BRPI0711266A2 - isolated non-recombinant bacterium, isolated nucleic acid molecule, isolated polypeptide, bacterial host cell, methods for producing a polypeptide, a non-recombinant bacterium, and atenol, kit, and, e. coli ah218 - Google Patents

Abstract

ISOLATED NON-RECOMBINANT BACTERIA, ISOLATED NUCLEIC ACID MOLECULE, ISOLATED POLYPEPTIDE, BACTERIAL HOSTING CELL, METHODS TO PRODUCE A NON-RECOMBINANT BACTERY, AND EATS, AND ETHANE, ETHANE KIT, primary fermentation product, associated nucleic acids and polypeptides, methods for producing ethanol using bacteria, and sets are disclosed.

Description

"ISOLATED NON-RECOMBINANT BACTERIA, ISOLATED NUCLEIC ACID MOLECULA, ISOLATED POLYPEPTIDE, BACTERIAL STONE CELL, METHODS FOR PRODUCING UMPOLIPEPTIDE, A NON-RECOMBINANT BETTER AND E. EFFOLI, CE KOL,

Related Request

This application claims the priority of U.S. Conditional Application Serial No. 60 / 796,652, filed May 1, 2006 and 60 / 848,234, filed September 29, 2007, the full disclosures of which are incorporated herein by reference.

Government Sponsored Research

This work was supported in part by Grant No. DE-FG36-04G014019 of the U.S. Department of Energy. Consequently, the government may have certain rights in the invention.

Fundamentals of the invention

Ethanol is an alternative transport fuel to replace at least a part of petroleum (Kheshgi, et al, 2000, Wooley, et al, 1999). Although ethanol is currently produced in the US by fermentation of glucose from cornstarch using Saccharomyces cerevisíae (Bothast, et al., 2005), which expands its process to produce a large fraction of the automotive fuel requirement and may adversely impact the industry. food and nutrition.

Lignocellulosic biomass is an attractive alternative food supply that can be fermented to ethanol after appropriate pretreatment without impacting food and nutritional supply (Wyman, etal., 2003, Zaldivar, et al, 2001). In contrast to maize starch, abiomass contains significant amounts of pentose sugars that are recalcitrant for yeast fermentation.

Converting complex sugars to ethanol requires microbial biocatalysts that effectively ferment both dehexose and pentose sugars. In this regard, recombinant organisms have been developed in which heterologous genes have been added to platform organisms such as yeast, Z. mobilis and E. coli. For example, recombinant ethanogenic Escherichia coli containing the Zymomonas mobilis pdc and adh genes ferment both quantopentose hexoses to ethanol at a high rate and yield (Ingram, et al., 1999). In addition, the genetic engineering of yeast and Z. mobilis by the addition of genes for pentose use should still produce a biocatalyst that matches the characteristics of the pseudose fermentation of recombinant Eseheriehia colietanologico (Kuyper, et al., 2005, Mohagheghi, et al. , 2004).

Summary of the Invention

As noted above, converting lignocellulosic feedstock to ethanol requires microbial biocatalysts that effectively ferment both hexose and pentose sugars. Such microbial biocatalysts include recombinant organisms in which heterologous genes have been added to platform organisms such as yeast and bacteria, for example Zymomonas mobilis and Escheriehiacoli.

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

Accordingly, the invention is based, at least in part, on the discovery of a mutation that redirects glycolysis through a homoethanol pathway in otherwise non-ethanogenic microorganisms and the development of non-recombinant ethanolic microorganisms that ferment glucose and xylose to ethanolsob. Anaerobic conditions based on that finding, in particular, the pdh operator was identified as the origin of the homoethanol pathway. More specifically, the Ipd gene within the pdh operon has been identified as responsible for the fermentation of homoethanol, for example by E. coli under anaerobic conditions.

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

In another aspect, the invention provides an isolated non-recombinant bacterium comprising an Ipd 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 nucleic acid molecules are expressed in a cell, for example 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 that 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 a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a complement thereof;

c) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of 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 encoding a fragment of a polypeptide comprising an amino acid sequence of SEQLD 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 encoding a naturally occurring allelic variant of a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, wherein the nucleic acid molecule hybridizes to a complement of an acid molecule. nucleic acid comprising SEQID No. 1 or SEQID No. 3 under severe conditions;

f) a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a complement thereof; and

g) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 2or SEQ ID NO: 4;

wherein the nucleic acid molecule when expressed in a cell makes the cell capable of producing ethanol as the primary defermentation product.

The invention also provides dihydrolipoamidashydrogenase polypeptides or functional fragments thereof. When polypeptides are expressed in a cell, for example 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 an 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 ID NO: 2 or SEQ ID NO: 4 ;

b) a naturally occurring allelic variant of a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, wherein the polypeptide is encoded by a nucleic acid molecule that hybridizes to the complement of a nucleic acid molecule comprising SEQ ID NO; 1 or SEQ ID NO: 3, under the above conditions;

c) a polypeptide that is encoded by a nucleic acid molecule that is at least 50% identical to a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3;

d) a polypeptide comprising an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and

e) an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and wherein the polypeptide, when expressed in a cell, makes the cell capable of producing ethanol as the primary fermentation product.

In one embodiment, the ethanol produced by the cell comprises more 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. In a further embodiment, the cell is a bacterial cell.

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 another aspect, the invention provides a method for producing a polypeptide selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence SEQ ED NO: 2 or SEQ ID NO: 4;

b) a fragment of a polypeptide comprising an 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 ID NO: 2 or SEQ ID NO: 4 and

c) a naturally occurring allelic variant of a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4,

wherein the polypeptide is encoded by a nucleic acid molecule that hybridizes to a complement of a nucleic acid molecule which comprises SEQ ID NO: 1 or SEQ ID NO: 3 under severe conditions;

which comprises culturing bacterial host cells containing isolated nucleic acid molecules encoding dihydrolipoamide dehydrogenase polypeptides or fragments thereof under conditions under which the nucleic acid molecule is expressed.

In a further aspect, the invention provides non-recombinant bacteria as described above comprising an isolated nucleic acid molecule described above.

Another aspect of the invention provides non-recombinant bacterium comprising an Ipd 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 in which the bacterium is prepared by a mutant. a process comprising the steps of: a) developing a candidate mutant strain of a bacterium under anaerobic development conditions in a sugar-rich medium and

b) select ethanol-producing mutants as the main fermentation product.

In another aspect, the invention provides a method of producing non-recombinant ethanogenic bacteria of the invention which comprises the steps of:

(a) develop a candidate mutant strain of a bacterium under anaerobic development conditions in a sugar-rich medium, and

b) select ethanol-producing mutants as the main fermentation product.

In one embodiment, the invention provides recombinant bacterium not isolated from any of the above-mentioned aspects, wherein the mutation in the lpd gene causes insensitivity to NADH.

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

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 bacteria 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 the non-recombinant bacterium or host cell of the invention as described above and instructions for producing ethanol according to the methods and methods described herein. In one embodiment, the kit comprises a sugar source.

In yet another aspect, the invention provides novel E. coli strains, which include 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 filed on September 27, 2006 with 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 descriptions and claims.

Brief Description of the Drawings

Figure 1 (A) shows the nucleic acid sequence for Ipd gene as a mutation in 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 Ipd gene as a base mutation 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 wild type Ipd gene (SEQ ID NO: 5) and (B) the corresponding amino acid sequence (SEQ ID NO: 6).

Figure 4 shows a graph depicting the development and fermentation characteristics of wild-type E. coli, ethanogenic migrating strain W3110, strain SE2378, in LB medium with glucose or xylose (50g L "1) at 37 ° C and pH 7.0 Panel (A) shows a W3110 strain of glucose type, development in glucose, panel (B) shows strain SE2378, glucose development, panel (C) shows a strain W3110 of glucose type, development xylose; panel (D) shows strain SE2378, xylose development

Figure 5 shows the deletion mutant (aroP-aceEF) transduction (A) (DaroP-aceEF) transduction in strain SE2378 (B). The mutations in strain SE2378 were mapped by co-transduction with zac :: Tn10. When the aroP-pdhR-aceEF genes were annulled by co-transduction, the transducer (C) loses its ability to develop in LB containing 1% glucose under anaerobic conditions, while the same wild type-based deletion does not affect anaerobic development.

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

Figure 7 shows the plasmids used for the expression of W3110 or SE2378 Ipd in YK100 host. Plasmid pKY32 (A) contains the wild-type Ipd gene, strain W3110. Plasmid pKY33 (B) contains the ethanological mutant SE2378 Ipd gene. Figure 8 (A to C) is a schematic showing a proposed pathway for the production of ethanol from pyruvate in the E.coliSE2378 strain, natural E.coli and other ethanogenic microorganisms.

Figure 9 shows a multiple amino acid alignment (using the multiple sequence alignment programCLUSTAL) of Escherichia coli K12 MG1655 LPD with selected LPD sequences, ie Clostridium tetani E88, Thermoanaerobaeterethanolieus, Bacillus eereus ATCC 10987, Lactobac WC871, Lactobac Lactoeoceus laetis Subspeeies eremoris SKI 1, Oenoeoeeus oeni MCW PSU-1, Salmonella typhimurium LT2, Vibrio fiseheri ATCC 700601, Shewanellasp ANA-3, Pseudomonas aeruginosa PAOl (ATCC15692), Rhodobaetersphaeroides 2.4all. ADP1, Glueonobaeter oxydans 621H, Corynebaeterium glutamieum DSM20300, Lactobacillus casei ATCC334, Streptomyces coelicolor M145 / A3 (2), Streptococcus mutans ATCC 700610, Methanosareina barkeri Fusaro. The histidine residue at amino acid 322, the proline residue at amino acid355 and the glutamate residue at amino acid 356 are highlighted with a ester (*).

Figure 10 is a graph showing inhibition of PDH activity by NADH. Wild type E. coli, strain W3110 or the mutant ethanogenic, strain SE2378. In the upper panel, the concentration of NAD was 2 mM for both strain W3110 and strain SE2378. In the lower panel, the NAD concentration was 2 mM NAD for the natural strain W3110 enzyme and 1 mM for the mutated form of the SE2378 strain enzyme.

Figure 11 is a graph showing inhibition of LPD by NADH. The ratio of NADH to NAD in enzyme activity was determined for both natural and mutated LPD forms.

Detailed Description of the Invention

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

I. Definitions

As used herein, the terms "non-recombinant bacterium" and "bacterium" are intended to include a bacterial cell that does not contain the heterologous polynucleotide sequence and is suitable for further amodification using the compositions and methods of the invention, for example, suitable. for genetic manipulation, for example, which may incorporate heterologous polynucleotide sequences, for example, which may be transfected. The term is intended to include the originally transfected cell progeny. In particular embodiments, the cell is a Gram negative bacterial cell or a Gram positive cell. The term "derived from" as in "bacterial-derived polynucleotide or gene" is intended to include the isolation (in whole or in part) of a polynucleotide segment from the indicated source (i.e. a bacterium) or purification of a polypeptide from from an indicated source (ie abacteria). In consideration, the term is intended to include, for example, direct cloning, PCR amplification, or artificial synthesis of or ground in a sequence associated with the indicated polynucleotide source.

The term "anaerobic conditions" is intended to include conditions that do not include oxygen; that is, conditions in which oxygen is substantially absent. In certain embodiments of the invention, anaerobic conditions comprise a closed container or container, for example a closed container with a plug. To create conditions that do not include oxygen, the gas phase is removed from the container or container using a vacuum pump and replaced with nitrogen gas. Oxygen is substantially absent when oxygen level is too low to be detected. The term "aerobic conditions" is intended to include conditions that include oxygen; that is, conditions in which oxygen is present.

The term "ethanological" 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 ethanological organisms and naturally occurring or induced ethanological organisms.

The term "non-ethanogenic" 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 ethanol-producing microorganisms as the secondary defermentation product comprising less than 40% of total non-gaseous defermentation products.

The terms "ferment" and "fermentation" are intended to include the degradation or depolymerisation of a complex sugar and bioconversion of that sugar residue to ethanol, acetate and succinate. The terms are intended to include the enzymatic process (e.g., cellular or acellular, for example a lysate or purified polypeptide mixture) by which ethanol is produced from a carbohydrate, in particular, as a primary fermentation product.

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

The term "secondary fermentation product" as used herein is intended to include non-gaseous fermentation products comprising less than 40% of the total non-gaseous product. In certain embodiments of the invention, the secondary fermentation product is ethanol.

The term "homoethanol fermentation pathway" as used herein is intended to include a fermentation pathway in an organism, e.g. a bacterium, which facilitates the production of ethanol as the primary defermentation product.

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

A "gene," as used herein, is a nucleic acid that can direct the synthesis of an enzyme or other peptide molecule, for example, may comprise coding sequences, for example, a contiguous open reading frame (ORF) encoding a polypeptide. or may, by itself, be functional in the body. A gene in an organism may be grouped into 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 may overlap without intergenic DNA between individual genes. In addition, the term "gene" is intended to include a specific gene for a selected purpose. A gene may be endogenous to the host cell or may be recombinantly introduced into the host cell, for example, as an episomally maintained plasmid 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 natural to the cell.

The terms "pdh operon" and "pdh site" are used interchangeably and are intended to mean the pdhR, lpd, eaceEF group of genes that are expressed as a group and its associated promoter and operator. By convention, the term "operon pdh" refers to the genes encoding the operon, whereas the term "PDH" refers to the complex of proteins that are coded for the operon. The activity of pyruvate dehydrogenase is responsible for the production of acetyl CoA for occlo TCA and energy production. The term operon pdh may include an operon pdh from any aerobic organism. All aerobic organisms, from eukaryotes to humans, contain the three components of PDH. Many bacteria have the genes that encode the PDH contained in an operon. The three genes, aceE, aceF, and Ipd are essential for PDH activity, and these genes are observed in all aerobic organisms when they are organized as an operon or as independent genes.

The term "dihydrolipoamide acetyltransferase" (aceF) is intended to include the E2 acetyltransferase enzymes from the depiruvate dehydrogenase gene site. By convention, the term "aceF" refers to a dihydrolipoamide acetyltransferase genide since the term "aceF" refers to an aceF genetic product, that is, a dihydrolipoamideacetyltransferase polypeptide or enzyme.

The term "PDH complex pyruvate decarboxylase / dehydrogenase" (aceE) is intended to include a pyruvate dehydrogenase gene dolocal El decarboxylase enzyme. By convention, the term "aceE" refers to a pyruvate decarboxylase / dehydrogenase gene as "AceE" refers to an aceE gene product, that is, a polypeptide or pyruvate decarboxylase / dehydrogenase enzyme.

The term "pyruvate dehydrogenase repressor" (pdhR) is intended to include a transcriptional pdh operon repressor. Accordingly, the term "pdhR" refers to a pyruvate dehydrogenase repressor gene since the term "PdhR" refers to a depdhR genetic product, that is, a pyruvate dehydrogenase repressor polypeptide.

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

The term "lactate dehydrogenase" (IdhA) is intended to include an enzyme that converts pyruvate to lactate under fermentative conditions. By convention, the term "IdhA" refers to a lactate dehydrogenase gene referred to as the term "LDHA". IdhA gene product, that is, a polypeptide or lactate dehydrogenase enzyme.

The term "pyruvate formate lyase" (pfl) is intended to include an enzyme that converts pyruvate to acetyl-CoA and formate under fermentation conditions. By convention, the term "pfl" refers to a pyruvatoformatiate lyase gene as the term "PFL" refers to a pfl gene product, that is, a pyruvate formatiate lyase polypeptide or enzyme. The term "alcohol dehydrogenase "(adhE) is intended to include an enzyme that converts Acetyl-CoA to ethanol under fermentative conditions. By convention, the term" adhE "refers to a dehydrogenase alcohol gene whereas the term" ADHE "refers to a product of adhE gene, that is, a polypeptide or alcohol dehydrogenase enzyme.

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

The term "nucleic acid" is intended to include nucleic acid molecules, for example polynucleotides which include an open deletion structure encoding a polypeptide and may further include non-coding and intron regulatory sequences. In addition, terms are intended to include one or more genes that map to a functional site. In addition, the terms are intended to include a specific gene for a selected purpose. In one embodiment, the polynucleotide segment gene is involved in at least one step in carbohydrate conversion to ethanol. Accordingly, the term is intended to include any gene encoding a polypeptide such as a pyruvate decarboxylase, an alcohol dehydrogenase, a polypeptide or a polysaccharase, for example a glucanase or a combination thereof. A gene in an organism can be grouped into an operon, as defined in it, where the operon is separated from genes and / or operons by the intergenic DNA. Individual genes contained within a pdh operon that may overlap without intergenic DNA between individual genes.

The term "homologous" is intended to include a first amino acid or nucleotide sequence that contains a sufficient or minimal number of identical or equivalent amino acid residues or nucleotide equivalents, for example, an amino acid residue that has a similar backbone, to a second amino acid or sequence of amino acids. nucleotide, such that the first and second amino acid or nucleotide sequences divide common structural domains and / or a common functional activity.

The term "heterologous polypeptide" is intended to include a polypeptide or fragment thereof which may be encoded by a heterologous nucleic acid derivative from any source, for example eukaryotes, prokaryotes, archaea, viruses or synthetic nucleic acid fragments.

The term an "isolated polypeptide" (for example, an isolated or purified enzyme biosynthetic enzyme) is substantially free of cellular material or other microorganism contaminating polypeptides from which the polypeptide is derived or substantially free of 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 the sequence from which it is derived and the polypeptide retains. the biological activity of 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 standard technique, the term pH is used to define solutions. The usual range of pH values is between 0 and 14, with 0 being the value for concentrated hydrochloric acid (1 M HCl), 7 being the value for pure water (neutral pH) and 14 being the value for concentrated sodium hydroxide (1 M NaOH). ).

The term "pK" is intended to mean a measure of proton binding affinity and is often used interchangeably as pH. One skilled in the art will recognize that the term pK is used to define proteins, amino acids and peptides. A skilled person will also recognize that the acidic strength of carboxyl, amino, and amino acid Rionizable groups can be defined by the constant de-coupling, Ka or more commonly the negative logarithm of Ka, the pKa.

The term "vector" is intended to include any suitable plasmid vector for ligation of the nucleotide sequence of interest and transformation in the host cell.

The term "sugar" is intended to include any carbohydrate source comprising sugar molecules. 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. Sugar sources include starch, the main form of fuel storage in most plants and cellulose, the main extracellular structural component of rigid cell walls, and fibrous and woody plant tissues. The term is intended to include monosaccharides, also called simple sugars, oligosaccharides and polysaccharides. In certain embodiments, sugars include, for example, glucose, xylose, arabinose, mannose, galactose, sucrose and lactose. In other embodiments, the sugar is glucose.

The term "Gram-negative bacterial cell" is intended to include a definition recognized in the art of this term. Exemplary Gram-negative bacteria include Acinetobacter, Glueonobaeter, Eseheriehia, Geobaeter, Shewanella, Salmonella, Eneterobaeter and Klebsella.

The term "Gram negative bacteria" is intended to include the definition recognized in the art of this term. Gram-negative bacteria examples include Baeillus, Clostridium, Corynebaeterium, Lactobacillis, Laetococeus, Oenocoeeus, Streptoeoceus and Eubaeterium.

The phrase "mutant nucleic acid molecule" or "genemutant" is intended to include a nucleic acid molecule or gene having a nucleotide sequence that includes at least one alteration (e.g., substitution, insertion, deletion) such that the polypeptide or polypeptide may be The mutant-encoded activity exhibits an activity that differs from the polypeptide or polypeptide encoded by the wild-type nucleic acid molecule or genotype.

The term "amino acid" is intended to include 20 alpha amino acids that occur regularly in proteins. Basic charged amino acids include arginine, asparagine, glutamine, histidine and lysine.

Neutrally charged amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Acid amino acids include aspartic acid and glutamic acid.

The term "mutagenizing agent" is intended to include any agent that may be used according to the inventive method of modifying a nucleotide sequence.

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

II. Non-recombinant bacteria

During mixed acid fermentations, glycolysis enzymes convert each mol of glucose into 2 mol of pyruvate plus 2 mol of NADH and a 2 mol network of ATP. Lower compound production than opiruvate (ethanol, lactate, etc.) serves as a mechanism for oxidizing NADH and regenerating NAD +, essential for continued glycolysis. In the known ethanol pathway that has only evolved into yeast, plants and bacteria (ie Zmobilis), pyruvate is decarboxylated to produce carbon dioxide and acetaldehyde by non-oxidative pyruvate decarboxylase. The resulting acetaldehyde serves as the electron receptor for the oxidation of NADH by alcohol dehydrogenase during the production of an ethanol.

A completely different ethanol pathway exists in many other bacterial types, where pyruvate is first converted to acetyl-CoA and pyruvate formate-lyase formate, an oxidative decarboxylation whereby the reduction equivalents are contained in the formate and dissipated as hydrogen gas (and CO2) by the hydrogen lyase formate. Acetyl-CoA is subsequently used as the electron receptor for the oxidation of NADH demolecules by adhE-decoded alcohol-dehydrogenase activities. Due to the requirement of 2 NADH for ethanol, acetyl-CoA methadone remains and is converted to acetate and an additional ATP. Thus, the natural E. coli to acetyl-CoA ethanol pathway cannot support fermentation of homoethanol due to the need for 2 NADH porethanol produced from acetyl-CoA. The 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 decarboxyl oxidatively opiruvate to acetyl-CoA and retains the associated reductant as NADH. In contrast to PFL, the associated reductant is dissipated as hydrogen gas through the formate as an intermediate and is not available for metabolic activity in the presence of glucose. By metabolizing pyruvate with PDH, additional pyruvate NADH is made available and can be used to fully reduce each acetyl-CoA to ethanol. Although genes encoding pyruvate dehydrogenase are typically expressed under both aerobic and anaerobic conditions in E. coli, the activity of this complex during anaerobic development is very low.

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

Thus, in one aspect, the invention provides the non-recombinant bacterium comprising a mutation, wherein the mutation renders non-recombinant abacteria capable of producing 4 mol of NADH per sugar mol under anaerobic conditions. In one embodiment, the mutation is located in a pdh operon. In a particular embodiment, the pdh operator comprises pdhR genes, aceEF gene Ipds. In an additional derealization form, the mutation is in the Ipd gene.

In another embodiment, the production of 4 mol of NADH per mol of sugar results in the production of ethanol as the primary defermentation 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 non-recombinant bacteria comprising an Ipd 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 more than 50% of total non-gaseous fermentation products under anaerobic conditions.

In an additional embodiment, non-recombinant bacteria, in the absence of mutation, is non-ethanogenic. Also, in another embodiment, the nonethanogenic bacterium produces ethanol as a secondary fermentation product. In one embodiment, the ethanol produced is less than 40% of non-gaseous total fermentation products.

Still, in another embodiment of the invention, mutation in the Ipd gene provides an ethanol pathway whereby ethanol is produced by a bacterium as the primary fermentation product.

In a further embodiment, one or more alternative routes for fermentation in a bacterium are inactivated. In one embodiment, 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 Idh gene, for example, the IdhA gene. In yet another embodiment, the mutation is in the pfl gene, for example, the pflB gene. In yet another embodiment, alternative routes for fermentation include lactate production by lactate dehydrogenase (Jdh), acetate, ethanol, formate or conversion of H2 and CO2 by pyruvate formate lyase (pfl) or succinate production.

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 negative bacteria. In certain embodiments, the bacteria are Gram negative bacteria. In particular embodiments, Gram-negative bacteria are selected from the group consisting of Acinetobacter, Glueonobaeter, Eseheriehia, Geobaeter, Shewanella, Salmonella, Eneterobaeter and Klebsella. In other embodiments, the bacteria are Gram negative bacteria. In particular embodiments, Gram-negative asbacteria are selected from the group consisting of Baeillus, Clostridium, Corynebaeterium, Lactobacillis, Laetococeus, Oenoeoceus, Streptoeoceus and Eubaeterium. In yet other derealization forms, the bacteria are Escherichia coli.

As described above, non-recombinant bacteria of the invention comprise one or more mutations, for example a mutation in an lpd gene. In one embodiment, the mutation comprises substituting one amino acid for another amino acid such that substitution changes the pK of the polypeptide expressed by the mutated Ipd gene. In certain embodiments, the mutation in the Ipd gene causes NADH insensitivity. That is, the cell carrying such a mutation manifests a decrease in the sensitivity of the PDH enzyme to NADH. In this way, a NADH-insensitive cell produces four NADH molecules per glucose (2 glucose and 2 PDH reaction), and all four NADHs can be used to reduce two acetyl-CoA to ethanol. In certain embodiments, PDH's NADH insensitivity to NADH and its ability to function even at a high NADH / NAD ratio allows a cell, for example, a bacterial cell, to be a homoethanol product.

In one embodiment, the polypeptide comprises SEQID No. 6 and the mutation comprises the substitution of one wild type amino acid for another amino acid in:

(a) heading 322 or any position within about 50 positions on the side of heading 322 in SEQID N ° 6 or

(b) heading 354 or any position within about 50 positions on the side of heading 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 dearginine, asparagine, glutamine, histidine and lysine.

In one embodiment, the mutation comprises H-substitution at position 322 with any amino acid, such that amino acid substitution increases the acidity of the mutated Ipd expressed polypeptide. In a particular embodiment, the non-recombinant bacterium has a mutation comprising the substitution of H to Y in position 322 in SEQ ID NO: 6. In one embodiment, the non-recombinant bacterium is the E. coli strain SE2377, represented by a deposit with the Agricultural Research Culture Collection and indicated as deposit number NRRL B-30970. In another embodiment, the non-recombinant bacterium is the E. coli strain SE2383, represented by a depot with the Agricultural Research Culture Collection and indicated as deposition number NRRL B-30973. In yet another embodiment, non-recombinant abacteria is the E. coli strain SE2384, represented by a deposit with the Agricultural Research Culture Collection and indicated as deposit number NRRL B-30974. In a further embodiment, strain SE2377 comprises SEQ ID NO: 1 or a fragment thereof. In another embodiment, strain SE2383 comprises SEQ ID NO: 1 or a fragment thereof. In yet another embodiment, strain SE2384 comprises SEQ ID NO: 1 or a fragment thereof.

In another embodiment, the mutation comprises replacing E at position 354 with any amino acid, such that amino acid substitution reduces the acidity of the mutated Ipd expressed polypeptide. In a particular embodiment, the non-recombinant bacterium has a mutation comprising the substitution of EtoK in position 354 in SEQ ID NO: 6. In one embodiment, the non-recombinant bacterium is the E. coli strain SE2378, represented by a deposit with the Agricultural Research Culture Collection and indicated as deposit number NRRL B-30971. In another embodiment, the non-recombinant bacterium is the E. coli strain SE2382, represented by a depot with the Agricultural Research Culture Collection and indicated as deposition number NRRL B-30972. Still, in another embodiment, non-recombinant abacteria is the E. coli strain SE2385, represented by a deposit with the Agricultural Research Culture Collection and indicated with deposit number NRRL B-30975. In a further embodiment, strain SE2378 comprises SEQ ED No. 3 or a fragment thereof. In another embodiment, strain SE2382 comprises SEQ ED No. 3 or a fragment thereof. In yet another embodiment, strain 2385 comprises SEQ ID NO: 3 or a fragment thereof.

Non-recombinant bacteria comprising one or more of the mutations described above are suitable for the production of ethanol from sugar. According to the invention, the mutation provides a homoethanol defermentation pathway. In certain embodiments, the ethanol produced comprises more than 50% of total non-gaseous fermentation products under anaerobic conditions.

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

In another embodiment, one or more alternative routes for fermentation in a bacterium are inactivated. Alternative routes for fermentation include lactate production by lactate dehydrogenase (ldh), acetate, ethanol, formate, H2 and CO2 starting from compiruvate formate lyase (pfO and succinate. In one embodiment, the alternative routes for fermentation are mutation-inactivated. In particular embodiments, alternative fermentation pathways are inactivated by introducing deletion mutations in a bacterium.III 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 Ipd gene with one or more mutations which when present in the bacterium of the invention results in the production by a bacterium of ethanol as the primary fermentation product under anaerobic conditions.

Nucleic acid molecules of the invention include DNA molecules and RNA molecules and DNA or RNA analogs generated using nucleotide analogs. The nucleic acid molecule may be single or double stranded, but advantageously is double-stranded DNA.

In one aspect, the invention provides isolated isolated nucleic acid molecules selected from the group consisting of:

(a) a nucleic acid molecule comprising a nucleotide sequence that 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 a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a complement thereof;

c) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of 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 encoding a fragment of a polypeptide comprising an amino acid sequence of SEQID 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 encoding a naturally occurring allelic variant of a polypeptide comprising an amino acid sequence of SEQ LD N0: 2 or SEQ ID NO: 4, wherein the nucleic acid molecule hybridizes to a complement of a nucleic acid molecule which comprises SEQ ID NO: 1 or SEQ ID NO: 3 under severe conditions;

f) a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a complement thereof; and

g) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 2or SEQ ID NO: 4;

wherein the nucleic acid molecule when expressed in a cell makes the cell capable of producing ethanol as the primary defermentation product.

In one embodiment, the ethanol produced by the cell comprises more 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 a nucleic acid molecule, is not ethanogenic. In a particular embodiment, the non-ethanogenic bacterial cell produces ethanol as the secondary fermentation product, that is, less than about 40% of 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 a bacterial cell provides a dehomoethanol fermentation pathway in a bacterial cell by 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 SEQID heading No 997: 1.

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 SEQID NO. : 1.

In one embodiment, the delpd 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 (for example, as compared to the total length of the nucleotide sequence) shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a complement thereto. SEQ ID NO: 1 and SEQ ID NO: 3 are shown in Figures I (A) and 3 (A), respectively.

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

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

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

The Ipd genes, as described herein (and in italics by convention), include a nucleic acid molecule (for example, a DNA molecule or segment thereof), for example, an RNA-encoding nucleic acid polypeptide or molecule that, in an organism, It is separated from another gene or genes by intergenic DNA (ie, intervention DNA or spacer that naturally flanks the gene and / or separate genes in the organism's chromosomal DNA). A gene may direct the synthesis of an enzyme or other peptide molecule (for example, it may comprise coding sequences, for example, a contiguous open reading frame (ORF) encoding a polypeptide) or may itself be functional in the body. A gene in an organism may be grouped into an operon, as defined therein, where the operon is separated from other genes and / or operons by intergenic DNA. Individual genes contained within an operon may overlap without intergenic DNA between individual genes.

One embodiment of the present invention features nucleic acid molecules or mutant Ipd genes. Typically, the mutant nucleic acid molecule or mutant gene as described herein includes a nucleic acid molecule or gene having a nucleotide sequence that includes at least one alteration (e.g., substitution, insertion, deletion) such that the polypeptide or polypeptide may be The mutant encoded 9 activity differs from the polypeptide or polypeptide encoded by a wild type nucleic acid molecule or gene. Advantageously, the mutant or genemuting nucleic acid molecule (e.g., an Ipd mutant gene) encodes an LPD polypeptide having enhanced activity, e.g., dihydrolipoamidashydrogenase activity.

In one embodiment, a nucleic acid molecule of the invention hybridizes under severe conditions to a nucleic acid molecule having a nucleotide sequence shown as SEQ ID NO: 1 or SEQ ID NO: 3. Such severe conditions are known to those skilled in the art. can be observed in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. A particular non-limiting example of harsh hybridization conditions (eg, high severity) is 6X sodium chloride / citrate disodium (SSC) hybridization at about 45 ° C, followed by one or more washes in 0.2X SSC. 0.1% SDS at 50 to 65 ° C. Advantageously, an isolated nucleic acid molecule of the invention that hybridizes under severe conditions with respect to the sequence of SEQ ID SEQ ID 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 naturally occurring nucleotide sequence.

A nucleic acid molecule of the present invention (e.g., a nucleic acid molecule having a denucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3) may be isolated using standard molecular biology techniques and sequence information. provided in this. For example, nucleic acid molecules may be isolated using standard hybridization techniques and cloning techniques (for example, 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 polymerase chain reaction using synthetic oligonucleotide initiators named based on the sequence of SEQ ID NO: 1 SEQ ED NO: 3. A nucleic acid of the invention may be amplified using cDNA, mRNA or alternatively, genomic DNA as a standard 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 that is a complement to the nucleotide sequence shown in SEQID No. 1, SEQID No.: 3.

Additional Ipd nucleic acid sequences are those comprising a nucleotide sequence of SEQ ID NO: 1, SEQ IDN: 3, which encodes a polypeptide homologue having an amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID N0: 4 (for example, encodes a polypeptide having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more identity to the polypeptide having an amino acid sequence shown in SEQ SEQ ID NO: 4, having substantially identical activity as the polypeptide), hybridizes under severe conditions to all or a fragment of a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1. , SEQID No. 3 or all or a fragment of a nucleic acid molecule that encodes a polypeptide having an amino acid sequence of SEQ ID NO: 2, or SEQ ID NO: 4, or are complementary to an Ipd nucleotide sequence as shown in this and such that a sequence of Nucleic acid ipd, when expressed in a cell, results in ethanol cell production as the primary fermentation product under anaerobic conditions.

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

In another embodiment, an Ipd nucleic acid molecule or gene encodes an LPD polypeptide homolog having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Typically, the "homologous" includes a polypeptide or polypeptide that divides at least about 30 to 35%, advantageously at least about 35 to 40%, more advantageously at least about 40 to 50%, and even more advantageously at least about 60%. 70%, 80%, 90% or more identity to an amino acid sequence of a wild type polypeptide or polypeptide described herein and having a substantially equivalent biological or functional activity as the wild type or polypeptide polypeptide. For example, an LPD counterpart divides at least about 60%, advantageously at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, or more opolipeptide identity having an amino acid sequence shown as SEQ ID NO: 2 or SEQ ID NO: 4, and has substantially equivalent functional or biological activity (that is, it is a functional equivalent) dopolipeptide having an amino acid sequence shown as SEQ ID NO: 2 or SEQ ID NO: 4 (for example, has a substantially equivalent dihydrolipoamide dehydrogenase activity).

In one embodiment, an Ipd nucleic acid molecule or gene comprises a nucleotide sequence encoding a polypeptide shown as SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, an Ipd nucleic acid molecule hybridizes to all or a fragment of a nucleic acid molecule having the nucleotide sequence set forth in SEQ LD No. 1 or SEQ ID No.: 3 or hybridizes to all or a portion of a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having an amino acid sequence of either SEQ ED No.: 2 or SEQ ID No.: 4.

Such hybridization conditions are known to those skilled in the art and can be observed in Current Protocols in Molecular Biology, Ausubel, et al., Eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additional severe conditions can be seen in Molecular Cloning: A Laboratory Manual, Sambrook, et al., Cold Spring Harbor Press, Cold Spring Harbor, ΝΎ (1989), chapters 7, 9 and 11. A particular non-limiting example of severe hybridization includes sodium chloride / citrate hybridization. 4X Sodium (SSC) at about 65 to 70 ° C (4X SSC hybridization plus 50% formamide at about 42 to 50 ° C) followed by one or more IX SSC washes at about 65 to 70 ° C ° C. A particular non-limiting example of highly severe hybridization conditions includes IX SSC hybridization at about 65 to 70 ° C (IX SSC hybridization plus 50% formamide at about 42-50 ° C) followed by one or more washes at 0 ° C. .3X SSC at about 65 to 70 ° C. A particular non-limiting example of low stringency hybridization conditions includes 4X SSC hybridization at about 50 to 60 ° C (or alternatively 6X SSC hybridization plus 50% about 40 to 45 ° C formamide) followed by one or more washes at 2X SSC, at about 50 to 60 ° C. intermediate ranges for the values cited above, for example from 65 to 70 ° C or from 42 to 50 ° C are also intended to be encompassed by the present invention. SSPE (IX SSPE is 0.15 M NaCl, 10 mM NaCl 2 PO4 and 1.25 mM EDTA, pH 7.4) can be replaced by SSC (IXSSC is 0.15 M NaCl and 15 mM sodium citrate) in hybridization and wash buffers; washes are performed for 15 minutes each after hybridization is complete. The hybridization temperature for anticipated hybrids to be less than 50 base pairs in length should be 5 to 10 ° C lower than the fusion temperature (Tm) of hybrids, where aTn is determined according to the following equations. For hybrids smaller than 18 base pairs in length, Tm (° C) = 2 (# of A + Tbases) + 4 (# of G + C bases). For hybrids between 18 and 49 base-length pairs, Tm (° C) = 81.5 + 16.6 (log10 [Na +]) + 0.41 (% G + C) - (600 / N), where N is the base number in the hybrid and [Na +] is the concentration of disodium ions in the hybridization buffer ([Na +] for IX SSC = 0.165 M).

It will be appreciated by the skilled practitioner that additional reagents may be added for hybridization and / or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example nitrocellulose or nylon membranes, which include but are not limited to blocking agents. (e.g. BSA or salmon or herring sperm DNA), detergents (eg SDS), chelating agents (eg EDTA), Ficoll, PVP and others when using nylon membranes, in particular a non-limiting example In addition to severe hybridization conditions is the 0.25 to 0.5 M hybridization of NaH2PO4, 7% SDS at about 65 ° C, followed by one or more washes in 0.02 M NaH2PO4, 1% SDS at 65 ° C. C, see, for example, Church and Gilbert (1984) Proc. Natl. Acad. Know. USA 81: 1991-1995, (or alternatively 0.2X SSC, 1% SDS). In another embodiment, an isolated nucleic acid molecule comprises a nucleotide sequence that is complementary to an lpd nucleotide sequence as set forth herein (for example, is the full complement of nucleotide sequence shown as SEQ ID NO: 1 or SEQ ID No.: 3).

III. SSC polypeptides are 0.15 M NaCl and 15 mM sodium citrate) in hybridization and wash buffers; washes are performed for 15 minutes each after hybridization is complete. The hybridization temperature for anticipated hybrids to be less than 50 base pairs in length should be 5 to 10 ° C lower than the fusion temperature (Tm) of hybrids, where aTn is determined according to the following equations. For hybrids smaller than 18 base pairs in length, Tm (° C) = 2 (# of A + Tbases) + 4 (# of G + C bases). For hybrids between 18 and 49 base-length pairs, Tm (° C) = 81.5 + 16.6 (log10 [Na +]) + 0.41 (% G + C) - (600 / N), where N is the base number in the hybrid and [Na +] is the concentration of disodium ions in the hybridization buffer ([Na +] for IX SSC = 0.165 M).

It will be appreciated by the skilled practitioner that additional reagents may be added for hybridization and / or wash buffers to decrease nonspecific hybridization of nucleic acid molecules to membranes, for example nitrocellulose or nylon membranes, which include but are not limited to blocking agents. (e.g. BSA or salmon or herring sperm DNA), detergents (eg SDS), chelating agents (eg EDTA), Ficoll, PVP and others when using nylon membranes, in particular a non-limiting example In addition to severe hybridization conditions is the 0.25 to 0.5 M hybridization of NaH2PO4, 7% SDS at about 65 ° C, followed by one or more washes in 0.02 M NaH2PO4, 1% SDS at 65 ° C. C, see, for example, Church and Gilbert (1984) Proc. Natl. Acad. Know. USA 81: 1991-1995, (or alternatively 0.2X SSC, 1% SDS). In another embodiment, an isolated nucleic acid molecule comprises a nucleotide sequence that is complementary to an lpd nucleotide sequence as set forth herein (for example, is the full complement of nucleotide sequence shown as SEQ ID NO: 1 or SEQ ID No.: 3).

III. PolypeptidesFeatures of the invention of polypeptides (e.g., mutant ethanological enzymes, e.g., dihydrolipoamide dehydrogenase (LPD)). When polypeptides are expressed in a cell, for example a bacterium, the cell produces ethanol as the primary defermentation 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 ID NO: 4, wherein the fragment comprises at least 15 continuous 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 that hybridizes to the complement of a nucleic acid molecule which comprises SEQ ID NO; 1 or SEQ ID NO: 3 under severe conditions;

c) a polypeptide that is encoded by a nucleic acid molecule that is at least 50% identical to the nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3;

d) a polypeptide comprising an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; and

e) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; and wherein the polypeptide, when expressed in a cell, yields 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 non-gaseous fermentation products under anaerobic conditions. In another embodiment of this embodiment, the polypeptide has dihydrolipoamidase dehydrogenase activity under anaerobic conditions. In a further embodiment, the cell is a bacterial cell.

In yet another embodiment, the bacterial cell, in the absence of polypeptide expression, is not ethanogenic. In a particular embodiment, nonethanogenic bacterial cells produce ethanol as the minor fermentation product; that is, 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 yet an additional embodiment the ethanol produced comprises greater than 50% of the non-gaseous fermentation products under anaerobic conditions. In a particular embodiment, expression of the polypeptide in the bacterial cell provides a homoethanol defermentation 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 ID NO: 4, wherein the fragment comprises at least 15, 25, 35, 45. , 55, or 65 continuous amino acid residues of an 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 total length of a sequence of amino acid) to an 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 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 which 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 ID NO: 4; wherein the fragment comprises at least 15 continuous 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 that hybridizes to a complement of a nucleic acid molecule which comprises SEQ ID NO: 1 or SEQ ID NO: 3 under severe conditions;

which comprises cultured bacterial host cells containing the isolated nucleic acid molecules encoding dihydrolipoamide dehydrogenase polypeptides or fragments thereof, under conditions under which the nucleic acid molecule is expressed.

In one embodiment, the LPD polypeptide or gene product is derived from a non-recombinant Gram-positive or ethanogenic Gram-negative bacterium. In exemplary embodiments, the LPD polypeptide or gene product is derived from an ethanological 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 a gram-positive ethanogenic 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 from polypeptides or gene products encoded by the naturally occurring bacterial genes. Still included within the scope of the present invention are bacterial derived polypeptides or product of the naturally occurring differing gene of differing and / or Escherichia coli genes (e.g., lpd), for example, genes having nucleic acids that are mutated, inserted or deleted, but encoding polypeptides substantially similar to the naturally occurring gene products of the present invention, for example, comprising dihydrolipoamide dehydrogenase activity.

It is then understood that a person skilled in the art can (for example, substitute) nucleic acids encoding conservative amino acid substitutions. It is well understood that a person skilled in the art can replace, add or delete amino acids to some degree without substantially affecting the function of the gene product (e.g., dihydrolipoamide dehydrogenase) as compared to a naturally occurring gene product, each example being intended to be included within the scope of the present invention.

Included within the scope of the invention are non-matching bacteria comprising an lpd gene comprising a mutation, wherein the substitution is a H-position 322, or E-position354 mutation in the wild type of the Ipd gene (SEQ DD N °: 6 ), for any amino acid, such that the amino acid alters the acidity of the region. In further embodiment forms, the amino acid is a physiologically neutral charge amino acid. In still further embodiments, the amino acid is a base charge amino acid at physiological pH.

In one embodiment, an isolated polypeptide of the present invention (for example, an isolated dihydrolipoamide dehydrogenase enzyme) has an amino acid sequence shown in SEQID No. 2 or SEQ ED No. 4. In other embodiments, an isolated polypeptide is isolated of the present invention is a homologue of at least one of the polypeptides set forth in SEQ ID NO: 2 or SEQ ID NO: 4 (for example, comprises an identical at least about 30 to 40% amino acid sequence, advantageously about 40 to 50 % identical, more advantageously about 50 to 60% identical, and even more advantageously about 60 to 70%, 70 to 80%, 80 to 90%, 90 to 95% or more identical to an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and has an activity that is substantially similar to that of the polypeptide encoded by the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, respectively.

To determine the percent identity of two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison purposes (for example, slits can be introduced in the sequence of a first amino acid or nucleic acid sequence by optimal alignments with a second amino acid or nucleic acid sequence). When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical in that position. The percent identity between their sequences is a function of several identical positions divided by the sequences (ie% identity = #of identical positions / total # of positions χ 100), advantageously made into account the various slots and size of slots needed to produce a great deal. alignment.

Sequence comparison and determination of the percent identity between two sequences can be performed using a mathematical algorithm. In particular, a non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Know. USA 87: 2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Know. USA90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST (version 2.0) programs of Altschul, et al. (1990) J. Mol. Biol. 215: 403-410. BLAST nucleotide scavenging can be performed with the NBLAST program, count = 100, word length = 12 to obtain nucleotide sequence homologs for the inventive nucleic acid molecule. BLAST polypeptide search can be performed with the XBLAST program, count = 50, word length = 3 to obtain amino acid sequence homologs for invention polypeptide molecules. To obtain slotted alignments for the purpose of comparison, slotted BLAST may be used as described in Altschul, et al. (1997) Nucleic Acids Research 25 (17): 3389-3402. When using BLAST and BLAST with slot programs, the lack of the respective program parameters (for example, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another particular, nonlimiting example of a mathematical algorithm used for a comparison of sequences is the algorithm of Myers and Miller (1988) Comput Appl Biosci.4: 11-17. Such an algorithm is incorporated into the available ALIGN program, for example, on the GENESTREAM, IGH Montpellier, FRANCE or server or ISREC network server. When using the ALIGN program for amino acid sequence comparison, a PAM120 weight residue table, a slit length penalty of 12, and a slit penalty of 4 can be used.

For example, in one embodiment of the opercent invention the identity between two amino acid sequences is determined using the Blast server in NCBI or ClustalW in the European Biotechnology Institute. For example, the amino acid sequence of dihydrolipoamide dehydrogenase from various organisms is compared to that of E. coli protein Lpd, and the percent identity of a specific sequence to that of the E. coli sequence that can be obtained from the databases. Dice. Table 7 and Figure 9 illustrate these comparisons. The value in parenthesis represents the total similarity of the protein specific to that of E. coli Lpd and including amino acid positions that are identical to the positions at which a conservative substitution occurs. For Bacilus subtilis, two dihydrolipoyl dehydrogenase, one from the PDH complex and one from acetoin dehydrogenase, were included for comparison.

A person skilled in the art will be recognized on the basis of the above calculations, there is a high degree of conservation in LPD among a range of bacterial species, including bacteria which are philogently removed from each other (based on 16S RNA (DNA) ribosome) , such as, for example, Gram-positive and Gram-negative bacteria, archaea and Streptomyces.

Purulent enzyme dehydrogenase is observed in all aerobic organisms and is the essential enzyme in glucose conversion for energy. Dihydrolipoamide dehydrogenase (Lpd) is one of three subunits of the PDH complex. Lpd contains two unique motifs: a flavin-binding motif (amino acids 15 to 45) and a pyridine bisulfide-nucleotide oxidoreductase motif (amino acids 347 to 456) (E.coli Lpd numbering). The amino acid sequence of the Lpd proteins of the various organism has significant homology due to their unique role in an amino acid identity PDH complex sequence between Lpd E. coli and other bacterial Lpd ranges from 30% to 99%. In the extreme case of E. coli eHuman Lpd, 42% of the amino acids in the sequence are identical. In the Lpd flavin deletion region (amino acids 15 to 45), this sequence identity increases to 67%. In a subsection of this 18 amino acid sequence (positions 28 to 55), but all of one amino acid is conserved in the human E. coli mouse sequences. Histidine at 322 and glutamate at 354 are also conserved among these proteins. Due to the higher degrees of sequence conservation, the E. coli Lpd mutations that are described in the present finding are expected to have similar phenotypes in introducing Lpd proteins from other organisms. Thus, the methods of the invention are not limited to the strains shown herein.

IV. Non-Recombinant Bacteria Manufacturing Methods

In a further aspect of the invention there are provided non-recombinant bacteria comprising an Ipd gene having one or more mutations, wherein the mutation yields non-recombinant bacteria capable of producing ethanol as the primary fermentation product under anaerobic conditions, and wherein the bacterium is prepared. a process that comprises the steps of:

(a) development of a mutant candidate strain of a bacterium under anaerobic development conditions in a rich sugar medium and

b) selection mutants that produce ethanol as the major fermentation product.

In one embodiment of the method, ethanol produced comprises greater than 50% of non-gaseous fermentation products under anaerobic conditions.

In another aspect, the invention provides a method of producing the ethanological non-recombinant bacteria of the invention which comprises the steps of: a) developing the candidate mutant bacterial strain under conditions of anaerobic development in rich sugar medium; and

b) selection mutants that produce ethanol as the major fermentation product.

In embodiments of the foregoing methods and processes of the invention, 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 bacteria non-matching having the above mentioned attributes are also ethanogenic. Accordingly, the invention provides methods for the manufacture of ethanological non-recombinant bacteria. Furthermore, the invention provides methods for evaluating the desired ethanological phenotype.

The precursor strain of the invention is characterized by a low level of ethanol production under anaerobic conditions when developed in sugar rich medium. An example of such a strain should be strain AH242; however, any strain that is characterized by low levels of ethanol production under anaerobic conditions is suitable for the particular use. Further mutation of a precursor strain according to methods known in the art (Dastenko and Wanner, 2000. Proc. Natl. Acad.Sei. USA 97: 6640-6645.) Is performed to yield the precursor strain capable of anaerobic (defective) development. In addition, a cassette for antibiotic resistance is added for selection purposes in accordance with well-known practice in the art.

Typically, selection is performed by cultivating the development of the defective strain under aerobic conditions under the average developmental phase and is achieved by expanding the emagar culture and exposing the culture by mutagenesis agent. One skilled in the art will recognize that various mutation agents may be used, including ethyl methane sulfonate, 2-aminopurine, ICR-191, methyl methane sulfonate, N-methyl-1SP-nitro-N-nitrosoguanidine, or any other agent known as cause a change in nucleotide sequence. After exposure to the mutated agent under anaerobic conditions, the cultures are changed by aerobic conditions, then back to the anaerobic conditions. Enlarged colonies were picked and plotted on the fresh plates and developed under anaerobic conditions. Each colony can be separately grown and grown on the appropriate antibiotic plate to confirm that mutant carriers of the doprecursor antibiotic resistance. One skilled in the art will understand that bacterial culture procedures are performed according to the standard protocol for the technique.

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 may be detected by HPLC.

One skilled in the art may recognize that based on the examples mentioned above, and based on homology between bacterial strains, the methods of the instant invention are not limited to those shown in the instant application.

V. Methods to Produce Ethanol

In another aspect, the invention provides a method for producing ethanol from an oligosaccharide source. The method comprises contacting the oligosaccharide with non-recombinant bacteria or host cells of the invention as described above, thereby producing 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 produce ethanol and acetate at a ratio of 1: 1 during anaerobic development. During the stationary phase of development, wild-type E.coli produces lactate as a major product, and the fraction of ethanol in total fermentation products is about 20%. The products in all of these fermentations comprise various acids, thus leading to the term, mixed acid fermentation. In one aspect, the present invention provides non-recombinant bacteria comprising an Ipd gene having one or more mutations, wherein the mutation yields non-recombinant asbacteria capable of producing ethanol as the primary defermentation product of broad anaerobic conditions. The primary defermentation product is intended to include non-gaseous fermentation products comprising greater than 50% of the total non-gaseous product. The primary fermentation product is the most abundant non-gaseous product.

Typically, fermentation conditions are selected to provide optimum pH and temperature to promote developmental kinetic improvements of the produced host cell strains and catalytic conditions for the enzymes produced by the culture medium (Doran, et al., (1993) Biotechnol. Progress. 9: 533-538). For example, for Klebsiella, for example acepa P2, optimal conditions were determined to be between 35 to 37 ° C and pH 5.0 to pH 5.4. Under these conditions, yet exogenously added endoglucanasefungica and exoglucanase are completely stable and continue to function for long periods of time. Other conditions are discussed in the examples. In addition, it will be appreciated by the skilled artisan that only routine experimentation is required, using practices known in the art, to optimize a fermentation arrangement of the invention. See, for example, U.S. Patent No. 5,424,202 and 5,916,787, which are specifically incorporated herein by reference.

In yet another aspect, the invention provides a kit comprising non-recombinant bacteria or inventive host cells as described above, and instructions for producing ethanol according to the methods and methods described herein. In one embodiment, okit comprises a sugar source.

SAW. Exemplification

The invention is further illustrated by the following examples, which are not to be construed as limiting. By all 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 ResearchCulture Collection and designated as deposit number NRRL B-30967 (AldhA and E (focA pflB)), were used in this study. Strain SE2378 is an ethanological mutant of strain AH242. The deletion of the pflB, adhE, mgsA and aceF genes were as per Dastenko, et al., The IdhA deletion strain was constructed after the introduction of Tn10 transposon into IdhA followed by the selection in fusaric acid medium (Klekner, et al. 1991; Maloy, et al., 1981). Construction of other strains utilizes standard egenetic molecular biological techniques (Maniatis, et al. 1982; Miller, et al. 1972). The genotypes of the parasites used in this are listed in Table 1, shown below.

Table 1: Bacterial strains and relevant genotype

<table> table see original document page 46 </column> </row> <table> Development and fermentation media

The rich medium (Broth L) contained (per liter), peptone tryptase (10g), yeast extract (5g) and NaCl (5g) (Lee, et al. 1985). Saline minerals have been previously described (Lee, et al. 1985). Glucose or xylose has been added as needed. Fermentations were conducted at 37 ° C as previously described (Hasona, et al. 2004). Culture pH was maintained at 7.0 by the addition of KOH. Batching as fermentations were conducted in 13 χ100 mm screw-capped tubes deposited on top as previously described (Patel, et al. 2006).

Vectors and transformation

Cloning and expression of Ipd as well as genes in the dph region is performed according to standard procedures described in the art. Vectors used in the transformation may include pTrc99a (GE), pCR2, l-TOP, pBR322, pUC19, pACYC184, pBAD24, in addition to other commonly known vectors. A CaCl2 based technical transformation method was used according to the standard procedure observed in Mantistis, et al. (1989).

Analytical Methods

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

EXAMPLE 1

ISOLATION OF THE ETHANOLOGICAL NON-RECOMBINANT E. COLI CEPAS SE23 77, SE23 78, SE2382, SE2383, SE2384, SE2385

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

The starting AH242 strain was used for isolation of the described homoethanogenic mutants of Escherichia coli. The AH242 strain is unable to anaerobic development in rich media containing sugars due to mutations in the Idh and pflB genes encoding dehydrogenase delactate (LDH) and pyruvate formate lyase (PFL), respectively (Mat-Jan, et al. 1989). Despite these mutations, aerobic development of AH242 remains unaffected. The anaerobic developmental defect in AH242 is a result of a deficiency in the re-oxidation of NADH to NAD +, an essential substrate for key enzymatic glyceraldehyde-3-phosphate dehydrogenase, and associated ATP production. The absence of LDH eliminates NADH oxidation by reducing lactate pyruvate. In the absence of acetyl CoAde which is normally produced by PFL, here is sufficient acetyl CoA available for effective oxidation of NADH by native aldehyde, and alcohol dehydrogenase activities.

At the time of the invention, starting with strain AH242, focA- and -pflB (pyruvate formate lyase) cannulation was constructed using previously described methods (Datsenko and Wanner, 2000). The single deletion mutants, AH240, - (focA-pflB) and AH241 - (IdhA) were precursor strains of the double mutant AH242 strains. At the annulment site, the FRT-Km-FRT cassette was inserted, thereby yielding acepa AH242 kanamycin resistance. Due to two mutations, cepaAH242 is of defective anaerobic development in total media. Table 2 below lists the developmental characteristics of E. coli mutants with mutations in anaerobic fermentation pathways. Table 2: Developmental characteristics of E.coli mutants commutations in anaerobic fermentation pathways.

<table> table see original document page 49 </column> </row> <table>

LB - Broth L; Minimum - minimum means of glucose supplemented with or without esuccinate acetate (1 mg / ml each). NG - no development.

Parenthesis value was the half-development rate of glucose with acetate, succinate and glutamate (1 mg / ml).

The resulting defective anaerobic development strain AH242 was grown in 5 ml of broth L under aerobic conditions at 37 ° C on a shaker at 200 RPM. In the exponential medium phase of development, the culture medium was removed from a shaker and expanded on L-glucose agar, or L-glucose agar plus a neutral redox dye. A Whatman paper filter was replaced on the surface of each medium. agar. The ethyl methane sulfonate mutagenesis agent (EMS) was added to the disc, and the plates were transferred to an anaerobic jar containing a palladium catalyst covered H2 + CO2 generator to create an O2 free environment. Other standard agents suitable for use in mutagenesis may be used in the invention. The anaerobic jar with the plates was incubated at 37 ° C for 5 days.

After 5 days of non-visible development, the indicated name was detected. Subsequently, both plates were incubated under aerobic conditions for 20 hours. At the end of this incubation, a bacterial cell layer was observed in both media and all but the adjacent area where the EMS paper disc was placed. Cells on the surface of each medium were transferred to a fresh medium of the same composition by replica plating and placed in the jar for 5 days. After 5 days, each plate had 100 colonies in all areas except where the EMS was placed. 31 colonies were chosen from one of glucose (15 colonies) and glucose + neutral red plates (16 colonies) and fresh L + agar layers. The plates were developed under anaerobic conditions. All colonies increased anaerobic conditions.

31 mutants were inoculated into the L + glucose culture broth, and after development each was transferred to the surface of L + kanamycin plates. All mutants increased in the presence of kanamycin, indicating that they perform antibiotic resistance of AH242 precursor strain.

The 31 mutants were transferred to L + glucose broth in screw-capped tubes, and incubated at 37 ° C without mixing. After visible development 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 ethanol-producing mutants as the primary, or largest fermentation product (73%). The remaining product was a combination of succinate and acetate.

These results show that non-recombinant mutants from the AH242 strain of origin are incapable of development in an anaerobic environment, have been isolated and are capable of development in anaerobic conditions, and produce ethanol as the major defermentation product. Table 3: Derivative fermentation profiles of ethanogenic mutants dacepa of E. coli AH242

<table> table see original document page 51 </column> </row> <table>

EXAMPLE 2

DEVELOPMENT RATE AND PROFILE DEFERMENTATION OF NON RECOMBINANTESETANOLOGICAL BACTERIA

In this example, the developmental rate and defermentation profile of ethanological non-recombinant bacteria are described.

From the above studies, the mutant SE2378 strain, which is capable of development under anaerobic conditions, and produces ethanol as the largest fermentation product, was selected for further study.

Development Features

Developmental characteristics of mutants of E.colicom mutations in anaerobic fermentation pathways were examined. The aerobic development of SE2378 strain was comparable to that of wild type E.coli strain W3110 or either single or double mutant (focA-pflB) or IdhA when grown in rich medium as described in Table 2, above. At a minimum, the aerobic development rate of dacepa SE2378 was about half of an AH242 precursor strain. Supplementing the development medium with acetate and succinate re-stores the development rate for that next precursor. Although the SE2378 strain increased anaerobically, the development rate, still half rich, was only about 50% than that of the single mutants AH240 and AH241 (see Table 2, above). Strain SE2378 did not increase anaerobically in minimal glucose medium, a phenotype associated with the pflB mutation (Clark, et al. 1989). Supplementation of the minimal medium with acetate supports the development of the pflB mutant, strain AH240, but not that derived from the ethanological strain SE2378. SE2378 strain also requires glutamate in addition to acetate for anaerobic development in minimal glucose medium. Previous studies have shown that the KOlletanologian Escherichia coli strain also requires glutamate for optimal xylose fermentation (Underwood, et al. 2004). This need for glutamate can be overcome by adding a protective osmolyte, betaine, to the medium. However, glutamate's need for anaerobic development of strain SE2378 in minimal medium was not precluded by betaine, indicating a biosynthetic flow deficiency of acetyl-CoA to 2-ketoglutarate, a glutamate precursor, rather than an osmotic requirement. And by which acetyl-CoA was rapidly converted to ethanol by this ethanogen, and that acetyl-CoA is the limiting rate for biosynthesis. With these needs, the rate of development of the SE2378 strain in minimal medium is reached from that of the pflB source strain, AH240. Baby maceration liquid, a low-cost medium supplement, replaces glutamate for the development of strain SE2378 in minimal glucose medium. Glucose fermentations

In pH-controlled fermentations with 50 gl "1 glucose (Hasona, et al., 2004), strain SE2378 increased with a specific development rate of 0.46 h" 1 after a delay of about 6 hours, and produced ethanol as the product. primary (Figure 3 and Table 4, below). Targeting the strain of immediate origin, AH242 is not able to increase aerobically, fermentation of strain SE2378 was compared to wild type strain W3110. W3110 supplemented with a 50 g l-1 fermentation of glucose in 24 hours, while the mutant strain requires about 72 hours. This difference can be primarily attributed to a difference in cell density (2.5 mg / ml dry weight for wild type). against 1.7 mg / ml dry weight for one mutant) and the maximum specific sugar metabolism rate of two strains (4.1 to 3.3 g glucose h-1 g cells-1 for one wild type and the mutant, respectively ; Table 5 below). CepaSE2378 produces about 480 mmol l-1 ethanol (22 g l-1), 88% of the total products for which small amounts of acetate, lactate and succinate are included. This is a contrast to the W3110 strain fermentations in which ethanol represented only 27 % of products at 6.6 g l-1 (Table 4). The specific maximum yield observed by the SE2378 strain was 1.34 g h -1 g cells (Table 5) compared to the 1.6 gh -1 -1 g cell value reported by yeast batch fermentations (Smits, et al., 2000) .Table 4: Fermentation characteristics of E.coli SE2378 strain and wild type W 3110a strain

<table> table see original document page 53 </column> </row> <table> Fermentations were conducted in L broth supplemented with 50 g 1 "sugar at pH 7.0 and 37 ° C.

b Ethanol yield as a theoretical maximum fraction (0.51 g ethanol per g sugar) .c Ethanol as a molar fraction of total products per mole of fermented glucose.Table 5: Development and production of ethanol by development of

E. coli strain SE2378 in glucose or xylose.

<table> table see original document page 54 </column> </row> <table>

Abbreviations: μΜaχ =, specific development rate, h-1; Yx / s cells, g cells (substrate g) -1;

Qs, g of sugar consumed L "1 h" 1; Qp, g of ethanol L -1 h -1; Yp / s: g ethanol (substrate g) -1; qs, g sugar consumed (g dry cell weight) -3 h-3; qp g ethanol (g dry cell weight) -1 h -1

Xylose Fermentations

Both wild-type strain W3110 and mutant-SE2378 increased the similar rate during anaerobic fermentation with 50g l-1 xylose, although the slow-moving strain SE2378 for approximately 8 hours (Figure 3-and Tables 4 and 5 above). xylose-specific development were 80% glucose-like, consistent with previously published reports (Gonzalez, et al. 2002) The fermented-xylose-mutant strain is faster than wild-type W3110.After 48 hours, xylose autilization exceeded glucose utilization for strain SE2378.About 88% of the fermentation products recovered with strain SE2378 were ethanol, 20g 1-1 of 50g Vi of xylose.Maximum ethanol yield by strain SE2378 with xylose was 2.23 g h-1 g cell cells "1.

The specific ethanol yield of both W3110 and SE2378 quantities was higher with xylose than with glucose, as shown in Table 5. This may be indicative of lower energy yields from xylose metabolism (Hasona, et al., 2004). For wild type, the yield of the xylose ATP network is only about 1.5 per xylose, as compared to 3.0 per glucose. This required that the use of the xylose cells produce the same amount of cell mass. However, the specific rate of wild-type xylose consumption was only slightly higher than that of glucose (4.93 versus 4.10 g h -1 g-1 cells), as seen in Table 5 above, considering the lower cell yield. and the longer defermentation period compared to glucose fermentation. In contrast, it lacks strain SE2378 of pyruvate formalin lyase, an enzyme that is critical for minimal-medium xylose fermentation (Hasona, et al. 2004). Due to stamutation, the yield of the ATP network calculated from the dexylose fermentation in strain SE2378 is only 0.67 per xylose. This is apparently lower than the ATP yield which is higher than the highest xylose flow in this ethanogenic mutant. Specific ethanol yield from xylosede 2.23 g h -1 g cells "is greater than 1.6 g g cells" in glucose yeast (Smits, et al. 2000) and for glucose and xylose in the E.coli strain E.coli KO11 strain carrying the Z.mobilis pdc and adh genes (about 2 g h-1 g cells-1).

These results show that the non-matching SE2378 mutant produces ethanol as the primary fermentation product for both glucose and xylose. Moreover, the ethanol production rate is comparable to other ethanological organisms.

EXAMPLE 3

Identification of the E. colinon mutant ethanological LPD mutant LPD

In this example, identification of the LPD mutant gene from E. coli strains SE2377, SE2378, SE2382, SE2383, SE2384, SE2385 is described.

Mutations in the non-recombinant etiological mutant E.coli strains were mapped by co-transduction with zac :: Tn10. When the aroP-pdhR-aceEF gene was deleted by co-transduction (Figure 5), the transducer to this ability to increase in LB containing 1% glucose under anaerobic conditions, while the same cancellation on the basis of wild type does not affect anaerobic development. These results suggest a role for the pyruvate dehydrogenase enzyme in the anaerobic development of the SE2378 strain. The mutation responsible for a phenological phenotype in the SE2377, SE2378, SE2382 mutant strains identified is mapped to the pyruvate dehydrogenase gene site.

} Pyruvate dehydrogenase complex (PDH) consists of three enzymes, pyruvate dehydrogenase / decarboxylase (enzyme 1, E1), transacetyldehyde lipoate (enzyme 2, E2), and dihydrolipoamide dehydrogenase subunits (enzyme 3, E3). The pdhR promoter is known to be the promoter for transcription of the pdhR-aceEF-lpd genes, despite the presence of independent promoters for aceEF genes and IpdA genes (Quail et al, 1995). Since PDH expression is negatively regulated by the pdhR protein (Quail, et al., 1995), pdhR genes from SE2377, SE2378, SE2382 were sequenced (Figure 6A). Sequence analysis of the SE2378 strain reveals two mutations within the pdhR coding region: 1 an amino acid substitution (S 12P) and 1 leucine amino acid insertion as amino acid 118. Other nucleotide substitution G a A was observed in the intergenic region between the gene pdhR and the aceE gene (Figure 6B). Strain SE2377 and SE2382 do not mutate in the pdhR-aceEF region of genomic DNA. However, these strains, as well as strains SE2383, SE2384 and SE2385, all have simple mutations in the Ipd gene (Figure 5A). The PdhR protein is a responsive regulator of pdhR-Ipd operon pyruvate and thus mutations in this protein are not expected. The aceEF may contain this having the transcription starting site for aceEF-lpd in addition to the starting site at the beginning of pdhR for pdhR-lpd transcription. Thus, mutations in the intergenic region may also support a high level of aceEF-lpd expression in the anaerobic cell. It has been previously reported that the level of pyruvate dehydrogenase / PDH complex complex decarboxylase activity in E.coli is about 5 fold higher in aerobically versus anaerobically developing cells (deGraef, et al., 1999).

These results provide the location of mutations in the E strains. ethanilogenic coli identified from the instant invention.

EXAMPLE 4

LPD GENE MUTATION IS RESPONSIBLE FOR THE ETHANOLOGICAL PHENOTYPE

In this example, mutation in the PDH5 complex and specifically the 1pd gene is shown to be causative for an ethanological phenotype.

Preliminary genetic analysis of strain SE2378 reveals that mutations responsible for anaerobic development and homoethanol production are located at or near the gene encoding the PDH complex (pdh: pdhR, aceF, lpd site). To confirm that PDH is required by the ethanogenic phenotype of the SE2378 strain, a mutation in the aceF (dihydrolipile acetyltransferase; PDH E2 enzyme) gene was transduced into Nacepa YK1, a derivative of the SE2378 strain that lacks the akanamycin resistant gene. The YK93 transducer strain loses anaerobically developing capacity, as shown in Table 6, below.Table 6: Developmental Characteristics of SE2378 Ethanogenic E.coli Strain with a pdh Site Mutation (aceF)

<table> table see original document page 57 </column> </row> <table>

Minimum - Minimum of glucose supplemented with or without acetate and succinate (1 mg / mlcada). NG- No development.

* Both ethanogenic derivatives require acetate and glutamate for minimal aerobic development such as strain SE2378.

This less anaerobic phenotype of strain YK93 was similar to that of strain AH242, the origin of strain SE2378. Although an aceF mutant is less aerobic than minimal due to cellular inability to produce acetyl co-A by biosynthesis, under conditions of anaerobic development this function is catalyzed by PFL and thus an aceF mutation does not affect the anaerobic development of E. coli (strain YK153, W3110 with mutationaceF, as shown in Table 6). Anaerobic development of cepaYK93 was defective in all media tested. The aceF nacepa YK1 52 mutation was transduced by the aceF + Pl phage with the W3110 (wild type) or SE2378 (ethanogen) gene and transducers were selected for minimal medium development under aerobic conditions. Ostraductors have also been tested for anaerobic development and fermentation products. Translators who received the aceF + gene from wild-type strain W3110 increased aerobically by half but lack to increase anaerobically in either half due to the presence of the IdhA and pflB mutations. All translators wanted the aceF + gene from the SE2378 strain that increased aerobically and all translators tested produced ethanol as the main fermentation product. These results show that the SE2378 strain phenotype requires the intact pdh site and PDH activity, and agrees with a PDH-dependent pathway for ethanol production (see Figure 8 C). In this pathway for homoethanol production, pyruvate is oxidatively decarboxylated to acetyl-coA by PDH and further reduced to acetylaldehyde and ethanol by dehydrogenase alcohol (Figure 8C). The aceF (minus PDH; strain YK93) annulment that is required for acetyl-coA or adhE production (minus ADH; strain YK91), required for ethanol production, results in the anaerobic development of negative phenotype supporting the distortion role for homoethanol production. and redox equilibrium in strain SE2378 which is fermentative dehydrogenase in need of lactate and pyruvate formate lyase.

In this next series of experiments, the Ipd gene is shown to cause the ethanological phenotype. The wild type strain Ip31 gene W3110, and from an ethanogenic strain mutant SE2378 were cloned into an expression vector for the production of Lpd protein from the trc promoter with IPTG as an inducer. These plasmids were transformed into strain YK100 which performs three deletions: IdhA5 (focA-pflB), and lpd. In addition to the three mutations, strain YK100 is similar to strain W3110. Due to three cancellations, the YK100 strain is defective for aerobic development in total media tested, and is defective for aerobic development in minimal medium. As previously discussed, the pyruvate dehydrogenase (PDH) complex consists of three enzymes, depiruvate dehydruvenase / decarboxylase (enzyme 1), lipoate transacetylase (enzyme 2), lipoamide edehydrogenase (enzyme 3). Aerobic development of E. coli is slowed by a mutation in any of the three components of the PDH complex.

Plasmid pKY32 (Figure 7A), containing the lpd (Lpd +) gene from strain W3110, or plasmid pKY33 (Figure 7B) containing the mutant genelpd (Lpd *) from strain SE2378, was transformed into strain 1 and strain resistant transformants. ampicillin were selected. These transformers were positive for PDH as seen by aerobic development in minimal medium; enzyme 1 and enzyme 2 of the PDHa complex approaches the chromosome and approaches Lpd from the plasmid.

Only plasmid transformers pKY33 performed the lpd gene from the ethanogenic SE2378 strain were able to develop anaerobic conditions. Ethanol as the largest or largest fermentation product I name spent from strain YK100 / pKY33 (named YKl 29). In contrast, strain YK100 with plasmid pKY32 carries the W3110 natural lpd gene not developed under anaerobic conditions.

Taken together, these results show that the LPD protein is responsible for the observed activity of the depiruvate dehydrogenase complex under anaerobic development conditions, and that the mutated forms of Lpd is sufficient to aid E. coli homoethanol production. The reason for the E. coli Ipd mutant is ethanogenic and this ability to produce4 NADH by glucose.

EXAMPLE 5

LPD GENE CHANGE IS RESPONSIBLE FOR PELONADH IMPASSIBLE

In this example, the mutation in the Ipd gene is shown to cause impassive NADH. More particularly, it has been observed that dihydrolipoamide (LPD) dehydrogenase (LPD) activity which is sensitive to NADH in the wild type (natural) enzyme is changed to impassive NADH in the mutant, as shown in Figure 10 and Figure 11. Why LPD is a component of the dehydrogenase complex of pyruvate (PDH) this impassive Lpd NADH performed through the PDH from the ethanological mutant.

Wild type E. coli, strain W3110, or the mutant ethanogenic strain SE2378 was cultured in glucose and demineral salts media for exponential phase of the development medium. The cells were then collected and an extract prepared. Enzyme activity in the cellular extract was determined with pyruvate and NAD as substrates, and concentrations varied from NADH as the inhibitor of enzyme activity. Figure 10 shows inhibition of PDH activity by NADH. In the upper panel, the NAD concentration was 2 mM NAD for both wild type and W3110 strain, and the ethanological mutant, strain SE2378. In the background panel, the concentration of NAD was 2 mM NAD for natural enzyme from strain W3110, and 1 mM for mutated forms of enzyme from strain SE2378.

The wild type E. coli Ipd gene, strain W3110, and ethanological omutant, strain SE2378, was amplified by PCR and cloned protein expression novector, pET15b. The Lpd DNA gene sequence in the selected plasmid was verified by sequencing the inserted DNA. The Lpd expression gene in the plasmid was induced and the protein was purified. Enzyme activity was determined in the reverse reaction where the two substrates were lipoamide (3 mM) and NADH (0.1 mM) in K5 phosphate buffer O5IM, pH 8.0 comi, 5 mM EDTA. Figure 11 shows the inhibition of LPD by NADH. Under these conditions, the undetectable natural enzyme detectable, as shown in the graph in Figure 11. NAD, the reaction product is an activator that requires enzyme activity and increased activity with increased NAD concentration. The ratio of NADH to NAD in enzyme activity was determined by both natural and mutated from the enzyme and the results shown in Figure 11.

PDH is produced by all aerobic organisms (dabacteria to man). This enzyme oxidatively decarboxylates depyruvate to acetyl CoA, CO2 and NADH and acetyl CoA is then fed to the TCA cycle for further oxidation and subsequent energy production. In E.coli, PDH is produced under aerobic as well as anaerobic conditions. However, under anaerobic conditions the enzyme is inactive due to inhibition of PDH by NADH. NADH is usually present at a high concentration in the anaerobic cell, and thus avoids the generation of NADH that cannot be oxidized by the cell that is required from external electron acceptors. As a result, the cell produces only 2 NADH per glucose, and the second reductase series is released as hydrogen gas. Because a deacetyl-CoA reduction by ethanol requires two NADH, the wild-type cell cannot produce two ethanols per glucose.

In the ethanogenic mutant strains of the instant invention, PDH is less sensitive to NADH. This decreased sensitivity allows enzyme function even under anaerobic conditions with the largest groups of NADH. Due to this biochemical change, the cell can produce four NADH glucose molecules (2 from glycolysis and 2 from PDH reaction). All four NADHs are used to reduce two acetyl-CoA by ethanol, making the mutant a homoethanol producer. Biochemically and physiologically, the cell is a homoethane producer due to the decreased sensitivity of PDH to NADH, and this ability to still function with a high NADH / NAD ratio.

Finally, in an additional experiment (data not shown), the LPD mutation (E354K) observed in the SE2378 strain is not introduced into the B. subtilis LPD, an aerobic organism, at the analogous site. The E356K mutation that helps anaerobic mutant development (MRO) ).

EXAMPLE 6

COMPARISON OF ALIGNMENT OF LPD SEQUENCES FROM OTHER ORGANIZATIONS

In this example, comparison of the amino acid sequence alignments of 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 is present in both the PDH complex and the 2-oxoglutarate dehydrogenase complex. In E.coli, the Ipd is separated by these two enzyme complexes, and due to the tightness, the Ipd gene is transcribed from an independent promoter, and a promoter that is in an amount of the pdhR gene is matched.

Lpd counterparts are observed in all life domains. Among bacterial strains, the Lpd protein ranges from 458 to 581amino acids, as an anhydrous molecular weight of 49,000 to 62,000 Da. The homologous amino acid identity of 20 Lpd from the bacterium due to phylogenetic arrangements 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 serves the NCBI or ClustalW at the EuropeanBiotechnology Institute. The percent identity of a specific sequence to that of the E. coli sequence was obtained from two databases. The parenthesis value represents the total similarity of the specific protein to that of E. coli Lpd and includes both amino acid positions that are identical as well as the positions at which a conservative substitution occurs. By Bacilus subtilis, two dihydrolipoil dehydrogenases, one from the PDH complex and one from acetoin dehydrogenase, were included for comparison.

Sequence identity ranges from a 24% drop by Methanosarcinia barkeri, an archaeon, to 98% by Salmonellatyphimurium LT2 strain, a Gram-negative bacterium. That the protein of Salmonella typhimurium LT2 LPD is most finally reported to E.coli LPD is consistent with the classification of two bacteria as Gram-negative bacteria in the same enterobacteriaceae family. The Escherichia coliW3110 strain or MG1655 Lpd amino acid sequence was aligned with the known Acinetobacter sp. ADP 1, Bacillus cereusATCC 10987, Bacillus subtilis 168 strain, Clostridium tetaniMassachusetts / E88 strain, Corynebacterium glutamicum strain ATCC13 032, Geobacter metallireducens GS-15, Gluconobacter oxydans 621H, Lactobacillus casei cisoccus Leptobacococcus leptocarsis Ccusacarcis ciliacis Leptobacillus caseiocis Fusaro barkeri, Oenococcus oeni MCW PSU-1, Pseudomonas aeruginosa PAOl (ATCC15692), Rhodobacter sphaeroides 2.4.1, Salmonella typhimurium LT2, Shewanella sp ANA-3, Streptococcus mutans ATCC 700610, Streptomycescoelicolor M1451, Thermoaeribacter caterpillar 700 Homology alignment is shown in Figure 9 [this Figure will have to be re-numbered]. When comparing total percent identity, E.coli LPD appears to be more similar to other Gram-negative LPDs; however, when percent identity calculations are made on the basis of conservative substitution, Table 4 reflects a high percentage of homology among LPD proteins in the diverse organisms examined. For example, the amino acid sequence of cepa Bacillus subtillis 168 LPD protein compared to that protein. E.coli LPD is 34%. However, taking into account only conservative substitutions, the counting identity increases to 57%. As can be seen from the alignment of the Figure, the various amino acids are highly conserved among the group of 20 LPD homologues from many diverse groups of organisms. Residues that are separated between organisms are illuminated with an asterisk. The regions of interest are underlined. Among the sequences of the various organisms analyzed, the identity sequence is higher in the região terminal region. Sequence identity can be seen between amino acids 40 and 55 (E. coli LPD numbering), which may represent a possible flavin site. Another region of similarity is entreamino acids 180 and 190. Although the sequence here are in various positions where the amino acid residue is conserved in all 20 LPDs from the various organisms analyzed. Notably, amino acid position 322 encodes Histidine (H), and in three strains of the instant invention (SE2377, SE2383 and SE2382), here was a histadine mutation in position32 by tyrosine (Y). Histidine at position 322 is conserved in all 20 LPDs of Gram positive, Gram negative by archaea bacteria. Other residues that are conserved crossed in this diverse range include aproline at position 355 (18/20 LPDs) and glutamate in position 356 (17/20 LPDs) .64

Table 7: Identity of the amino acid sequence of the E. coli LPD protein by LPD homologs from other organisms.

<table> table see original document page 65 </column> </row> <table>

Represents amino acids that are identical and also conservative amino acid changesReferences

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Equivalents

Those skilled in the art will be recognized, or capable of for sure using more than routine experimentation, any equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

Embed reference

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

sequence no: 1

seqID no: 2

msteiktqw vlgagpagys aafrcadlgl etvíverynt lggvclnvgc

ipskallifva kvieeakala ehgjvfgbpk tdidkirtwk ekvinqltgglagmakgrkv kwnglgkft gantlevege ngktvinfdn aiiaagsrpiqlpfephedf riwdstdalb lkewe rllv mgggiiglem gtvyhalgsqπ> wemfdqvifaadkdivk vftkriskkf nlmletkvta veakedgfyvtmegkkapae fqrydavlvaigrvpngknl dagkagvevd drgfirvdkqlrtnvrphifaigdivgqfml aykgvheghv aájbvíagkkh yfdpkvffsiaytefevawv glffekeakekl g1syetatfp waasgraias dcadgmtklifdkeshrvig gajvgthgge llgeiglaie mgcbaedial tffiahftlhesvglaaevfe gshidlpnfk akkkSEQ ID NO: 3

SEQ ID NO: 4

MSTEIKTQW VLGAGPAG YS AAFRCADLGL ETVTVERYNT LGGVCLNVGCIfSKALLHVA KVIEEAKALA EHGIVFGEPK TDIDKntTWKEKVlNQLTGGLAGMAKGftKV KWNGLGKFT GANTLB VEGE NGKTVTNFDN AIIAAGSRPIQLPflPHEDP RIWDSTDALE LKEVPERLLV MGGGIIGLEM GTVYHALGSQIDWEMFDQVIP AADKDIVK VFTKRISKKF NLMLETKVTA VEAKEDGIYVTMEGKKAPAE PQRYDAVLVAIGRVPNGKNL DAGKAGVEVDSlQ ID NO: S

SBQ ID NO: 6

IGDÍVGQPML AHKGVHBGHV AAEVIAGitCKH YFDPKVÍPSIAYTEPEVAWVGLTEKEAKEK GISYETATFP WAASGRAIAS DCADGMTKLIFDKESHRVIGGA WGTNGGE LLGEIGLAIE MGCDAEDIAL TWAHPTEKKLKKKKKKKK

Claims (106)

1. Isolated non-recombinant bacterium characterized by the factor comprising a mutation wherein the mutation renders the non-recombinant bacterium capable of producing 4 mol of NADH per mol of sugar under anaerobic conditions. A non-recombinant bacterium isolated according to claim 1, characterized in that the mutation is located in a pdh operon. A non-recombinant bacterium isolated according to claim 2, characterized in that the pdh operon comprises pdhR, aceEF and Ipds. 4. Non-recombinant bacterium isolated according to claim 3, characterized in that the mutation is in the Ipd gene. Isolated non-recombinant bacterium according to any one of claims 1 to 4, characterized in that the production of -4 mol of NADH per mol of sugar results in the production of ethanol as the primary fermentation product. Isolated non-recombinant bacterium according to any one of claims 1 to 5, characterized in that the sugar is selected from the group consisting of: glucose, xylose, arabinose, mannose, galactose, sucrose and lactose. 7. Isolated non-recombinant bacterium characterized by the factor comprising an Ipd gene having a mutation, wherein the mutation renders non-recombinant bacteria capable of producing ethanol as the primary defermentation product under anaerobic conditions. An isolated non-recombinant bacterium according to any one of claims 5 or 7, characterized in that the ethanol produced comprises more than 50% of total non-gaseous fermentation products under anaerobic conditions. Isolated non-recombinant bacterium according to any one of claims 1 to 8, characterized in that the bacterium, in the absence of mutation, is nonethanogenic. Isolated non-recombinant bacterium according to claim 9, characterized in that ethanol is the secondary defermentation product and comprises less than 40% of the total non-gaseous defermentation products. Isolated non-recombinant bacterium according to any one of claims 1 to 8, characterized in that the mutation provides a homoethanol fermentation pathway. Isolated non-recombinant bacterium according to any one of claims 1 to 8, characterized in that one or more alternatives for fermentation in a bacterium are inactivated. Isolated non-recombinant bacterium according to claim 12, characterized in that alternative avenues for affermentation include lactate production by lactate dehydrogenase (JdhA), acetate, ethanol, formate, H2 and CO2 starting with pyruvate formate lyase ( mp) and succinate. A non-recombinant bacterium isolated according to claim 13, characterized in that alternative pathways for affermentation are inactivated by mutation. Isolated non-recombinant bacterium according to claim 13, characterized in that the mutation is in the IdhA gene. A non-recombinant bacterium isolated according to claim 13 characterized in that the mutation is in the pflgene gene. Isolated non-recombinant bacterium according to claim 15 or claim 16, characterized in that the mutation is in the IdhA or pflB genes. 18. Isolated nucleic acid molecule, characterized in that the factor is selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence that is at least 60% homologous to the nucleotide sequence of SEQ ED N °: 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 a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3or (c) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of at least about 50% homologous to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; a nucleic acid molecule encoding a fragment of a polypeptide comprising an amino acid sequence of SEQID 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 encoding a naturally occurring allelic variant of a polypeptide comprising a 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 that comprises SEQ ID NO: 1 or SEQ ID NO: 3, under severe conditions; a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a complement thereof eg) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: Wherein the nucleic acid molecule when expressed in a cell makes the cell capable of producing ethanol as the primary defermentation product. An isolated nucleic acid molecule according to claim 18, characterized in that the ethanol produced comprises more than 50% of total non-gaseous fermentation products under anaerobic conditions. An isolated nucleic acid molecule according to claim 18, characterized in that the cell is a bacterial cell. 21. Isolated nucleic acid molecule according to claim 20, characterized in that the bacterial cell, in the absence of expression of a nucleic acid molecule, is nonethanogenic. 22. Isolated nucleic acid molecule according to claim 21, characterized in that ethanol is the secondary defermentation product and comprises less than 40% of total non-gaseous defermentation products. An isolated nucleic acid molecule according to claim 19, characterized in that the bacterial cell produces ethanol as the primary fermentation product under anaerobic conditions. An isolated nucleic acid molecule according to claim 19, characterized in that the expression of a nucleic acid molecule in a bacterial cell provides a homoethanol fermentation pathway in a bacterial cell. An isolated nucleic acid molecule according to claim 18, characterized in that 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 one. position corresponding to heading 997 of SEQ ID NO: 1. An isolated nucleic acid molecule according to claim 18, characterized in that 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 one Gem. a position corresponding to position 1023 of SEQ ID NO: 1. 27. Isolated polypeptide characterized in that it is selected from the group consisting of: a) a fragment of a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, wherein the fragment comprises at least 15 amino acids b) a naturally occurring allelic variant of a polypeptide comprising an 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 the conditions c) a polypeptide that is encoded by a nucleic acid molecule that is at least 50% identical to a nucleic acid that comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID D) a polypeptide comprising an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4e) an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, wherein the polypeptide when expressed in a cell makes the cell capable of producing ethanol as the primary fermentation product. An isolated polypeptide according to claim 27, characterized in that the ethanol produced comprises more than -50% of total non-gaseous fermentation products under anaerobic conditions. An isolated polypeptide according to claim 27, characterized in that the polypeptide has dihydrolipoamide dehydrogenase activity under anaerobic conditions. An isolated polypeptide according to claim 27, characterized in that the cell is a bacterial cell. An isolated polypeptide according to claim 30, characterized in that the bacterial cell, in the absence of polypeptide expression, is nonethanogenic. An isolated polypeptide according to claim 30, characterized in that ethanol is the secondary fermentation product and comprises less than 40% of non-gaseous fermentation products. An isolated polypeptide according to claim 27, characterized in that the bacterial cell produces ethanol as the primary fermentation product under anaerobic conditions. An isolated polypeptide according to claim 30, characterized in that the ethanol produced comprises more than -50% of total non-gaseous fermentation products under anaerobic conditions. An isolated polypeptide according to claim 30, characterized in that expression of the polypeptide in a bacterial cell provides a homoethanol fermentation pathway in a bacterial cell. Bacterial host cell, characterized in that it comprises the nucleic acid molecule as defined in any one of claims 18 to 26. Bacterial host cell according to claim 36, characterized in that it comprises a vector comprising a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment thereof. 38. Bacterial host cell according to claim 30, characterized in that the vector is pKY33. 39. Bacterial host cell according to claim 36, characterized in that it was genetically engineered to express the nucleic acid molecule. Bacterial host cell, characterized in that it comprises the polypeptide according to any one of claims 27 to 35. 41. A method for producing a polypeptide, characterized in that it is selected from the group consisting of: a) a polypeptide comprising an amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 4, b) a fragment of a polypeptide which comprises an 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 ID NO: 2 or SEQ ID NO: 4 and c) a naturally occurring allelic variant of a polypeptide comprising an amino acid sequence of SEQ ID NO: 2or SEQ ID NO. Wherein the polypeptide is encoded by a nucleic acid molecule that hybridizes to a complement of a nucleic acid molecule which comprises SEQ ID NO: 1 or SEQ ID NO: 3, which comprises culturing host cells of a nucleic acid. according to claim 30 under conditions wherein the nucleic acid molecule is expressed. An isolated non-recombinant bacterium according to any one of claims 1 to 5 and 7, comprising the nucleic acid molecule as defined in claim 18. An isolated non-recombinant bacterium according to any one of claims 1 to 4, characterized in that ethanol is produced from sugar. 44. Isolated non-recombinant bacterium according to claim 42 or 43, characterized in that sugar is selected from the group consisting of glucose, xylose, arabinose, mannose, galactose, sucrose and lactose. An isolated non-recombinant bacterium according to any one of claims 1 to 7, an isolated nucleic acid molecule according to claim 18, an isolated polypeptide according to claim 27 or a bacterial host cell according to claim 36, characterized in ( o) the fact that the bacterium is selected from the group consisting of Gram negative bacteria and Gram negative bacteria. An isolated non-recombinant bacterium according to any one of claims 1 to 7, an isolated nucleic acid molecule as defined in claim 18, an isolated polypeptide as defined in claim 27 or a bacterial host cell as defined in claim 36, wherein: The bacterium is a gram-negative bacterium. An isolated non-recombinant bacterium according to any one of claims 1 to 7, an isolated nucleic acid molecule as defined in claim 18, an isolated polypeptide as defined in claim 27 or a bacterial host cell as defined in claim 36 wherein the bacterium is present. Gram negative is selected from the group consisting of Acinetobacter, Glueonobaeter, EscherichiQ, Geobaeter, Shewanella, Salmonella, Enterobaeter and Klebsiella. An isolated non-recombinant bacterium according to any one of claims 1 to 7, an isolated nucleic acid molecule according to claim 18, an isolated polypeptide according to claim 27 or a bacterial host cell according to claim 36, characterized in ( o) the fact that the bacterium is a gram positive bacterium. An isolated non-recombinant bacterium according to any one of claims 1 to 7, an isolated nucleic acid molecule according to claim 18, an isolated polypeptide according to claim 27 or a bacterial host cell according to claim 36, characterized in ( o) the fact that Gram-positive bacteria are selected from the group consisting of Bacillus, Clostridium, Corynebaeterium, Lactobacillis, Lactoeoceus, Oenoeoeeus, Streptoeoeeus and Eubacterium. An isolated non-recombinant bacterium according to any one of claims 1 to 7, an isolated nucleic acid molecule according to claim 18, an isolated polypeptide according to claim 27 or a bacterial host cell according to claim 36, characterized in ( o) the fact that the bacterium is Eseheriehia eoli. 51. The isolated non-recombinant bacterium according to claim 4 or 7, characterized in that the mutation comprises the substitution of one amino acid for another amino acid in a polypeptide expressed by the mutated Ipd gene, wherein the substitution changes the pK dopolipeptide. Isolated non-recombinant bacterium according to claim 51, characterized in that the polypeptide comprises a SEQ ID NO: 6 and the mutation comprises the substitution of one wild type amino acid for another amino acid at: a) position 322 or any position within about 50 positions on the side of position 322 in SEQ ID NO: 6 orb) position 354 or any position within about 50 positions on the side of position 354 in SEQ ID NO: 6. Isolated non-recombinant bacterium according to claims 51 or 52, characterized in that the other amino acid is a neutral amino acid selected from the group consisting of dealanin, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine. , tryptophan, tyrosine and valine. Isolated non-recombinant bacterium according to claims 51 or 52, characterized in that the other amino acid is a basic amino acid selected from the group consisting of dearginine, asparagine, glutamine, histidine and lysine. 55. Isolated non-recombinant bacterium according to claim 52, characterized in that the mutation comprises H-substitution at position 322 with any amino acid, such that amino acid substitution increases the acidity of the mutated Ipd expressed polypeptide. 56. Isolated non-recombinant bacterium according to claim 55, characterized in that the mutation comprises the substitution of H to Y at position 322 in SEQ ID NO: 6. 57. Isolated non-recombinant bacterium according to claim 52, characterized in that the mutation comprises the substitution of E at position 354 with any amino acid, such that the amino acid substitution reduces the acidity of the mutated Ipd expressed polypeptide. 58. Isolated non-recombinant bacterium according to claim 57, characterized in that the mutation comprises the substitution of E to K at position 354 in SEQ ID NO: 6. 59. Isolated non-recombinant bacterium according to claim 56, characterized in that the bacterium is the strain of E. coliSE2377. 60. Isolated non-recombinant bacterium according to claim 59, characterized in that the bacterium comprises SEQLD No. 1 or a fragment thereof. 61. Isolated non-recombinant bacterium according to claim 58, characterized in that the bacterium is the strain of E. coliSE2378. 62. The isolated non-recombinant bacterium according to claim 61, characterized in that the bacterium comprises SEQID No. 3 or a fragment thereof. 63. Isolated non-recombinant bacterium according to claim 58, characterized in that the bacterium is the strain of E. coliSE2382. 64. Isolated non-recombinant bacterium according to claim 63, characterized in that the bacterium comprises SEQID No. 3 or a fragment thereof. 65. Isolated non-recombinant bacterium according to claim 56, characterized in that the bacterium is the strain of E. coliSE2383. 66. Isolated non-recombinant bacterium according to claim 65, characterized in that the bacterium comprises SEQID No. 1 or a fragment thereof. 67. Isolated non-recombinant bacterium according to claim 56, characterized in that the bacterium is the strain of E. coliSE2384. 68. Isolated non-recombinant bacterium according to claim 67, characterized in that the bacterium comprises SEQID No. 1 or a fragment thereof. 69. Isolated non-recombinant bacterium according to claim 58, characterized in that the bacterium is the strain of E. coliSE2385. 70. Isolated non-recombinant bacterium according to claim 69, characterized in that the bacterium comprises SEQID No. 3 or a fragment thereof. Isolated non-recombinant bacterium according to any one of claims 56 to 70, characterized in that the bacterium is suitable for producing ethanol from sugar. 72. Isolated non-recombinant bacterium, characterized in that the fat comprising an Ipd gene having one or more mutations, wherein mutation renders the non-recombinant bacterium capable of producing ethanol as the primary fermentation product under anaerobic conditions, wherein the abacteria is prepared by a a process comprising the steps of: a) developing a candidate mutant strain of a bacterium under anaerobic development conditions in a sugar rich medium and b) selecting ethanol-producing mutants as the primary fermentation product. A method according to claim 72, characterized in that the ethanol produced comprises more than 50% of total non-gaseous fermentation products under anaerobic conditions. A method for producing a non-recombinant bacterium as defined in any one of claims 1 to 8, characterized in that it comprises the steps of: a) developing a candidate mutant strain of a bacterium under conditions of anaerobic development in sugar rich medium and b) select ethanol-producing mutants as the main fermentation product. 75. The method of claim 72, characterized in that the ethanol produced comprises more than 50% of total non-gaseous fermentation products under anaerobic conditions. Isolated non-recombinant bacterium according to claim 72 or method according to claim 74, characterized in that the mutants result from spontaneous mutation. Isolated non-recombinant bacterium according to claim 72 or method according to claim 74, characterized in that the bacterium is exposed to a demutagenesis agent. Isolated non-recombinant bacterium according to claim 72 or method according to claim 74, characterized in that 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. Isolated non-recombinant bacterium or method according to claim 78, characterized in that the demutagenesis agent is ethyl methane sulfonate. Isolated non-recombinant bacterium according to claim 72 or method according to claim 74, characterized in that 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 according to claim 72 or the method according to claim 74, characterized in that it further comprises the method of inactivating alternative fermentation pathways in a bacterium. An isolated non-recombinant bacterium according to claim 72 or a method according to claim 74, characterized in that alternative fermentation pathways are inactivated by the introduction of nullification mutations into a bacterium. 83. Isolated non-recombinant bacterium according to claim 72, characterized in that the bacterium, in the absence of mutation, is nonethanogenic. 84. Isolated non-recombinant bacterium according to claim 83, characterized in that ethanol is the secondary defermentation product and comprises less than 40% of total non-gaseous defermentation products. 85. Isolated non-recombinant bacterium according to claim 72, characterized in that the bacterium produces ethanol as the primary fermentation product under anaerobic conditions. 86. Isolated non-recombinant bacterium according to claim 85, characterized in that the ethanol produced comprises more than 50% of total non-gaseous fermentation products under anaerobic conditions. 87. Isolated non-recombinant bacterium according to claim 72, characterized in that the mutation provides a homoethanol defermentation pathway. 88. Isolated non-recombinant bacterium according to claim 87, characterized in that one or more alternative pathways for fermentation in a bacterium are inactivated. 89. Isolated non-recombinant bacterium according to claim 88, characterized in that the alternative pathways for affermentation are inactivated by mutation. 90. Isolated non-recombinant bacterium according to claim 88, characterized by the fact that alternative routes for affermentation include lactate dehydrogenase (Jdh) lactate production, acetate, ethanol, formate, H2 and CO2 starting with pyruvate formate lyase ( mp) and succinate. A method for producing ethanol from a deoligosaccharide source, comprising contacting the oligosaccharide with the isolated non-recombinant bacterium as defined in any one of claims 1 to 8 or the bacterial host cell as defined in claim 36, thereby producing ethanol. of an oligosaccharide source. A method according to claim 91, characterized in that the oligosaccharide is selected from the group consisting of delignocellulose, hemicellulose, cellulose, pectin and any combination thereof. 93. Kit comprising the recombinant non-isolated bacterium as defined in any one of claims 1 to 8 and instructions for the production of ethanol. A kit according to claim 93, characterized in that it further comprises a sugar source. 95. E. coli strain AH218, characterized in that it is represented by a deposit with the Agricultural Research Culture Collection indicated as deposit number NRRL B-30967. 96. E. coli strain AH241 strain, characterized in that it is represented by a deposit with the Agricultural Research Culture Collection indicated with deposit number NRRL B-30968. 97. E. coli strain AH242, characterized in that it is represented by a deposit with the Agricultural Research Culture Collection indicated under deposit number NRRL B-30969. 98. E. coli strain SE2377, characterized in that it is represented by a deposit with the Agricultural Research Culture Collection indicated under deposit number NRRL B-30970. 99. E. coli strain SE2378, characterized in that it is represented by a deposit with the Agricultural Research Culture Collection indicated under deposit number NRRL B-30971. 100. E. coli strain SE2382, characterized in that it is represented by a filing with the Agricultural Research Culture Collection indicated under filing number NRRL B-30972. E. coli strain SE2383, characterized in that it is represented by a filing with the Agricultural Research Culture Collection indicated under filing number NRRL B-30973. 102. E. coli strain SE2384, characterized in that it is represented by a filing with the Agricultural Research Culture Collection indicated under filing number NRRL B-30974. 103. E. coli strain SE2385, characterized in that it is represented by a deposit with the Agricultural Research Culture Collection indicated under deposit number NRRL B-30975. 104. Isolated non-recombinant bacterium according to claim 4, 7 or 72, characterized in that the Ipd gene mutation causes insensitivity to NADH. 105. The method according to claim 72, 74 or 91 oukit according to claim 93, characterized in that the mutant results from mutation in the Ipd gene. 106. The method or kit according to claim 105, characterized in that the mutation in the Ipd gene causes aNADH insensitivity.