WO2008018930A2 - Ethanol production in non-recombinant hosts - Google Patents

Abstract

Non-recombinant bacteria that produce ethanol as the primary fermentation product, associated nucleic acids and polypeptides, methods for producing ethanol using the bacteria, and kits are disclosed.

Description

ETHANOL PRODUCTION IN NON-RECOMBINANT HOSTS

Related Application

This application claims priority to U.S. provisional application Ser. Nos. 60/796,652, filed May 1, 2006, and 60/848,234, filed September 29, 2007, the entire disclosures of which are incorporated herein by this reference.

Government Sponsored Research

This work was supported, in part, by Grant No. DE-FG36-04GO14019 from the U.S. Department of Energy. Accordingly, the government may have certain rights to the invention.

Background of the Invention

Ethanol is an attractive alternate transportation fuel to replace at least a part of petroleum (Kheshgi, et al, 2000, Wooley, et al, 1999). Although ethanol is currently produced in the U.S. by fermenting glucose from cornstarch using Saccharomyces cerevisiae (Bothast, et al, 2005), expanding this process to produce a large fraction of the automotive fuel requirement would adversely impact the food and feed industry. Lignocellulosic biomass is an attractive alternative feedstock that can be fermented to ethanol after appropriate pretreatment without impacting food and feed supply (Wyman, et al, 2003, Zaldivar , et al, 2001). In contrast to cornstarch, biomass contains significant amounts of pentose sugars that are recalcitrant to fermentation by yeast.

Conversion of complex sugars to ethanol requires microbial biocatalysts that effectively ferment both hexose and pentose sugars. Towards this goal, recombinant organisms have been developed in which heterologous genes were added to platform organisms such as yeasts, Z. mobilis and E. coli. For example, recombinant ethanologenic Escherichia coli containing the pdc and adh genes from Zymomonas mobilis ferment both hexoses and pentoses to ethanol at high rate and yield (Ingram, et al, 1999). In addition, genetic engineering of yeast and Z. mobilis by adding genes for pentose utilization has yet to yield a biocatalyst that matches the pentose fermentation characteristics of the recombinant ethanologenic Escherichia coli (Kuyper , et al, 2005, Mohagheghi , et al, 2004). Summary of the Invention

As noted above, conversion of lignocellulosic feedstocks to ethanol requires microbial biocatalysts that effectively ferment both hexose and pentose sugars. Such microbial biocatalysts include recombinant organisms in which heterologous genes were added to platform organisms such as yeasts and bacteria, e.g., Zymomonas mobilis and Escherichia coli.

However, the use of a recombinant organism for large-scale fuel production is perceived by some as a barrier to commercialization. Development of a non- recombinant ethanologen may reduce one of the perceived barriers to commercial ethanol production from lignocellulosic substrates.

Accordingly, the invention is based, at least on part, on the discovery of a mutation that redirects glycolysis via a homoethanol pathway in microorganisms that are otherwise non-ethanologenic and the development of non-recombinant ethanologenic microorganisms that ferment glucose and xylose to ethanol under anaerobic conditions based on that discovery. In particular, the pdh operon has been identified as the origin of the homoethanol pathway. More specifically, the lpd gene within the pdh operon has been identified as responsible for homoethanol fermentation by, e.g., E. coli under anaerobic conditions.

Thus, in one aspect, the invention provides an isolated non-recombinant bacterium comprising a mutation, wherein the mutation renders the non-recombinant bacterium capable of producing 4 moles of NADH per mole of sugar under anaerobic conditions.

In another aspect, the invention provides an isolated non-recombinant bacterium comprising a lpd gene having one or more mutations, wherein the mutation renders the non-recombinant bacterium capable of producing ethanol as the primary fermentation product under anaerobic conditions.

The invention also provides isolated nucleic acid molecules encoding dihydrolipoamide dehydrogenase (LPD) polypeptides or functional fragments thereof. When the nucleic acid molecules are expressed in a cell, e.g., a bacterium, the cell produces ethanol as the primary fermentation product.

Thus, in another aspect, the invention provides isolated nucleic acid molecules selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60 % homologous to the nucleotide sequence of SEQ ID NO: 1 or SEQ ED NO: 3, or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 100 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ED NO:

3, or a complement thereof; c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 50% homologous to the amino acid sequence of SEQ ED NO: 2 or SEQ ED NO: 4; d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; wherein the fragment comprises at least 15 contiguous amino acid residues of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; e) a nucleic acid which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ED NO: 4, wherein the nucleic acid molecule hybridizes to a complement of a nucleic acid molecule comprising SEQ ED NO: 1 or SEQ ED NO: 3, under stringent conditions; f) a nucleic acid molecule comprising the nucleotide sequence of SEQ ED NO: 1 or SEQ ID NO: 3, or a complement thereof; and g) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ED NO: 4; wherein the nucleic acid molecule when expressed in a cell, renders the cell capable of producing ethanol as the primary fermentation product.

The invention also provides dihydrolipoamide dehydrogenase polypeptides or functional fragments thereof. When the polypeptides are expressed in a cell, e.g., a bacterium, the cell produces ethanol as the primary fermentation product.

Thus, in another aspect, the invention provides polypeptides selected from the group consisting of: a) a fragment of a polypeptide comprising the amino acid sequence of SEQ ED NO: 2 or SEQ ED NO: 4, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ED NO: 2 or SEQ ED NO: 4; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO; 2 or SEQ ED NO: 4, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule comprising SEQ ID NO; 1 or SEQ ED NO: 3, under stringent conditions; c) a polypeptide which is encoded by a nucleic acid molecule which is at least 50% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3; d) a polypeptide comprising an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ DD NO: 4; and e) an isolated polypeptide comprising the amino acid sequence of SEQ DD NO: 2 or SEQ DD NO: 4; and wherein the polypeptide when expressed in a cell, renders the cell capable of producing ethanol as the primary fermentation product.

In one embodiment, the ethanol produced by the cell comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions, hi another embodiment of this aspect, the polypeptide has dihydrolipoamide dehydrogenase activity under anaerobic conditions, hi a further embodiment, the cell is a bacterial cell. hi a further aspect, the invention provides a bacterial host cell comprising the isolated nucleic acid molecules encoding dihydrolipoamide dehydrogenase polypeptides or fragments thereof.

In another aspect, the invention provides a method for producing a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence SEQ DD NO: 2 or SEQ

DD NO: 4; b) a fragment of a polypeptide comprising the amino acid sequence of SEQ

DD NO: 2 or SEQ DD NO: 4; wherein the fragment comprises at least 15 contiguous amino acids of SEQ DD NO: 2 or SEQ DD NO: 4; and c) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ DD NO: 2 or SEQ DD NO: 4, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a complement of a nucleic acid molecule comprising SEQ DD NO: 1 or SEQ DD NO: 3, under stringent conditions; comprising culturing bacterial host cells containing the isolated nucleic acid molecules encoding dihydrolipoamide dehydrogenase polypeptides or fragments thereof, under conditions in which the nucleic acid molecule is expressed. In a further aspect, the invention provides non-recombinant bacteria as described above, which comprise an isolated nucleic acid molecule described above.

A further aspect of the invention provides a non-recombinant bacterium comprising a lpd gene having one or more mutations, wherein the mutation renders the non-recombinant bacterium capable of producing ethanol as the primary fermentation product under anaerobic conditions, and wherein the bacterium is prepared by a process comprising the steps of: a) growing a candidate mutant strain of the bacterium under anaerobic growth conditions in sugar-rich medium; and b) selecting mutants that produce ethanol as the major product of fermentation.

In another aspect, the invention provides a method of producing ethanologenic non-recombinant bacteria of the invention comprising the steps of: a) growing a candidate mutant strain of the bacterium under anaerobic growth conditions in sugar-rich medium; and b) selecting mutants that produce ethanol as the major product of fermentation.

In one embodiment, the invention provides the isolated non-recombinant bacterium of any of the above-mentioned aspects, wherein the mutation in the lpd gene causes NADH insensitivity.

In another embodiment of the above-described aspects, the mutants result from mutation in the lpd gene. In a particular embodiment, the mutation in the lpd gene causes NADH insensitivity.

In another aspect, the invention provides a method for producing ethanol from an oligosaccharide source. The method comprises contacting the oligosaccharide with a non-recombinant bacterium or host cell of the invention as described above, to thereby produce ethanol from an oligosaccharide source. In a particular embodiment of the method, the oligosaccharide is selected from the group consisting of lignocellulose, hemicellulose, cellulose, pectin and any combination thereof. In yet another aspect, the invention provides a kit comprising a non-recombinant bacterium or host cell of the invention as described above, and instructions for producing ethanol in accordance with the methods and processes described herein. In one embodiment, the kit comprises a sugar source. In still another aspect, the invention provides novel E. coli strains, including strains AH218 (NRRL B-30967), AH241 (NRRL B-30968), AH242 (NRRL B-30969), SE2377 (NRRL B-30970), SE2378 (NRRL B-30971), SE2382 (NRRL B-30972), SE2383 (NRRL B-30973), SE2384 (NRRL B-30974), and SE2385 (NRRL B-30975), which were deposited on September 27, 2006 with the Agricultural Research Culture Collection (NRRL), 1815 N. University Street, Peoria, IL, USA.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

Brief Description of the Drawings

Figure 1 (A) shows the nucleic acid sequence for the lpd gene with a mutation at base 997 (SEQ ID NO: 1) and (B) the corresponding amino acid sequence (SEQ ID NO: 2).

Figure 2 (A) shows the nucleic acid sequence for the lpd gene with a mutation at base 1093 (SEQ ID NO: 3) and (B) the corresponding amino acid sequence (SEQ ID NO: 4).

Figure 3 (A) shows the nucleic acid sequence for the wild type lpd gene (SEQ ID NO: 5) and (B) the corresponding amino acid sequence (SEQ ID NO: 6).

Figure 4 shows a graph depicting the growth and fermentation characteristics of E. coli wild type, strain W3110, and ethanologenic mutant, strain SE2378, in LB-medium with glucose or xylose (50 g L^"1) at 37⁰C and pH 7.0. Panel (A) shows the wild type W3110 strain, grown in glucose; Panel (B) shows the SE2378 strain, grown in glucose; Panel (C), shows the wild type W3110 strain, grown in, xylose; Panel (D) shows the SE2378 strain, grown in xylose

Figure 5 shows the transduction of deletion mutant (aroP-aceEF) (A) (ΔaroP-aceEF) into strain SE2378 (B). Mutations in strain SE2378 were mapped by co-transduction with zac::Tnl0. When aroP-pdhR-aceEF genes were deleted by co-transduction, the transductant (C) lost its ability to grow in LB containing 1% glucose under anaerobic conditions, while the same deletion in wild type background did not affect anaerobic growth.

Figure 6 shows the amino acid sequence of the pdhR gene product from the wild type W3110 strain and the SE2378 mutant (A). The nucleic acid sequence of the intergenic region of strain SE2378 is shown in (B).

Figure 7 shows plasmids used for expression of W3110 or SE2378 lpd in YKlOO host. Plasmid pKY32 (A) contains the lpd gene from the wild type, strain W3110. Plasmid pKY33 (B) contains the lpd gene from the ethanologenic mutant SE2378.

Figure 8 (A - C) is a schematic that shows the proposed pathway for ethanol production from pyruvate in E.coli strain SE2378, native E.coli, and other ethanologenic microorganisms.

Figure 9 shows a multiple amino acid sequence alignment (using the CLUSTAL multiple sequence alignment program) of the LPD of Escherichia coli Kl 2 MGl 655 with selected LPD sequences, i.e. Clostridium tetani E88, Thermoanaerobacter ethanolicus, Bacillus cereus ATCC 10987, Lactobacillus plantarum WCFSl, Lactococcus lactis Subspecies cremoris SKl 1, Oenococcus oeni MCW PSU-I,

Salmonella typhimuήum LT2, Vibrio fischeri ATCC 700601, Shewanella sp ANA-3, Pseudomonas aeruginosa PAOl (ATCC15692), Rhodobacter sphaeroides 2.4.1, Geobacter metallireducens GS- 15, Acinetobacter sp. ADPl, Gluconobacter oxydans 621H, Corynebacterium glutamicum DSM20300, Lactobacillus casei ATCC334, Streptomyces coelicolor M 145/A3 (2), Streptococcus mutans ATCC 700610,

Methanosarcina barkeri Fusaro. The histadine residue at amino acid 322, the proline residue at amino acid 355, and the glutamate residue at amino acid 356 are highlighted with an asterisk (*).

Figure 10 is a graph showing inhibition of PDH activity by NADH. E. coli wild type, strain W3110 or the ethanologenic mutant, strain SE2378. hi the top panel, NAD concentration was 2 mM for both strain W3110 and strain SE2378. In the bottom panel, NAD concentration was 2 mM NAD for the native enzyme from strain W3110 and 1 mM for the mutated form of the enzyme from strain SE2378.

Figure 11 is a graph showing inhibition of LPD by NADH. The ratio of NADH to NAD on the activity of the enzyme was determined for both the native and mutated form of LPD.

Detailed Description of the Invention

In order for the full scope of the invention to be clearly understood, the following definitions are provided.

/. Definitions

As used herein, the terms "non-recombinant bacterium" and "bacterium" are intended to include a bacterial cell that does or does not contain heterologous polynucleotide sequence, 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. In particular embodiments, the cell is a Gram-negative bacterial cell or a Gram-positive cell. The term "derived from" as in "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). In this regard, 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.

The term "anaerobic conditions" in intended to include conditions that do not include oxygen; i.e., conditions in which oxygen is substantially absent. In certain embodiments of the invention, anaerobic conditions comprise a closed vessel or container, for example a vessel closed with a stopper . To create conditions that do not include oxygen, the gas phase is removed from the vessel or container using a vacuum pump, and replaced with nitrogen gas. Oxygen is substantially absent when the oxygen level is too low to be detected. The term "aerobic conditions" is intended to include conditions that include oxygen; i.e., conditions in which oxygen is present. The term "ethanologenic" is intended to include the ability of a microorganism 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. The term "non-ethanologenic" is intended to include the inability of a microorganism 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 40% of total non-gaseous fermentation products. The terms "fermenting" and "fermentation" are intended to include 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.

The terms "primary fermentation product" and "major fermentation product" are used herein interchangeably and are intended to include non-gaseous products of fermentation that comprise greater than about 50% of total non-gaseous product. The primary fermentation product is the most abundant non-gaseous product. In certain embodiments of the invention, the primary fermentation product is ethanol.

The term "minor fermentation product" as used herein is intended to include non-gaseous products of fermentation that comprise less than 40% of total non-gaseous product. In certain embodiments of the invention, the minor fermentation product is ethanol. The term "homoethanol fermentation pathway" as used herein is intended to include the fermentation pathway in an organism, e.g., a bacterium, that facilitates production of ethanol as the primary fermentation product.

The term "alternative fermentation pathway" as used herein is intended to include the fermentation pathway wherein ethanol is not the primary fermentation product.

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, 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. In addition, 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. The terms "pdh operon" and "pdh locus" are used interchangeable and are intended to mean the pdhR, Ipd, and aceEF cluster of genes that are expressed as a group, and their associated promoter and operator. By convention, the term "pdh operon " refers to the genes which encode the operon, whereas the term "PDH" refers to the complex of proteins that are encoded by the operon. Pyruvate dehydrogenase activity is responsible for the production of acetyl CoA for the TCA cycle and energy production. The term pdh operon can include a pdh operon from any aerobic organism. All aerobic organisms, from eukaryotes to humans, contain the three components of PDH. Many bacteria have the genes encoding PDH contained in an operon. The three genes, αceE, αceF and Ipd are essential for the activity of PDH, and these three genes are found in all aerobic organisms whether they are organized as an operon or as independent genes.

The term "dihydrolipoamide acetyltransferase" (αceF) is intended to include the E2 acetyltransferase enzymes of the pyruvate dehydrogenase gene locus. By convention, the term "αceF" refers to a dihydrolipoamide acetyltransferase gene whereas the term "AceF" refers to an αceF gene product, i.e., a dihydrolipoamide acetyltransferase polypeptide or enzyme.

The term "pyruvate decarboxylase/dehydrogenase of the PDH complex" (αceE) is intended to include the El decarboxylase enzyme of the pyruvate dehydrogenase gene locus. By convention, the term "αceE" refers to a pyruvate decarboxylase/dehydrogenase gene whereas the term "AceE" refers to an αceE gene product, i. e. , a pyruvate decarboxylase/dehydrogenase polypeptide or enzyme.

The term "pyruvate dehydrogenase repressor" (pdhR) is intended to include the transcriptional repressor of the pdh operon. By convention, the term "pdhR " refers to a pyruvate dehydrogenase repressor gene whereas the term "PdhR" refers to apdhR gene product, i.e., a pyruvate dehydrogenase repressor polypeptide.

The term "dihydrolipoamide dehydrogenase" (Ipd) is intended to include the enzyme that is part of the pyruvate dehydrogenase gene locus or "pdh operon". By convention, the term "Ipd" refers to a dihydrolipoamide dehydrogenase gene whereas the term "LPD" refers to a Ipd gene product, i.e., a dihydrolipoamide dehydrogenase polypeptide or enzyme. The nucleotide sequence of the wild-type Ipd gene is represented by SEQ ID NO: 5, shown in Figure 3(A), and the amino acid sequence of the polypeptide expressed by the wild- type Ipd gene is represented by SEQ ID NO: 6, shown in Figure 3 (B) .

The term "lactate dehydrogenase" (JdhA) is intended to include the enzyme that converts pyruvate to lactate under fermentative conditions. By convention, the term "idhA " refers to a lactate dehydrogenase gene whereas the term "LDHA" refers to a ldhA gene product, i.e., a lactate dehydrogenase polypeptide or enzyme. The term "pyruvate formate lyase" (pfl) is intended to include the enzyme that converts pyruvate to Acetyl-CoA and formate under fermentative conditions. By convention, the term "pfl " refers to a pyruvate formate lyase gene whereas the term "PFL" refers to a pfl gene product, i.e., a pyruvate formate lyase polypeptide or enzyme. The term "alcohol dehydrogenase" (adhE) is intended to include the enzyme that converts Acetyl-CoA to ethanol under fermentative conditions. By convention, the term "adhE" refers to an alcohol dehydrogenase gene whereas the term "ADHE" refers to a adhE gene product, i.e., a alcohol dehydrogenase polypeptide or enzyme.

The term "NADH insensitivity" means a decrease in sensitivity of the PDH enzyme to NADH. The term is intended to include a partial decrease insensitivity or a complete lack of sensitivity.

The term "nucleic acid" is intended to include nucleic acid molecules, e.g., polynucleotides which include an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences, and introns. In addition, the terms are intended to include one or more genes that map to a functional locus. In addition, the terms are intended to include a specific gene for a selected purpose. In one embodiment, the gene of polynucleotide segment is involved in at least one step in the bioconversion of a carbohydrate to ethanol. Accordingly, the term is intended to include any gene encoding a polypeptide such as a pyruvate decarboxylase, an alcohol dehydrogenase, a secretory polypeptide/s, or a polysaccharase, e.g., a glucanase, or a combination thereof. 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 a pdh operon can overlap without intergenic DNA between the individual genes.

The term "homologous" is intended to include a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent amino acid residues or nucleotides, e.g., an amino acid residue which has a similar side chain, to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains and/or a common functional activity.

The term "heterologous polypeptide" is intended to include a polypeptide or fragment thereof that can be encoded by a heterologous nucleic acid derived from any source, e.g., eukaryotes, prokaryotes, archaea, virii, or synthetic nucleic acid fragments. The term an "isolated polypeptide" {e.g. , an isolated or purified biosynthetic enzyme) is substantially free of cellular material or other contaminating polypeptides from the microorganism from which the polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.

The term "fragment" as in "nucleotide fragment" or "polypeptide fragment" is intended to mean a portion of the nucleotide sequence or polypeptide sequence that is substantially identical to at least a portion of sequence from which it is derived, and where the polypeptide retains the biological activity from the sequence from which it is derived.

The term "pH" is intended to mean a measure of the molar concentration of hydrogen ions in a solution, and as such is a measure of the acidity or basicity of the solution. According to the standard in the art, the term pH is used to define solutions. The usual range of pH values encountered is between 0 and 14, with 0 being the value for concentrated hydrochloric acid (I M HCl), 7 the value for pure water (neutral pH), and 14 being the value for concentrated sodium hydroxide (I M NaOH). The term "pK" is intended to mean a measure of proton binding affinity, and is often used interchangeably with pH. One skilled in the art will recognize that the term pK is used to define proteins, amino acids and peptides. One skilled in the art will also recognize that the acidic strength of the carboxyl, amino and ionizable R-groups in amino acids can be defined by the association constant, K_a or more commonly the negative logarithm of K_a, the pK*.

The term "vector" is intended to include any plasmid vector suitable for ligation of nucleotide sequence of interest and transformation into host cell. The term "sugar" is intended to include any carbohydrate source comprising a sugar molecule(s). Such sugars are potential sources of sugars for depolymerization (if required) and subsequent bioconversion to acetaldehyde and subsequently to ethanol by fermentation according to the products and methods of the present invention. Sources of sugar include starch, the chief form of fuel storage in most plants, and cellulose, the main extracelluloar structural component of the rigid cell walls and the fibrous and woody tissues of plants. The term is intended to include monosaccharides, also called simple sugars, oligosaccharides and polysaccharides. In certain embodiments, sugars include, e.g., glucose, xylose, arabinose, rhannose, galactose, sucrose, and lactose, hi other embodiments, the sugar is glucose. The term "Gram-negative bacterial cell" is intended to include the art-recognized definition of this term. Exemplary Gram-negative bacteria include Acinetobacter, Gluconobacter, Escherichia, Geobacter, Shewanella, Salmonella, Eneterobacter and Klebsella.

The term "Gram-positive bacteria" is intended to include the art-recognized definition of this term. Exemplary Gram-positive bacteria include Bacillus, Clostridium, Corynebacterium, Lactobacillis, Lactococcus, Oenococcus, Streptococcus and Eubacterium.

The phrase "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 that differs from the polypeptide or polypeptide encoded by the wild-type nucleic acid molecule or gene. The term "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. The term "mutagenizing agent" is intended to include any agent that can be used according to the method of the invention to modify a nucleotide sequence.

The term "spontaneous mutation" is intended to include a mutation that occurs in the absence of mutagens. The term can include a mutation that occurs in the method of the invention without the addition of a mutagenizing agent.

//. Non-Recombinant Bacteria

During mixed acid fermentations, the enzymes of glycolysis convert each mole of glucose into 2 moles of pyruvate plus 2 moles of NADH and a net 2 moles of ATP. The production of compounds more reduced than pyruvate (ethanol, lactate, etc.) serves as a mechanism to oxidize NADH and regenerate NAD⁺, essential for continued glycolysis. In the only known homoethanol pathway that evolved in yeast, plants, and bacteria {i.e., Z. mobilis), pyruvate is decarboxylated to yield carbon dioxide and acetaldehyde by the non-oxidative pyruvate decarboxylase. The resulting acetaldehyde serves as the electron acceptor for NADH oxidation by alcohol dehydrogenase during production of one ethanol.

A completely different ethanol pathway exists in many other types of bacteria, in which pyruvate is first converted to acetyl-CoA and formate by pyruvate formate-lyase, an oxidative decarboxylation in which reducing equivalents are contained in the formate and dissipated as hydrogen gas (and CO₂) by formate hydrogen-lyase. Acetyl-CoA is subsequently used as the electron acceptor for the oxidation of two NADH molecules by adhE-encoded aldehyde-alcohol dehydrogenase activities. Due to the requirement of 2 NADH per ethanol, half of the acetyl-CoA remains and is converted to acetate and an additional ATP. Thus the native E. coli pathway for ethanol from acetyl-CoA cannot support homoethanol fermentation due to the need for 2 NADH per ethanol produced from acetyl-CoA. Redox balance is preserved by acetate production. This is the main reason that wild type E. coli produces equimolar amounts of acetate and ethanol during fermentation.

Pyruvate dehydrogenase oxidatively decarboxylates pyruvate to acetyl-Co A and conserves the associated reductant as NADH. This is in contrast to PFL in which the associated reductant is dissipated as hydrogen gas through formate as an intermediate and is not available for metabolic activity in the presence of glucose. By metabolizing pyruvate with PDH, an additional NADH per pyruvate is made available that can be used to fully reduce each acetyl-CoA to ethanol. Although genes coding for pyruvate dehydrogenase are typically expressed under both aerobic and anaerobic conditions in E. coli, the activity of this complex during anaerobic growth is very low.

The invention is based, at least on part, on the discovery of a mutation that redirects glycolysis via a homoethanol pathway in microorganisms that are otherwise non-ethanologenic and the development of non-recombinant ethanologenic microorganisms that ferment glucose and xylose to ethanol under anaerobic conditions based on that discovery. In accordance with this redirected glycolysis, the non- recombinant bacteria of the invention produce 4 moles of NADH per mole of sugar, or 2 NADH per pyruvate, under anaerobic conditions.

Thus, in one aspect, the invention provides a non-recombinant bacterium comprising a mutation, wherein the mutation renders the non-recombinant bacterium capable of producing 4 moles of NADH per mole of sugar under anaerobic conditions. In one embodiment, the mutation is located in apdh operon. In a particular embodiment, the pdh operon comprises pdhR, αceEF and lpd genes. In a further embodiment, the mutation is in the lpd gene.

In another embodiment, the production of 4 moles of NADH per mole of sugar results in the production of ethanol as the primary fermentation product. In a particular embodiment, the sugar is selected from the group consisting of: glucose, xylose, arabinose, mannose, galactose, sucrose, and lactose.

In another aspect, the invention provides a non-recombinant bacterium comprising a lpd gene having one or more mutations, wherein the mutation renders the non-recombinant bacterium capable of producing ethanol as the primary fermentation product under anaerobic conditions. In one embodiment, the ethanol produced comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions.

In a further embodiment, the non-recombinant bacterium, in the absence of the mutation, is non-ethanologenic. In yet a further embodiment, the non-ethanologenic bacterium produces ethanol as a minor fermentation product. In one embodiment, the ethanol produced is less than 40% of the total non-gaseous fermentation products.

In yet a further embodiment of the invention, the mutation in the lpd gene provides a homoethanol pathway by which ethanol is produced by the bacterium as the primary fermentation product. In a further embodiment, one or more alternative pathways for fermentation in the bacterium are inactivated. In one embodiment the alternative pathways are inactivated by mutation. Such a mutation includes deletion, substitution or addition of nucleotides in one or more genes in the alternative pathway. In another embodiment, the mutation is in an ldh gene, e.g. , the ldhA gene. In yet another embodiment, the mutation is in thepfl gene, e.g., the pflB gene. In still another embodiment, the alternative pathways for fermentaion include lactate production by lactate dehydrogenase (JdK), acetate, ethanol, formate or H₂ and CO₂ conversion by pyruvate formate-lyase (pfl) or production of succinate. In various embodiments of the non-recombinant bacteria and bacterial cells described herein, the bacteria are selected from the group consisting of Gram-negative bacteria and Gram-positive bacteria. In certain embodiments, the bacteria are Gram- negative bacteria. In particular embodiments, the Gram-negative bacteria are selected from the group consisting of Acinetobacter, Gluconobacter, Escherichia, Geobacter, Shewanella, Salmonella, Eneterobacter and Klebsella. In other embodiments, the bacteria are Gram-positive bacteria. In particular embodiments, the Gram-positive bacteria are selected from the group consisting of Bacillus, Clostridium, Corynebacterium, Lactobacillis, Lactococcus, Oenococcus, Streptococcus and Eubacterium. In still further embodiments, the bacteria are Escherichia coli. As described above, the non-recombinant bacteria of the invention comprise one or more mutations, e.g., a mutation in an lpd gene. In one embodiment, the mutation comprises substitution of an amino acid with another amino acid, such that the substitution changes the pK of the polypeptide expressed by the mutated lpd gene. In certain embodiments, the mutation in the lpd gene causes NADH insensitivity. That is, a ' cell carrying such a mutation manifests a decrease in sensitivity of the PDH enzyme to NADH. Thus, an NADH insensitive cell produces four NADH molecules per glucose (2 from glycolysis and 2 from PDH reaction), and all four NADHs may be used to reduce two acetyl-CoA to ethanol. In certain embodiments, NADH insensitivity of the PDH to NADH and its ability to function even with a high NADH/NAD ratio enables a cell, e.g., a bacterial cell, to be a homoethanol producer.

In one embodiment, the polypeptide comprises SEQ ID NO: 6 and the mutation comprises a substitution of a wild type amino acid with another amino acid at: a) position 322 or any position within about 50 positions on either side of position 322 in SEQ ID NO: 6; or b) position 354 or any position within about 50 positions on either side of position 354 in SEQ ID NO: 6. In certain embodiments, the other amino acid is a neutral amino acid selected from the group consisting of alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In other embodiments, the other amino acid is a basic amino acid selected from the group consisting of arginine, asparagine, glutamine, histidine and lysine. In one embodiment, the mutation comprises a substitution of H at position 322 with any amino acid, such that the amino acid substitution increases the acidity of the polypeptide expressed by the mutated lpd gene. In a particular embodiment, the non- recombinant bacterium has a mutation that comprises a substitution of H to Y at position 322 in SEQ ID NO: 6. In one embodiment, the non-recombinant bacterium is E. coli strain SE2377, represented by a deposit with the Agricultural Research Culture

Collection and designated as deposit number NRRL B-30970. In another embodiment, the non-recombinant bacterium is E. coli strain SE2383, represented by a deposit with the Agricultural Research Culture Collection and designated as deposit number NRRL B-30973. In yet another embodiment, the non-recombinant bacterium is E. coli strain SE2384, represented by a deposit with the Agricultural Research Culture Collection and designated as deposit number NRRL B-30974. In a further embodiment, strain SE2377 comprises SEQ ED NO: 1, or a fragment thereof. In another further embodiment, strain SE2383 comprises SEQ ID NO: 1, or a fragment thereof. In still another further embodiment, strain SE2384 comprises SEQ ID NO: 1, or a fragment thereof. In another embodiment, the mutation comprises a substitution of E at position

354 with any amino acid, such that the amino acid substitution reduces the acidity of the polypeptide expressed by the mutated lpd gene. In a particular embodiment, the non- recombinant bacterium has a mutation that comprises a substitution of E to K at position 354 in SEQ ID NO: 6. In one embodiment, the non-recombinant bacterium is E. coli strain SE2378, represented by a deposit with the Agricultural Research Culture

Collection and designated as deposit number NRRL B-30971. In another embodiment, the non-recombinant bacterium is E. coli strain SE2382, represented by a deposit with the Agricultural Research Culture Collection and designated as deposit number NRRL B-30972. In yet another embodiment, the non-recombinant bacterium is E. coli strain SE2385, represented by a deposit with the Agricultural Research Culture Collection and designated as deposit number NRRL B-30975. In a further embodiment, strain SE2378 comprises SEQ ID NO: 3, or a fragment thereof. In another further embodiment, strain SE2382 comprises SEQ ID NO: 3, or a fragment thereof. In still a further embodiment, strain 2385, comprises SEQ ID NO: 3, or a fragment thereof.

The non-recombinant bacteria comprising one or more of the mutations described above are suitable for producing ethanol from sugar. In accordance with the invention, the mutation provides a homoethanol fermentation pathway, hi certain embodiments, the ethanol produced comprises greater than 50% of total non- gaseous fermentation products under anaerobic conditions.

In one embodiment, the mutation result from spontaneous mutation, hi another embodiment, the bacterium is exposed to a mutagenizing agent, hi a particular embodiment, the mutagenizing agent is selected from the group consisting of ethyl methane sulfonate, 2-aminopurine, ICR- 191, methyl methane sulfonate, N-methyl-N ' - nitro-N-nitrosoguanidine. hi a further particular embodiment, the mutagenizing agent is ethyl methane sulfonate (EMS).

In another embodiment, one or more alternative pathways for fermentation in the bacterium are inactivated. Alternative pathways for fermentation include lactate production by lactate dehydrogenase (Idh), acetate, ethanol, formate, H₂ and CO₂ starting with pyruvate formate-lyase (pfl) and succinate, hi one embodiment, the alternative pathways for fermentation are inactivated by mutation, hi particular embodiments, the alternative fermentation pathways are inactivated by introducing deletion mutations in the bacterium.

//. Isolated Nucleic Acid Molecules and Genes

The invention also provides isolated nucleic acid molecules encoding dihydrolipoamide dehydrogenase (lpd) polypeptides or fragments thereof. The nucleic acid molecules of the invention comprise an lpd gene with one or more mutations that when present in bacterium of the invention results in the production by the bacterium of ethanol as the primary fermentation product under anaerobic conditions. The nucleic acid molecules of the invention include DNA molecules and RNA molecules and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but advantageously is double-stranded DNA. In one aspect, the invention provides isolated nucleic acid molecules selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60 % homologous to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 100 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or a complement thereof; c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 50% homologous to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; wherein the fragment comprises at least 15 contiguous amino acid residues of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; e) a nucleic acid which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, wherein the nucleic acid molecule hybridizes to a complement of a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO: 3, under stringent conditions; f) a nucleic acid molecule comprising the nucleotide sequence of SEQ ED NO: 1 or SEQ ID NO: 3, or a complement thereof; and g) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ED NO: 4; wherein the nucleic acid molecule when expressed in a cell, renders the cell capable of producing ethanol as the primary fermentation product. In one embodiment, the ethanol produced by the cell comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions. In another embodiment, the cell is a bacterial cell. In yet another embodiment, the bacterial cell, in the absence of expression of the nucleic acid molecule, is non-ethanologenic. In a particular embodiment, the non-ethanologenic bacterial cell produces ethanol as the minor fermentation product; i.e., less than about 40% of total non-gaseous fermentation products.

In another embodiment, the bacterial cell produces ethanol as the primary fermentation product under anaerobic conditions. In a particular embodiment, expression of the nucleic acid molecule in the bacterial cell provides a homoethanol fermentation pathway in the bacterial cell through which ethanol is produced as the primary fermentation product.

In yet another embodiment of this aspect of the invention, the nucleic acid molecule comprises a fragment of SEQ ID NO: 1 wherein the nucleic acid molecule is at least 100 nucleotides in length and contains a T at a position corresponding to position 997 ofSEQ ID NO:l.

In another embodiment, the nucleic acid molecule comprises a fragment of SEQ ID NO: 3 wherein the nucleic acid molecule is at least 100 nucleotides in length and contains a G at a position corresponding to position 1023 of SEQ ID NO: 1.

In one embodiment, the lpd nucleic acid molecule of the invention is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more identical to the nucleotide sequence (e.g., when compared to the overall length of the nucleotide sequence) shown in SEQ ID NO: 1 or SEQ ID NO: 3, or a complement thereof. SEQ ID NO: 1 and SEQ ID NO: 3 are shown in Figures l(A) and 3(A), respectively.

In another embodiment, the invention provides an isolated nucleic acid molecule comprises a fragment of at least 100, 150, 200, 250, or 300 nucleotides of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or a complement thereof.

In another particular embodiment, the invention provides a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, shown in Figure l(B) and Figure 2 (B).

In another embodiment, the nucleic acid molecule encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ED NO: 4, wherein the fragment comprises at least 15, 25, 35, 45, 55, 65 contiguous amino acid residues of the amino acid sequence of SEQ ID NO: 2 or SEQ ED NO: 4.

In a further aspect, the invention provides non-recombinant bacteria as described above, which comprise an isolated nucleic acid molecule described above. In one embodiment, the non-recombinant bacterium produces ethanol from a sugar. In another embodiment, the sugar is selected from the group consisting of glucose, xylose, arabinose, mannose, galactose, sucrose, and lactose.

The lpd genes, as described herein (and italicized by convention), 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. 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. An embodiment of the present invention features mutant lpd nucleic acid molecules or genes. Typically, a mutant nucleic acid molecule or mutant gene as described herein, includes 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 that differs from the polypeptide or polypeptide encoded by the wild-type nucleic acid molecule or gene. Advantageoulsy, a mutant nucleic acid molecule or mutant gene {e.g., a mutant lpd gene) encodes a LPD polypeptide having improved activity, e.g., dihydrolipoamide dehydrogenase activity.

In one embodiment, a nucleic acid molecule of the invention hybridizes under stringent conditions to a nucleic acid molecule having a nucleotide sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 3. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. A particular, non-limiting example of stringent {e.g. high stringency) hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65⁰C. Advantageously, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID SEQ ED NO: 1, SEQ ID NO: 3 corresponds to a naturally occurring nucleic acid molecule. Typically, a naturally occurring nucleic acid molecule includes an RNA or DNA molecule having a nucleotide sequence that occurs in nature.

A nucleic acid molecule of the present invention (e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3) can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) or can be isolated by the polymerase chain reaction using synthetic oligonucleotide primers designed based upon the sequence of SEQ ED NO: 1, SEQ ID NO: 3. A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3.

Additional lpd nucleic acid sequences are those that comprise the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, that encode a homologue of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 (e.g., encode a polypeptide having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more identity to the polypeptide having the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, and having a substantially identical activity as the polypeptide), hybridize under stringent conditions to all or a fragment of a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3 or to all or a fragment of a nucleic acid molecule that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or are complementary to a lpd nucleotide sequence as set forth herein, and such that the lpd nucleic acid sequences, when expressed in a cell, result in the production by the cell of ethanol as the primary fermentation product under anaerobic conditions

In one embodiment, the nucleic acid molecule encodes a polypeptide or a biologically active fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, wherein the polypeptide or the biologically active fragment retains the ability to produce ethanol in a host cell.

In another embodiment, an lpd nucleic acid molecule or gene encodes a homologue of the LPD polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4,. Typically, the term "homologue" includes a polypeptide or polypeptide sharing at least about 30-35%, advantageously at least about 35-40%, more advantageously at least about 40-50%, and even more advantageously at least about 60%, 70%, 80%, 90% or more identity with the amino acid sequence of a wild-type polypeptide or polypeptide described herein and having a substantially equivalent functional or biological activity as the wild-type polypeptide or polypeptide. For example, a LPD homologue shares at least about 60%, advantageously at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more identity with the polypeptide having the amino acid sequence set forth as SEQ ID NO: 2 or SEQ ID NO: 4, and has a substantially equivalent functional or biological activity (i.e., is a functional equivalent) of the polypeptide having the amino acid sequence set forth as SEQ ID NO: 2 or SEQ ID NO: 4 (e.g., has a substantially equivalent dihydrolipoamide dehydrogenase activity).

In an embodiment, an lpd nucleic acid molecule or gene comprises a nucleotide sequence that encodes a polypeptide as set forth as SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, an lpd nucleic acid molecule hybridizes to all or a fragment of a nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, or hybridizes to all or a portion of a nucleic acid molecule having a nucleotide sequence that encodes a polypeptide having the amino acid sequence of any of SEQ ID NO: 2 or SEQ ID NO: 4.

Such hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel , et al., eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additional stringent conditions can be found in Molecular Cloning: A Laboratory Manual, Sambrook , et al., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), chapters 7, 9 and 11. A particular, non-limiting example of stringent hybridization conditions includes hybridization in 4X sodium chloride/sodium citrate (SSC), at about 65-70⁰C (or hybridization in 4X SSC plus 50% formamide at about 42-50°C) followed by one or more washes in IX SSC, at about 65- 7O⁰C. A particular, non-limiting example of highly stringent hybridization conditions includes hybridization in IX SSC, at about 65-70⁰C (or hybridization in IX SSC plus 50% formamide at about 42-50⁰C) followed by one or more washes in 0.3X SSC, at about 65-70⁰C. A particular, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4X SSC, at about 50-60⁰C (or alternatively hybridization in 6X SSC plus 50% formamide at about 40-45⁰C) followed by one or more washes in 2X SSC, at about 50-60⁰C. Ranges intermediate to the above-recited values, e.g., at 65-70⁰C or at 42-5O⁰C are also intended to be encompassed by the present invention. SSPE (IX SSPE is 0.15 M NaCl, 1OmM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (IX SSC is 0.15 M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes each after hybridization is complete. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10⁰C less than the melting temperature (T_m) of the hybrid, where T_m is determined according to the following equations. For hybrids less than 18 base pairs in length, T₀₁(⁰C) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in length, T_m(°C) = 81.5 + 16.6(1Og₁₀[Na⁺]) + 0.41(%G+C) - (600/N), where N is the number of bases in the hybrid, and [Na⁺] is the concentration of sodium ions in the hybridization buffer ([Na⁺] for IX SSC = 0.165 M).

It will also be recognized by the skilled practitioner that additional reagents can be added to hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like. When using nylon membranes, in particular, an additional, non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65⁰C, followed by one or more washes at 0.02M NaH₂PO₄, 1% SDS at 65⁰C, see e.g., Church and Gilbert (1984) Proc. Natl. Acad. ScL USA 81:1991-1995, (or, alternatively, 0.2X SSC, 1% SDS). hi another embodiment, an isolated nucleic acid molecule comprises a nucleotide sequence that is complementary to a lpd nucleotide sequence as set forth herein (e.g., is the full complement of the nucleotide sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 3).

///. Polypeptides

The invention features polypeptides (e.g., mutant ethanologenic enzymes, for example, dihydrolipoamide dehydrogenase (LPD)). When the polypeptides are expressed in a cell, e.g., a bacterium, the cell produces ethanol as the primary fermentation product under anaerobic conditions. Thus, in another aspect, the invention provides polypeptides selected from the group consisting of: a) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ED NO: 4, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 4; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO; 2 or SEQ ID NO: 4, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule comprising SEQ ID NO; 1 or SEQ ID NO: 3, under stringent conditions; c) a polypeptide which is encoded by a nucleic acid molecule which is at least 50% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: l or SEQ ID NO: 3; d) a polypeptide comprising an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ED NO: 4; and e) an isolated polypeptide comprising the amino acid sequence of SEQ ED NO: 2 or SEQ ED NO: 4; and wherein the polypeptide when expressed in a cell, renders the cell capable of producing ethanol as the primary fermentation product.

In one embodiment, the ethanol produced by the cell comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions. In another embodiment of this aspect, the polypeptide has dihydrolipoamide dehydrogenase activity under anaerobic conditions. En a further embodiment, the cell is a bacterial cell.

In yet another embodiment, the bacterial cell, in the absence of expression of the polypeptide, is non-ethanologenic. In a particular embodiment, the non-ethanologenic bacterial cell produces ethanol as the minor fermentation product; i.e., less than about 40% of total non-gaseous fermentation products.

In a further embodiment, the bacterial cell produces ethanol as the primary fermentation product under anaerobic conditions, and in yet a further embodiment the. the ethanol produced comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions. In a particular embodiment, expression of the polypeptide in the bacterial cell provides a homoethanol fermentation pathway in the bacterial cell.

In another embodiment, the isolated polypeptide of the invention is a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ E) NO: 4, wherein the fragment comprises at least 15, 25, 35, 45, 55, or 65 contiguous amino acid residues of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4

In another embodiment, the invention provides an isolated polypeptide having at least about 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more identity (e.g. , when compared to the overall length of the amino acid sequence) to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:4.

In a further aspect, the invention provides a bacterial host cell comprising the isolated nucleic acid molecules encoding dihydrolipoamide dehydrogenase polypeptides or fragments thereof.

In one embodiment, the bacterial host cell comprises a vector comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or a fragment thereof. In another embodiment, the bacterial host cell comprises the vector is pKY33.

The invention also provides a method for producing a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 4; b) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ED NO: 4; wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 4; and c) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a complement of a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO: 3, under stringent conditions; comprising culturing bacterial host cells containing the isolated nucleic acid molecules encoding dihydrolipoamide dehydrogenase polypeptides or fragments thereof, under conditions in which the nucleic acid molecule is expressed.

In one embodiment, the LPD polypeptide or gene product is derived from a non- recombinant ethanologenic Gram-positive or Gram-negative bacterium. In exemplary embodiments, the LPD polypeptide or gene product is derived from an ethanologenic Gram-negative microorganism selected from the group consisting of

Acinetobacter.Gluconobacter, Escherichia, Geobacter, Shewanella, Salmonella, Eneterobacter and Klebsella.

In another embodiment, the LPD polypeptide or gene product is derived from an ethanologenic Gram-positive microorganism selected from the group consisting of Bacillus, Clostridium, Corynebacterium, Lactobacillis, Lactococcus, Oenococcus, Streptococcus and Eubacterium.

Included within the scope of the present invention are LPD polypeptides or gene products that are Escherichia coli derived polypeptides or gene products encoded by naturally occurring bacterial genes. Further included within the scope of the present invention are bacterial-derived polypeptides or gene products which differ from naturally-occurring bacterial and/or Escherichia coli genes {e.g., Ipd), for example, genes which have nucleic acids that are mutated, inserted or deleted, but which encode polypeptides substantially similar to the naturally-occurring gene products of the present invention, e.g., comprise a dihydrolipoamide dehydrogenase activity. It is well understood that one of skill in the art can mutate {e.g. , substitute) nucleic acids which encode for conservative amino acid substitutions. It is further well understood that one of skill in the art can substitute, add or delete amino acids to a certain degree without substantially affecting the function of a gene product {e.g., dihydrolipoamide dehydrogenase) as compared with a naturally-occurring gene product, each instance of which is intended to be included within the scope of the present invention.

Included within the scope of the invention are non-recombinant bacterium comprising an Ipd gene comprising a mutation, wherein the substitution is a mutation of H at position 322, or E at position 354, in the wild type lpd gene (SEQ DD NO: 6), to any amino acid, such that the amino acid alters the acidity of the region. In further embodiments, the amino acid is a neutral charged amino acid at physiological pH. In yet further embodiments, the amino acid is a basic charged amino acid at physiological pH. In an embodiment, an isolated polypeptide of the present invention {e.g. , an isolated dihydrolipoamide dehydrogenase enzyme) has an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. In other embodiments, an isolated polypeptide of the present invention is a homologue of at least one of the polypeptides set forth as SEQ ID NO: 2 or SEQ ED NO: 4 (e.g., comprises an amino acid sequence at least about 30- 40% identical, advantageously about 40-50% identical, more advantageously about 50- 60% identical, and even more advantageously about 60-70%, 70-80%, 80-90%, 90-95% or more identical to the amino acid sequence of SEQ DD NO: 2 or SEQ DD NO: 4, and has an activity that is substantially similar to that of the polypeptide encoded by the amino acid sequence of SEQ DD NO: 2 or SEQ DD NO: 4, respectively. To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions x 100), advantageously taking into account the number of gaps and size of the gaps necessary to produce an optimal alignment. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A particular, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. ScL USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul , et al. (1990) J. MoI. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST polypeptide searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to polypeptide molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul , et al. (1997) Nucleic Acids Research 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another particular, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) Comput Appl Biosci. 4:11-17. Such an algorithm is incorporated into the ALIGN program available, for example, at the GENESTREAM network server, IGH Montpellier, FRANCE or at the ISREC server. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

For example, in one embodiment of the invention the percent identity between two amino acid sequences is determined using the Blast server at NCBI or ClustalW at the European Biotechnology Institute.. For instance,the amino acid sequence of dihydrolipoamide dehydrogenase from various organisms is compared to that of E. coli Lpd protein, and the percent identity of a specific sequence to that of the E. coli sequence can be obtained from either of the two databases. Table 7 and Figure 9 illustrate these comparisons. The values in parenthesis represent the total similarity of the specific protein to that of the E. coli Lpd and include both the amino acid positions that are identical as well as the positions at which a conservative substitution occurred. For Bacilus subtilis, two dihydrolipoyl dehydrogenases, one from the PDH complex and the other from acetoin dehydrogenase, were included, for comparison. One of ordinary skill in the art will recognize that based on the aforementioned calculations, there is a high degree of conservancy in LPD among a range of bacterial species, including bacteria that are far removed from each other phylogentically (based on 16S ribosomal RNA(DNA) sequence), such as, e.g., Gram-positive and Gram- negative bacteria, archaea and Streptomyces.

The enzyme puruvate dehydrogenase is found in all aerobic organisms and is the pivotal enzyme in the conversion of glucose to energy. Dihydrolipoamide dehydrogenase (Lpd) is one of the three subunits of the PDH complex. The Lpd contains two unique motifs: a flavin binding motif (amino acids 15-45) and a pyridine nucleotide-disulfide oxidoreductase motif (amino acids 347-456) (E. coli Lpd numbering). The amino acid sequences of the Lpd proteins from several organisms have significant homology due to their unique role in PDH complex The amino acid sequence identity between E. coli Lpd and other bacterial Lpds ranges from 30 % to 99 %. In the extreme case of the Lpd from E. coli and Human, 42% of the amino acids in the sequence are identical. In the flavin binding region of the Lpd (amino acids 15-45), this sequence identity increases to 67%. hi one sub-section of this sequence of 18 amino acids (positions 28-55), all but one amino acid is conserved in the Lpd sequences of E. coli, human and mouse. Histidine at 322 and glutamate at 354 are also conserved among these proteins. Due to this very high degree of sequence conservation, E. coli Lpd mutations that are described in the present disclosure are expected to have similar phenotypes upon introduction into the Lpd proteins from other organisms. Thus, the methods of the invention are not limited to the strains taught herein.

IV. Methods of Making Non-Recombinant Bacterium

A further aspect of the invention provides a non-recombinant bacterium comprising a lpd gene having one or more mutations, wherein the mutation renders the non-recombinant bacterium capable of producing ethanol as the primary fermentation product under anaerobic conditions, and wherein the bacterium is prepared by a process comprising the steps of: a) growing a candidate mutant strain of the bacterium under anaerobic growth conditions in sugar-rich medium; and b) selecting mutants that produce ethanol as the major product of fermentation.

In one embodiment of the method, the ethanol produced comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions, hi another aspect, the invention provides a method of producing the ethanologenic non-recombinant bacteria of the invention comprising the steps of: a) growing a candidate mutant strain of the bacterium under anaerobic growth conditions in sugar-rich medium; and b) selecting mutants that produce ethanol as the major product of fermentation. In embodiments of the foregoing methods and processes of the invention, the sugar in the sugar-rich medium is selected from the group consisting of glucose, xylose, arabinose, mannose, galactose, sucrose, and lactose. In one embodiment of the present invention, the non-recombinant bacterium having the aforementioned attributes is also ethanologenic. Accordingly, the invention provides methods for making the ethanologenic non-recombinant bacterium. Further, the invention provides methods for screening for the desired ethanologenic phenotype.

The parent strain of the invention is characterized by a low level of ethanol production under anaerobic conditions, when grown in sugar rich medium. An example of such a strain could be strain AH242; however, any strain that is characterized by low levels of ethanol production under anaerobic conditions is suitable for use in the method. Further mutation of the parent strain according to known methods in the art (Dastenko and Wanner, 2000. Proc. Natl. Acad. Sci. USA 97:6640-6645.) are carried out to render the parent strain incapable of anaerobic growth (defective) in all media. Additionally, a cassette for antibiotic resistance is added for selection purposes, according to practice well known in the art.

Typically, selection is carried out by culturing the growth defective strain in aerobic conditions until mid exponential phase of growth is reached, spreading the culture on agar, and exposing the culture to mutagenizing agent. One of ordinary skill in the art will recognize that a number of mutagenizing agents can be used, including ethyl methane sulfonate, 2-aminopurine, ICR-191, methyl methane sulfonate, N-methyl-N'- nitro-N-nitrosoguanidine, or any other agent known to cause a change in nucleotide sequence. After exposure to mutagenizing agents in anaerobic conditions, the cultures are switched to aerobic conditions, then back to anaerobic conditions. Colonies that grew were chosen and streaked on to fresh plates and grown under anaerobic conditions. Each colony can be separately cultured and grown on the appropriate antibiotic plate to confirm that the mutant carries the antibiotic resistance of the parent. One of ordinary skill in the art will understand that bacterial culture procedures are carried out according to protocol standard to the art. High performance liquid chromatography can be used to determine the yield of fermentation products in the spent medium of the isolated mutants. For example, ethanol, acetate, formate and succinate can be detected by HPLC. One of ordinary skill in the art can recognize that based on the aforementioned examples, and based on homology among bacterial strains, the methods of the instant invention are not limited to the strains taught in the instant application.

V. Methods for Producing Ethanol

In another aspect, the invention provides a method for producing ethanol from an oligosaccharide source. The method comprises contacting the oligosaccharide with a non-recombinant bacterium or host cell of the invention as described above, to thereby produce ethanol from an oligosaccharide source. In a particular embodiment of the method, the oligosaccharide is selected from the group consisting of lignocellulose, hemicellulose, cellulose, pectin and any combination thereof.

The host cell of the invention is characterized by a low level of ethanol production under anaerobic conditions. Wild type E. coli produces ethanol and acetate at a ratio of 1 : 1 during anaerobic growth. During stationary phase of growth, wild type E.coli produces lactate as the main product, and the fraction of ethanol in the total fermentation products is about 20%. The products in all these fermentations comprise various acids, thus leading to the term, mixed acid fermentation. In one aspect, the instant invention provides a non-recombinant bacterium comprising an lpd gene having one or more mutations, wherein the mutation renders the non-recombinant bacterium capable of producing ethanol as the primary fermentation product under anaerobic conditions. The primary fermentation product is intended to include non-gaseous products of fermentation that comprise greater than 50% of total non-gaseous product. The primary fermentation product is the most abundant non-gaseous product.

Typically, 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 al., (1993) Biotechnol. Progress. 9:533-538). For example, for Klebsiella, e.g., the P2 strain, optimal conditions were determined to be between 35-37° C and pH 5.0- pH 5.4. Under these conditions, even exogenously added fungal endoglucanases and exoglucanases are quite stable and continue to function for long periods of time. Other conditions are discussed in the Examples. Moreover, it will be appreciated by the skilled artisan, that only routine experimentation is needed, using techniques known in the art, for optimizing a given fermentation reaction of the invention. See, for example, U.S. patent Nos. 5,424,202 and 5,916,787, which are specifically incorporated herein by this reference.

In yet another aspect, the invention provides a kit comprising a non-recombinant bacterium or host cell of the invention as described above, and instructions for producing ethanol in accordance with the methods and processes described herein. In one embodiment, the kit comprises a sugar source.

VL Exemplification

The invention is further illustrated by the following examples, which should not be construed as limiting. Throughout the examples, the following materials and methods are used unless otherwise stated.

Materials and Methods Bacterial Strains E. coli K- 12 strain W3110 (ATCC 27325) and a derivative, strain AH242, represented by a deposit with the Agricultural Research Culture Collection and designated as deposit number NRRL B-30967 (ΔldhA and A(focA-pβB)), were used in this study. Strain SΕ2378 is an ethanologenic mutant of strain AH242. Deletion of the genes pflB, adhE, mgsA and aceF were as per Dastenko , et al . The ldhA deletion strain was constructed after introduction of transposon TnIO into ldhA followed by selection in fusaric acid medium (Klekner , et al. 1991; Maloy , et al.1981). Construction of the other strains utilized standard genetic and molecular biology techniques (Maniatis , et al. 1982; Miller , et al. 1972). The genotypes of the strains used herein are listed in Table 1, shown below.

Table 1 : Bacterial strains and Relevant Genotype

Strain Relevant Genotype Source

W3110 ATCC 27325

AH240

This study

AH241 Δ(M/L4) This study

AH242 A[IdIiA) Δ(focΛ-pflB)-ΕRτ-Km-TKT This study

SE2378 AH242. Anaerobic growth-plus This study

YKl SE237S, Kni^S This study

YK29 AH242. Km⁵ This study

YK91 YKl. Δ(<κtø£)-FRT-Km-FRT This study

YK93 YKl, Δ(αceF)-FRT-Kni-FRT This study

YK96 YKl, Δ(>HgsΛ)-FRT-Km-FRT This study

YK152 YK29, Δ(flceF)-FRT-Km-FRT This study

YK153 W3110, Δ(øcef>FRT-Km-FRT This study

YK157 YK152. αc4?F"(W311O) YK152 x Pl(W3110)

YK158 YK152, <7c'<?Fⁱ(SE2378) YK152 x Pl(SE237S)

Growth Medium and fermentation

Rich medium (L-broth) contained (per liter), trypticase peptone (10 g), yeast extract (5g) and NaCl (5g) (Lee, et al. 1985). Mineral salts medium was described previously (Lee, et al. 1985) Glucose or xylose was added as needed. Fermentations were conducted at 37⁰C as described previously (Hasona, et al. 2004). Culture pH was maintained at 7.0 by the addition of KOH. Batch fermentations were conducted in 13 x 100 mm screw cap tubes filled to the top as previously described (Patel , et al. 2006)

Vectors and transformation

Cloning and expression of lpd as well as the genes in the p dh region is accomplished according to standard procedures described in the art. Vectors employed in transformation can include pTrc99a (GE), pCR2.1 -TOPO, pBR322, pUC 19, pACYC 184, pBAD24, in addition to other commonly known vectors. A CaCl₂ based chemical transformation method was used, according to standard procedure found in Maniatis, et al. (1989). Analytical Methods

Sugars and fermentation products were determined by HPLC (Underwood, et al. 2002). Pyruvate decarboxylase activity was measured in disrupted cell preparations as previously described (Talarico, et al. 2001).

EXAMPLE 1

ISOLA TION OF ETHANOLOGENIC NON-RECOMBINANT E. COLI STRAINS

SE2377, SE2378, SE2382, SE2383, SE2384, SE2385

In this example, the isolation of non-recombinant ethanologenic strains of the bacterium E. coli is described.

The starting strain AH242 was used for isolation of the described homoethanologenic mutants of Escherichia coli. Strain AH242 is incapable of anaerobic growth in rich medium containing sugars due to mutations in the ldh and pflB genes encoding lactate dehydrogenase (LDH) and pyruvate formate lyase (PFL), respectively (Mat- Jan, et al. 1989). Despite these mutations, the aerobic growth of AH242 remains unaffected. The anaerobic growth defect in AH242 is a result of a deficiency in the re-oxidation of NADH to NAD⁺, an essential substrate for the key glycolytic enzyme glyceraldehyde-3 -phosphate dehydrogenase, and the associated ATP production. The absence of LDH eliminates NADH oxidation by the reduction of pyruvate to lactate. In the absence of acetyl CoA that is normally produced by PFL, there is insufficient acetyl CoA available for effective NADH oxidation by native aldehyde, and alcohol dehydrogenase activities. hi the instant invention, starting with the AH242 strain, tiiefocA- and -pflB (pyruvate formate lyase) deletion was constructed using previously described methods (Datsenko and Wanner, 2000). The single deletion mutants, AH240, -(focA-pflB) and AH241 -(idhA) were the parent strains of the double mutant AH242 strain. At the location of deletion, the FRT-Km-FRT cassette was inserted, thus rendering strain AH242 kanamycin-resistant. Due to the two mutations, strain AH242 is anaerobic growth defective in all media. Table 2, below, lists the growth characteristics of the E. coli mutants with mutations in anaerobic fermentation pathways. Table 2: Growth characteristics oϊE.coli mutants with mutations in anaerobic fermentation pathways.

Strain Genotype Specific growth rate (h^'s)

Aerobic Anaerobic

LB Minimal Minimal LB Minimal Minimal (+Ace, Slice) (÷Ace, Slice)

W3110 wild type. 1.31 1.05 0.97 0.98 0.51 0.51

AH240 1.23 0.95 0.99 0.79 NG 0.26

AH241 IdM 1.35 0.96 0.94 0.81 0.39 0.30

AH242 pflB, MM 1.21 1.18 0.97 NG NG NG

SE2378 pβB, IdM, Ana^* 1.18 0.51 0.82 0.46 NG NG (0.21)

LB₁ L-broth; Minimal - glucose minimal medium supplemented without or with acetate and succinate (1 mg/ml each).

NG-No growth.

Value in parenthesis, was the growth rate in glucose-minimal medium with acetate, succinate and glutamate (1 mg/ml).

The resulting anaerobic growth defective strain AH242 was cultured in 5 ml L- broth in aerobic conditions, at 37⁰C, in a shaker at 200 RPM. At mid-exponential phase of growth, the culture was removed from the shaker and spread on L-agar with glucose, or L-agar with glucose plus a redox dye of neutral red. A Whatman paper filter was placed on the surface of each of the agar medium. The mutagenizing agent ethyl methane sulfonate (EMS) was added to the disc, and the plates were transferred to an anaerobic jar containing an H₂ + CO₂ generator envelope with palladium catalyst to create an O2 free environment. Other standard agents suitable for use in mutagenesis can be employed in the invention. The anaerobic jar with the plates was incubated at 37° C for 5 days. After 5 days no visible growth was detected on either of the indicated media.

Subsequently, both dishes were incubated under aerobic conditions for~20 hours. At the end of this incubation, a lawn of bacterial cells was observed in both media in all areas except the area surrounding where the paper disc with EMS was placed. Cells on the surface of each media were transferred to fresh media of the same composition by replica plating and placed in an anaerobic jar for 5 days. After 5 days, each plate had over 100 colonies in all areas except for where the EMS was placed. 31 colonies were chosen from each of the glucose (15 colonies) and glucose + neutral red plates (16 colonies) and streaked on fresh L-agar + glucose. The plates were grown under anaerobic conditions. All colonies grew under anaerobic conditions.

31 mutants were inoculated to L-broth + glucose cultures, and after growth each was transferred to the surface of L-agar + kanamycin plates. All mutants grew in the presence of kanamycin, indicating they carried the antibiotic resistance of the parent strain AH242.

The 31 mutants were transferred to L-broth + glucose in screw cap tubes, and incubated at 37⁰C without mixing. After visible growth was detected, the medium was separated from the cells, and the fermentation products in the spent medium were determined using high performance liquid chromatography (Underwood , et al, 2002). Table 3 shows that thirty of the thirty-one mutants produced ethanol as the primary, or major fermentation product (73%). The remaining product was a combination of succinate and acetate.

These results show that non-recombinant mutants from the AH242 parent strain, that is incapable of growth in an anaerobic environment, have been isolated that are capable of growth in anaerobic conditions, and produce ethanol as the major fermentation product.

Table 3: Fermentation profiles of ethanologenic mutant derivatives of E. coli strain AH242

Isolate Glucose Fermentation Products (mM) Ethanol Total Product

Number Consumed Succinate Lactate Formate Acetate Ethanol % Yield (%) fmMΪ

SE2138 19.7 3.7 22.0 3.5 7.2 8.2 20

1 19.7 2.8 3.1 31.1 84 90

2 18.7 2.1 3 1 28.4 84 94

3 19.7 2.7 3.6 30.6 83 92

4 19.7 - 5.4 30.7 85 92

5 rSE2383ϊ 19.7 3.3 3.9 29.4 80 93

6 19.7 2.9 3.0 30.8 84 93

7 19.7 1.7 4.8 30.3 82 93

8 19.7 2.8 0.5 3.3 3.4 26.8 81 85

9 19.7 2.4 3 1 30.4 85 91 10 CSE2384) 5.6 2.0 2.6 5.8 56 93

11 19.7 2.8 3.2 32.0 84 96 12 19.7 3.3 0.2 3.0 30.7 83 94 13 19.7 2.5 0.2 2.4 32.4 86 95 14 (SE2376) 19.7 2.3 2.9 33.3 86 98

15 (SE2385) 19.7 2.6 3.1 32.7 85 98

17 15.0 1.8 2.4 24. 85 96 18 (SE2377Ϊ 19.7 2.3 2.7 4.0 31.2 83 95

19 CSE2378) 19.7 2.6 2.7 33. 86 98

20 9.9 0.9 7.6 2.2 11.4 52 100 21 19.5 1.9 2.6 31.9 88 93 22 19.7 2.5 4.9 2.9 28.2 73 98 23 19.7 2.6 2.9 33.2 86 98 24 19.7 2.5 3.8 3.0 29.2 76 98 25 19.7 3.1 2.7 32.6 85 98 26 19.7 2.4 4.5 2.7 27.4 74 93 27 19.7 2.1 0.2 2.5 32.0 87 93 28 18.7 3.3 0.6 3.5 4.9 26.3 78 90 29 19.7 3.1 3.6 32.2 81 100 30 14.5 2.1 2.8 23.5 83 98 3HSE2382L 19.7 2.3 2.8 32.8 87 96

32 19.7 2.9 3.0 32.0 84 96

EXAMPLE 2 GROWTHRATE AND FERMENTATION PROFILE OF ETHANOLOGENIC

NON-RECOMBINANT BA CTERIUM

In this example, the growth rate and fermentation profile of ethanologenic non- recombinant bacterium are described. From the above studies, mutant strain SE2378, which is capable of growth in anaerobic conditions, and produces ethanol as the major fermentation product, was selected for further study.

Growth Characteristics

Growth characteristics of E.coli mutants with mutations in anaerobic fermentation pathways were examined. Aerobic growth of strain SE2378 was comparable to the wild type E.coli strain W3110 or any of the single or double (focA- pflB) or ldhA mutants when cultured in rich medium as described in Table 2, above. In minimal medium, the aerobic growth rate of strain SE2378 was about half of the parent strain AH242. Supplementation of the growth medium with acetate and succinate restored the growth rate to near that of the parent. Although strain SE2378 grew anaerobically, the growth rate, even in rich medium, was only about 50% of that of the AH240 and AH241 single mutants (see Table 2, above). Strain SE2378 did not grow anaerobically in glucose-minimal medium, a phenotype associated with the pflB mutation (Clark , et al. 1989). Supplementation of the minimal medium with acetate supported the growth of the pflB mutant, strain AH240, but not the ethanologenic derivative strain SE2378. Strain SE2378 also required glutamate in addition to acetate for anaerobic growth in glucose-minimal medium. Previous studies have shown that the ethanologenic Escherichia coli strain KOl 1 also requires glutamate for optimum fermentation of xylose (Underwood , et al. 2004). This glutamate requirement can be overcome by the addition of a protective osmolyte, betaine, to the medium. However, the glutamate requirement for anaerobic growth of strain SE2378 in minimal medium was not suppressed by betaine, indicating a biosynthetic deficiency in acetyl-CoA flux to 2-ketoglutarate, a precursor of glutamate, rather than an osmotic requirement. It is thought that the acetyl-CoA was rapidly converted to ethanol by this ethanologen, and that acetyl-CoA is rate-limiting for biosynthesis. With these supplements, the growth rate of strain SE2378 in minimal medium reached that of the pflB parent strain, AH240. Corn steep liquor, a low cost medium supplement, replaced glutamate for growth of strain SE2378 in glucose-minimal medium. Glucose Fermentations

In pH controlled fermentations with 50 g I^*1 glucose (Hasona , et al., 2004), strain SE2378 grew with a specific growth rate of 0.46 h ^A after a lag of about 6 hours, and produced ethanol as the primary product (Figure 3 and Table 4, below). Since the immediate parent strain, AH242 is unable to grow anaerobically, the fermentation of strain SE2378 was compared to that of wild type strain W3110. W3110 completed the fermentation of 50g I^"1 glucose in 24 hours, while the mutant strain required about 72 hours. This difference can be primarily attributed to a difference in cell density (2.5 mg/ml dry wt for the wild type versus 1.7 mg/ml dry wt for the mutant) and the maximum specific rate of sugar metabolism of the two strains (4.1 to 3.3 g glucose h ^~λ g cells ^for the wild type and the mutant, respectively; Table 5 below). Strain SE2378 produced about 480 mmol I^'1 ethanol (22 g I^"1), 88 % of the total products which included small amounts of acetate, lactate and succinate. This is in contrast to strain W3110 fermentations in which ethanol represented only 27% of the products at 6.6 g 1^" '(Table 4) The maximum specific productivity observed for strain SE2378 was 1.34 g h ^"' g cell ^Λ (Table 5) comparable to the value of 1.6 g h ^'' g cell -¹ reported for batch fermentations with yeast (Smits , et al, 2000).

Table 4: Fermentation characteristics of E.coli strain SE2378 and wild type strain W 3110^a

Strain Glucose Product (rnM) Eώanol Total Consumed Ethanol Acetate Formate Lactate Succinate Yield" Product (inM) Yield⁵

Glucose fermentation

W3110 298 ±19 142 ±6 1(52 ±6 20δ-=ll 206±U 18 ±0.7 0.24 ±0.01 0.89 ±005

SE2378 296 = 4 478 ±15 27 ±2 0 13 = 2 27±2 0.813:0.02 0.92 ±0.04

Xylose fermentation

W3110 333 ±8 191 ±7 215 ±10248±53 32±3 57 ±1 0.34 =-0.00 0.89 ±0.02

SE2378 325 ±2 444±9 25 ±2 0 0 33±5 0.S2±001 093 ±0.02

* Fermentations were conducted in L-broth supplemented with 50 g I^"1 sugar atpH 7.0 aad 37⁰C. b Ethanol yieldi as a fraction of the theoretical maximum (0.51 g ethanol per g sugar). 1 Ethanol as a molar fraction of total products per mole of glucose fermented.

Table 5: Growth and Ethanol production by E.coli strain SE2378 grown on Glucose or Xylose.

W3110 SE2378 Glucose Xylose Glucose Xylose

0.44 0.37 0.46 0.38

Yx* 0.04 0.04 0.04 0.04

Qs 2.94 1.58 1.29 1.65

YΛS 0.12 0.18 0.41 0.42 qs 4.10 4.93 3.26 5.33 q? 0.49 0.S9 1.34 2.24

Abbreviations:

rate, h^"! = g cells (g substrate)^'1; Qs, g sugar consumed L^'! h ';Qp. g ethanol L^'1 h^"1; YRIS_* g ethanol (g substrate)^"1; qs, g sugar consumed (g cell dry weight)^'1 h^"1: q_?, g ethanol (g cell dry weight)^'1 h^'1

Xylose Fermentations

Both the wild type W3110 and the mutant SE2378 strain grew at similar rates during anaerobic fermentation with 5Og I^"1 xylose, although strain SE2378 lagged by approximately 8 hours (Figure 3 and Tables 4 and 5, above). Specific growth rates on xylose were 80% of those with glucose, consistent with previously published reports (Gonzalez , et al. 2002). The mutant strain fermented xylose more rapidly than the wild type W3110. After 48 hours, xylose utilization exceeded glucose utilization for strain SE2378. Approximately 88% of the fermentation products recovered with strain

SE2378 was ethanol; 2Og I^"1 from 50 g I^"1 of xylose. The maximum specific productivity of ethanol for strain SE2378 with xylose was 2.23 g h^"1 g cells^"1.

The specific ethanol productivity of both W3110 and SE2378 was higher with xylose than with glucose, as shown in Table 5. This may be indicative of the lower energy yields from xylose metabolism (Hasona , et al, 2004). For wild type, the net ATP yield from xylose is only about 1.5 per xylose, as compared to 3.0 per glucose. This would require that cells use more xylose to produce the same amount of cell mass. However the specific rate of xylose consumption by the wild type was only slightly higher than that of glucose (4.93 vs. 4.10 g h^'1 g cells^'1), as seen in Table 5 above, thus accounting for the lower cell yield and the longer fermentation time compared to glucose fermentation. In contrast, strain SE2378 lacks pyruvate formate lyase, an enzyme that is critical for xylose fermentation in minimal medium (Hasona , et al. 2004). Due to this mutation, the net calculated ATP yield from xylose fermentation in strain SE2378 is only 0.67 per xylose. It is apparently this lower ATP yield that is driving the high xylose flux in this ethanologenic mutant. The specific productivity of ethanol from xylose of 2.23 g h^'1 g cells^'1 is higher than the value of 1.6 g h^"1 g cells^"1 on glucose for yeast (Smits , et al. 2000) and for glucose and xylose in the ethanologenic E.coli strain KOl 1 carrying the Z.mobilis pdc and adh genes (about 2 g h^"1 g cells^'1).

These results show that the non-recombinant SE2378 mutant produces ethanol as the primary fermentation product from both glucose and xylose. Further, the rate of ethanol production is comparable to other ethanologenic organisms.

EXAMPLE 3

IDENTIFICATION OF MUTANT LPD GENE FROM NON-RECOMBINANT

ETHANOLOGENICE. COLI

In this example, the identification of mutant LPD gene from E.coli strains

SE2377, SE2378, SE2382, SE2383, SE2384, SE2385 is described.

Mutations in the non-recombinant ethanologenic E .coli mutant strains were mapped by co-transduction with zac: :Tn70. When aroP-pdhR-aceEF genes were deleted by co-transduction (Figure 5), the transductant lost its ability to grow in LB containing 1% glucose under anaerobic conditions, while the same deletion in wild type background did not affect anaerobic growth. These results suggest a role for pyruvate dehydrogenase enzyme in the anaerobic growth of strain SE2378. The mutation responsible for the ethanologenic phenotype in the SE2377, SE2378, SE2382, mutant strains identified mapped in the pyruvate dehyrogenase gene locus.

The pyruvate dehydrogenase complex (PDH) consists of three enzymes, pyruvate dehydrogenase/ decarboxylase (enzyme 1, El), lipoate transacetylase (enzyme 2, E2), and dihydrolipoamide dehydrogenase (enzyme 3, E3) subunits. It is known that the pdhR promoter is the promoter for the transcription of the pdhR-aceEF-lpd genes, despite the presence of independent promoters for aceEF and lpdA genes (Quail et al, 1995). Since the expression of the PDH operon is negatively regulated by pdhR protein (Quail , et al, 1995), the pdhR genes of SE2377, SE2378, SE2382 were sequenced (Figure 6A). The sequence analysis of strain SE2378 revealed two mutations within the coding region of pdhR: 1 an amino acid substitution (S 12P) and 1 amino acid insertion of leucine as amino acid 118. Another nucleotide substitution of G to A was found in the intergenic region between the pdhR gene and the aceE gene (Figure 6B). Strain SE2377 and SE2382 did not carry any mutation in the pdhR-aceEF region of genomic DNA. However, these strains, as well as strains SE2383, SE2384 and SE2385, all had single mutations in the lpd gene (Figure 5A). The PdhR protein is a pyruvate-responsive regulator of the pdhR-lpd operon and thus mutations in this protein are not unexpected. The aceEF may contain its own transcription start site for aceEF-lpd in addition to the start site at the beginning of the pdhR for transcription ofpdhR-lpd. Thus, the mutations in the intergenic region may also support an elevated level of aceEF-lpd expression in the anaerobic cell. It has previously been reported that the level of pyruvate dehydrogenase/ decarboxylase activity of PDH complex in E.coli is about 5-fold higher in cells grown aerobically vs. anaerobically (deGraef , et al, 1999).

These results provide location of the mutations in the identified ethanolo genie E. coli strains of the instant invention.

EXAMPLE 4

MUTATION IN THE LPD GENE IS RESPONSIBLE FOR ETHANOLOGENIC

PHENOTYPE

In this example, mutation in the PDH complex, and specifically the lpd gene is shown to be causative for the ethanologenic phenotype.

Preliminary genetic analysis of strain SE2378 revealed that the mutation(s) responsible for anaerobic growth and homoethanol production are located in or near the genes coding for the PDH complex (pdh locus: pdhR, aceF, lpd). To confirm that PDH is required for the ethanologenic phenotype of strain SE2378, a mutation in the aceF gene (dihydrolipyl acetyltransferase; E2 enzyme of PDH) was transduced into strain YKl, a derivative of strain SE2378 that lacks the kanamycin-resistance gene. The transductant strain YK93, lost the ability to grow anaerobically, as shown in Table 6, below. Table 6: Growth characteristics of ethanologenic E.coli strain SE2378 with a mutation in the pdh locus, (aceF)

Strain Genotype Specific growth rate (h ')

Aerobic Anaerobic

LB Minimal Minimal LB Minimal (+Ace, Succ)

W3110 wild type 1 31 1 05 0 97 0 98 0 51

YK1₅3 W3110, aceF 0 46 NG 0 55 1 07 0 44

YK29 pflB, IdhA 1 29 099 0 99 NG NG

YKl 52 YK29, aceF 0 83 NG 0 50 NG NG

YKl pflB. idhA, Ana⁺ 1 14 0 51 0 83 041 NG*

YK93 YKl, aceF 0 68 NG 046 NG NG

YK 157 YK.152, aceF* (W31 IQ) 1 32 0 96 0 87 NG NG

YK158 YKl₅2, σcef* (SE2378) 1 17 0 51 0 80 0 45 NG*

Minimal - glucose minimal medium supplemented without or with acetate and succinate (1 mg/ml each) NG-No growth *The two ethanologenic derivatives require acetate and glutamate for anaerobic growth m minimal medium as of strain SE2378

This anaerobic-minus phenotype of strain YK93 was similar to that of strain AH242, the parent of strain SE2378. Although an aceF mutant is aerobic-minus in minimal medium due to the cell's inability to produce acetyl co-A for biosynthesis, under anaerobic growth conditions this function is catalyzed by the PFL and thus, an aceF mutation does not affect anaerobic growth of E.coli (strain YKl 53, W3110 with aceF mutation, as shown in Table 6). Anaerobic growth of strain YK93 was defective in all of the media that was tested. The aceF mutation in strain YKl 52 was transduced to aceF* by phage Pl with the gene from either W3110 (wild type) or SE2378 (ethanologen) and the transductants were selected for growth in minimal medium under aerobic conditions. The transductants were also tested for anaerobic growth and fermentation products. The transductants that received the aceF* gene from the wild type strain W3110, grew aerobically in minimal medium but failed to grow anaerobically in any of the media tested due to the presence of IdhA and pflB mutations. All the transductants that received the aceF* gene from strain SE2378 grew anaerobically and all the tested transductants produced ethanol as the main fermentation product. These results show that the ethanologenic phenotype of strain SE2378 requires intact pdh locus and PDH activity, and agree with a PDH-dependent pathway for ethanol production (see Figure 8 C). hi this pathway for homoethanol production, pyruvate is oxidatively decarboxylated to acetyl-coA by PDH and further reduced to acetylaldehyde and ethanol by the alcohol dehydrogenase (Figure 8C). Deletion of either aceF (PDH -minus; strain YK93) that is required for acetyl-coA production or adhE (ADH minus; strain YK91), needed for ethanol production, resulted in anaerobic growth negative phenotype supporting the role of this pathway for homoethanol production and redox balance in strain SE2378 that is lacking fermentative lactate dehydrogenase and pyruvate formate lyase.

In the next set of experiments, the lpd gene is shown to be causative for the ethanologenic phenotype. The lpd gene from the wild type strain W3110, and from the ethanologenic mutant strain SE2378 were cloned in to an expression vector for the production of the LPD protein from the trc promoter with DPTG as inducer. These plasmids were transformed in to strain YKlOO that carries three deletions: idhA, (focA- pflB), and lpd. Beyond the three mutations, strain YKlOO is similar to W3110 strain. Due to the three deletions, strain YKlOO is defective for anaerobic growth in all media tested, and is defective for aerobic growth in minimal medium. As discussed previously, the pyruvate dehydrogenase complex (PDH) consists of three enzymes, pyruvate dehydrogenase/ decarboxylase (enzyme 1), lipoate transacetylase (enzyme 2), and lipoamide dehydrogenase (enzyme 3). Aerobic growth of E. coli is impaired by a mutation in any one of the three components of the PDH complex.

Plasmid pKY32 (Figure7A), containing the lpd gene (Lpd+) from strain W3110, or plasmid pKY33 (Figure 7B) containing the mutant lpd gene (Lpd*) from strain SE2378, was transformed in to strain YKlOO, and ampicillin resistant transformants were selected. These transformants were PDH-positive as seen by aerobic growth in minimal medium; enzyme 1 and enzyme 2 of the PDH complex came from the chromosome and the Lpd came from the plasmid. Only the transformants with plasmid pKY33 carrying the lpd gene from the ethanologenic SE2378 strain were able to grow under anaerobic conditions. Ethanol was the major fermentation product in the spent medium from strain YK100/pKY33 (named YKl 29). In contrast, strain YKlOO with plasmid pKY32 carrying the native lpd gene from W3110 did not grow under anaerobic conditions. Taken together, these results show that the LPD protein is responsible for the observed activity of the pyruvate dehydrogenase complex under anaerobic growth conditions, and further that the mutated form of Lpd is sufficient to support homoethanol production by E. coli. The reason the lpd mutant of E. coli is ethanologenic is its ability to produce 4 NADH per glucose.

EXAMPLE 5 MUTA TION IN THE LPD GENE IS RESPONSIBLE FOR NADH INSENSITIVTY

In this example, mutation in the lpd gene is shown to cause NADH insensitivity. More particularly, it was found that dihydrolipoamide dehydrogenase (LPD) activity that is NADH sensitive in the wild type (native) enzyme is changed to NADH-insensitive in the mutant, as shown in Figure 10 and Figure 11. Because LPD is a component of the pyruvate dehydrogenase complex (PDH) this NADH insensitivity of the LPD is carried through to the PDH from the ethanologenic mutant.

E. coli wild type, strain W3110, or the ethanologenic mutant, strain SE2378 were cultured in glucose-mineral salts medium to mid-exponential phase of growth. The cells were then harvested and an extract was prepared. Enzyme activity in the cell extract was determined with pyruvate and NAD as substrates, and varying concentrations of NADH as the inhibitor of enzyme activity. Figure 10 shows inhibition of PDH activity by NADH. In the top panel, NAD concentration was 2 mM NAD for both wild type, strain W3110, and the ethanologenic mutant, strain SE2378. In the bottom panel, NAD concentration was 2 mM NAD for native enzyme from strain W3110, and 1 mM for the mutated form of the enzyme from strain SE2378.

The lpd gene from E. coli wild type, strain W3110, and the ethanologenic mutant, strain SE2378, was amplified by PCR and cloned into a protein expression vector, pET15b. The DNA sequence of the lpd gene in the selected plasmid was verified by sequencing the insert DNA. Expression of the lpd gene in the plasmid was induced and the protein was purified. Enzyme activity was determined in the reverse reaction in which the two substrates were lipoamide (3 mM) and NADH (0.1 mM) in 0.1 M K- phosphate buffer, pH 8.0 with 1.5 mM EDTA. Figure 11 shows inhibition of LPD by NADH. Under these conditions, the native enzyme had no detectable activity, as shown in the graph in Figure 11. NAD, the product of the reaction is a required activator of the enzyme activity and the activity increased with increasing NAD concentration.. The ratio of NADH to NAD on the activity of the enzyme was determined for both the native and mutated from of the enzyme and the results are presented in Figure 11. PDH is produced by all aerobic organisms (from bacteria to man). This enzyme oxidatively decarboxylates pyruvate to acetyl-CoA, CO2 and NADH and the acetyl-CoA is then fed into the TCA cycle for further oxidation and subsequent energy production. In E. coli, PDH is produced under both aerobic and anaerobic conditions. However, under anaerobic conditions the enzyme is inactive due to inhibition of PDH by NADH. NADH is usually present at a higher concentration in the anaerobic cell, and thus prevents generation of NADH that cannot be oxidized by the cell that is lacking external electron acceptors. As a consequence the cell produces only 2 NADH per glucose, and the second set of reductant is released as hydrogen gas. Because one acetyl-CoA reduction to ethanol requires two NADH, the wild type cell cannot produce two ethanols per glucose.

In the ethanologenic mutant strains of the instant invention, PDH is less sensitive to NADH. This decreased sensitivity allows the enzyme to function even under anaerobic conditions with a higher NADH pool. Due to this biochemical change, the cell can produce four NADH molecules per glucose (2 from glycolysis and 2 from PDH reaction). All four NADHs are used to reduce two acetyl-CoA to ethanol, making the mutant a homoethanol producer. Biochemically and physiologically, the cell is a homoethanol producer due to the decrease in sensitivity of the PDH to NADH, and its ability to function even with a high NADH/NAD ratio. Finally, in an additional experiment (data not shown), the mutation (E354K) in the LPD found in strain SE2378 was introduced into the LPD of B. subtilis, an aerobic organism, at the analogous location. The E356K mutation supported anaerobic growth of the mutant (MRl).

EXAMPLE 6

COMPARISON ALIGNMENT OFLPD SEQUENCES FROM OTHER

ORGANISMS

In this example, comparison alignments of the amino acid sequences of the dihydrolipoamide dehydrogenase (LPD) enzymes from different organisms are compared and contrasted.

Pyruvate dehydrogenase (PDH) is present in all aerobic organisms from bacteria to humans. LPD is an essential component of the PDH enzyme complex, and it is present in both the PDH complex and 2-oxoglutarate dehydrogenase complex. In E. coli, the lpd is shared by these two enzyme complexes, and due to this requirement, the lpd gene is transcribed from an independent promoter, in addition to a promoter lying upstream of the pdhR gene.

Lpd homologs are found in all domains of life. Among bacterial strains, Lpd protein ranges from 458 to 581 amino acids, with an anhydrous molecular weight of 49 000 to 62 000 Da. Amino acid sequence identity of 20 Lpd homologs from bacteria from various phylogenetic groupings is shown in Table 7 below.

The amino acid sequence of dihydrolipoamide dehydrogenase from various organisms was compared to that of E. coli LPD protein using Blast server at NCBI or ClustalW at the European Biotechnology Institute. Percent identity of a specific sequence to that of the E. coli sequence was obtained from either of the two databases. The values in parenthesis represent the total similarity of the specific protein to that of the E. coli Lpd and include both the amino acid positions that are identical as well as the positions at which a conservative substitution occurred. For Bacilus subtilis, two dihydrolipoyl dehydrogenases, one from the PDH complex and the other from acetoin dehydrogenase, were included, for comparison.

Sequence identity varies from a low of 24% for Methanosarcinia barken, an archaeon, to 98% for Salmonella typhimurium strain LT2, a Gram-negative bacterium. That Salmonella typhimurium LT2 LPD protein is most closely related to the E. coli LPD is consistent with the classification of the two bacteria as Gram-negative bacteria in the same family enterobacteriaceae. The Escherichia coli strain W3110 or MG1655 Lpd amino acid sequence was aligned with known Lpd sequences from Acinetobacter sp. ADPl, Bacillus cereus ATCC 10987, Bacillus subtilis strain 168, Clostridium tetani strain Massachusetts/E88, Corynebacterium glutamicum strain ATCC13032, Geobacter metallireducens GS-15, Gluconobacter oxydans 621H, Lactobacillus casei ATCC334, Lactococcus lactis subspecies cremoris SKIl, Lactobacillus plantarum WCFSl, Methanosarcinia barkeri strain Fusaro, Oenococcus oeni MCW PSU-I, Pseudomonas aeruginosa PAOl (ATCC15692), Rhodobacter sphaeroides 2.4.1, Salmonella typhimurium LT2, Shewanella sp ANA-3, Streptococcus mutans ATCC 700610, Streptomyces coelicolor M145, Thermoanaerobacter ethanolicus, Vibrio fischeri strain ATCC 700601. Homology alignment is shown in Figure 9 [this figure will have to be re-numbered]. When comparing total percent identity, the E. coli LPD appears to be most similar to other Gram- negative LPDs; however, when calculations of percent identity are made based on conservative substitution, Table 4 reflects a higher percent homology among the LPD proteins in the diverse organisms examined. For example, the amino acid sequence identity of Bacillus subtillis strain 168 LPD protein compared to that of E. coli LPD protein is 34%. However, taking in to account only conservative substitutions, the identity score increases to 57%. As can be seen from the alignment figure, several amino acids are highly conserved among the group of 20 LPD homologs from a very diverse group of organisms. Residues that are shared among the organisms are highlighted with an asterisk. Regions of interest are underlined. Among the sequences of the diverse organisms analyzed, the sequence identity is highest in the N- terminal region. Sequence identity can be seen between amino acids 40 and 55 (E.coli LPD numbering), which could represent a possible Flavin site. Another region of similarity is between amino acids 180 and 190. Throughout the sequence there are several positions at which the amino acid residues are conserved in all 20 LPDs from the diverse organisms analyzed. Notably, amino acid position 322 encodes histidine (H), and in three strains of the instant invention (SE2377, SE2383 and SE2382), there was a mutation in the histadine at position 322 to tyrosine (Y). Histidine at position 322 is conserved in all 20 LPDs from Gram-positive, Gram-negative bacteria to archaea. Other residues that are conserved across this diverse range include the proline at position 355 (18/20 LPDs) and the glutamate at position 356 (17/20 LPDs).

Table 7: Amino acid sequence identity of E. coli LPD protein to LPD homologs from other organisms.

Organism No. of amino acids % Identity

(Adjusted %)^a

Acinetobacter sp. strain ADPl 468 35 (55) Bacillus cereus strain ATCC 10987 470 44 (62) Bacillus subtilis strain 168 Ε3 protein of Pyruvate DH 470 47 (64) Bacillus subtilis strain 168 E3 protein of Acetoin DH 458 35 (57) Clostridium tetani strain Massachusetts/E88 589 35 (58) Corynebacterium glutamicum strain ATCC 13032 469 34(53) Escherichia coli strain W3110 or MG1655 474 100 Geobacter metallireducens GS- 15 476 35 (57) Gluconobacter oxydans strain 62 IH 468 32(51) Lactobacillus casei strain ATCC 334 471 30 (52) Table 7: (continued)

Organism No. of amino acids % Identity (Adjusted %)^a

Lactococcus lactis subsp. cremoris strain SKl 1 472 40 (59) Lactobacillus plantarum strain WCFSl (NCIMB 8826) 470 39 (58) Methanosarcina barken strain Fusaro 476 24 (49) Oenococcus oeni strain MCW PSU-I 473 39 (59) Pseudomonas aeruginosa strain PAOl ATCC 15692 467 37(57) Rhodobacter sphaeroides strain 2.4.1 462 40 (58) Salmonella typhimurium LT2 ATCC 700720 474 98 (99) Shewanella sp. strain ANA-3 475 85 (94) Streptococcus mutans strain ATCC 700610 581 36 (56) Streptomyces coelicolor strain M145 486 36(55) Thermoanaerobacter ethanolicus 479 40 (58) Vibrio fischeri strain ATCC 700601 475 86 (94)

^a Represents the amino acids that are identical and also conservative amino acid changes

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Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

Incorporation by Reference

All publications, patent applications and patents identified herein are expressly incorporated herein by reference in their entirety.

Claims

ClaimsWhat is claimed is:

1. An isolated non-recombinant bacterium comprising a mutation, wherein the mutation renders the non-recombinant bacterium capable of producing 4 moles of

NADH per mole of sugar under anaerobic conditions.

2. The isolated non-recombinant bacterium of claim 1 , wherein the mutation is located in &pdh operon.

3. The isolated non-recombinant bacterium of claim 2, wherein the pdh operon comprises pdhR, aceEF and lpd genes.

4. The isolated non-recombinant bacterium of claim 3, wherein the mutation is in the Ip d gene.

5. The isolated non-recombinant bacterium of any one of claims 1 - 4, wherein the production of 4 moles of NADH per mole of sugar results in the production of ethanol as the primary fermentation product.

6. The isolated non-recombinant bacterium of any one of claims 1 - 5, wherein the sugar is selected from the group consisting of: glucose, xylose, arabinose, mannose, galactose, sucrose, and lactose.

7. An isolated non-recombinant bacterium comprising an lpd gene having a mutation, wherein the mutation renders the non-recombinant bacterium capable of producing ethanol as the primary fermentation product under anaerobic conditions.

8. The isolated non-recombinant bacterium of any one of claims 5 or 7, wherein the ethanol produced comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions.

9. The isolated non-recombinant bacterium of any one of claims 1 - 8, wherein the bacterium, in the absence of the mutation, is non-ethanologenic.

10. The isolated non-recombinant bacterium of claim 9, wherein ethanol is the minor fermentation product and comprises less than 40% of total non gaseous fermentation products.

11. The isolated non-recombinant bacterium of any of claims 1 - 8, wherein the mutation provides a homoethanol fermentation pathway.

12. The isolated non-recombinant bacterium of any of claims 1 - 8 wherein one or more alternative pathways for fermentation in the bacterium are inactivated.

13. The isolated non-recombinant bacterium of claim 12 wherein the alternative pathways for fermentation include lactate production by lactate dehydrogenase (idhA), acetate, ethanol, formate, H₂ and CO₂ starting with pyruvate formate-lyase (pfl) and succinate.

14. The isolated non-recombinant bacterium of claim 13, wherein the alternative pathways for fermentation are inactivated by mutation.

15. The isolated non-recombinant bacterium of claim 13 wherein the mutation is in the IdhA gene.

16. The isolated non-recombinant bacterium of claim 13 wherein the mutation is in the pfl gene.

17. The isolated non-recombinant bacterium of claim 15 or claim 16, wherein the mutation is in the IdhA oτpflB genes.

18. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60 % homologous to the nucleotide sequence of SEQ E) NO: 1 or SEQ ID NO: 3, or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 100 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:

3, or a complement thereof; c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 50% homologous to the amino acid sequence of SEQ ED NO: 2 or SEQ ID NO: 4; d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ED NO: 2 or SEQ ED NO: 4; wherein the fragment comprises at least 15 contiguous amino acid residues of the amino acid sequence of SEQ ED NO: 2 or SEQ ED NO: 4; e) a nucleic acid which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ED NO: 2 or SEQ ED NO: 4, wherein the nucleic acid molecule hybridizes to a complement of a nucleic acid molecule comprising SEQ ED NO: 1 or SEQ ED NO: 3, under stringent conditions; f) a nucleic acid molecule comprising the nucleotide sequence of SEQ ED NO: 1 or SEQ ED NO: 3, or a complement thereof; and g) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ED NO: 2 or SEQ ED NO: 4; wherein the nucleic acid molecule when expressed in a cell, renders the cell capable of producing ethanol as the primary fermentation product.

19. The isolated nucleic acid molecule of claim 18, wherein the ethanol produced comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions.

20. The isolated nucleic acid molecule of claim 18, wherein the cell is a bacterial cell.

21. The isolated nucleic acid molecule of claim 20, wherein the bacterial cell, in the absence of expression of the nucleic acid molecule, is non-ethanologenic.

22. The isolated nucleic acid molecule of claim 21 , wherein ethanol is the minor fermentation product and comprises less than 40% of total non gaseous fermentation products.

23. The isolated nucleic acid molecule of claim 19, wherein the bacterial cell produces ethanol as the primary fermentation product under anaerobic conditions.

24. The isolated nucleic acid molecule of claim 19, wherein expression of the nucleic acid molecule in the bacterial cell provides a homoethanol fermentation pathway in the bacterial cell.

25. An isolated nucleic acid molecule according to claim 18 wherein the nucleic acid molecule comprises a fragment of SEQ ID NO: 1 wherein the nucleic acid molecule is at least 100 nucleotides in length and contains a T at a position corresponding to position 997 of SEQ ID NO: 1.

26. An isolated nucleic acid molecule according to claim 18 wherein the nucleic acid molecule comprises a fragment of SEQ ID NO: 3 wherein the nucleic acid molecule is at least 100 nucleotides in length and contains a G at a position corresponding to position 1023 ofSEQ ID NO: l.

27. An isolated polypeptide selected from the group consisting of: a) a fragment of a polypeptide comprising the amino acid sequence of SEQ TD NO; 2 or SEQ ID NO: 4, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 4; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ TD NO; 2 or SEQ ID NO: 4, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule comprising SEQ TD NO; 1 or SEQ ID NO: 3, under stringent conditions; c) a polypeptide which is encoded by a nucleic acid molecule which is at least 50% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: l or SEQ ID NO: 3; d) a polypeptide comprising an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ TD NO: 2 or SEQ ID NO: 4; and e) an isolated polypeptide comprising the amino acid sequence of SEQ BD NO: 2 or SEQ ID NO: 4; wherein the polypeptide when expressed in a cell, renders the cell capable of producing ethanol as the primary fermentation product.

28. The isolated polypeptide of claim 27, wherein the ethanol produced comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions.

29. The isolated polypeptide of claim 27, wherein the polypeptide has dihydrolipoamide dehydrogenase activity under anaerobic conditions.

30. The isolated polypeptide of claim 27, wherein the cell is a bacterial cell.

31. The isolated polypeptide of claim 30, wherein the bacterial cell, in the absence of expression of the polypeptide, is non-ethanologenic.

32. The isolated polypeptide of claim 30, wherein ethanol is the minor fermentation product and comprises less than 40% of total non gaseous fermentation products.

33. The isolated polypeptide of claim 27, wherein the bacterial cell produces ethanol as the primary fermentation product under anaerobic conditions.

34. The isolated polypeptide of claim 30, wherein the ethanol produced comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions.

35. The isolated polypeptide of claim 30, wherein expression of the polypeptide in the bacterial cell provides a homoethanol fermentation pathway in the bacterial cell.

36. A bacterial host cell comprising the nucleic acid molecule of any one of claims 18 - 26.

37. The bacterial host cell of claim 36 comprising a vector comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or a fragment thereof.

38. The bacterial host cell of claim 30, wherein the vector is pKY33.

39. The bacterial host cell of claim 36, which has been genetically engineered to express the nucleic acid molecule.

40. A bacterial host cell comprising the polypeptide of any one of claims 27 - 35.

41. A method for producing a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 4; b) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; wherein the fragment comprises at least 15 contiguous amino acids of SEQ ED NO: 2 or SEQ ID NO: 4; and c) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a complement of a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO: 3, under stringent conditions; comprising culturing host cells of claim 30 under conditions in which the nucleic acid molecule is expressed.

42. The isolated non-recombinant bacterium of any one of claims 1 - 5 and 7, comprising the nucleic acid molecule of claim 18.

43. The isolated non-recombinant bacterium of any one of claims 1 -4, wherein ethanol is produced from the sugar.

44. The isolated non-recombinant bacterium of claim 42 or 43, wherein the sugar is selected from the group consisting of glucose, xylose, arabinose, mannose, galactose, sucrose, and lactose.

45. The isolated non-recombinant bacterium of any one of claims 1 - 7, the isolated nucleic acid molecule of claim 18, the isolated polypeptide of claim 27 or the bacterial host cell of claim 36, wherein the bacterium is selected from the group consisting of Gram-negative bacteria and Gram-positive bacteria.

46. The isolated non-recombinant bacterium of any one of claims 1 - 7, the isolated nucleic acid molecule of claim 18, the isolated polypeptide of claim 27 or the bacterial host cell of claim 36, wherein the bacterium is a Gram-negative bacterium.

47. The isolated non-recombinant bacterium of any one of claims 1 - 7, the isolated nucleic acid molecule of claim 18, the isolated polypeptide of claim 27 or the bacterial host cell of claim 36, wherein the Gram-negative bacterium is selected from the group consisting of Acinetobacter.Gluconobacter, Escherichia, Geobacter, Shewanella, Salmonella, Enterobacter and Klebsiella.

48. The isolated non-recombinant bacterium of any one of claims 1 - 7, the isolated nucleic acid molecule of claim 18, the isolated polypeptide of claim 27 or the bacterial host cell of claim 36, wherein the bacterium is a Gram-positive bacterium.

49. The isolated non-recombinant bacterium of any one of claims 1 - 7, the isolated nucleic acid molecule of claim 18, the isolated polypeptide of claim 27 or the bacterial host cell of claim 36, wherein the Gram-positive bacterium is selected from the group consisting of Bacillus, Clostridium, Corynebacterium, Lactobacillis, Lactococcus, Oenococcus, Streptococcus and Eubacterium.

50. The isolated non-recombinant bacterium of any one of claims 1 - 7, the isolated nucleic acid molecule of claim 18, the isolated polypeptide of claim 27 or the bacterial host cell of claim 36, wherein the bacterium is Escherichia coli.

51. The isolated non-recombinant bacterium of claim 4 or 7, wherein the mutation comprises substitution of an amino acid with another amino acid in a polypeptide expressed by the mutated lpd gene, wherein the substitution changes the pK of the polypeptide.

52. The isolated non-recombinant bacterium of claim 51 , wherein the polypeptide comprises SEQ K⁾ NO: 6 and the mutation comprises a substitution of a wild type amino acid with another amino acid at: a) position 322 or any position within about 50 positions on either side of position 322 in SEQ ID NO: 6; or b) position 354 or any position within about 50 positions on either side of position 354 in SEQ ID NO: 6.

53. The isolated non-recombinant bacterium of claims 51 or 52, wherein the another amino acid is a neutral amino acid selected from the group consisting of alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

54. The isolated non-recombinant bacterium of claims 51 or 52, wherein the another amino acid is a basic amino acid selected from the group consisting of arginine, asparagine, glutamine, histidine and lysine.

55. The isolated non-recombinant bacterium of 52, wherein the mutation comprises a substitution of H at position 322 with any amino acid, such that the amino acid substitution increases the acidity of the polypeptide expressed by the mutated lpd gene.

56. The isolated non-recombinant bacterium of claim 55, wherein the mutation comprises a substitution of H to Y at position 322 in SEQ ID NO: 6.

57. The isolated non-recombinant bacterium of 52, wherein the mutation comprises a substitution of E at position 354 with any amino acid, such that the amino acid substitution reduces the acidity of the polypeptide expressed by the mutated lpd gene.

58. The isolated non-recombinant bacterium of claim 57, wherein the mutation comprises a substitution of E to K at position 354 in SEQ ID NO: 6.

59. The isolated non-recombinant bacterium of claim 56, wherein the bacterium is E. coli strain SE2377.

60. The isolated non-recombinant bacterium of claim 59, wherein the bacterium comprises SEQ ID NO: 1, or a fragment thereof.

61. The isolated non-recombinant bacterium of claim 58, wherein the bacterium is E. coli strain SE2378.

62. The isolated non-recombinant bacterium of claim 61 , wherein the bacterium comprises SEQ E) NO: 3, or a fragment thereof.

63. The isolated non-recombinant bacterium of claim 58, wherein the bacterium is E. coli strain SE2382.

64. The isolated non-recombinant bacterium of claim 63, wherein the bacterium comprises SEQ E) NO: 3, or a fragment thereof.

65. The isolated non-recombinant bacterium of claim 56, wherein the bacterium is E. coli strain SE2383.

66. The isolated non-recombinant bacterium of claim 65, wherein the bacterium comprises SEQ E) NO: 1, or a fragment thereof.

67. The isolated non-recombinant bacterium of claim 56, wherein the bacterium is E. coli strain SE2384.

68. The isolated non-recombinant bacterium of claim 67, wherein the bacterium comprises SEQ E) NO: 1 , or a fragment thereof.

69. The isolated non-recombinant bacterium of claim 58, wherein the bacterium is E. coli strain SE2385.

70. The isolated non-recombinant bacterium of claim 69, wherein the bacterium comprises SEQ ID NO: 3, or a fragment thereof.

71. The isolated non-recombinant bacterium of any one of claims 56 - 70, wherein the bacterium is suitable for producing ethanol from sugar.

72. An isolated non-recombinant bacterium comprising a lpd gene having one or more mutations, wherein the mutation renders the non-recombinant bacterium capable of producing ethanol as the primary fermentation product under anaerobic conditions, wherein the bacterium is prepared by a process comprising the steps of: a) growing a candidate mutant strain of the bacterium under anaerobic growth conditions in sugar-rich medium; and b) selecting mutants that produce ethanol as the major product of fermentation.

73. The method of claim 72, wherein the ethanol produced comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions.

74. A method of producing the non-recombinant bacterium of any one of claims 1 - 8, comprising the steps of: a) growing a candidate mutant strain of the bacterium under anaerobic growth conditions in sugar-rich medium; and b) selecting mutants that produce ethanol as the major product of fermentation.

75. The method of claim 72, wherein the ethanol produced comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions.

76. The isolated non-recombinant bacterium of claim 72 or the method of claim 74, wherein the mutants result from spontaneous mutation.

77. The isolated non-recombinant bacterium of claim 72 or the method of claim 74, wherein the bacterium is exposed to a mutagenizing agent.

78. The isolated non-recombinant bacterium of claim 72 or the method of claim 74, wherein the mutagenizing agent is selected from the group consisting of ethyl methane sulfonate, 2-aminopurine, ICR-191, methyl methane sulfonate, N-methyl-N'-nitro-N- nitrosoguanidine.

79. The isolated non-recombinant bacterium or the method of claim 78, wherein the mutagenizing agent is ethyl methane sulfonate.

80. The isolated non-recombinant bacterium of claim 72 or the method of claim 74, wherein the sugar in the sugar-rich medium is selected from the group consisting of glucose, xylose, arabinose, mannose, galactose, sucrose, and lactose.

81. The isolated non-recombinant bacterium of claim 72 or the method of claim 74, further comprising the step of inactivating alternative fermentation pathways in the bacterium.

82. The isolated non-recombinant bacterium of claim 72 or the method of claim 74, wherein the alternative fermentation pathways are inactivated by introducing deletion mutations in the bacterium.

83. The isolated non-recombinant bacterium of claim 72, wherein the bacterium, in the absence of the mutation, is non-ethanologenic.

84. The isolated non-recombinant bacterium of claim 83, wherein ethanol is the minor fermentation product and comprises less than 40% of total non gaseous fermentation products.

85. The isolated non-recombinant bacterium of claim 72, wherein bacterium produces ethanol as the primary fermentation product under anaerobic conditions.

86. The isolated non-recombinant bacterium of claim 85, wherein the ethanol produced comprises greater than 50% of total non-gaseous fermentation products under anaerobic conditions.

87. The isolated non-recombinant bacterium of claim 72, wherein the mutation provides a homoethanol fermentation pathway.

88. The isolated non-recombinant bacterium of claim 87, wherein one or more alternative pathways for fermentation in the bacterium are inactivated.

89. The isolated non-recombinant bacterium of claim 88, wherein the alternative pathways for fermentation are inactivated by mutation.

90. The isolated non-recombinant bacterium of claim 88, wherein the alternative pathways for fermentation include lactate production by lactate dehydrogenase (Idh), acetate, ethanol, formate, H₂ and CO₂ starting with pyruvate formate-lyase (pfl) and succinate.

91. A method for producing ethanol from an oligosaccharide source comprising, contacting the oligosaccharide with the isolated non-recombinant bacterium of any one of claims 1 - 8 or the bacterial host cell of claim 36, thereby producing ethanol from an oligosaccharide source.

92. The method of claim 91 wherein the oligosaccharide is selected from the group consisting of lignocellulose, hemicellulose, cellulose, pectin and any combination thereof.

93. A kit comprising the isolated non-recombinant bacterium of any one of claims 1 - 8 and instructions for producing ethanol.

94. The kit of claim 93 further comprising a sugar source.

95. The E. coli strain AH218 represented by a deposit with the Agricultural Research Culture Collection designated as deposit number NRRL B-30967.

96. The E. coli strain AH241 represented by a deposit with the Agricultural Research Culture Collection designated as deposit number NRRL B-30968.

97. The E. coli strain AH242 represented by a deposit with the Agricultural Research Culture Collection designated as deposit number NRRL B-30969.

98. The E. coli strain SΕ2377 represented by a deposit with the Agricultural Research Culture Collection fdesignated as deposit number NRRL B-30970.

99. The E. coli strain SE2378 represented by a deposit with the Agricultural Research Culture Collection designated as deposit number NRRL B-30971.

100. The E. coli strain SE2382 represented by a deposit with the Agricultural Research Culture Collection designated as deposit number NRRL B-30972.

101. The E. coli strain SE2383 represented by a deposit with the Agricultural Research Culture Collection designated as deposit number NRRL B-30973.

102. The E. coli strain SE2384 represented by a deposit with the Agricultural Research Culture Collection designated as deposit number NRRL B-30974.

103. The E. coli strain SE2385 represented by a deposit with the Agricultural Research Culture Collection designated as deposit number NRRL B-30975.

104. The isolated non-recombinant bacterium of claim 4, 7, or 72, wherein the mutation in the lpd gene causes NADH insensitivity.

105. The method of claim 72, 74 or 91 or the kit of claim 93, wherein the mutant results from mutation in the lpd gene.

106. The method or the kit of claim 105, wherein mutation in the lpd gene causes NADH insensitivity.