C. Cultures (0.5 ml) were centrifuged and the cells washed with 2x ZH salts solution. The cell pellets were suspended in 1.0 ml 0.5% [2-13C]xylose (99% enrichment; Omicron Biochemicals Inc, IN) in ZH minimal medium. Similar fermentations were also prepared with
1.0 ml 0.5% [l-13C]glucose, or 1.0 ml 0.5% [6-13C]glucose ZH minimal medium using D-[I- 13C]glucose or D-[6-13C]glucose (99% enrichment; Cambridge Isotope Laboratories, Andover, MA). Fermentations were carried out under anaerobic conditions at 37°C for 8 hours. Cells were removed by centrifugation, and the supernatants analyzed by HPLC (after filtration) and 13C-NMR spectrometry. 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 μl) containing 13C at natural abundance in 700 μl sample was used as an internal reference of 31.07 ppm for the 13C 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 (13C at natural abundance) of known concentrations.
Determination of molar cell dry weight yield For molar growth yield experiments (Smalley et al. J Bacteriol 96:1595-1600
(1968); Bauchop & Elsden J Gen Microbiol 23:457-469 (1960); Gunsalus & Shuster in The Bacteria (1961)), 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 600C 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).
Results
Identification and characteristics of E. asburiae JDR-I A novel bacterial strain able to grow on MeGAX minimal medium was obtained and subsequently identified with three tests. The partial 16S rRNA sequence (accession number EUl 17142, Gene Bank, NCBI), amplified using primers corresponding to E. coli 16S rRNA positions 005 and 531 (526bp), provided an alignment with 99.5% identity within the sequence of Enterobacter asburiae (MIDI Aerobic Bacteria Database version 4.0, January 1999). Results of FAME (fatty acid methyl ester) analysis indicated this strain had the greatest similarity index with Enterobacter asburiae species (0.766) compared with any other entry in the MIDI database. 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.
When exponential phase cultures were observed by optical DIC microscopy, E. asburiae JDR-I appeared as short motile rods. Negative stain electron microscopy revealed 3 μm x 1 μm 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)). When grown on LB agar plates, colonies of E. asburiae JDR-I were morphologically indistinguishable from E. coli colonies.
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. To evaluate this potential, E. asburiae JDR-I was grown aerobically in minimal medium comprised of neutralized MeGAXn acid hydrolysate and Zucker and Hankin mineral salts. Based upon HPLC analysis of media samples taken at different stages of growth, E. asburiae JDR-I utilized MeGAX completely in minimal media containing MeGAXn hydrolysate after it depleted xylose (Fig. 2A). Biphasic growth occurred as E. asburiae JDR-I switched from utilizing xylose to MeGAX (Fig. 2B). In contrast to E. asburiae JDR-I, E. coli B consumed only the free xylose with the MeGAX concentration in the medium remaining constant. Concentrations of xylose and MeGAX in MeGAXn hydrolysate medium, as determined by HPLC, were 0.206% w/v and 0.036% w/v, respectively. Therefore, E. asburiae JDR-I utilized 17.5% more substrate (mass amount) than E. coli B, which was unable to utilize MeGAX (Fig. 2B). Under aerobic conditions, 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 MeGAXn hydrolysates. E. asburiae JDR-I was also able to grow in xylobiose and xylotriose minimal medium, which E. coli B could not utilize. However, E. asburiae JDR-I was unable to utilize MeGAX2 and MeGAX3 (data not shown).
Substrate preference of E. asburiae JDR-I
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 MeGAX2 generated by acid hydrolysis, or MeGAX3 generated by a GHlO endoxylanase. When growing in a minimal medium containing an eqimolar mixture of glucose and xylose, 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.
To study the process by which MeGAX was utilized, E. asburiae JDR-I was grown in minimal medium containing both xylose and glucuronate, products that might be generated from MeGAX. A single phase growth curve was observed in which both substrates were consumed by 15 hours (Fig. 3B). This is similar to its single phase growth curve on MeGAX, in which the 6.5 mM substrate was depleted in about 11 hours (Fig. 3C). The similarity in growth pattern with MeGAX and the combination of xylose and glucuronate as carbon sources supports the possibility that free glucuronate and free xylose may be released during the metabolism of MeGAX.
Fermentation characteristics Fermentation experiments were performed to evaluate the potential of E. asburiae
JDR-I as a biocatalyst for the production of biobased products, and define the processes involved in the metabolism of MeGAX. Using limiting amounts (0.25% w/v) of substrates and cultivation under anaerobic standing conditions, 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). Small amounts of formic acid and very small amounts of fumaric and succinic acids were detected in most fermentations. The HPLC profiles indicate that E. asburiae JDR-I performs mixed acid fermentation as does E, coli, but with preferential formation of acetate and ethanol over lactate.
With sweetgum MeGAXn hydrolysatc as substrate, E. asburiae JDR-I consumed 99% of the substrate when the pH was maintained above 5, giving the major products acetic acid and ethanol (Table 2). With glucuronic acid as carbon source, acetic acid was the major fermentation product. To study the process of MeGAX metabolism, the presumed degradation products of MeGAX, xylose and glucuronate, both at 11 mM, were used as substrates. The predominant products were 20.4 mM acetate and 5.25 mM ethanol. Major fermentation products from 4.0 mM MeGAX were 8.1 mM acetic acid, 1.2 mM ethanol, and 4.3 mM methanol (Table T).
Central metabolic pathways determined by 13C-NMR
The total quantities of ethanol, acetate and lactate were determined by HPLC and the quantities of ' C labeled products were quantified from integration of differentially labeled compounds detected in the 13C-NMR spectra. This allowed determination of the fraction of each fermentation product that was differentially labeled with 13C, which helped to illustrate the central metabolic pathways E. asburiae JDR-I uses. The quantities of each product and the fractions labeled with 13C are presented in Table 3. To determine the primary pathway of xylose metabolism by E. asburiae JDR-I, comparisons were made for the fermentation of [2-13C]xylose with cultures of E. coli B. For E. coli B, employing only the pentose-phosphate pathway to metabolize xylose, the prominent shift signals in the C-NMR spectrum of the fermentation products were assigned to [l-13C]ethanol at 57.6 ppm, [2-BC]lactate at 68.8 ppm, and [l -13C]acetate at 181.0 ppm. Shift signals at 71.0 ppm and 74.5 ppm were assigned to the a- and β- anomers of unused [2- 13C]xylose, and the signal at 30.6 ppm to the methyl carbons of the acetone standard (Fig. 4B). Fractions of labeled versus total acetate, ethanol, and lactate with E. coli B were 0.26, 0.27, and 0.31, respectively, which was slightly less than the theoretical fraction 0.4 expected for metabolism through the pentose-phosphate pathway (Table 3). The lower quantities of labeled products as fractions of the total found for E. coli may reflect accuracy limitations for integration against the 13C-acetone standard, as these products all showed similar fractions (0.26-0.31 ) were labeled.
When E. asburiae JDR-I fermented [2-13C]xylose, a 13C-NMR spectrum for fermentation products was obtained with prominent signals for [l ~13C]ethanol, [2- 13C] lactate, and [1-13C] acetate at expected shift positions of 57.8 ppm, 68.8 ppm and 181.0 ppm respectively (Fig. 4A). The fractions of labeled ethanol, labeled acetate and labeled lactate to their total amounts were 0.43, 0.4 and 0.45, respectively (Table 3), and nearly identical to the theoretical fraction of 0.4. Moreover, the fractions of labeled acetate and ethanol were not higher than the fraction of labeled lactate (Table 3). These results establish that the pentose phosphate pathway is the main metabolic pathway for xylose utilization in E. asburiae JDR- 1.
To determine the primary pathway E. asburiae JDR-I utilizes to metabolize glucose, [l-13C]glucose and [6-13C]glucose were used as fermentation substrates. Similar 13C-NMR spectra of fermentation products were obtained from [6- C] glucose and [1 - C] glucose (Fig.
4C, 4D). Shift signals at 92.4 and 96.2 ppm were assigned to the a- and β- anomers of unused [l-13C]glucose (Fig. 4C); signals at 60.9 and 60.1 ppm were assigned to the a- and β- anomers of unused [6-13C]glucose (Fig. 4D). The signal at 30.6 ppm was assigned to the methyl carbons of the acetone standard. Excepting the shift signals for reference and unused substrates, the prominent signals in both spectra were for [2-13C]etlianol at 17.1 ppm, [2- 13C]acetate at 22.2 ppm and [3-13C]lactate at 20.3 ppm with similar distributions for both substrates. The absence of [1-13C] lactate indicates that no [l-13C]glucose was metabolized through the Entner-Douderoff (ED) pathway. Moreover, the fractions of all labeled products of their total amounts were similar for fermentation of [6-13C]glucose and [l-13C]giucose; and these fractions for [6-l3C]glucose were not higher than those found for [l-13C]glucose (Table 3), indicating little or no [I-1 C]glucose went through the pentose-phosphate pathway. Collectively, these results establish that the Embden-Meyerhof (EM) pathway is the main metabolic pathway for glucose utilization in E. asburiae JDR-I.
Growth and projected ATP yields with different substrates
To understand the bioenergetics in the process of MeGAX fermentation by E. asburiae JDR-I, molar cell dry weight yields were determined after 24 hours of growth with glucose, xylose, glucuronate and MeGAX as sole carbon sources in Zucker-Hankin minimal medium. The experiment was performed three times and the average approximate YM values were about 1O g per mole of substrate for growth on xylose and glucuronate, 20 for growth on glucose, and 30 for growth on MeGAX (Table 4). The experimental YATP in anaerobic growth has been reported in the range of 8 to 12 gram cell dry weight per mole of ATP for bacteria (Russell & Cook Microbiol Rev 59:48-62 (1995)). An estimated YAI P value at the lower end of this range, 8, was used here since this is for anaerobic growth in batch cultures in minimal medium with a relatively low concentration of carbon source (Bauchop & Elsden J Gen Microbiol 23:457-469 (1960); Gunsalus & Shuster in The Bacteria (1961)). The apparent ATP yields per mole of substrate were calculated based on the estimated YATP of 8 as 1.3 mole of ATP produced from either xylose or glucuronate, 2.6 from glucose and 4.0 from MeGAX (Table 4). These apparent ATP yields allow an estimate of the relative ATP yields obtained for the different substrates without considerations of maintenance energy or overflow metabolism (Russell & Cook Microbiol Rev 59:48-62 (1995)), providing insight into the metabolism of MeGAX. The ratios of the molar growth yields obtained with xylose, glucuronate, and MeGAX as carbon sources are 1.0:1.0:3.2 (Table 4), indicating that the requirement for MeGAX transport is less than that for separate transport of xylose and glucuronate.
EXAMPLE 2 GENETIC ENGINEERING FOR LACTIC ACID PRODUCTION
Bacterial strains, media, and fermentation conditions The bacterial strains constructed and used in these studies are listed in Table 5. The E. asburiae JDR-I served as a starting point for genetic modification.
Sweetgum methylglucuronoxylan (MeGAXn) was prepared from sweetgum stem wood (Liquidambar styracifluά) as previously described and characterized by C13-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 HoSO4 at 121 0C for 60 min, followed by neutralization with BaCO3. 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. 2009). 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 0C 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.
Genetic methods Standard methods were used for most of the genetic manipulations. Qiagen kits were used for genomic DNA and plasmid extractions (Qiagen, Valencia, CA). Polymerase chain reaction (PCR) amplification was performed with an I-cycler thermal cycler (Biorad, Hecules, CA) with primers synthesized by Operon (Huntsville, Alabama). Topo cloning kits were used for cloning (Invitrogen, Carlsbad, CA). Electroporation was performed on a Gene pulser Xcell instrument (Biorad, Hecules, CA). Restriction endonucleases were purchased from New England Biolabs (Ipswich, MA). DNA sequencing was provided by the University of Florida Interdisciplinary Center for Biotechnology Research. The plasmids constructed are listed in Table 5.
The methods for gene deletion have been previously described (Jantama et al. 2008), with minor modifications made to apply to E. asburiae JDR-I. The partial sequence of E. asburiae JDR-I pflB gene (gene bank accession number: EU719655) was determined on a DNA fragment amplified by PCR using specific primers based on E. coli pflB gene sequence. A segment of the E. asburiae JDR-I als gene (FJ008982) 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.
Determination of lactate isomers produced by E. asburiae Ll
To determine the isomers of lactate formed, fermentation products were assayed with D-lactate or L-lactate dehydrogenases (Taguchi and Ohta 1991). The conditions of the colorimetric enzyme assays were similar to those used to measure lactate dehydrogenase activity (Babson and Babson 1973). NAD+ was obtained from Research Products International Corp, Chicago IL. All other reagents, substrates, and enzymes were obtained from Sigma. Iodonitrotetrazolium chloride (40 mg), 100 mg IN1AD+ and 10 mg PMSF were dissolved in 20 ml 0.2 M Tris/HCl (pH 8.2) to obtain the colorimetric reagent. Reactions were initiated by adding 4 Kunitz units (1 μmol/min) of either L-lactate dehydrogenase (rabbit muscle, 140 U/mg protein) or D-lactate dehydrogenase {Lactobacillus leichmanii, 232 U/mg protein) in 100 μl colorimetric reagent and 100 μl 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.
Results and discussion
Fermentation characteristics of the wild type strain E, asburiae JDR-I
When growing with either 0.8% glucose, 0.5% arabinose or 0.5% xylose as the sole carbon source, the wild type strain produced several products including succinate, lactate, acetate, 2,3-butanediol and ethanol. Glucose fermentations resulted in the formation of 2,3- butanediol, ethanol and acetate as major products. Larger amount of acetate and no 2,3- butanediol was detected in 0.5% xylose and 0.5% arabinose fermentations (Table 6).
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 MeGAX2. Previous studies indicated that MeGAX was metabolized by E. asburiae JDR-I into methanol, glucuronate and xylose (Bi et al. 2009). In these previous studies glucuronate fermentation by E. asburiae JDR-I generated acetate in nearly 100% yield, indicating fermentation products more reduced than acetate could only be produced from the free xylose and the xylose released from MeGAX in the hydrolysate. The theoretical maximum yield of lactate from this hydrolysate medium was 35.7 mM based on the total xylose initially present. In the fermentation of methylglucuronoxylan hydrolysate, E. asburiae JDR-I utilized all of the MeGAX within 3O h and xylose within 40 h. Similar amounts of ethanol (15.6 mM) and acetate (20 mM) were produced but no 2,3-butanediol or lactate was detected (Table 6, Fig. 5A). When supplemented with LB, E. asburiae JDR-I fermented the 0.5% hydrolysate more rapidly than with ZH minimal salts. Substrates were utilized within 15 h, producing 16.2 mM ethanol, 22 mM acetate, and 3.2 mM succinate, again with no 2,3-butanediol or lactate detected. (Table 6, Fig. 5C).
Fermentation characteristics of the engineered strains E. asburiae El and Ll
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. In order to convert more carbon flux from pyruvate to lactate, 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).
Both 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). In xylose and arabinose fermentations, 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.
After 60 h, only a portion of free xylose in the hydrolysate was utilized and the MeGAX portion was not utilized (Fig. 5B). Within 100 h, 22.2 mM lactate was produced (Table 6). The low fomentation rate of Ll in hydrolysate medium may be due to a limiting activity of lactate dehydrogenase. The absence of detectable lactate formation in the parent strain during fermentation of xylan hydrolysates also indicates a limitation in lactate dehydrogenase activity of E. asburiae JDR-I . The E. asburiae Ll strain fermented more rapidly in the xylan hydrolysate supplemented with LB, with the complete consumption of both MeGAX as well as xylose in 65 h (Fig. 5D) with the formation of 36.4 mM lactate as well as very small amount of acetate and succinate (Table 6). Both El and Ll were able to produce lactate at 100% of the theoretical maximum yield. The small amounts of acetate were likely derived from the glucuronate group of the 1.4 mM MeGAX present in the hydrolysate substrate.
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
The 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 μmol 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. When the same sample was assayed under the same conditions with 4 units L-lactate dehydrogenase, there was no detectable increase in A503. Therefore the lactate produced by E. asburiae Ll was D-lactate with an apparent optical purity 100%.
Conclusion
The fermentations of dilute acid hydrolysates of methylglucuronoxylan by E. asburiae strains El and Ll provide the first examples of lactate formation from the aldouronate as well as the xylose present in these hydrolysates. The efficient formation of the D(-)entantiomer demonstrates a metabolic potential for the efficient production optically pure lactate from the most predominant polysaccharide components in the hemicellulose fractions derived from woody biomass and agricultural residues. Although the relatively low production rate and dependence on rich media limit direct application of E. asburiae Ll, metabolic evolution by adaptive culturing and further genetic engineering may overcome these limitations. EXAMPLE 3 GENETIC ENGINEERING FOR ETHANOL PRODUCTION
Bacterial strains, media, and growth conditions The bacterial strains constructed and used in these studies are listed in Table 8. The E. asburiae JDR-I served as a starting point for genetic engineering. During strain construction, cultures were grown aerobically at 300C, 37°C, or 39°C in Luria broth (10 g I" 1 Difco tryptone, 5 g F ' Difco yeast extract, and 5 g 1" ' NaCl) containing either 2% (w/v) glucose, 5% sucrose or 3% (w/v) arabinose. Ampicillin (50 mg l' 1), tetracycline (12.5 mg I" 1), kanamycin (20 mg F and 50 mg F ), apramycin (20 mg F ]) or chloramphenicol (10 mg F l and 40 mg F ') were added as needed.
Sweetgum methylglucuronoxylan (MeGAXn) was prepared from sweetgum stem wood (Liquidambar styraciflua) as previously described and characterized by C13-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 H2SO4 at 121 0C for 60 min. followed by neutralization with BaCO3. 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.
Genetic methods
Standard methods were used for most of the genetic manipulations. Qiagen kits were used for genomic DNA and plasmid extraction (Qiagen, Valencia, CA). Polymerase chain reaction (PCR) amplification was performed with an I-cycler thermal cycler (Biorad. Hecules, CA) with primers synthesized by Operon (Huntsville, Alabama). Topo cloning kits were used for cloning (Invitrogen, Carlsbad, CA). Electroporation was performed on Gene pulser Xccll (Biorad, Hercules, CA). Restriction endonucleases were purchased from New England Biolabs (Ipswich, MA). DNA sequencing was provided by the University of Florida Interdisciplinary Center for Biotechnology Research. Fermentation
Batch fermentations were carried out in 16- by 100-mm screw-cap tubes filled with nitrogen and sealed with rubber stoppers. The tubes were set in a Glas-Col minirotator at 60 rpm in a 300C incubator. Neutralized sweetgum xylan acid hydrolysate (0.5% w/v) was added to 2x ZH salts directly as growth medium buffered by 100 rnM phosphate buffer or MOPS buffer at pH 7.0. Fermentations in hydrolysates were inoculated to an initial optical density at 600 nm of 1.0 (determined using a Beckman DU500 series spectrophotometer). For analysis of fermentation products, cultures were centrifuged, and the supernatants were passed through 0.22 um filters and subjected to HPLC. Products were resolved on a Bio-Rad HPX-87H column with 0.01 N H2SO4 at 65 ° C. Samples were delivered with a 710B WlSP automatic injector and chromatography controlled with a Waters 610 solvent delivery system at a flow rate of 0.5 ml/min. Products were detected by differential refract ometry with a Waters 2410 RI detector. Data analysis was performed with Waters Millennium Software. A quantitative relationship was determined between E. asburiae JDR-I cell dry weight and culture OD at 600nm. For calculation of specific consumption rates and specific production rates, the cell dry weight was determined based on the OD600 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.
Transformation of E. asburiae JDR-I with plasmids carrying PET operon
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 μl cuvette under the condition of 1.8kV, 25 μF capacitance and 200Ω resistance. For electroporation competent cells from 25 ml exponential phase cultures were washed 3 times by suspension and centrifugation with cold 10% glycerol. Cultures were plated on LB agar containing 2% glucose and tetracycline (12.5 mg I" 1) or chloramphenicol (40 mg I" 1) to select E. asburiae JDR-I and El carrying pLOI297 or pLOI555 respectively. Plasmids were extracted confirming their presence in E. asburiae cells. Deletion of the pflB gene in E. ashuriae JDR-I
The method for gene deletion in E.cυli 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. 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. The pflB gene fragment amplified from E. 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. After electroporation of the step 1 fragment into E. asburiae JDR-I containing pLO13240, cells were incubated for 2 hr at 300C. 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 300C for 4 h and then transferred into a 250-ml flask containing 100 ml of LB minus NaCl with 10% sucrose. Following an overnight incubation (300C), colonies were streaked on LB minus NaCl plates containing 6% w/v sucrose (390C, 16 h). Colonies were tested for loss of apramycin and chloramphenicol resistance and confirmed by PCR. The resulting strain E. asbuήae El had a disrupted pfIB gene without detectable heterogenous DNA sequences.
PIasmid stability in E. asburiae JDR-I E. asburiae JDR-I harboring either pLOI555 or pLOI297 was serially transferred in
Luria broth containing 2% glucose without antibiotics for more than 72 generations at 3O0C. 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. Ten colonies retaining antibiotic resistance (and therefore presumed to retain pLOI555 or pLOI297) after 72 generations were subjected to fermentation to test their ethanol producing ability.
Assay of PDC activity Pyruvate decarboxylase activity was assayed in engineered E. asburiae JDR-I strains by monitoring the pyruvate-dependent oxidation of NADH with alcohol dehydrogenase as a coupling enzyme (Conway et al. J Bacteriol 169:2591-2597 (1987); Ohta et al. Appl Environ Microbiol 57:2810-2815 (1991)). Exponential phase anaerobic cultures were harvested and cells were disrupted using the FastPrep bead mill MP system (MP Biomedicals, Irvine, CA) in 0.05 M phosphate buffer. The supernatant was collected after 15 min centrifugation at 1.8k rpm (Eppendorf centrifuge 5414). The entire process was carried out at 40C. Heat treatment for 15 min at 600C 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 MgCl2, 0.40 mM NADH, 20 mM sodium pyruvate and
0.05 M sodium phosphate buffer, pH 6.5. The assay was started by adding 20 μl crude cell extract. Protein concentration of the crude extract was determined with BCA protein assay reagent kit (Pierce Chemical Co., Rockford, IL).
Results
Fermentation characteristics of the wild type strain E. asburiae JDR-I
E. asburiae JDR-I performed a mixed-acid fermentation in low substrate concentration. When growing in 2.5% (w/v) glucose or 2% (w/v) xylose, the wild type strain produced a wide range of products, including succinate, lactate, acetate, formate, 2,3- butanediol and ethanol (Table 9). In glucose fermentation, 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). In both batch fermentations buffered with 0.1 M sodium phosphate (pH 7.0), the wild type strain failed to utilize all the substrates during the 48 h allotted . Even in the buffered medium the pH after fermentation decreased to 4.8, which suggested that acid production might be the main factor preventing the cells from utilizing all the substrate. The components 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 MeGAX2 (Fig. 7). Previous studies suggested that MeGAX was metabolized by E. asburiae JDR-I into methanol, glucuronate and xylose. Glucuronate fermentation by E. asburiae JDR- 1 generated acetate in nearly 100% yield, indicating more reduced fermentation products (ethanol and lactate) could only come from the free xylose and the xylose released from
MeGAX (Bi et al. Appl Envron Microbiol 75:395-404 (2009)). Therefore, the theoretical maximum yield of ethanol from this hydrolysate was calculated to be 35.7 mM based on the total amount of xylose present in hydrolysate. E. asburiae JDR-I was able to completely utilize MeGAX in the 0.5% hydrolysate in about 12 hours and xylose in 20 hours after a period of several hours for adaptation to the hydrolysate medium. Similar amounts of ethanol (15.6 mM) and acetate (20 mM) were produced with small a amount of formate and no detectable 2,3-butanediol; the ethanol yield was 44.2% of the theoretical maximum (Table 10, Fig. 7, Fig. 8A). The specific consumption rates of xylose and MeGAX in the hydrolysate and specific production rates of acetate and ethanol are included in Table 11.
Fermentation characteristics of E. asburiae JDR-I (pLOI297) and E. asburiae JDR-I
(pLOI555)
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. ashuriae JDR-I (pLOI555) and JDR-I (pLOI297) were tested for the fermentation of dilute acid hyrolysates of sweetgum MeGAXn. Both strains consumed MeGAX as well as xylose within 18 hr and fermentation was complete within 25 hr (Fig. 8C for JDR-I
(pLOI555); data for JDR-I (pLOI297) was not shown). The xylose specific consumption rate of JDR-I (pLOI555) was similar to the parent strain but the MeGAX specific consumption rate was lower. Ethanol was the major fermentation product, and the yield was much higher than the parent strain. However, both strains produced substantial amount of acetate (approximately 10 mM) and had lower yields of ethanol than with either xylose or glucose as substrates (Table 11).
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). This result indicated that only the acetate production pathway initiated from pyruvate formate lyase competed for pyruvate and lowered the ethanol yield. In order to direct greater carbon flux from pyruvate to ethanol, the/?/7B gene of E. asburiae JDR-I was deleted to obtain strain E. asburiae El, followed by pLOI555 transformation. When testing this strain in hydrolysate fermentations, no formic acid was produced, and only small amount of acetate was produced (4.5 mM). After several hours of adaption, the MeGAX portion was consumed in 12 hr and the xylose portion was consumed in 20 hr (Fig. 8D). While the specific consumption rates of the substrates were close to the parent strain and JDR-I (pLOI555), E. ashuriae El (pLOI555) had a much higher specific production rate of the ethanol (0.11 ±0.0 Ig ethanol/g DCW/h) and a much lower specific production rate of the acetate (0.022±0.003 g ethanol/g DCW/h). Most of the carbon sources in the hydrolysates were converted to ethanol, achieving 99% of maximal theoretical yield (Table 10, Table 11, Fig. 7).
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). This result indicated that 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
(pLO1555) (O.l l±O.Ol g ethanol/g DCW/h) (Table 11). Compared with E. coli KOl L E. asburiae El (pLO1555) utilized more substrate in sweetgum hydrolysate and was able to produce 57.8% more ethanol at higher rate.
PDC Activities in E. asburiae strains
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 650C 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)). While crude extracts from both strains showed pyruvate-dependent NADH oxidase activity before heat treatment (data not shown), the wild type strains were unable to oxidize NADH after the heat treatment. However, all three strains carrying plasmid with the PET operon showed substantial PDC activities after heat treatment, indicating the presence of PDC encoded by pdc genes derived from Zymomonas mobilis in E. asburiae strains which carry pLOI297 and pLOI555 plasmids and produce ethanol as the predominant fermentation product.
Piasmid stability in E. asburiae JDRl
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, however, 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
A wild type Enterobacter asburiae strain with limited knowledge of its genetic and physiological properties was genetically engineered for a new metabolic potential. The methodology and protocols developed in this study may provide reference value for engineering other wild type Enterobacter spp. While E. asburiae JDR-I was determined to be relatively resistant to ampicillin and probably other β-lactam antibiotics, it was sensitive to tetracycline (12.5 mg 1" ), kanamycin (20 mg 1" l and 50 mg F [), apramycin (20 mg F l) and chloramphenicol (10 mg F 1 and 40 mg I" 1). To deteπnine if a plasmid-based system developed for use in E. coli could be maintained and function in 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.
To decrease the formation of organic acids, acetate and formate, the pflB gene was then deleted. The convenient one-step gene inactivation method successfully applied to E. coli (Datsenko and Wanner Proc Nat Acad Sci USA 97:6640-6645 (2000)) 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. coli recombinants and required lower concentrations, 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. By maximizing DNA concentration to approximately
5μg/μl and cell concentrations of 1010 cells/1 OOμl 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. In this respect it has been able to outperform E. coli KOI l in medium of sweetgum xylan hydrolysate, which has been developed as a commercial ethanologenic biocatalyst.
The specific PDC activities measured in transformed E. asburiae strains were noticeably lower than those measured in the engineered Klebsiella oxytoca M5Al(Ohta et al. Appl Environ Microbiol 57:2810-2815 (1991)), possibly due to lower copy number of the plasmids pLOI297 and pLOI555 in E. asburiae JDR-I . However, as found with engineered Klebsiella oxytoca strains, E. asburiae JDR-I pLOI297 had higher activity than pLOI555, which may be due to the presence of the colEl replicon in pLOI297 resulting in a higher copy number than in the strain transformed with pLOI555. It was found that E. asburiae El (pLO1555) with highest ethanol yield in hydrolysate had the lowest PDC activity in the glucose culture.
The contribution of the adh gene from pLOI1555 is likely critical to homoethanol production in E. asburiae El as it was in initially generating the ethanologenic strains in E. coli (Ingram and Conway Appl Environ Microbiol 54:397-404 (1988); Ingram et al. Appl Environ Microbiol 53:2420-2425 (1987)). When selected genes were deleted in E. asburiae JDR-I to produce lactate as the predominant product from E. asburiae Ll, fermentations were slow and incomplete without supplementation with Luria Bertani medium (Bi et al. Biotechnol Lett, in press, DOl 10.1007/sl0529-009-0044-z (2009)), supporting the conclusion that efficient fermentation to a targeted product requires high level of expression of the gene encoding the oxido-reductase responsible for generating that final fermentation product during the reoxidation of NADH.
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)). Similar to the studies in Klebsiella oxytoca, 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 (pLOI555) 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 λ 08:237 -61 (2007)).
All patents, patent applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.
Table 1. Fermentation products formed by E. asburiae JDR-I from monosaccharides derived from hemicellulose. Anaerobic cultures were allowed to consume each carbon source, initially at 0.25% w/v. Concentrations of components resolved by HPLC were determined for duplicate cultures by differential refractometry.
Fermentation products (mM) Substrate (0.25% w/v)
Acetic Acid Ethanol Lactic Acid . , acid
D-Xylose lO.l±O.l 10.2±0.7 0 1.6±0.3
D-Glucose 7.2±0.3 9.7±0.5 1.8±0.1 1.6±0.4
D-Mannose 7.5±0.1 9.2±0.2 0.9±0.2 3.2±0.8
D-Galactose 9.0±0.4 9.0±0.3 0 0.7±0.3
L-Arabinose 8.1±0.2 9.5±0.1 0.8±0.2 1.3±0.3
Figure imgf000049_0001
a: Composition of the acid hydrolysate was determined by HPLC and differential refractometry. b: Due to background noise and very small product amounts, accurate data was not obtained for quantification of methanol in the MeGAXn hydrolysate.
°: None detected. Table 3. Distribution of "C in fermentation products formed in anaerobic cultures of E. asburiae JDR-I and E. coli B grown with differentially 13C labeled xylose and glucose. Carbons enriched in 13C in different fermentation products were determined and quantified by 13C-NMR (Fig. 3) and are noted by *. Total Products were quantified by HPLC. The fractions of labeled products to their total products were calculated and noted parenthetically in the table.
Labeled products, mM, and (fraction) labeled with "1C
Fermentation Acetate Ethanol Lactate
CH3C*HOHCOOH+CH3C*
CH3C*OOH CH3C*H2OH H0HC*00H
[2- 13/ CJxylose,
4.8 (0.40) 5.8 (0.43) 0.9 (0.45) E. asburiae JDR-I
[2- 13 Cjxylose, £. coli B 3.0 (0.26) 1.9 (0.27) 2.8 (0.31)
C*H3COOH C*H3CH2OH C*H3CHOHCOOH
[1- 13 C, ]glucose, 2.3 (0.34) 4.6 (0.37) 4.8 (0.38) E. asburiae JDR-I
[6-1 CJglucose, 1.9 (0.28) 4.7 (0.35) 5.4 (0.40) E. asburiae JDR-I
Table 4. Anaerobic molar cell dry weight and ATP yield from different substrates calculated based on estimated YATP, 8, for all substrates in is. asburiae JDR-I.
Fermentation substrates
Glucose Xylose Glucuronate MeGAX
Yivi-substrate (g/mole)a 20.5±1.4 10.2±0.7 10.4±0.3 32.0±] .l
Estimated ATP yield per mole
2.6 1.3 4.0 of substrate 1.3
a: Y]vrsubstrate: molar cell dry weight yields for different substrates, determined in triplicate with indicated standard deviations. Table 5. Bacterial strains and plasmids.
Strain and plasmid Relevant characteristics Source or reference
Strains
E. coli ToplO For general cloning Invitrogen
E. asburiae JDR-I Wild type Described herein
E. asburiae El E. asburiae JDR-I ΔpflB Described herein
E. asburiae Ll E. asburiae JDR-I ΔpflB AaIs Described herein
Plasmids
PLOI3240 Amr red, red recombinase protein Wood et al. (2005) pLOI4162 bla cat; cat-sacB cassette Jantama et al. (2008) pCR 4-TOPO bla kan amp; TOPO TA cloning vector Invitrogen pTOPOpfl pflB (PCR) amplified from E. asburiae Described herein JDR-I and cloned into PCR4-TOPO vector pTOPO4162pfl cai-sacB cassette cloned into pflB in Described herein pTOPOpfl pTOPODpfl PCR fragment amplified from pTOPOpfl, Described herein kinase treated, and self-ligated pTOPOals als (PCR) amplified from E. asburiae Described herein JDR-I and cloned into PCR4-TOPO vector pTOPO4162als cai-sacB cassette cloned into ah in Described herein pTOPOals pTOPODals PCR fragment amplified from pTOPOals, Described herein kinase treated, and self-ligated
Table 6. Comparing fermentation products of wild type and genetically engineered E. asburiae JDR-I strainsa
Fermentation products ( mM)
Ethanol Acetate 2,3- Succinate Lactate Lactate Butanediol %yieldb
E. asburiae JDR-I
0.8% glucose 26.8 11. 5 12.9 5.2 3.9 4.6
0.5% xylose 20.9 17. 5 0 4.1 1 1.9
0.5% arabinose 24.0 17. 1 0 4.2 1 1.9
0.5% xylan hydrolysate 15.6 20 0 0 0 0
0.5% xylan hydrolysate 16.2 22 0 3.2 0 0 with LB
E. asburiae El
0.8% glucose 5.6 0 2.9 2.7 77 91.7
0.5% xylose 3.4 2.8 0 3.2 46 7 89.8
0.5% arabinose 6.5 2.9 0 2.1 41 3 78
0.5% xylan hydrolysate 0 2 0 0 36 2 100.4 with LB
E. asburiae Ll
0.8% glucose 4.4 0 0 1.7 78 9 94.1
0.5% xylose 1.5 2.9 0 1.3 47 .2 90.8
0.5% arabinose 5.0 2.8 0 2.1 49 6 93.6
0.5% xylan hydrolysate0 0 0 0 0 22 2 96
0.5% xylan hydrolysate 0 3 0 1.0 36 4 101.2 with LB a Fermentations were completed within 72 h with minimal media, or otherwise as indicated footnote c. The initial concentrations of 0.8% glucose, 0.5% xylose and 0.5% arabinose media were determined by HPLC to be 42 mM. 31 mM and 31.5 mM, respectively. The 0.5% xylan hydrolysate medium was measured to contain 20 mM xylose and 1.4 mM MeGAX. b Percent of actual yield of lactate to theoretical maximum yield. Maximum yield is defined as 2 mol lactate/mol glucose or 5 mol lactate/3 mol xylose. c This result was obtained after fermentation for 100 h at which time 65 % of the xylose in the hydrolysate was utilized. Table 7. Specific consumption rates and specific production rates of E. asburiae Ll in 5 g/1 acid hydrolysate of swectgum xylana q MeGAX q Xylose (g MeGAX/g q Lactate
Strains (g xylose/g DCW/h) DCW/h) (g lactate/g DCW/h)
E. asburiae Ll in ZH 0.067 ± 0.006 0 0.049 ± 0.003 salts
E. asburiae Ll in 0.13 ± 0.01 0.019 ± 0.002 0.13 ± 0.005
0.12% LB a q Xylose and q MeGAX: Xyose and MeGAX specific consumption rate respectively, as grams of substrate consumed per gram dry cell weight per hour, q Lactate: Lactate specific production rate, products generated per gram dry cell weight per hour.
Table 8. Bacterial strains and plasmids for engineering ethanolgenic E. asburiae.
Strain and plasmid Relevant characteristics Source or reference
Strains
E.coli Topl O For general cloning Invitrogen
E. col i KOU pfl::(pdc adhB' cat) Afrd Ohta et al. Appl Environ Microbiol 57:893-900 (1991)
E. asburiae JDR-I Wild type Described herein
E. asburiae El Enterobacter asburiae JDR-I ΔpflB Described herein
Plasmids
PLOI3240 Am1 red, red recombinase protein Wood et al. Biotechnol Progr
21 :1366-1372 (2005) pLOI297 Tcr pdc+ adhB" Ingram et al. Appl Environ Microbiol 55:1943-1948 (1989) pLO1555 Cmr pdc adhB+ Ohta et al. Appl Environ Microbiol 57:2810-2815 (1991) pLO14162 bla cat; cat-sacB cassette Jantama et al. Biotechnol Bioeng 99:1140-53 (2008) Table 8. Bacterial strains and plasmids for engineering ethanolgenic E. asburiae. pCR 4-TOPO bla kan amp; TOPO TA cloning vector Invitrogen pTOPOpfl pflB (PCR) amplified from E. asburiae. Described herein JDR-I and cloned into PCR4-TOPO vector pTOPO4162pfl cat-sacB cassette cloned into pflB in Described herein pTOPOpfl pTOPODpfl PCR fragment amplified from pTOPOpfl, Described herein kinase treated, and self-ligated
Table 9. Comparison of sugar fermentation products of wild type and genetically engineered E. asburiae JDR-I . Fermentations were earned out at 300C in ZH minimal media for 48 hours as described in the Materials and Methods section.
Fermentation products (mM)
Fermentations Ethanol Succinate Lactate Formate Acetate 2,3- Ethanol yield(% of butanediol theoretical)8
Glucose (2.5% w/v)
E. asburiae JDR- lb 2.0 9.6 39.1 1.0 45.9 45.0 25.6
E. asburiae JDR-I 1 „ . _ 94.1
9.4 3.8 ND 261.6 4^ (pLOI297)
E. asburiae JDR-I
7.7 3.4 ND 265 95.3 (pLO1555)
Xylose (2% w/v)
E. asburiae JOR-lb 12.7 5.6 15.0 25.2 13.4 42.6 32.8
E. asburiae JDR-I ^ - . -
3.6 4.2 ND 217.4 98.0 (pLOI555) a) Percentage of amount of ethanol produced to a theoretical maximal amount. A yield of 100% is defined as 2 mole ethanol/ mole glucose or 5 mole ethanol/ 3 mole xylose. b) E. asburiae JDR-I did not completely utilize the substrates within 48 hours.
Table 10. Fermentation products from acid hydrolysates of sweetgum xylan. Fermentations were carried out at 300C in ZH minimal media for 48 hours as described in the Materials and Methods section. Results were averages of 3 experiments.
Fermentation products (rnM)
Formic Acetic Ethanol yield
Ethanol acid acid (% of theoretical) 4
E. asbnriae JDR-I 4.9±0.4 20.0±0.7 15.6±0.8 44±2 E.coli KO 11 5.9±1.0 10.6±0.3 22.5±0.2 63±1
E. asburiae .TDR-I
4.0±0.4 13.5±0.5 (pLOI555) 26.7±1.0 75±3
E. asburiae JDR-I
3.8±0.3 9.9±0.3 30.0±1.5 (pLO1297) 84±5
E. asburiae El
0 4.5±0.2 35.5±1.1 (pLOI555) 99±3 a: percentage of amount of ethanol produced relative to the theoretical maximum. A yield of 100% is defined as 2 mole ethanol/ mole glucose or 5 mole ethanol/ 3 mole xvlose.
Table 11. Specific consumption rates and specific production rates in acid hydrolysates of sweetgum xylan (5g /liter)a. Results were averages of 3 experiments.
Strains q Xylose q MeGAX q Acetate q Ethanol
E. asburiae JDR-I 0.33±0.04 0.087±0.012 0.13+0.01 0.060±0.009
E.coli KOW 0.38±0.04 ND O.l l±O.Ol 0.074±0.006
E. asburiae JDR-I (pLOI555) 0.29±0.03 0.058±0.012 0.14±0.02 0.052±0.004
E. asburiae El (pLOI555) 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.
Strains Specific Activity(Ua/mg of cell protein)
E. asburiae JDR-I 0
E. asburiae JDR-I (pLOI297) 1.02±0.12
E. asburiae JDR-I (pLOI555) 0.77=0.13
E. asburiae El (pLOI555) 0.53±0.10 a) One U is defined as that amount of the enzyme that catalyzes the conversion of 1 μmole of substrate per minute at room temperature.
Table 13. Plasmid stability of pLOI297 and ρLOI555 in E. asburiae JDR-I . Results were averages of 3 experiments.
Plasm ids % cells retaining antibiotic resistance
After 36 generations After 72 generations pLOI297 29.5±1.3 10.7+2.6 pLOI555 100.0±2.8 98.Id=I 1.8
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Claims

CLAIMSI claim:
1. A process for fermenting MeGAX comprising:
(a) forming a substrate from biomass materials;
(b) subjecting the substrate to acid hydrolysis;
(c) selecting and isolating a strain of Enterobacter asbiiriae that has the ability to ferment MeGAX;
(d) inoculating the acid hydrolyzed substrate with the selected strain of Enterobacter asburiae to ferment MeGAX under conditions favorable for cell viability and conversion of MEGAX to a fermentation product; and
(e) optionally, recovering said fermentation product.
2. The process of claim 1. wherein the Enterobacter asburiae an Enterobacter asburiae strain according to any one of claims 15-27.
3. The process of claim 1, wherein the biomass materials contain hemicellulose.
4. The process of claim 1, wherein the biomass materials comprise sweetgum.
5. The process of claims 1, 3 or 4, wherein the acid hydrolysis is dilute acid hydrolysis.
6. A process for fermenting MeGAX comprising:
(a) selecting and/or isolating a strain of Enterobacter asburiae that has the ability to ferment MeGAX in a biomass;
(b) inoculating culture media comprising MeGAX with the selected strain of Enterobacter asburiae to ferment MeGAX under conditions favorable for cell viability and conversion of MEGAX to a fermentation product; and
(c) optionally, recovering fermentation product from the substrate.
7. The process of claim 6, wherein the Enterobacter asburiae an Enterobacter asburiae strain according to any one of claims 15-27.
8. The process of claim 6, wherein the culture media contains hemicellulose.
9. The process of claim 6, wherein the culture media comprises sweetgum or other biomass.
10. The process according to claim 6. wherein said fermentation product is acetate/acetic acid; ethanol; methanol; succinate/succinic acid; lactate/lactic acid; formate/formic acid; acetate/acetic acid; 2,3-tmtanediol; or combinations thereof.
11. A process for fermenting a substrate comprising:
(a) selecting and isolating a strain of Enterobacter asburiae that has the ability to ferment a biomass substrate;
(b) inoculating culture media comprising said substrate with the selected strain of Enterobacter asburiae and fermenting said substrate under conditions favorable for cell viability and conversion of the substrate to a fermentation product; and
(c) optionally, recovering fermentation product from the substrate.
12. The process of claim 11, wherein the Enterobacter asburiae an Enterobacter asburiae strain according to any one of claims 15-27.
13. The process of claim 1 1 , wherein said fermentation product acetate/acetic acid; ethanol; methanol; succinate/succinic acid; lactate/lactic acid; formate/formic acid; acetate/acetic acid; 2,3-butanediol; or combinations thereof.
14. The process of claim 1 1, wherein said substrate is D-glucose, D-xylose, D- mannose, L-arabinose. D-galactose, glucuronate, or various combinations thereof.
15. An isolated strain of Enterobacter asburiae.
16. The isolated E. asburiae strain of claim 15, wherein said strain is selected from the group consisting of JDR-I, El, and Ll.
17. The isolated E. asburiae strain of any preceding claim, wherein said strain comprises one or more genetic modifications selected from the group consisting of: incorporation and/or overexpression 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.
18. The isolated E. ashuriae strain of any preceding claim, wherein said strain comprises one or more genetic modifications selected from the group consisting of: incorporation and/or overexpression and/or inactivation of a gene encoding L- lactate dehydrogenase; incorporation and/or overexpression and/or inactivation of a gene encoding D- lactate dehydogenase; inactivation of a gene encoding fumarate reductase (frd); inactivation of a gene encoding alcohol/aldehyde dehydrogenase (adh); inactivation of a gene encoding pyruvate formate lyase (pfl); inactivation of a gene encoding acetate kinase (ack); and inactivation of a gene encoding methylglyoxal synthase (mgs).
19. The isolated E. asburiae strain of any preceding claim, wherein said strain 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; inactivation of a gene encoding phosphoenolpyruvate carboxylase; inactivation of a gene encoding acetate kinase; and inactivation of a gene encoding pyruvate formate lyase.
20. The isolated E. asburiae strain of any preceding claim, 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.
21. The isolated E. asburiae strain of any preceding claim, wherein said strain comprises one or more further genetic modifications selected from the group consisting of: inactivation of a gene encoding acetate kinase; inactivation of a gene encoding alcohol dehydrogenase; inactivation of a gene encoding aspartate aminotransferase; inactivation of a gene encoding citrate lyase; inactivation of a gene encoding lactate dehydrogenase; inactivation of a gene encoding methylglyoxal synthase; inactivation of a gene encoding pyruvate oxidase; inactivation of a gene encoding phosphate acety transferase; inactivation of a gene encoding malic enzyme; and inactivation of a gene encoding threonine dehydratase.
22. The isolated E. asburiae strain of any preceding claim, wherein said strain comprises one or more genetic modifications selected from the group consisting of: incorporation and/or overexpression of a gene encoding alanine dehydrogenase; inactivation of a gene encoding alanine racemase; inactivation of a gene encoding lactate dehydrogenase; inactivation of a gene encoding alcohol dehydrogenase; inactivation of a gene encoding fumarate reductase; inactivation of a gene encoding pyruvate formate lyase; inactivation of a gene encoding acetate kinase; and inactivation of a gene encoding methylglyoxal synthase.
23. The isolated E. asburiae strain of any preceding claim, wherein said strain comprises one or more genetic modifications selected from the group consisting of: incorporation and/or overexpression of a gene encoding cellobiose utilizing enzyme; incorporation and/or overexpression of a gene encoding phospho-β- glucosidase; and incorporation and/or overexpression of a gene encoding an endoglucanase or cellulase.
24. The isolated E. asburiae strain of any preceding claim, wherein said strain comprises one or more genetic modifications selected from the group consisting of: inactivation of a gene encoding lactate dehydrogenase; inactivation of a gene encoding pyruvate formatelyase; inactivation of a gene encoding fumarate reductase; inactivation of a gene encoding (FiFo)H+-ATP synthase; inactivation of a gene encoding alcohol/aldehyde dehydrogenase; and inactivation of a gene encoding 2-ketoglutarate dehydrogenase.
25. The isolated E. asburiae strain of any preceding claim, wherein said strain comprises one or more further genetic modifications selected from the group consisting of: inactivation of a gene encoding acetate kinase; and inactivation of a gene encoding pyruvate oxidase.
26. The isolated E. asburiae strain of any preceding claim, wherein said strain comprises one or more genetic modifications selected from the group consisting of: incorporation and/or overexpression of a gene encoding glycerol-3-phosphate dehydrogenase; incorporation and/or overexpression of a gene encoding glycerol-3- phosphatase; incorporation and/or overexpression of a gene encoding glycerol dehydratase: incorporation and/or overexpression of a gene encoding 1,3 -propanediol oxidoreductase; incorporation and/or overexpression of a gene encoding aldose reductase; and incorporation and/or overexpression of a gene encoding glycerol dehydrogenase.
27. The isolated E. asburiae strain of any preceding claim, wherein said strain comprises one or more genetic modifications selected from the group consisting of: inactivation of a gene encoding pyruvate formate lyase; and inactivation of a gene encoding acetolactate synthase.
28. The process of claims 1, 6 or 11, wherein said biomass comprises sweetgum, wood preproccssed 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, com stover, wheat straw and/or combinations thereof.
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