WO2011100571A1 - Bactéries capables d'utiliser du cellobiose, et procédés d'utilisation de ces bactéries - Google Patents

Bactéries capables d'utiliser du cellobiose, et procédés d'utilisation de ces bactéries Download PDF

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WO2011100571A1
WO2011100571A1 PCT/US2011/024561 US2011024561W WO2011100571A1 WO 2011100571 A1 WO2011100571 A1 WO 2011100571A1 US 2011024561 W US2011024561 W US 2011024561W WO 2011100571 A1 WO2011100571 A1 WO 2011100571A1
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bacterium
seq
set forth
coli
acid sequence
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PCT/US2011/024561
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Gregory Mckenzie
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Bp Corporation North America Inc.
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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
    • C12P7/02—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
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • 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
    • 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
    • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
    • 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
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • 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

  • Lignocellulosic biomass represents a renewable source of carbohydrate for biological conversion into fuels and chemicals and, as such, presents an attractive alternative to petroleum-based technology. It is recognized, however, that to reach its full potential, commodity production of ethanol from biomass will require high rates and efficiencies, simple processes, and inexpensive media.
  • Bacteria such as Escherichia coli, have the native ability to metabolize all sugar constituents contained in lignocellulose.
  • the present invention is based, at least in part, on the discovery of new strains of E. coli that use cellobiose as their carbon source. Accordingly, the present invention provides new methods for ethanol production using these novel recombinant bacteria. Thus, the invention described herein provides microorganisms which can effectively transport cellobiose, thereby permitting the microorganism to utilize both cellobiose and cellulose in, for example, the production of ethanol.
  • the bacteria of the invention are recombinant or isolated.
  • the bacteria are ethanologenic.
  • the bacteria are Gram-positive or Gram-negative.
  • Examplary Gram- positive bacteria include Bacillus, Clostridium,
  • Exemplary Gram-negative bacteria include Acinetobacter, Gluconobacter, Escherichia, Zymomonas, Geobacter, Shewanella, Salmonella, Shigella, Enterobacter, Citrobacter, Erwinia, Serratia, Proteus, Hafnia, Yersinia, Morganella, Edwardsiella, and Klebsiella.
  • One specific Gram-negative bacteria of the invention is Escherichia coli.
  • the invention also provides recombinant bacteria derived from Escherichia coli, e.g., KOl l (ATCC55124) or SD7.
  • the invention provides bacteria comprising a porin.
  • Exemplary porins are encoded by the nucleic acid sequence set forth as SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, or a subsequence thereof, and has a polypeptide sequence set forth as SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18, respectively.
  • the bacteria comprise a
  • the phosphotransferase system comprises PTS component IIA, IIB, and/or IIC.
  • the PTS Component IIA is encoded by the nucleic acid sequence set forth as SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or a subsequence thereof, and has a polypeptide sequence set forth as SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21, respectively.
  • the PTS Component IIB is encoded by the nucleic acid sequence set forth as SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a subsequence thereof and has a polypeptide sequence set forth as SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, respectively.
  • the PTS Component IIC is encoded by the nucleic acid sequence set forth as SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, or a subsequence thereof, and has a polypeptide sequence set forth as SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27, respectively.
  • the bacteria of the invention comprise a
  • the beta-glucosidase is encoded by the nucleic acid sequence set forth as SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or a subsequence thereof and has a polypeptide sequence set forth as SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO: 30, respectively.
  • the bacteria of the invention comprise a porin, a
  • the bacteria of the invention may comprise an operon encoding a porin, a phosphotransferase system, and a beta glucosidase, e.g., the nucleic acid sequence set forth as SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33.
  • the bacteria of the invention are E. coli as deposited as NRRL B-50344 (Strain Designation 4e3) as NRRL B-50345 (Strain Designation 6el) or as NRRL B-50346 (Strain Designation 1E1).
  • the bacteria of the invention are E. coli SD7 further comprising SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33.
  • the invention provides methods of producing ethanol by culturing an ethanologenic bacterium in the presence of cellobiose, thereby producing ethanol.
  • the cellobiose is present as a result of the breakdown of cellulose.
  • the bacterium is cultured in minimal media, e.g., AM6.
  • kits comprising a bacterium described herein and instructions for use.
  • the bacterium is ethanologenic.
  • the kits further comprise minimal media, e.g., AM6.
  • Figure 1 is a schematic that shows the genes involved in conferring cellobiose metabolism on a cell.
  • Figure 2 depicts the growth of the cloned operons in ethanologenic E. coli SD7 using the indicated carbon source (either 0.5% glucose or 5% glucose).
  • Figure 3 depicts the growth of the cloned operons in ethanologenic E. coli SD7 using the indicated carbon source (either 0.5% cellobiose or 5% cellobiose).
  • Figure 4 depicts the growth of isolated organisms themselves, grown in AM5 supplemented with either glucose or cellobiose (g) or (c). Detailed Description of the Invention
  • bacterium may include “non-recombinant bacterium” “recombinant bacterium” and “mutant bacterium”.
  • cellobiose is meant to refer to a disaccharide of glucose obtained by partial hydrolysis of cellulose.
  • host and "host bacterium” are used interchangeably and are intended to include a bacterium, e.g., a naturally occurring bacterium or a recombinant bacterium, which serves as a host cell from which a recombinant bacterium of the invention is produced. Hence the recombinant bacterium of the invention is said to be “derived from” the host bacterium.
  • non-recombinant bacterium includes a bacterial cell that does not contain heterologous polynucleotide sequences, and is suitable for further modification using the compositions and methods of the invention, e.g. suitable for genetic manipulation, e.g., which can incorporate heterologous polynucleotide sequences, e.g., which can be transfected.
  • the term is intended to include progeny of the cell originally transfected.
  • the cell is a Gram-negative bacterial cell or a Gram-positive cell.
  • mutant as it refers to bacterium, means a bacterial cell that contains a heterologous polynucleotide sequence, or that has been treated such that a native polynucleotide sequence has been mutated or deleted.
  • mutant as it refers to bacterium, means a bacterial cell that is not identical to a reference bacterium, as defined herein below.
  • a “mutant” bacterium includes a “recombinant” bacterium.
  • ethanologenic means the ability of a bacterium to produce ethanol from a carbohydrate as a primary fermentation product.
  • the term is intended to include naturally occurring ethanologenic organisms and ethanologenic organisms with naturally occurring or induced mutations or ethanologenic organisms with genetic alterations.
  • non-ethanologenic means the inability of a bacterium to produce ethanol from a carbohydrate as a primary fermentation product.
  • the term is intended to include microorganisms that produce ethanol as the minor fermentation product comprising less than about 40% of total non-gaseous fermentation products.
  • ethanol production means the production of ethanol from a carbohydrate as a primary fermentation product.
  • capable of producing ethanol means capable of "ethanol production” as defined herein.
  • the terms “fermenting” and “fermentation” mean the degradation or depolymerization of a complex sugar and bioconversion of that sugar residue into ethanol, acetate and succinate.
  • the terms are intended to include the enzymatic process (e.g. cellular or acellular, e.g. a lysate or purified polypeptide mixture) by which ethanol is produced from a carbohydrate, in particular, as a primary product of fermentation.
  • primary fermentation product is intended to include non-gaseous products of fermentation (e.g., ethanol) that comprise greater than about 40%, 50%,
  • the primary fermentation product is the most abundant non-gaseous product.
  • the primary fermentation product is ethanol.
  • minor fermentation product as used herein is intended to include non-gaseous products of fermentation (e.g., ethanol) that comprise less than 40%, for example 20%, 30%, or 40%, of total non-gaseous product.
  • non-gaseous products of fermentation e.g., ethanol
  • anaerobic conditions in intended to include conditions in which there is significantly less oxygen than is present in an aerobic environment. In particular embodiments, there is 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% less oxygen in the anaerobic environment than in the aerobic environment.
  • SSF simultaneous saccharification and fermentation
  • SSF is a well-known process that can be used for breakdown of biomass to polysaccharides that are ultimately convertible to ethanol by bacteria.
  • SSF combines the activities of fungi (or enzymes such as cellulases extracted from fungi) with the activities of ethanologenic bacteria (or enzymes derived therefrom) to break down sugar sources such as lignocellulose to simple sugars capable of ultimate conversion to ethanol. SSF reactions are typically carried out at acid pH to optimize the use of the expensive fungal enzymes.
  • saccharide “saccharide source,” “oligosaccharide source,” “oligosaccharide,” “complex cellulose,” “complex carbohydrate,” “complex sugar,” “polysaccharide,” “sugar source,” “source of a fermentable sugar” and the like are intended to include any carbohydrate source comprising more than one sugar molecule.
  • Sugars include glucose, xylose, arabinose, mannose, galactose, sucrose, and lactose.
  • saccharide also includes, e.g., disaccharides, trisaccharides, oligosaccharides, and polysaccharides. These carbohydrates may be derived from any unprocessed plant material or any processed plant material. Examples are wood, paper, pulp, plant derived fiber, or synthetic fiber comprising more than one linked
  • lignocellulose represents approximately 90% of the dry weight of most plant material and contains carbohydrates, e.g., cellulose, hemicellulose, pectin, and aromatic polymers, e.g., lignin.
  • carbohydrates e.g., cellulose, hemicellulose, pectin, and aromatic polymers, e.g., lignin.
  • Cellulose makes up 30%-50% of the dry weight of lignocellulose and is a homopolymer of cellobiose (a dimer of glucose).
  • hemicellulose makes up 20%-50% of the dry weight of lignocellulose and is a complex polymer containing a mixture of pentose (xylose, arabinose) and hexose (glucose, mannose, galactose) sugars which contain acetyl and glucuronyl side chains.
  • Pectin makes up l%-20% of the dry weight of lignocellulose and is a methylated homopolymer of glucuronic acid.
  • CMC carboxymethyl cellulose
  • amorphous cellulose e.g., acid-swollen cellulose
  • cellooligosaccharides cellobiose, cellotriose, cellotetraose, and cellopentaose.
  • Cellulose, e.g., amorphous cellulose may be derived from a paper or pulp source (including, e.g., fluid wastes thereof) or, e.g., agricultural byproducts such as corn stalks, soybean solubles, or beet pulp. Any one or a combination of the above carbohydrate polymers is a potential source of sugars for depolymerization and subsequent bioconversion to ethanol by fermentation according to the products and methods of the present invention.
  • the disaccharide is cellobiose.
  • porin is meant to refer to a beta barrel protein that crosses a cellular membrane and acts as a pore through which molecules can diffuse.
  • porin refers to a protein that moves cellobiose across the outer membrane of E. coli.
  • the nucleic acid sequence of exemplary porin genes are set forth as SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3, and encode polypeptides with the amino acid sequences set forth as SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.
  • PTS phosphotransferase system
  • the relevant portion of the phosphotransferase system comprises PTS component IIA, IIB and/or IIC.
  • PTS IIA is meant to refer to the IIA component of the PTS system.
  • the nucleic acid sequence of exemplary PTS IIA genes are set forth as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 and encode polypeptides with the amino acid sequences set forth as SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively.
  • PTS IIB is meant to refer to the IIB subunit of the PTS system.
  • the nucleic acid sequence of exemplary PTS IIB genes are set forth as SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and encode polypeptides with the amino acid sequences set forth as SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively.
  • PTS IIC is meant to refer to the IIC component of the PTS system.
  • the nucleic acid sequence of exemplary PTS IIC genes are set forth as SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 and encode polypeptides with the amino acid sequences set forth as SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively.
  • beta-glucosidase is meant to refer to a glucosidase enzyme that acts upon ⁇ 1->4 bonds linking two glucose or glucose-substituted molecules, for example cellobiose.
  • the nucleic acid sequence of exemplary beta-glucosidase genes are set forth as SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, and encode polypeptides with the amino acid sequcnes set forth as SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO: 30, respectively.
  • mutant nucleic acid molecule or “mutant gene” is intended to include a nucleic acid molecule or gene having a nucleotide sequence which includes at least one alteration (e.g., substitution, insertion, deletion) such that the polypeptide or polypeptide that can be encoded by the mutant exhibits an activity or property that differs from the polypeptide encoded by the wild-type nucleic acid molecule or gene, or wherein a polypeptide is not produced from the mutant gene.
  • alteration e.g., substitution, insertion, deletion
  • mutants refers to a nucleic acid molecule or gene means deletion of a nucleic acid or a gene, or a decrease in the level of expression of a nucleic acid or a gene, wherein the deletion or decrease in expression results in a deletion or decrease in the expression of the polypeptide that can be encoded by the nucleic acid molecule or gene.
  • a mutation also means a nucleic acid molecule or gene having a nucleotide sequence which includes at least one alteration (e.g., substitution, insertion, deletion) such that the polypeptide that can be encoded by the mutant exhibits an activity or property that differs from the polypeptide encoded by the wild-type nucleic acid molecule or gene.
  • polynucleotide or gene derived from a bacterium is intended to include the isolation (in whole or in part) of a polynucleotide segment from the indicated source (i.e., the bacterium) or the purification of a polypeptide from an indicated source (i.e., the bacterium).
  • the term is intended to include, for example, direct cloning, PCR amplification, or artificial synthesis from, or based on, a sequence associated with the indicated polynucleotide source.
  • isolated means partially or completely free from
  • An isolated bacterium can exist in the presence of a small fraction of other bacteria which do not interfere with the properties and function of the isolated bacterium.
  • An isolated bacterium will generally be at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% pure.
  • an isolated bacterium according to the invention will be at least 98% or at least 99% pure.
  • fragment or “subsequence” is intended to include a portion of parental or reference nucleic acid sequence or amino acid sequence, or a portion of polypeptide or gene which encodes or retains a biological function or property of the parental or reference sequence, polypeptide or gene.
  • a portion means exhibits at least 50% of the reference nucleic acid, amino acid sequence, polypeptide or gene (for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) of the function or property of a parental or reference nucleic acid sequence, amino acid sequence, polypeptide or gene.
  • "retains” means exhibits at least 50% (for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) of the function or property of a parental or reference sequence, polypeptide or gene.
  • a "mutant" bacterium includes a bacterium comprising a “mutation” as defined hereinabove.
  • reference or “reference bacterium” includes, at least, a wild-type bacterium or a parental bacterium.
  • wild-type means the typical form of an organism or strain, for example a bacterium, gene, or characteristic as it occurs in nature, in the absence of mutations. "Wild-type” refers to the most common phenotype in the natural population. Wild type is the standard of reference for the genotype and phenotype.
  • parental or “parental bacterium” refers to the bacterium that gives rise to a bacterium of interest.
  • a “gene,” as used herein, is a nucleic acid that can direct synthesis of an enzyme or other polypeptide molecule, e.g., can comprise coding sequences, for example, a contiguous open reading frame (ORF) that encodes a polypeptide, a subsequence or fragment thereof, or can itself be functional in the organism.
  • a gene in an organism can be clustered in an operon, as defined herein, wherein the operon is separated from other genes and/or operons by intergenic DNA. Individual genes contained within an operon can overlap without intergenic DNA between the individual genes.
  • the term "gene" is intended to include a specific gene for a selected purpose.
  • a gene can be endogenous to the host cell or can be recombinantly introduced into the host cell, e.g., as a plasmid maintained episomally or a plasmid (or fragment thereof) that is stably integrated into the genome.
  • a heterologous gene is a gene that is introduced into a cell and is not native to the cell.
  • nucleic acid is intended to include nucleic acid molecules, e.g., polynucleotides that include an open reading frame encoding a polypeptide, a subsequence or fragment thereof, and can further include non-coding regulatory sequences, and introns.
  • the terms are intended to include one or more genes that map to a functional locus.
  • the terms are intended to include a specific gene for a selected purpose.
  • the term gene includes any gene encoding a porin, a phosphotransferase system or a beta-glucosidase, e.g. a
  • the gene or polynucleotide fragment is involved in at least one step in the bioconversion of a carbohydrate to ethanol.
  • a gene in an organism can be clustered in an operon, as defined herein, wherein the operon is separated from other genes and/or operons by intergenic DNA.
  • activity refers to the activity of a gene, for example the level of transcription of a gene.
  • Activity also refers to the activity of an mRNA, for example, the level of translation of an mRNA.
  • Activity also refers to the activity of a protein, for example porin, phosphotransferase system, or beta-glucosidase.
  • An “increase in activity” includes an increase in the rate and/or the level of activity.
  • expression refers to the expression of the protein product of the porin, a phosphotransferase system, and beta-glucosidase, respectively.
  • expression as in "expression of porin” or “expression of a phosphotransferase system gene” or “expression of beta-glucosidase” also refers to the expression of detectable levels of the mRNA transcript corresponding to the porin, a phosphotransferase system, and beta-glucosidase genes, respectively.
  • Gram-negative bacterium is intended to include the art-recognized definition of this term.
  • Exemplary Gram-negative bacteria include Acinetobacter, Gluconobacter, Zymomonas, Escherichia, Geobacter, Shewanella, Salmonella, Enterobacter and Klebsiella.
  • Gram-positive bacterium is intended to include the art-recognized definition of this term.
  • Exemplary Gram-positive bacteria include Bacillus,
  • amino acid is intended to include the 20 alpha-amino acids that regularly occur in proteins.
  • Basic charged amino acids include arginine, asparagine, glutamine, histidine and lysine.
  • Neutral charged amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • Acidic amino acids include aspartic acid and glutamic acid.
  • selecting refers to the process of determining that an identified bacterium produces ethanol in the presence of furfural.
  • obtaining as in "obtaining the recombinant bacterium” is intended to include purchasing, preparing, engineering or otherwise acquiring the recombinant bacterium.
  • providing as in “providing the recombinant bacterium” is intended to include selling, distributing or otherwise making available the recombinant bacterium.
  • NRL followed by a number refers to the organism deposited with the United
  • the present invention provides cells, e.g. bacteria, e.g.
  • ethanologenic bacteria and in particular recombinant bacteria, capable of utilizing cellobiose as a carbon source.
  • the bacteria comprise one or more of a porin, a phosphotransferase system, or a beta-glucosidase, e.g. a phospho-beta-glucosidase.
  • the cell can also be a cell of a single-celled or multi-cellular microorganism, such as a fungus, yeast, or bacterium.
  • the recombinant host cells and recombinant cells derived thereform are intended to include cells suitable for, or subjected to, genetic manipulation, or to incorporate heterologous polynucleotide sequences by transfection.
  • Recombinant host cells include progeny of the host cell originally transfected.
  • the host cell can be a non-recombinant or recombinant bacterial host cell.
  • bacterial host cells in accordance with the invention include Gram-positive bacteria, e.g., Bacillus, Clostridium, Corynebacterium,
  • bacterial host cells include Gram-negative bacteria and include, for example, Acinetobacter, Gluconobacter, Escherichia, Zymomonas, Geobacter, Shewanella, Salmonella, Shigella, Enterobacter, Citrobacter, Erwinia, Serratia, Proteus, Hafnia, Yersinia, Morganella, Edwardsiella, and Klebsiella.
  • Exemplary bacterial host cells in accordance with the invention include non-recombinant bacteria such as, e.g., Escherichia coli B or Escherichia coli W.
  • the invention provides recombinant cells, in particular recombinant bacteria, comprising one or more of porin, a phosphotransferase system, or a beta-glucosidase, e.g. a phospho-beta-glucosidase.
  • the recombinant bacteria of the invention are able to utilize cellobiose as a carbon source.
  • the recombinant bacteria of the invention can be used to produce ethanol. In one embodiment, ethanol is produced as the primary fermentation product.
  • heterologous porin includes heterologous porin, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids
  • beta-glucosidase genes derived from yeast and/or Gram-positive or Gram-negative bacteria.
  • One or more of the genes can be derived from different organisms or from the same organism.
  • the bacterium comprises a porin.
  • the porin is encoded by the nucleic acid sequence set forth as SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3, encoding the polypeptide sequence set forth as SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively.
  • the bacterium comprises a
  • the phosphotransferase system preferably comprises PTS component IIA, IIB, and IIC.
  • the PTS Component IIA is encoded by the nucleic acid sequence set forth as SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6
  • the PTS Component IIB is encoded by the nucleic acid sequence set forth as SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9 encoding the polypeptide sequence set forth as SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively.
  • the PTS Component IIC is encoded by the nucleic acid sequence set forth as SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12 encoding the polypeptide sequence set forth as SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively.
  • the bacterium comprises a
  • the beta-glucosidase is encoded by the nucleic acid sequence set forth as SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 encoding the polypeptide sequence set forth as SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively.
  • the isolated or recombinant bacteria of the invention may comprise one or more of the polypeptides set forth as SEQ ID NO: 16-30. Included within the scope of the present invention are heterologous genes, e.g. heterologous porin, PTS or
  • beta-glucosidase or gene products which differ from naturally-occurring genes, for example, genes which have nucleic acids that are mutated, inserted or deleted, but which encode polypeptides substantially similar and functionally equivalent to the
  • genes can comprise, for example, the naturally occurring porin, PTS or beta-glucosidase genes, one or more genes can be a mutated form of the naturally occurring gene.
  • recombinant E. coli strains are produced comprising the nucleic acid sequences set forth as SEQ ID NOs: 1-8.
  • recombinant E. coli capable of producing ethanol are provided by the invention.
  • the recombinant E. coli is E. coli SD7 further comprising SEQ ID NO:31.
  • SEQ ID NO: 31 was isolated from isolate 6E1 and is shown below.
  • SEQ ID NO:31 comprises a Lacl-like repressor (nucleic acid residues
  • a PTS component IIB (nucleic acid residues 1454-1777), a PTS component IIC (nucleic acid residues 1790-3121), a phospho-beta -glucosidase(nucleic acid residues 3121-4539), a PTS component IIA (nucleic acid residues 4521-4838) and a porin (nucleic acid residues 5023-6417).
  • the recombinant E. coli is E. coli SD7 further comprising SEQ ID NO:32.
  • SEQ ID NO: 32 was isolated from isolate 1E1 and is shown below.
  • SEQ ID NO:32 comprises a Lacl-like repressor (nucleic acid residues 1207-215), a PTS component IIB (nucleic acid residues 1454-1777), a PTS component IIC (nucleic acid residues 1790-3121), a phospho-beta-glucosidase (nucleic acid residues 3121-4539), a PTS component IIA (nucleic acid residues 4521-4838) and a porin (nucleic acid residues 5023-6417).
  • the recombinant E. coli is E. coli SD7 further comprising SEQ ID NO:33.
  • SEQ ID NO: 33 was isolated from isolate 4E3 and is shown below.
  • SEQ ID NO:33 comprises a Lacl-like repressor (nucleic acid residues 1279-287), a PTS component IIB (nucleic acid residues 1526-1849), a PTS component IIC (nucleic acid residues 1862-3193), a Phospho-beta-glucosidase (nucleic acid residues
  • the present invention provides methods of making the recombinant organisms having the aforementioned attributes. Accordingly, in another aspect, the invention provides a method for producing a recombinant bacterium that comprises porin, PTS or beta-glucosidase genes.
  • the genes include a nucleic acid molecule (e.g., a DNA molecule or segment thereof), for example, a polypeptide or RNA-encoding nucleic acid molecule that, in an organism, is separated from another gene or other genes, by intergenic DNA (i.e., intervening or spacer DNA which naturally flanks the gene and/or separates genes in the chromosomal DNA of the organism).
  • a gene can direct synthesis of an enzyme or other polypeptide molecule (e.g., can comprise coding sequences, for example, a contiguous open reading frame (ORF) which encodes a polypeptide) or can itself be functional in the organism.
  • ORF contiguous open reading frame
  • a gene in an organism can be clustered in an operon, as defined herein, wherein the operon is separated from other genes and/ or operons by intergenic DNA. Individual genes contained within an operon can overlap without intergenic DNA between the individual genes. Also included in the scope of the invention are promoterless operons, which are operons lacking the promoter portion (e.g., an frd or ure operon).
  • a gene as described herein includes a gene which is essentially free of sequences which naturally flank the gene in the chromosomal DNA of the organism from which the gene is derived (i.e.
  • an isolated gene includes predominantly coding sequences for a polypeptide (e.g., sequences which encode PTS component polypeptides).
  • microorganisms of the invention comprise nucleic acid molecules comprising nucleotide sequences which set forth herein.
  • the microorganisms of the invention comprise nucleic acid molecules comprising nucleotide sequences which set forth herein.
  • microorganisms can comprise nucleic acid molecules that share sequence identity with the disclosed molecules.
  • the nucleic acid sequences have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the disclosed sequences.
  • Preparation of mutant nucleotide sequences can be accomplished by methods known in the art as described in old, et al., Principles of Gene Manipulation, Fourth Edition, Blackwell Scientific Publications (1989), in Sambrook et al., and in Ausubel et al. Mutant sequences may also be isolated from microorganisms found in nature.
  • the invention also relates to microorganisms comprising proteins encoded by the nucleic acid molecules described above.
  • the proteins of the invention can also be recombinant proteins produced by heterologous expression of the nucleic acid molecules which encode the disclosed protein(s) or a silent mutation thereof, as discussed above.
  • the proteins can have an amino acid sequences which are homologous to the amino acid sequences disclosed herein.
  • the term "homologous”, as used herein, describes a protein having at least about 80% sequence identity or homology with the reference protein, and preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology, in an amino acid alignment.
  • the parent strain is a non-recombinant bacterium.
  • the parent strain can be a naturally occurring non-ethanologenic bacterium, e.g., E. coli W.
  • the parent strain can be a recombinant organism.
  • Exemplary host cells for use in the methods according to the invention include, e.g., E. coli strains B, W, K04 (ATCC 55123), KOl l (ATCC 55124), and K012
  • E. coli ATCC 11303
  • E. coli ATCC 11303
  • DH5cc E. coli K04 (ATCC 55123), E. coli LYOl (ATCC PTA-3466), E. coli W (ATCC
  • the invention provides E. coli SD7 comprising one of more of the genes set forth as SEQ ID NO: 1-8.
  • E. coli SD7 has been engineered as described below and as described in detail in US Provisional Application No.:
  • E. coli SD7 was constructed via the following modifications starting from E. coli W. Introducing the alcohol operon consisting of the Zymomonas mobilis pyruvate decarboxylase gene ipdc), Z. mobilis alcohol dehydrogenase II gene (adhB) and pBR325 chloramphenicol acetyltransferase (cat) gene into the pflB gene). Resulting in strain K03. Amplifying the alcohol operon copy number by selection for high-level chloramphenicol resistance, resulting in strain K04.
  • the method further comprises adding one or more selectable markers, e.g., a marker such as green fluorescent protein.
  • the recombinant bacteria of the invention produce ethanol from an
  • the bacteria of the present invention are capable of using cellobiose as a carbon source.
  • the recombinant bacteria catabolize the simpler sugar into ethanol by fermentation.
  • fermentation conditions are selected that provide an optimal pH and temperature for promoting the best growth kinetics of the producer host cell strain and catalytic conditions for the enzymes produced by the culture (Doran et ah, (1993) Biotechnol. Progress. 9:533-538).
  • a variety of exemplary fermentation conditions are disclosed in U.S. Patents 5,487,989 and 5,554,520.
  • optimal conditions included temperatures ranging from about 25 to about 43° C and a pH ranging from about 4.5 to 8.0. Other conditions are discussed in the Examples.
  • the conversion of a complex saccharide such as lignocellulose is a very involved, multi-step process.
  • the lignocellulose must first be degraded or depolymerized using acid hydrolysis. This is followed by steps that separate liquids from solids and these products are subsequently washed and detoxified to result in cellulose that can be further depolymerized and finally, fermented by a suitable ethanologenic host cell.
  • the fermenting of corn is much simpler in that amylases can be used to break down the corn starch for immediate bioconversion by an ethanologenic host in essentially a one-step process.
  • the present invention provides bacteria that use cellobiose as a carbon source. Accordingly, the invention can utilize a saccharide source that has been, heretofore, underutilized. Consequently, a number of complex saccharide substrates may be used as a starting source for depolymerization and subsequent fermentation using the recombinant bacteria and methods of the invention. Ideally, a recyclable resource may be used in the SSF process.
  • Mixed waste office paper is a preferred substrate (Brooks et al, (1995) Biotechnol. Progress. 11:619-625; Ingram et al, (1995) U.S.P.N. 5,424,202), and is much more readily digested than acid pretreated bagasse (Doran et al., (1994) Biotech.
  • lawn clippings husks, cobs, stems, leaves, fibers, pulp, hemp, sawdust, and newspapers, etc.
  • KOll-RDl a DNA fragment internal to the adhB Zm open reading frame was synthesized by PCR amplification and cloned into pMEV.
  • the pMEY-adhB Zm plasmid was used to remove the pdc Zm -adhB Zm -cat genes from KOll-RDl, by two recombination events with the secondary recombination occurring between the outermost duplicated pflB sequences, selection for sucrose-resistance and screening for chloramphenicol sensitivity.
  • the resultant strain
  • SD1 was shown to be absent the genes for pdc Zm -adhB Zm -cat and restored for the wild- type pflB by PCR and nucleotide sequencing. Deletion of the frdABCD operon from SD1
  • the fumarate reductase of E. coli carries out the reductive conversion of fumarate to succinate and represents a side product of fermentation that would be desirable to remove to increase conversion of biomass derived sugars to ethanol (Ohta, K. et al, 1991).
  • E. coli and other heterofermentative organisms can produce lactic acid from sugars by fermentation (August, B. et al, 1996).
  • a DNA sequence was designed and synthesized to delete the IdhA open-reading frame using E. coli K12 W3110 genome sequence as template. The fragment was cloned into pMEV and the deletion was introduced into the chromosome of SD2 and was verified by PCR and nucleotide sequencing. Because of the use of E. coli K12 sequence to design the DNA fragment for deletion of IdhA from SD2, the single base changes derived from the E. coli K12 sequence.
  • Codon-optimization (indicated as Ec ) resulted in a change in only the nucleotide sequences compared to the native Z. mobilis genes but encodes identical amino acid sequences.
  • a second alcohol dehydrogenase gene, adhA was added to the construct to improve conversion of acetaldehyde to ethanol.
  • an E. coli codon-optimized gene for a green fluorescent protein (gfp Ec° ) was added to the end of the operon to serve as a reporter for gene expression.
  • sequences of the 23S ribosomal gene were chosen to provide homologous sequences flanking the operon. This location was chosen as a site for integration of the alcohol operon because of its documented, high transcription rate (Gourse, R.L. et ah, 1996), making it a desirable location to drive the expression of the alcohol genes.
  • a DNA fragment containing the alcohol operon flanked by sequences of the 23S ribosomal gene was cloned into pMEV.
  • the site of initial integration can take place in any one of them.
  • kanamycin-resistant colonies were selected and assayed for fluorescence due to the expression of the gfp gene.
  • the highest expressing clones were then tested for the ability to ferment xylose to ethanol (xylose was used as substrate because this strain is designed for fermenting pentose sugars from biomass hydrolysate), and the best-performing strain was selected. After selecting the second recombination event by obtaining sucrose-resistant colonies, the complete integration of the new alcohol operon was verified by PCR analysis and nucleotide sequencing. Sequencing of the region adjacent to the integrated operon showed that the 23S ribosomal gene rrlH was the site of integration. The resultant strain is SD4.
  • E. coli strain W has been described in the literature as containing a lysogenic phage, W(
  • ) integrated into its chromosome and capable of producing and releasing phage at a low level (10 3 -104 pfu/ml of culture supernatant) that produce plaques on a E. coli C indicator strain
  • gpN a strategy was adopted to eliminate the expression of the major capsid protein by deletion of the encoding gene, gpN. This modification also resulted in a strain that is immune to further infection by W(
  • a fragment of DNA with sequences flanking the open reading frame of gpN was designed from the W(
  • the resultant strain is E. coli SD5. The strain was tested for its ability to produce phage on the indicator strain E. coli C and was found to be unable to produce detectable infectious particles. In addition, E. coli SD5 was tested based on its known immunity to superinfection with phage produced from E. coli W and was found to be resistant, with no plaque formation seen. Insertion of the Klebsiella oxytoca urease operon.
  • Urease urea amidohydrolase, EC3.5.1.5 converts urea into ammonia and carbon dioxide (Mobley and Hausinger, 1989- a review of microbial ureases). The ammonia can then be assimilated through regular nitrogen assimilation pathways. While E. coli W does not normally contain this enzyme, many aquatic and soil microorganisms do express ureases, including Klebsiella oxytoca M5A1. In one study, urease was found to be expressed by between 17-30% of the cultivated bacteria (Lloyd and Sheaffe, 1973).
  • the urease operon of Klebsiella was first described in a relative of K oxytoca, Klebsiella aerogenes, and consists of seven genes, ureDABCEFG, in a contiguous fragment or operon.
  • the same organization and a high degree of homology at both the nucleotide and protein level (ureD, 78%; UreD, 78%; ureA, 91%; UreA, 97%; ureB, 82%; UreB, 88%; ureC, 85%; UreC, 94%; ureE ,81%; UreE, 87%; ureF, 83%; UreF, 86%; ureG, 87%; UreG, 94%) are seen in K oxytoca M5A1.
  • the urease operon-encoded proteins consist of three structural subunits for the enzyme, UreA, -B and -C, while the remaining four proteins, UreD, -E, -F and -G are required for incorporation of the nickel cofactor of the enzyme (Lee et al, 1992).
  • a 5kb PCR product was generated from K oxytoca M5A1 chromosomal DNA and ligated into a plasmid consisting of the pMEV vector and flanking E. coli W sequences from the downstream end of the proVWX operon.
  • This operon is regulated by osmotic conditions (Lucht and Bremer, 2006) and has been found to be able to similarly regulate the express of heterologous genes (Herbst et al, 1994).
  • the constructed plasmid was used to introduce the urease operon into E. coli SD6. The successful introduction was confirmed by PCR and nucleotide sequencing. The resultant strain was E. coli SD6. Although a number of other sites in the E. coli chromosome were tested for urease expression, this location proved to be optimal for growth and performance.
  • methylglyoxal bypass (Russell and Cook, 1995), which serves to reduce intracellular ATP concentrations which result from exposure of bacteria to high sugar concentrations and inability to balance the flux of energy and carbon through anabolic and catabolic metabolism.
  • the effect of the methylglyoxal pathway is to discharge excess ATP resulting in the production of methylglyoxal from dihydroxyacetone phosphate.
  • Methylglyoxal is a reactive aldehyde and can inhibit the growth and reduce survival of the cell if it is not further metabolized (Booth et al, 2003; Grabar et al, 2006).
  • the methylgloxal synthase gene, mgsA was deleted from SD6.
  • a region of the E. coli W genome sequence flanking the mgsA open reading frame was used to design and synthesize a DNA fragment that was then cloned into pMEV. The deletion was introduced and confirmed by PCR analysis and DNA sequencing. The resultant strain is SD7.
  • Example 2 Example 2
  • Escherichia coli ethanologens are missing one major feature to make it an appropriate ethanologen for both C6 and C5 processes. It cannot use the major cellulose breakdown product cellobiose as a carbon source.
  • MacConkey media contains bile salts and crystal violet. The crystal violet selects against gram positive bacteria, and the bile salts select for enteric bacteria. MacConkey media turns red when the sugar is fermented, so any red colonies on MacConkey cellobiose are enteric gram negative bacteria that can use lactose.
  • Isolates were further screened for their appearance on EMB lactose (a green sheen is characteristic of E. coli) and for their failure to grow on Simmons citrate agar. Together, these features are characteristic metabolic markers of being E. coli.
  • E. coli Being E. coli, it was suspected that the genes that confer cellobiose metabolism on the cell would be easily cloned into other E. coli isolates. Large-insert libraries from the genomic DNA of these bacteria have been built, and these libraries have been transformed into an E. coli
  • K-12 cloning strain Selection for growth on a minimal medium with cellobiose as the sole carbon source provided an excellent way to isolate inserts that contain the genes of interest. As a result, several cosmid clones have been successfully isolated that contain the genes of interest. From the libraries, the genes involved have been cloned.
  • the genes consist of a porin (to move cellobiose across the outer membrane of the cell), a phospho-transferase system and a beta-glucosidase. These genes confer cellobiose-utilization on the host organism, e.g. a bacteria or an ethanologen.

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L'invention porte sur des bactéries recombinées qui sont capables d'utiliser du cellobiose en tant que source de carbone et sur des procédés d'utilisation du métabolisme du cellobiose pour la production d'éthanol.
PCT/US2011/024561 2010-02-12 2011-02-11 Bactéries capables d'utiliser du cellobiose, et procédés d'utilisation de ces bactéries WO2011100571A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014080069A1 (fr) * 2012-11-26 2014-05-30 Neste Oil Oyj Cellules bactériennes oléagineuses et procédés de production de lipides
CN111247250A (zh) * 2018-09-28 2020-06-05 Cj第一制糖株式会社 用于生产L-氨基酸的具有增强的α-葡萄糖苷酶活性的微生物和使用其生产L-氨基酸的方法

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Publication number Priority date Publication date Assignee Title
US4464471A (en) * 1982-02-01 1984-08-07 University Of Cincinnati Biologically engineered plasmid coding for production of β-glucosidase, organisms modified by this plasmid and methods of use
US20060110812A1 (en) * 2000-06-26 2006-05-25 University Of Florida Research Foundation, Inc Methods and compositions for simultaneous saccharification and fermentation
US20100028966A1 (en) * 2008-07-28 2010-02-04 Jeffrey Blanchard Methods and Compositions for Improving The production Of Products In Microorganisms
US20100035320A1 (en) * 2008-07-28 2010-02-11 Jeffrey Blanchard Methods and compositions for improving the production of products in microorganisms

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4464471A (en) * 1982-02-01 1984-08-07 University Of Cincinnati Biologically engineered plasmid coding for production of β-glucosidase, organisms modified by this plasmid and methods of use
US20060110812A1 (en) * 2000-06-26 2006-05-25 University Of Florida Research Foundation, Inc Methods and compositions for simultaneous saccharification and fermentation
US20100028966A1 (en) * 2008-07-28 2010-02-04 Jeffrey Blanchard Methods and Compositions for Improving The production Of Products In Microorganisms
US20100035320A1 (en) * 2008-07-28 2010-02-11 Jeffrey Blanchard Methods and compositions for improving the production of products in microorganisms

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014080069A1 (fr) * 2012-11-26 2014-05-30 Neste Oil Oyj Cellules bactériennes oléagineuses et procédés de production de lipides
WO2014080070A1 (fr) * 2012-11-26 2014-05-30 Neste Oil Oyj Cellules bactériennes oléagineuses et procédés de production de lipides
US9708593B2 (en) 2012-11-26 2017-07-18 Neste Oyj Oleaginous bacterial cells and methods for producing lipids
CN111247250A (zh) * 2018-09-28 2020-06-05 Cj第一制糖株式会社 用于生产L-氨基酸的具有增强的α-葡萄糖苷酶活性的微生物和使用其生产L-氨基酸的方法

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