CN101657541A

CN101657541A - Alcohol production in the non-recombinant hosts

Info

Publication number: CN101657541A
Application number: CN200780025274A
Authority: CN
Inventors: Y·金; K·桑穆加姆; L·O·因格拉姆
Original assignee: University of Florida Research Foundation Inc
Current assignee: University of Florida; University of Florida Research Foundation Inc
Priority date: 2006-05-01
Filing date: 2007-04-26
Publication date: 2010-02-24
Also published as: BRPI0711266A2

Abstract

Disclose and produced the non-recombinant bacteria of ethanol as basic tunning, relevant nucleic acid and polypeptide use described bacterium to produce the alcoholic acid method, and test kit.

Description

Alcohol production in the non-recombinant hosts

Related application

60/848,234 the right of priority that the application requires the U.S. Provisional Application series number of submitting on May 1st, 2,006 60/796,652 and submitted on September 29th, 2007, their all the elements are incorporated this paper into as a reference.

The research of government-funded

The present invention partly obtains the support of USDOE grant number DE-FG36-04GO14019.Therefore, government can have some right of the present invention.

Background of invention

Ethanol is a kind of attractive alternative transport fuel that is used for substituting at least a portion oil (people such as Kheshgi, 2000, people such as Wooley, 1999).Use fermentation by saccharomyces cerevisiae from the glucose producing and ethanol in next life of W-Gum people such as (, 2005) Bothast although cross at present, enlarge this technology and produce most automobile fuel demand and can impact food and fodder industry unfriendly at Americanologist.

The wood fibre biomass is a kind of attractive alternative materials, and it suitably can be fermented into ethanol after the pre-treatment, and does not impact food and feed supply (people such as Wyman, 2003, people such as Zaldivar, 2001).Different with W-Gum, biomass contains the pentose that a large amount of yeast are difficult to ferment.

Compounding sugar transform to need the microorganism biological catalyzer of zymohexose and pentose effectively to alcoholic acid.For this purpose, develop the reorganization biology, wherein heterologous gene has been added platform (platform) biology, for example yeast, zymomonas mobilis (Z.mobilis) and intestinal bacteria.For example, contain from the pdc of zymomonas mobilis and the reorganization producing and ethanol intestinal bacteria of adh gene and can with productive rate hexose be become ethanol (people such as Ingram, 1999) with pentose fermentation with two-forty.In addition, add the biological catalyst that pentose utilizes the yeast of gene and zymomonas mobilis genetically engineered to produce to be complementary with the colibacillary pentose fermentation feature of reorganization producing and ethanol (people such as Kuyper, 2005, people such as Mohagheghi, 2004).

Summary of the invention

As mentioned above, lignocellulose raw material transform to need the microorganism biological catalyzer of zymohexose and pentose effectively to alcoholic acid.Such microorganism biological catalyzer comprises that reorganization is biological, wherein heterologous gene is added the platform biology, for example yeast and bacterium, for example zymomonas mobilis and intestinal bacteria.

But the biological purposes that is used for extensive fuel production of reorganization is regarded as business-like barrier by some people.The exploitation in non-reorganization ethanol source (ethanologen) can reduce from lignocellulosic substrates commercially produces one of known barrier of alcoholic acid.

Therefore, the present invention at least in part based on, by microorganism otherwise not high ethanol (homoethanol) approach of producing and ethanol (otherwise non-ethanologenic) change the discovery of glucolytic sudden change, with the exploitation that under anaerobic glucose is become the non-reorganization producing and ethanol of alcoholic acid microorganism with wood-sugar fermentation based on this discovery.More specifically, the pdh operon has been differentiated and has been the origin of high ethanol approach.More specifically, the lpd gene in the pdh operon has been differentiated that described fermentation is for example under anaerobic carried out by intestinal bacteria in order to be responsible for high ethanol fermentation.

Thereby, in one aspect, the invention provides the isolating non-recombinant bacteria that comprises sudden change, wherein said sudden change makes this non-recombinant bacteria can under anaerobic produce 4 moles of NADH/ mole sugar.

In yet another aspect, the invention provides isolating non-recombinant bacteria, it comprises the lpd gene with one or more sudden changes, and wherein said sudden change makes this non-recombinant bacteria can under anaerobic produce ethanol as basic tunning.

The present invention also provides isolated nucleic acid molecule, its coding dihydrolipoamide dehydrogenase (LPD) polypeptide or its function fragment.When described nucleic acid molecule was expressed in cell (for example bacterium), described cells produce ethanol was as basic tunning.

Thereby, in yet another aspect, the invention provides and be selected from following isolated nucleic acid molecule:

A) nucleic acid molecule, it comprises the nucleotide sequence at least 60% homologous nucleotide sequence with SEQ ID NO:1 or SEQ ID NO:3, or its complementary sequence;

B) nucleic acid molecule, it comprises the fragment of at least 100 Nucleotide of the nucleic acid of the nucleotide sequence that contains SEQ ID NO:1 or SEQ ID NO:3, or its complementary sequence;

C) nucleic acid molecule, its coding comprise and the aminoacid sequence of SEQ ID NO:2 or the SEQ ID NO:4 polypeptide at least about 50% homologous aminoacid sequence;

D) nucleic acid molecule, its coding comprise the polypeptide fragment of the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4; Wherein said fragment comprises at least 15 adjacency (contiguous) amino-acid residue of the aminoacid sequence of SEQ ID NO:2 or SEQ IDNO:4;

E) nucleic acid, its coding comprises the naturally occurring allele variant of polypeptide of the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4, wherein said nucleic acid molecule under rigorous condition with the complementary sequence hybridization that comprises the nucleic acid molecule of SEQ ID NO:1 or SEQ ID NO:3;

F) comprise the nucleic acid molecule of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, or its complementary sequence; With

G) nucleic acid molecule, its coding comprise the polypeptide of the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4;

Wherein said nucleic acid molecule makes described cell can produce ethanol as basic (primary) tunning when expressing in cell.

The present invention also provides dihydrolipoamide dehydrogenase polypeptide or its function fragment.When described polypeptide was expressed in cell (for example bacterium), described cells produce ethanol was as basic tunning.

Thereby, in yet another aspect, the invention provides and be selected from following polypeptide:

A) comprise the polypeptide fragment of the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4, wherein said fragment comprises SEQ ID NO:2 or SEQ ID NO:4 at least 15 in abutting connection with amino acid;

B) comprise SEQ ID NO; 2 or the naturally occurring allele variant of the polypeptide of the aminoacid sequence of SEQ ID NO:4, wherein said polypeptide by under rigorous condition with comprise SEQ IDNO; 1 or the nucleic acid molecule encoding of the complementary sequence hybridization of the nucleic acid molecule of SEQ ID NO:3;

C) by the polypeptide of the nucleic acid molecule encoding consistent with the nucleic acid at least 50% that comprises SEQ ID NO:1 or SEQ ID NO:3 nucleotide sequence;

D) comprise the polypeptide of the aminoacid sequence consistent with the aminoacid sequence at least 90% of SEQ ID NO:2 or SEQ ID NO:4; With

E) comprise the isolated polypeptide of the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4; And when wherein said polypeptide is expressed in cell, make described cell can produce ethanol as basic tunning.

In one embodiment, comprise total non-gas tunning under anaerobic by the ethanol of cells produce greater than 50%.In another embodiment aspect this, described polypeptide under anaerobic has the dihydrolipoamide dehydrogenase activity.In another embodiment, described cell is a bacterial cell.

In yet another aspect, the invention provides bacterial host cell, it comprises coding dihydrolipoamide dehydrogenase polypeptide or its segmental isolated nucleic acid molecule.

In yet another aspect, the invention provides the method that is selected from following polypeptide of producing:

A) comprise the polypeptide of aminoacid sequence SEQ ID NO:2 or SEQ ID NO:4;

B) comprise the polypeptide fragment of the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4; Wherein said fragment comprises SEQ ID NO:2 or SEQ ID NO:4 at least 15 in abutting connection with amino acid; With

C) comprise the naturally occurring allele variant of polypeptide of the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4,

Wherein said polypeptide by under rigorous condition with the nucleic acid molecule encoding of complementary sequence hybridization of the nucleic acid molecule that comprises SEQ ID NO:1 or SEQ ID NO:3;

This method comprises, under the condition of express nucleic acid molecule, cultivates the bacterial host cell that contains coding dihydrolipoamide dehydrogenase polypeptide or its segmental isolated nucleic acid molecule.

In yet another aspect, the invention provides aforesaid non-recombinant bacteria, it comprises above-mentioned isolated nucleic acid molecule.

Another aspect of the present invention provides non-recombinant bacteria, it comprises the lpd gene with one or more sudden changes, wherein said sudden change makes this non-recombinant bacteria can under anaerobic produce ethanol as basic tunning, and wherein said bacterium prepares by the method that comprises following step:

A) in the sacchariferous substratum of richness, cultivating candidate's bacteria variants under the anaerobic growth condition; With

B) select to produce the mutant of ethanol as main tunning.

In yet another aspect, the invention provides the method for producing the non-recombinant bacteria of producing and ethanol of the present invention, it comprises following step:

B) select to produce the mutant of ethanol as main tunning.

In one embodiment, the invention provides the isolating non-recombinant bacteria of any above-mentioned aspect, wherein the sudden change in the lpd gene causes the NADH insensitivity.

In another embodiment aspect above-mentioned, described mutant is derived from the sudden change in the lpd gene.In a particular, the sudden change in the lpd gene causes the NADH insensitivity.

In yet another aspect, the invention provides from oligosaccharides source production alcoholic acid method.This method comprises, makes oligosaccharides contact aforesaid non-recombinant bacteria of the present invention or host cell, thereby produces ethanol from the oligosaccharides source.In a particular of this method, described oligosaccharides is selected from: lignocellulose, hemicellulose, Mierocrystalline cellulose, pectin and its arbitrary combination.

In yet another aspect, the invention provides test kit, it comprises aforesaid non-recombinant bacteria of the present invention or host cell and according to method as herein described and explained hereafter alcoholic acid specification sheets.In one embodiment, described test kit comprises sugared source.

In yet another aspect, the invention provides new coli strain, comprise strains A H218 (NRRL B-30967), AH241 (NRRL B-30968), AH242 (NRRLB-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), they are deposited in farming research culture collection center (Agricultural Research Culture Collection) (NRRL) on September 27th, 2006,1815 N.University Street, Peoria, IL, USA.

From following detailed description and claims, can understand other features and advantages of the present invention.

The accompanying drawing summary

Fig. 1 (A) has shown and has contained in the nucleotide sequence (SEQ ID NO:1) of the lpd gene of the sudden change at base 997 places and (B) corresponding amino acid sequence (SEQ ID NO:2).

Fig. 2 (A) has shown and has contained in the nucleotide sequence (SEQ ID NO:3) of the lpd gene of the sudden change at base 1093 places and (B) corresponding amino acid sequence (SEQ ID NO:4).

Fig. 3 (A) has shown the nucleotide sequence (SEQ ID NO:5) of wild-type lpd gene and (B) corresponding amino acid sequence (SEQ ID NO:6).

The figure that Fig. 4 shows has described intestinal bacteria wild type strain W3110 and producing and ethanol mutant strain SE2378 is containing glucose or wood sugar (50g L ^-1) LB-substratum (37 ℃, pH 7.0) in growth and fermenting characteristic.Figure (A) has shown the wild-type W3110 bacterial strain of growing in glucose; Figure (B) has shown the SE2378 bacterial strain of growing in glucose; Figure (C) has shown the wild-type W3110 bacterial strain of growing in wood sugar; Figure (D) has shown the SE2378 bacterial strain of growing in wood sugar.

Fig. 5 has shown (A) (Δ aroP-aceEF) transduction in bacterial strain SE2378 (B) of depletion mutant (aroP-aceEF).By with zac ∷ Tn10 cotransduction, draw the mutation map among the bacterial strain SE2378.When transduceing deletion aroP-pdhR-aceEF gene by the worker, the ability that transduttant (C) forfeiture is under anaerobic grown in the LB that contains 1% glucose, and the identical disappearance in the wild-type background does not influence anaerobic growth.

Fig. 6 has shown the aminoacid sequence (A) from the pdhR gene product of wild-type W3110 bacterial strain and SE2378 mutant.The nucleotide sequence of the intergenic region of bacterial strain SE2378 is shown in (B).

Fig. 7 has shown the plasmid that is used for expressing YK100 host W3110 or SE2378lpd.Plasmid pKY32 (A) contains the lpd gene from wild type strain W3110.Plasmid pKY33 (B) contains the lpd gene from producing and ethanol mutant SE2378.

The synoptic diagram of Fig. 8 (A-C) has shown that producing alcoholic acid from pyruvate salt in coli strain SE2378, natural intestinal bacteria and other producing and ethanol microorganism proposes approach.

Fig. 9 has shown the amino acids sequence alignment (using CLUSTAL multisequencing comparison program) of the LPD sequence of the LPD of e. coli k12 MG1655 and following selection: i.e. clostridium tetanus E88, the thermophilic anaerobic ethanol bacillus, bacillus cereus ATCC 10987, plant lactobacillus WCFS1, lactococcus lactis subsp SK11, drinks wine coccus MCWPSU-1, Salmonella typhimurium LT2, Fei Xiershi vibrios ATCC 700601, Shiva Salmonella ANA-3, Pseudomonas aeruginosa PAO1 (ATCC15692), Spherical red antibacterial 2.4.1, metallic reducing ground bacillus (Geobacter metallireducens) GS-15, acinetobacter calcoaceticus ADP1, oxidizing glucose acidfast bacilli 621H, corynebacterium glutamicum DSM20300, lactobacterium casei ATCC334, streptomyces coelicolor M145/A3 (2), streptococcus mutans ATCC 700610, Pasteur's sarcina methanica Fusaro.Highlight with asterisk (*) at the histidine residues at amino acid 322 places, proline residue and glutaminic acid residue at amino acid 356 places at amino acid 355 places.

Figure among Figure 10 has shown that NADH is to the active inhibition of PDH.Intestinal bacteria wild type strain W3110 or producing and ethanol mutant strain SE2378.In last figure, for bacterial strain W3110 and bacterial strain SE2378, NAD concentration is 2mM.In figure below, for for the natural enzyme of bacterial strain W3110, NAD concentration is 2mM NAD, and for for the mutant form of the enzyme of bacterial strain SE2378, NAD concentration is 1mM.

Figure among Figure 11 has shown the inhibition of NADH to LPD.The enzymic activity of the natural and mutant form of mensuration LPD and the relation of NADH/NAD ratio.

Detailed Description Of The Invention

In order to be expressly understood four corner of the present invention, provide following definition.

I. definition

Term used herein " non-recombinant bacteria " and " bacterium " are intended to comprise and contain or do not contain the heterologous polynucleotide sequence and be fit to use the further bacterial cell of modification of the compositions and methods of the invention, for example be fit to genetic manipulation, for example it can mix the heterologous polynucleotide sequence, and for example it can be transfected. This term is intended to comprise the offspring of the cell of initial transfection. In specific embodiments, described cell is gramnegative bacterium cell or gram-positive cell. Term in " being derived from polynucleotides or the gene of bacterium " " is derived from " and is intended to comprise that the polynucleotides section from the source that indicates (namely, bacterium) separation (completely or partially) or polypeptide are from the purifying in the source (that is, bacterium) that indicates. In this respect, this term is intended to comprise, for example, from or based on the sequence Direct Cloning relevant with the polynucleotides source that indicates, pcr amplification or artificial synthetic.

Term " anaerobic condition " is intended to comprise oxygen-free condition; That is the condition that, does not wherein basically have oxygen. In certain embodiments of the invention, anaerobic condition comprises vapor tight tank or container, for example uses the airtight tank of stopper. In order to set up the oxygen containing condition of not wrapping, use vavuum pump to remove gas phase from tank or container, and replace with nitrogen. When the oxygen level low when being difficult to detect, oxygen does not exist basically. Term " aerobic condition " is intended to comprise the condition that contains aerobic; That is the condition that, wherein has oxygen.

Term " producing and ethanol " is intended to comprise that microorganism produces ethanol as the ability of basic tunning from carbohydrate. The biology that this term is intended to comprise the biology of naturally occurring producing and ethanol and has the producing and ethanol of sudden change natural generation or that induce.

Term " not producing and ethanol " is intended to comprise that microorganism can not produce ethanol as basic tunning from carbohydrate. This term is intended to comprise produces ethanol as the microorganism of less important tunning, and described less important tunning comprises the total non-gas tunning less than 40%.

Term " fermentation " is intended to comprise that the degraded of glycoconjugate or depolymerization and saccharide residue are to the bio-transformation of ethanol, acetate and succinate. This term be intended to comprise from carbohydrate generate ethanol (more specifically, as basic tunning) enzymatic processes (for example cell or acellular, the mixtures of polypeptides of lysate or purifying for example).

Term " basic tunning " and " mainly tunning " be Alternate in this article, is intended to comprise the non-gas tunning that comprises greater than about 50% total non-gas product. Basic tunning is the abundantest non-gas product. In certain embodiments of the invention, described basic tunning is ethanol.

Term used herein " less important tunning " is intended to comprise the non-gas tunning that comprises less than 40% total non-gas product. In certain embodiments of the invention, described less important tunning is ethanol.

Term used herein " high alcohol fermentation approach " is intended to comprise in the biological for example bacterium and promotes ethanol as the fermentation approach of the production of basic tunning.

Term used herein " substitutes fermentation approach " and is intended to comprise that wherein ethanol is not the fermentation approach of basic tunning.

" gene " used herein is the synthetic nucleic acid that can instruct enzyme or other peptide molecule, for example can comprise coded sequence, for example, coded polypeptide in abutting connection with opening code-reading frame (ORF), or can be from function in the biology. Gene in the biology can be at bunch collection in the operon as herein defined, and wherein said operon is separated by intergenic DNA and other gene and/or operon. Be included in the operon individual gene can not have in the situation of intergenic DNA overlapping between individual gene. In addition, term " gene " is intended to comprise the purpose specific gene for selecting. Gene can be that host cell is endogenous, maybe can import in the host cell plasmid or the stable plasmid (or its fragment) that mixes in the genome for example kept as episome with recombinating. Heterologous gene is non-natural gene in the transfered cell and for this cell.

Term " pdh operon " and " pdh locus " are used interchangeably, and mean pdhR, lpd and aceEF gene cluster as colony's expression, with their relevant promoter and operator. According to routine, term " pdh operon " refer to the to encode gene of operon, and term " PDH " refers to the albumen composition by the operon coding. The acetyl coenzyme A that is used for tricarboxylic acid cycle and energy production is produced in the pyruvate dehydrogenase activity load. Term pdh operon can comprise the pdh operon from any aerobe. All aerobes from the eucaryote to the mankind, all contain 3 PDH components. Many bacteriums have the gene that is coded in the PDH that contains in the operon. 3 gene aceE, aceF and lpd are that the PDH activity is necessary, and these 3 genes are present in all aerobes, and no matter they are as operon or organize as separate gene.

Term " dihydrolipoamide acetyltransferase " (aceF) is intended to comprise the E2 transacetylase of pyruvic dehydrogenase gene locus. According to routine, term " aceF " refers to the dihydrolipoamide acetyltransferase gene, and term " AceF " refers to the aceF gene outcome, lipoyl transacetylase polypeptide or enzyme.

Term " pyruvate decarboxylase/PDH compound dehydrogenase " (aceE) is intended to comprise the E1 decarboxylase of pyruvic dehydrogenase gene locus. According to routine, term " aceE " refers to pyruvate decarboxylase/dehydrogenase gene, and term " AceE " refers to the aceE gene outcome, i.e. pyruvate decarboxylase/dehydrogenase polypeptide or enzyme.

Term " pyruvic dehydrogenase repressor " (pdhR) is intended to comprise the transcription repressor of pdh operon. According to routine, term " pdhR " refers to pyruvic dehydrogenase repressor gene, and term " PdhR " refers to the pdhR gene outcome, i.e. pyruvic dehydrogenase repressor polypeptide.

Term " dihydrolipoamide dehydrogenase " (lpd) is intended to comprise enzyme as the part of pyruvic dehydrogenase gene locus or " pdh operon ". According to routine, term " lpd " refers to the dihydrolipoamide dehydrogenase gene, and term " lpD " refers to the lpd gene outcome, i.e. dihydrolipoamide dehydrogenase polypeptide or enzyme. The nucleotide sequence of wild type lpd gene represents with the SEQ ID NO:5 shown in Fig. 3 (A), represented with the SEQ ID NO:6 shown in Fig. 3 (B) by the amino acid sequence of the polypeptide of wild type lpd gene expression.

Term " lactic dehydrogenase " (ldhA) is intended to comprise and under fermentation conditions acetonate is changed into Lactated enzyme. According to routine, term " ldhA " refers to lactate dehydrogenase gene, and term " LDHA " refers to the ldhA gene outcome, i.e. lactic dehydrogenase enzyme polypeptide or enzyme.

Term " pyruvic acid formic acid lyase " (pfl) is intended to comprise the enzyme that under fermentation conditions acetonate is changed into acetyl coenzyme A and formates. According to routine, term " pfl " refers to pyruvic acid formic acid lyase genes, and term " PFL " refers to the pfl gene outcome, i.e. pyruvic acid formic acid lyase polypeptide or enzyme.

Term " alcohol dehydrogenase " (adhE) is intended to comprise the enzyme that under fermentation conditions acetyl coenzyme A is changed into ethanol. According to routine, term " adhE " refers to alcohol dehydrogenase gene, and term " ADHE " refers to the adhE gene outcome, i.e. alcohol dehydrogenase polypeptide or enzyme.

Term " NADH insensitivity " refers to that the PDH enzyme is to the reduction of the sensitiveness of NADH. This term is intended to comprise that part reduces the fully disappearance of insensitivity or sensitiveness.

Term " nucleic acid " is intended to comprise nucleic acid molecules, for example comprises the polynucleotides of the opening code-reading frame of coded polypeptide, and may further include non-coding adjusting sequence and introne. In addition, this term is intended to comprise one or more genes of drawing into functional locus. In addition, this term is intended to comprise the purpose specific gene for selecting. In one embodiment, the gene of polynucleotides section participates in carbohydrate at least one step of the bio-transformation of ethanol. Therefore, this term is intended to comprise any gene of coded polypeptide, for example for example dextranase or its combination of pyruvate decarboxylase, alcohol dehydrogenase, secreted polypeptide or polysaccharase of described polypeptide. Gene in the biology can be at bunch collection in the operon as herein defined, and wherein said operon is separated by intergenic DNA and other gene and/or operon. Be included in the pdh operon individual gene can not have in the situation of intergenic DNA overlapping between individual gene.

Term " homology " is intended to comprise, first amino acid or nucleotide sequence contain amino acid residue or the nucleotides identical or of equal value with second amino acid or nucleotide sequence of enough or minimal amount, the amino acid residue that for example has similar side chain is so that first has common domain and/or common functional activity with second amino acid or nucleotide sequence.

Term " heterologous polypeptide " is intended to comprise can be by the heterologous nucleic acids that is derived from any source (eucaryote for example, prokaryotes, archeobacteria, bacterium (virii) beyond the highest heavens) or polypeptide or its fragment of nucleic acid fragment coding.

Term " polypeptide of separation " (for example biosynthetic enzyme that separate or purifying) is substantially devoid of from the cell material of polypeptide source microorganism or other and pollutes polypeptide, or is substantially devoid of precursor or other chemical reagent when chemical synthesis.

Term " fragment " in " nucleotide fragments " or " polypeptide fragment " is intended to refer at least a portion of coming source sequence same nucleotide sequence or the peptide sequence part basically with it, and wherein said polypeptide keeps its biologically active of coming source sequence.

Term " pH " is intended to refer to the tolerance of hydrogen ion molar concentration in the solution, so also be the tolerance of solution acidity or basicity. According to the standard of this area, term pH is used for limiting solution. Common pH value scope is 0 to 14,0th, the value of concentrated hydrochloric acid (1M HCl), the 7th, the value of pure water (neutral pH), the 14th, the value of concentrated sodium hydroxide (1M NaOH).

Term " pK " is intended to refer to the tolerance of proton binding affinity, frequent and pH Alternate. Those of skill in the art will recognize that term pK is used for limiting albumen, amino acid and peptide. Those skilled in the art will appreciate that also the acid strength of carboxyl, amino and ionogenic R-group can be used association constant K in the amino acid_aOr more commonly use K_aNegative logarithm pK_aLimit.

Term " carrier " is intended to comprise and is applicable to the linking objective nucleotide sequence and transforms any plasmid vector in the host cell.

Term " sugar " is intended to comprise any carbohydrate source that comprises glycan molecule. Such sugar is the potential glycosylation source, and it is used for depolymerization (if necessary), and product according to the present invention and method become acetaldehyde, change into ethanol again by the subsequently bio-transformation of fermenting. The sugar source comprises starch, the principal mode of fuel storage in the most plants, and cellulose, the main cell external structure component of sclereid wall and fiber and the lignum of plant. This term is intended to comprise monose (being also referred to as simple sugars), oligosaccharides and polysaccharide. In certain embodiments, described steamed bun stuffed with sugar is drawn together for example glucose, wood sugar, arabinose, mannose, galactolipin, sucrose and lactose. In other embodiments, described sugar is glucose.

Term " gramnegative bacterium cell " is intended to comprise the definition of this art-recognized term. Exemplary gramnegative bacterium comprise acinetobacter calcoaceticus, glucose bacillus, escherich's bacillus, bacillus (Geobacter), Xie Wanala bacterium (Shewanella), salmonella, enteric bacteria and klebsiella bacillus.

Term " gram-positive bacterium " is intended to comprise the definition of this art-recognized term. Exemplary gram-positive bacterium comprises bacillus, clostridium, corynebacteria, Bacillus acidi lactici, galactococcus, wine coccus (Oenococcus), streptococcus and eubacteria.

Phrase " mutant nucleic acid molecule " or " mutator " are intended to comprise nucleic acid molecules or the gene with the nucleotide sequence that comprises at least one change (for example replace, insert, lack), so that polypeptide or can be shown by the polypeptide of mutant code is different from described polypeptide or by the activity of the polypeptide of wild-type nucleic acid molecule or gene code.

Term " amino acid " is intended to be included in 20 kinds of common in albumen a-amino acids. The charged amino acid of alkalescence comprises arginine, asparagine, glutamine, histidine and lysine. Neutral charged amino acid comprises alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Acidic amino acid comprises aspartic acid and glutamic acid.

Term " mutagens " is intended to comprise any agent that can the method according to this invention be used for revising nucleotide sequence.

Term " spontaneous mutation " is intended to be included in does not have mutagen to have the lower sudden change that occurs. This term can be included in the sudden change that occurs in the method for the invention when not adding mutagens.

II. non-recombinant bacteria

In the mixed acid fermentation process, glycolytic ferment becomes 2 mole of acetone hydrochlorate+2 mole NADH and clean 2 moles of ATP with every mole of conversion of glucose. Chemical combination the production of material than the higher reduction of acetonate (ethanol, lactate etc.) is used as oxidation NADH and regeneration of NAD⁺Mechanism, this is that to continue glycolysis necessary. Relating in the unique known high ethanol approach of yeast, plant and bacterium (that is, zymomonas mobilis), acetonate is produced carbon dioxide and acetaldehyde by non-oxidizable pyruvate decarboxylase decarboxylation. The acetaldehyde that obtains is used as the electron acceptor that alcohol dehydrogenase carries out the NADH oxidation in ethanol production process.

In the bacterium of many other types, there is a kind of diverse ethanol approach, wherein acetonate is at first changed into acetyl coenzyme A and formates by pyruvic acid formic acid lyase, and this is that wherein reducing equivalent is included in the formates and by formic hydrogenase and is separated into hydrogen (and CO₂) oxidative deamination. The electron acceptor of the aldehyde that acetyl coenzyme A is encoded as adhE-subsequently-2 NADH molecules of alcohol dehydrogenase active oxidation. Because each ethanol needs 2 NADH, remaining half acetyl coenzyme A is converted to acetate and extra ATP. Thereby the natural Escherichia coli approach from the acetyl coenzyme A to ethanol can not be supported high alcohol fermentation, because need 2 NADH from 1 ethanol of the every generation of acetyl coenzyme A. The reduction-oxidation balance is generated to keep by acetate. This is the main cause that wild-type e. coli is produced acetate and the ethanol of equimolar amounts during the fermentation.

Pyruvic dehydrogenase becomes acetyl coenzyme A with acetonate oxidative deamination, and relevant reducing agent is changed into NADH. This is different from PFL, and wherein relevant reducing agent is separated into hydrogen by the formates as intermediate, can not be used for metabolic activity having in the presence of the glucose. By using PDH metabolism acetonate, each acetonate has an extra NADH can be used for each acetyl coenzyme A is reduced into ethanol fully. Although the gene of coding pyruvic dehydrogenase is all expressed under aerobic and anaerobic conditions usually in Escherichia coli, the activity of this compound is very low in the anaerobic growth process.

The present invention at least in part based on otherwise do not change the discovery of glucolytic sudden change in the microorganism of producing and ethanol by high ethanol approach, with based on the exploitation of the non-restructuring producing and ethanol microorganism that under anaerobic glucose and xylose is fermented into ethanol of this discovery. According to the glycolysis of this change, non-recombinant bacteria of the present invention is under anaerobic produced 4 moles of NADH/ mole sugar or 2 NADH/ acetonates.

Thereby, in one aspect, the invention provides the non-recombinant bacteria that comprises sudden change, wherein said sudden change makes this non-recombinant bacteria can under anaerobic produce 4 moles of NADH/ mole sugar. In one embodiment, described sudden change is arranged in the pdh operon. In a particular, described pdh operon comprises pdhR, aceEF and lpd gene. In another embodiment, described sudden change is in the lpd gene.

In another embodiment, the production of 4 moles of NADH/ mole sugar causes ethanol as the production of basic tunning. In a particular, described sugar is selected from: glucose, wood sugar, arabinose, mannose, galactolipin, sucrose and lactose.

In yet another aspect, the invention provides non-recombinant bacteria, it comprises the lpd gene with one or more sudden changes, and wherein said sudden change makes this non-recombinant bacteria can under anaerobic produce ethanol as basic tunning.

In one embodiment, the ethanol of production comprises the total non-gas tunning under anaerobic greater than 50%.

In another embodiment, described non-recombinant bacteria producing and ethanol not when not suddenling change.In another embodiment, the bacterium of described not producing and ethanol produces ethanol as less important tunning.In one embodiment, the ethanol of production is less than 40% of total non-gas tunning.

In another embodiment of the invention, the sudden change in the lpd gene provides high ethanol approach, and bacterium produces ethanol as basic tunning by this approach.

In another embodiment, the one or more alternative fermentation approach in the deactivation bacterium.In one embodiment, described alternative route is by the deactivation that suddenlys change.Such sudden change comprises disappearance, replacement or the interpolation of the Nucleotide of one or more genes in the alternative route.In another embodiment, described sudden change is at the ldh gene for example in the ldhA gene.In another embodiment, described sudden change is at the pfl gene for example in the pflB gene.In another embodiment, described alternative fermentation approach comprises the lactic acid-producing of serum lactic dehydrogenase (ldh), the acetate (acetate) of pyruvic acid formic acid lyase (pfl), ethanol, formic acid (formate) or H ₂And CO ₂The production of conversion or succsinic acid (succinate).

In the different embodiments of non-recombinant bacteria described herein and bacterial cell, described bacterium is selected from: gram negative bacterium and gram positive bacterium.In certain embodiments, described bacterium is a gram negative bacterium.In specific embodiments, described gram negative bacterium is selected from: acinetobacter calcoaceticus, gluconobacter suboxydans, escherich's bacillus, bacillus, Xie Wanala bacterium, Salmonellas, intestinal bacteria and klebsiella bacillus.In other embodiments, described bacterium is a gram positive bacterium.In specific embodiments, described gram positive bacterium is selected from: genus bacillus, clostridium, coryneform bacteria, lactobacillus, galactococcus, wine coccus, suis and eubacterium.In other embodiments, described bacterium is intestinal bacteria.

As mentioned above, non-recombinant bacteria of the present invention comprises one or more sudden changes, for example the sudden change in the lpd gene.In one embodiment, described sudden change comprises with another amino acid replaces an amino acid, thereby should replace the pK that changes by the polypeptide of the lpd genetic expression that suddenlys change.In certain embodiments, the sudden change in the lpd gene causes the NADH insensitivity.That is to say that the cell that carries such sudden change shows the reduction of PDH enzyme to the susceptibility of NADH.Thereby, 4 NADH molecule/glucose of the insensitive cells produce of NADH (2 from glycolysis-, and 2 from the PDH reaction), all 4 NADH can be used for 2 acetyl-CoAs are reduced into ethanol.In certain embodiments, PDH to the NADH insensitivity of NADH and it in addition high NADH/NAD than the time ability that works, make cell for example bacterial cell become high ethanol producer.

In one embodiment, described polypeptide comprises SEQ ID NO:6, and described sudden change comprises another amino acid replacement wild-type amino acid that is used in following position:

A) position 322 among the SEQ ID NO:6 or the optional position in about 50 positions of the either side of position 322; Or

B) position 354 among the SEQ ID NO:6 or the optional position in about 50 positions of the either side of position 354.

In certain embodiments, other amino acid is to be selected from following neutral amino acids: L-Ala, halfcystine, glycine, Isoleucine, leucine, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine and Xie Ansuan.In other embodiments, other amino acid is to be selected from following basic aminoacids: arginine, l-asparagine, glutamine, Histidine and Methionin.

In one embodiment, described sudden change comprises the H that replaces 322 places in the position with arbitrary amino acid, makes this amino acid replace the acidity of increase by the polypeptide of the lpd genetic expression of sudden change.In a particular, described non-recombinant bacteria has the H that comprises 322 places, position among the SEQ ID NO:6 and replaces with Y in interior sudden change.In one embodiment, described non-recombinant bacteria is coli strain SE2377, and it is the preserved material representative at the farming research culture collection center of NRRL B-30970 by specifying preserving number.In another embodiment, described non-recombinant bacteria is coli strain SE2383, and it is the preserved material representative at the farming research culture collection center of NRRLB-30973 by specifying preserving number.In another embodiment, described non-recombinant bacteria is coli strain SE2384, and it is the preserved material representative at the farming research culture collection center of NRRL B-30974 by specifying preserving number.In another embodiment, bacterial strain SE2377 comprises SEQ ID NO:1 or its fragment.In another embodiment, bacterial strain SE2383 comprises SEQ ID NO:1 or its fragment.In another embodiment, bacterial strain SE2384 comprises SEQ ID NO:1 or its fragment.

In another embodiment, described sudden change comprises the E that replaces 354 places in the position with arbitrary amino acid, makes this amino acid replace the acidity that reduces by the polypeptide of the lpd genetic expression that suddenlys change.In a particular, described non-recombinant bacteria has the E that comprises 354 places, position among the SEQ ID NO:6 and replaces with K in interior sudden change.In one embodiment, described non-recombinant bacteria is coli strain SE2378, and it is the preserved material representative at the farming research culture collection center of NRRL B-30971 by specifying preserving number.In another embodiment, described non-recombinant bacteria is coli strain SE2382, and it is the preserved material representative at the farming research culture collection center of NRRLB-30972 by specifying preserving number.In another embodiment, described non-recombinant bacteria is coli strain SE2385, and it is the preserved material representative at the farming research culture collection center of NRRL B-30975 by specifying preserving number.In another embodiment, bacterial strain SE2378 comprises SEQ ID NO:3 or its fragment.In another embodiment, bacterial strain SE2382 comprises SEQ ID NO:3 or its fragment.In another embodiment, bacterial strain 2385 comprises SEQ ID NO:3 or its fragment.

The non-recombinant bacteria that comprises above-mentioned one or more sudden changes is applicable to from sugar produces ethanol.According to the present invention, described sudden change provides high ethanol fermentation approach.In certain embodiments, the ethanol of production comprises the total non-gas tunning under anaerobic greater than 50%.

In one embodiment, described sudden change is derived from spontaneous mutation.In another embodiment, with bacterial exposure in mutagenic compound.In a particular, described mutagenic compound are selected from: ethyl methane sulfonate, 2-aminopurine, ICR-191, methylmethanesulfonate, N-methyl-N '-nitro-N-nitrosoguanidine.In another particular, described mutagenic compound are ethyl methane sulfonate (EMS).

In another embodiment, the one or more alternative fermentation approach in the bacterium is inactivated.Alternative fermentation approach comprises the lactic acid-producing of serum lactic dehydrogenase (ldh), the acetate, ethanol, formate, the H that begin from pyruvic acid formic acid lyase (pfl) and succinate ₂And CO ₂Transform.In one embodiment, described alternative fermentation approach is by the deactivation that suddenlys change.In specific embodiments, described alternative fermentation approach is come deactivation by import deletion mutantion in bacterium.

II. isolated nucleic acid molecule and gene

The present invention also provides isolated nucleic acid molecule, its coding dihydrolipoamide dehydrogenase (lpd) polypeptide or its fragment.Nucleic acid molecule of the present invention comprises the lpd gene with one or more sudden changes, and when existing in bacterium of the present invention, described sudden change causes bacterium under anaerobic to produce ethanol as basic tunning.

Nucleic acid molecule of the present invention comprises dna molecular and RNA molecule and uses the DNA of nucleotide analog deposits yields or the analogue of RNA.Nucleic acid molecule can be strand or double-stranded, but double-stranded DNA advantageously.

In one aspect, the invention provides and be selected from following isolated nucleic acid molecule:

B) nucleic acid molecule, it comprises at least 100 nucleotide fragments of the nucleic acid of the nucleotide sequence that contains SEQ ID NO:1 or SEQ ID NO:3, or its complementary sequence;

D) nucleic acid molecule, its coding comprise the polypeptide fragment of the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4; Wherein said fragment comprises at least 15 of aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4 in abutting connection with amino-acid residue;

Wherein said nucleic acid molecule makes described cell can produce ethanol as basic tunning when expressing in cell.

In one embodiment, under anaerobic comprise total non-gas tunning greater than 50% by the ethanol of cells produce.In another embodiment, described cell is a bacterial cell.In another embodiment, described bacterial cell producing and ethanol not when not expressing described nucleic acid molecule.In a particular, the bacterial cell of described not producing and ethanol is produced ethanol as less important tunning; That is, less than about 40% of total non-gas tunning.

In another embodiment, described bacterial cell is under anaerobic produced ethanol as basic tunning.In a particular, described nucleic acid molecule being expressed in bacterial cell provides high ethanol fermentation approach in the bacterial cell, by this approach, produces ethanol as basic tunning.

In another embodiment aspect this of the present invention, described nucleic acid molecule comprises the fragment of SEQID NO:1, and the length of wherein said nucleic acid molecule is at least 100 Nucleotide, and contain with the T of the 997 corresponding positions, position of SEQ ID NO:1.

In another embodiment, described nucleic acid molecule comprises the fragment of SEQ ID NO:3, and the length of wherein said nucleic acid molecule is at least 100 Nucleotide, and contain with the G of the 1023 corresponding positions, position of SEQ IDNO:1.

In one embodiment, the nucleotide sequence shown in lpd nucleic acid molecule of the present invention and SEQ ID NO:1 or the SEQID NO:3 (for example when the total length with nucleotide sequence compares) or its complementary sequence have at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or higher identity.SEQ IDNO:1 and SEQ ID NO:3 are presented at respectively among Fig. 1 (A) and 3 (A).

In another embodiment, the invention provides isolated nucleic acid molecule, it comprises the fragment of at least 100,150,200,250 or 300 Nucleotide of the nucleic acid molecule of the nucleotide sequence that contains SEQ ID NO:1 or SEQ ID NO:3, or its complementary sequence.

In another particular, the invention provides nucleic acid molecule, its coding comprises aminoacid sequence with SEQ ID NO:2 shown in Fig. 1 (B) and Fig. 2 (B) or SEQ ID NO:4 to has at least at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or the polypeptide of the aminoacid sequence of higher identity.

In another embodiment, described nucleic acid molecule encoding comprises the polypeptide fragment of the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4, and wherein said fragment comprises at least 15,25,35,45,55,65 of aminoacid sequence of SEQ IDNO:2 or SEQ ID NO:4 in abutting connection with amino-acid residue.

In yet another aspect, the invention provides aforesaid non-recombinant bacteria, it comprises above-mentioned isolated nucleic acid molecule.In one embodiment, described non-recombinant bacteria is produced ethanol from sugar.In another embodiment, described sugar is selected from: glucose, wood sugar, pectinose, seminose, semi-lactosi, sucrose and lactose.

The lpd gene of (and according to conventional italic demonstration) described herein comprises nucleic acid molecule (for example dna molecular or its section), for example, the nucleic acid molecule of coded polypeptide or RNA, its in biology and between another gene or other gene by intergenic DNA (promptly, insert or spacer DNA, its natively the described gene of side joint and/or in the chromosomal DNA of biology, separate gene) separate.Gene can instruct enzyme or other peptide molecule synthetic (for example can comprise encoding sequence, for example, coded polypeptide in abutting connection with opening code-reading frame (ORF)), or can self function be arranged in biology.Gene in the biology can be at bunch collection in the operon as herein defined, and wherein said operon is separated by intergenic DNA and other gene and/or operon.Be included in the operon individual gene can not have under the situation of intergenic DNA overlapping between individual gene.

One embodiment of the invention have characterized the lpd nucleic acid molecule or the gene of sudden change.Usually, mutant nucleic acid molecule as herein described or mutator gene comprise nucleic acid molecule or the gene with the nucleotide sequence that comprises at least one change (for example replace, insert, lack), make polypeptide or can be shown by the polypeptide of mutant code be different from described polypeptide or by the activity of the polypeptide of wild-type nucleic acid molecule or genes encoding.Advantageously, the activity for example dihydrolipoamide dehydrogenase active LPD polypeptide of mutant nucleic acid molecule or mutator gene (the lpd gene for example suddenlys change) coding with raising.

In one embodiment, nucleic acid molecule of the present invention under rigorous condition with have the making nucleic acid molecular hybridization of SEQID NO:1 or the described nucleotide sequence of SEQ ID NO:3.Rigorous condition like this is well known by persons skilled in the art, and can be referring to Current Protocols inMolecular Biology, John Wiley ﹠amp; Sons, N.Y. (1989), 6.3.1-6.3.6.A concrete limiting examples of rigorous (for example high preciseness) hybridization conditions is in about 45 ℃ of hybridization, to wash one or many at 50-65 ℃ subsequently in 0.2X SSC, 0.1%SDS in 6X sodium chloride/sodium citrate (SSC).Advantageously, corresponding with the isolated nucleic acid molecule of the present invention of the sequence hybridization of SEQ ID NO:1, SEQID NO:3 naturally occurring nucleic acid molecule under rigorous condition.Usually, naturally occurring nucleic acid molecule comprises RNA or the dna molecular with the nucleotide sequence that produces at occurring in nature.

Nucleic acid molecule of the present invention (nucleic acid molecule that for example has the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3) can use the Protocols in Molecular Biology of standard to separate with sequence information provided herein.For example, nucleic acid molecule can use the hybridization of standard and clone technology (for example to be described in Sambrook, J., Fritsh, E.F., and Maniatis, T.Molecular Cloning:A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) separate, maybe can use the synthetic oligonucleotide primer thing that on SEQ ID NO:1, SEQ ID NO:3 sequence basis, designs to separate by the polymerase chain reaction.According to the pcr amplification technology of standard, use cDNA, mRNA or genomic dna as template and suitable Oligonucleolide primers, nucleic acid of the present invention can increase.In another embodiment, isolated nucleic acid molecule of the present invention comprises the nucleic acid molecule as the complementary sequence of nucleotide sequence shown in SEQ ID NO:1, the SEQ ID NO:3.

Other lpd nucleotide sequence is such nucleotide sequence, it comprises SEQ ID NO:1, the nucleotide sequence of SEQ ID NO:3, and coding has the homologue of the polypeptide of SEQ ID NO:2 or the described aminoacid sequence of SEQ ID NO:4 and (for example encodes and have a SEQ ID NO:2, the polypeptide of the described aminoacid sequence of SEQID NO:4 has at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher identity and have the active polypeptide substantially the same with this polypeptide), its under rigorous condition with have a SEQ ID NO:1, whole or the fragment hybridization of the nucleic acid molecule of the nucleotide sequence of SEQ ID NO:3, or has SEQ ID NO:2 with coding, whole or the fragment hybridization of the nucleic acid molecule of the polypeptide of the aminoacid sequence of SEQID NO:4, or with lpd nucleotide sequence described herein complementation, make described lpd nucleotide sequence when in cell, expressing, cause cell under anaerobic to produce ethanol as basic tunning.

In one embodiment, described nucleic acid molecule encoding comprises the polypeptide of aminoacid sequence of SEQ ID NO:2 or SEQID NO:4 or the bioactive fragment of polypeptide, and wherein said polypeptide or bioactive fragment are retained in and produce the alcoholic acid ability in the host cell.

In another embodiment, lpd nucleic acid molecule or genes encoding have the homologue of LPD polypeptide of the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4.Usually, term " homologue " comprise with wild type peptide or amino acid sequence of polypeptide described herein have at least about 30-35%, advantageously at least about 35-40%, more advantageously at least about 40-50%, more advantageously at least about 60%, 70%, 80%, 90% or higher identity and have function or the bioactive polypeptide of equal value basically with wild type peptide.For example, LPD homologue and the polypeptide with SEQ ID NO:2 or the described aminoacid sequence of SEQ ID NO:4 have at least about 60%, advantageously at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or higher identity, and function that the polypeptide that has and have SEQ ID NO:2 or the described aminoacid sequence of SEQ ID NO:4 is of equal value basically or biological activity (for example having dihydrolipoamide dehydrogenase activity of equal value basically) (that is, being function equivalent).

In one embodiment, lpd nucleic acid molecule or gene comprise the nucleotide sequence of coding SEQ ID NO:2 or the described polypeptide of SEQ ID NO:4.

In another embodiment, the lpd nucleic acid molecule is hybridized with the whole or fragment of the nucleic acid molecule with SEQ ID NO:1 or the described nucleotide sequence of SEQID NO:3, or has all or part of hybridization of the nucleic acid molecule of the nucleotide sequence of the polypeptide of any aminoacid sequence among SEQ ID NO:2 or the SEQ ID NO:4 with coding.

Such hybridization conditions is well known by persons skilled in the art, and can be referring to CurrentProtocols in Molecular Biology, and people such as Ausubel compile., John Wiley; Sons, Inc. (1995), the 2nd, 4 and 6 parts.Other rigorous condition can be referring to MolecularCloning:A Laboratory Manual, people such as Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), the 7th, 9 and 11 chapters.A concrete limiting examples of rigorous hybridization conditions comprises, in 4X sodium chloride/sodium citrate (SSC) in about 65-70 ℃ of hybridization (or in 4X SSC+50% methane amide in about 42-50 ℃ hybridization), subsequently in 1X SSC at about 65-70 ℃ of washing one or many.A concrete limiting examples of high rigorous hybridization conditions comprises, in about 65-70 ℃ of hybridization (or in 1X SSC+50% methane amide in about 42-50 ℃ hybridization), washs one or many at about 65-70 ℃ subsequently in 0.3X SSC in 1X SSC.A concrete limiting examples of low rigorous hybridization conditions comprises, in about 50-60 ℃ of hybridization (or in 6X SSC+50% methane amide in about 40-45 ℃ hybridization), washs one or many at about 50-60 ℃ subsequently in 2X SSC in 4X SSC.The present invention also is intended to be included in above-mentioned value intermediary scope, for example at 65-70 ℃ or at 42-50 ℃.(1X SSPE is 0.15M NaCl to SSPE, 10mM NaH ₂PO ₄And 1.25mM EDTA, pH 7.4) can replace the SSC (1X SSC is 0.15M NaCl and 15mM Trisodium Citrate) in hybridization and the lavation buffer solution; After hybridization finished, washing was carried out 15 minutes separately.Expection length should be than the melting temperature(Tm) (T of heterozygote less than the hybridization temperature of the heterozygote of 50 base pairs _m) low 5-10 ℃, T wherein _mDetermine according to following equation.For the heterozygote of length less than 18 base pairs, T _m(℃)=2 (number of A+T base)+4 (number of G+C base).For the heterozygote of length at 18 to 49 base pairs, T _m(℃)=81.5+16.6 (log ₁₀[Na ⁺])+0.41 (%G+C)-(600/N), wherein N is the number of base in the heterozygote, and [Na ⁺] be the concentration ([Na of 1X SSC of sodium ion in the hybridization buffer ⁺]=0.165M).

The technician also will appreciate that, other reagent can be added in hybridization and/or the lavation buffer solution, to reduce nucleic acid molecule, include but not limited to blocker (for example BSA or salmon or herring sperm carrier DNA), stain remover (for example SDS), sequestrant (for example EDTA), ficoll, PVP etc. to the film non-specific hybridization of nitrocellulose or nylon membrane for example.When using nylon membrane, particularly, an other limiting examples of rigorous hybridization conditions is at 0.25-0.5M NaH ₂PO ₄, among the 7%SDS in about 65 ℃ of hybridization, subsequently at 0.02M NaH ₂PO ₄, among the 1%SDS at 65 ℃ of washing one or many, referring to for example Church and Gilbert (1984) Proc.Natl.Acad.Sci.USA 81:1991-1995 (perhaps, 0.2X SSC, 1%SDS).In another embodiment, isolated nucleic acid molecule comprises and lpd nucleotide sequence complementary nucleotide sequence described herein (for example being the fully-complementary sequence of SEQ ID NO:1 or the described nucleotide sequence of SEQ ID NO:3).

III. polypeptide

The present invention has characterized polypeptide (for example, the enzyme of the producing and ethanol of sudden change, for example, dihydrolipoamide dehydrogenase (LPD)).When described polypeptide was expressed in cell (for example bacterium), described cell was under anaerobic produced ethanol as basic tunning.

In another embodiment, described bacterial cell producing and ethanol not when not expressing this polypeptide.In a particular, the bacterial cell of described not producing and ethanol is produced ethanol as less important tunning; That is, less than about 40% of total non-gas tunning.

In another embodiment, described bacterial cell is under anaerobic produced ethanol as basic tunning, and in another embodiment, the ethanol of production comprises the total non-gas tunning under anaerobic greater than 50%.In a particular, described polypeptide being expressed in bacterial cell provides high ethanol fermentation approach in the described bacterial cell.

In another embodiment, isolated polypeptide of the present invention is the polypeptide fragment that comprises the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4, and wherein said fragment comprises at least 15,25,35,45,55 or 65 of aminoacid sequence of SEQ IDNO:2 or SEQ ID NO:4 in abutting connection with amino-acid residue.

In another embodiment, the invention provides isolated polypeptide, the aminoacid sequence shown in itself and SEQ ID NO:2 or the SEQ ID NO:4 has 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 higher identity (for example when the total length with aminoacid sequence compares).

In one embodiment, described bacterial host cell comprises nucleotide sequence or its segmental carrier that contains SEQ ID NO:1 or SEQ ID NO:3.In another embodiment, the carrier that comprises of described bacterial host cell is pKY33.

The present invention also provides the method that is selected from following polypeptide of producing:

A) comprise the polypeptide of aminoacid sequence SEQ ID NO:2 or SEQ ID NO:4;

In one embodiment, described LPD polypeptide or gene product are derived from the Gram-positive or the gram negative bacterium of non-reorganization producing and ethanol.In exemplary embodiment, described LPD polypeptide or gene product are derived from the gram-negative micro-organism that is selected from following producing and ethanol: acinetobacter calcoaceticus, gluconobacter suboxydans, escherich's bacillus, bacillus, Xie Wanala bacterium, Salmonellas, intestinal bacteria and klebsiella bacillus.

In another embodiment, described LPD polypeptide or gene product are derived from the gram-positive microorganism that is selected from following producing and ethanol: genus bacillus, clostridium, coryneform bacteria, lactobacillus, galactococcus, wine coccus, suis and eubacterium.

Comprise LPD polypeptide or gene product within the scope of the invention, they are intestinal bacteria polypeptides derived or by the gene product of naturally occurring bacterial gene coding.The polypeptide or the gene product that also comprise bacterial derivation within the scope of the invention, they are different from naturally occurring bacterium and/or bacillus coli gene (for example lpd), for example, have sudden change, that insert or disappearance nucleic acid, but the gene of the substantially similar polypeptide (for example comprising the dihydrolipoamide dehydrogenase activity) of coding and naturally occurring gene product of the present invention.

Understand easily, those skilled in the art can change the nucleic acid that (for example replacing) coding conserved amino acid is replaced.Also understand easily, those skilled in the art can replace, add or delete amino acid to a certain extent, and compare the function that does not influence gene product (for example dihydrolipoamide dehydrogenase) basically with naturally occurring gene product, every kind of situation is wherein had a mind within the scope of the present invention.

Comprise non-recombinant bacteria within the scope of the invention, it comprises the lpd gene that contains sudden change, wherein replace and be, the E at 354 places is mutated into arbitrary amino acid at the H at 322 places, position of wild-type lpd gene (SEQ ID NO:6) or in the position, makes the acidity that this amino acid change should the zone.In other embodiments, described amino acid is neutral charged amino acid under physiological pH.In other embodiments, described amino acid is alkaline charged amino acid under physiological pH.

In one embodiment, isolated polypeptide of the present invention (for example isolating dihydrolipoamide dehydrogenase) has the aminoacid sequence shown in SEQ ID NO:2 or the SEQ ID NO:4.In other embodiments, isolated polypeptide of the present invention is that (aminoacid sequence that for example comprises with SEQ ID NO:2 or SEQ ID NO:4 has the identity at least about 30-40% at least one homologue in SEQ ID NO:2 or the described polypeptide of SEQ IDNO:4, advantageously about 40-50% identity, more advantageously about 50-60% identity, more advantageously about 60-70%, 70-80%, 80-90%, the aminoacid sequence of 90-95% or higher identity, and have respectively and the substantially similar activity of amino acid sequences encoded polypeptide by SEQ ID NO:2 or SEQ ID NO:4).

In order to determine the identity per-cent of 2 aminoacid sequences or 2 nucleic acid, for the best contrast purpose aligned sequences (for example breach can be introduced first aminoacid sequence or nucleotide sequence, be used for and second amino acid or the best comparison of nucleotide sequence).When the position in first sequence was occupied by the amino-acid residue identical with correspondence position in second sequence or Nucleotide, then molecule was same in this position.Identity per-cent between 2 sequences changes (that is, the number of % identity=same position/total number of positions x 100) with the number of the total same position of sequence, advantageously considers to generate essential breach number and the breach size of best comparison.

Use mathematical algorithm, can contrast the identity per-cent between sequence and definite 2 sequences.A concrete limiting examples that is used for the correlated mathematical algorithm of sequence is the algorithm of Karlin and Altschul (1990) Proc.Natl.Acad.Sci.USA 87:2264-2268, improves as described in Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-5877.Such algorithm is incorporated among the NBLAST and XBLAST program (2.0 editions) of people such as Altschul (1990) J.Mol.Biol.215:403-410.Can carry out the BLAST nucleotide search with the NBLAST program, scoring=100, word length=12 are to obtain and nucleic acid molecule homologous nucleotide sequence of the present invention.Can carry out the search of BLAST polypeptide with the XBLAST program, scoring=50, word length=3 are to obtain and peptide molecule homologous aminoacid sequence of the present invention.In order to obtain being used to contrast the breach comparison of purpose, can use described breach BLAST as people such as Altschul (1997) nucleic acid Research25 (17): 3389-3402.When using BLAST and breach blast program, can use the default parameter of each program (for example, XBLAST and NBLAST).Referring to http://www.ncbi.nlm.nih.gov.Another the concrete limiting examples that is used for the correlated mathematical algorithm of sequence is the algorithm of Myers and Miller (1988) Comput Appl Biosci.4:11-17.Such algorithm is incorporated in the ALIGN program, and this program can be at for example GENESTREAM webserver, IGH Montpellier, FRANCE or obtain at the ISREC server.When using the ALIGN program to contrast aminoacid sequence, can use PAM120 weighting residue table, the notch length point penalty is 12, the breach point penalty is 4.

For example, in one embodiment of the invention, use, determine 2 identity per-cents between the aminoacid sequence at the Blast of NCBI server or at the ClustalW of European biotechnology research institute (European Biotechnology Institute).For example, will compare from the aminoacid sequence and the proteic aminoacid sequence of intestinal bacteria Lpd of the biological dihydrolipoamide dehydrogenase of difference, any from 2 databases can obtain the identity per-cent of particular sequence and intestinal bacteria sequence.Table 7 and Fig. 9 have explained these contrasts.Value in the bracket is represented total similarity of specific protein and intestinal bacteria Lpd, and comprises identical amino acid position and conservative position of replacing takes place.For subtilis, in contrast, comprise 2 kinds of dihydrolipoic acid dehydrogenases, a kind of from the PDH mixture, another kind of from the 3-acetoin dehydrogenase.

Those of ordinary skills will appreciate that, based on aforementioned calculating, there is high conservative degree in LPD in the various bacteria species, be included in bacterium (based on 16S ribosome-RNA(rRNA) (DNA) sequence) away from each other, for example Gram-positive and gram negative bacterium, archeobacteria and streptomycete in the phylogeny.

Pyruvic oxidase is present in all aerobes, is the key enzyme of glucose to Conversion of energy.Dihydrolipoamide dehydrogenase (LPD) is one of 3 subunits of PDH mixture.LPD contains the motif of 2 uniquenesses: flavine binding motif (amino acid/11 5-45) and pyridine nucleotide-disulphide oxydo-reductase motif (amino acid 347-456) (intestinal bacteria Lpd numbering).Because their unique effect in the PDH mixture have significant homology from the proteic aminoacid sequence of the Lpd of several biologies.Amino acid sequence identity scope between intestinal bacteria Lpd and other bacterium Lpd is from 30% to 99%.Under the extreme case from intestinal bacteria and people's Lpd, 42% amino acid is identical in the sequence.In the flavine land of Lpd (amino acid/11 5-45), this sequence identity increases to 67%.In one the 18 amino acid subdivision (position 28-55) of this sequence, except an amino acid, all amino acid are guarded in the Lpd of intestinal bacteria, people and mouse sequence.Also guard at the Histidine at 322 places with at the L-glutamic acid at 354 places in these albumen.Because the conservative degree of this very high sequence is expected at the intestinal bacteria Lpd sudden change described in the present invention and has similar phenotype after introducing the Lpd albumen biological from other.Thereby method of the present invention is not limited to the bacterial strain of this paper instruction.

IV. the method for preparing non-recombinant bacteria

B) select to produce the mutant of ethanol as main tunning.

In an embodiment of this method, the ethanol of production comprises the total non-gas tunning under anaerobic greater than 50%.

B) select to produce the mutant of ethanol as main tunning.

In preceding method of the present invention and implementation of processes scheme, the sugar in the rich sacchariferous substratum is selected from: glucose, wood sugar, pectinose, seminose, semi-lactosi, sucrose and lactose.In one embodiment of the invention, the non-recombinant bacteria with aforementioned attribute also is a producing and ethanol.Therefore, the invention provides the method for preparing the non-recombinant bacteria of producing and ethanol.In addition, the invention provides the method for the producing and ethanol phenotype of screening hope.

Parent strain of the present invention is characterised in that, when growing in the sacchariferous substratum of richness, under anaerobic hangs down the alcohol production level.An example of such bacterial strain is strains A H242; But, be characterised in that under anaerobic any bacterial strain of low alcohol production level is applicable to this method.(Dastenko and Wanner, 2000.Proc.Natl.Acad.Sci.USA 97:6640-6645.) carries out other sudden change to parent strain according to means known in the art, be provided in all substratum can not anaerobic growth (defective type) parent strain.In addition, according to practice well-known in the art, add the expression cassette of antibiotics resistance for selecting purpose.

Usually, the following selection: incubation growth deficient strain under aerobic condition, up to reaching mid-term exponential phase of growth, culture is coated on the agar, culture is exposed to mutagenic compound.Those of ordinary skills will appreciate that, can use many mutagenic compound, comprise ethyl methane sulfonate, 2-aminopurine, ICR-191, methylmethanesulfonate, N-methyl-N '-nitro-N-nitrosoguanidine, or notified any other reagent that causes nucleotide sequence to change.After under anaerobic being exposed to mutagenic compound, culture is transformed into aerobic condition, returns anaerobic condition then.Select the bacterium colony of growth, on fresh flat board, rule, and growth under anaerobic.Can separately cultivate each bacterium colony, and suitably grow on the microbiotic flat board, to confirm to carry the mutant of parent's antibiotics resistance.Those of ordinary skills will appreciate that, carry out the microbial culture operation according to the standard method of this area.

High performance liquid chromatography can be used for measuring the productive rate of the exhausted substratum tunning of isolating mutant.For example, can detect ethanol, acetate, formate and succinate by HPLC.

Those skilled in the art will realize that based on previous examples with based on the homology between the bacterial isolates bacterial strain that method of the present invention is not limited to instruct among the application.

V. produce the alcoholic acid method

Host cell of the present invention is characterised in that, under anaerobic low alcohol production level.Wild-type e. coli ratio with 1: 1 in the anaerobic growth process is produced ethanol and acetate.In the stationary phase of growth, wild-type e. coli is produced lactic acid salt as primary product, and the alcoholic acid ratio is about 20% in total tunning.The product of all these fermentations comprises various acid, thereby produces term " mixed acid fermentation ".In one aspect, the invention provides non-recombinant bacteria, it comprises the lpd gene with one or more sudden changes, and wherein said sudden change makes this non-recombinant bacteria can under anaerobic produce ethanol as basic tunning.Basic tunning is intended to comprise the non-gas tunning that comprises greater than 50% total non-gas product.Described basic tunning is the abundantest non-gas product.

Usually, selection can provide the optimal pH of catalytic condition of the enzyme that the best growth kinetics that promotes host cell to produce bacterial strain and culture generate and the fermentation condition of temperature people such as (, (1993) Biotechnol.Progress.9:533-538) Doran.For example, for klebsiella, P2 bacterial strain for example, optimum condition is determined at 35-37 ℃ and pH 5.0-pH 5.4.Under these conditions, even exogenous fungi endoglucanase and the exoglucanase that adds be very stable, and the long-time section that continues to work.Other condition has been discussed in an embodiment.In addition, the technician can understand, only needs routine test, uses technology known in the art, optimizes specifically fermentation reaction of the present invention.Referring to, for example, U.S. Patent number 5,424,202 and 5,916,787, they provide with reference to incorporating this paper hereby into.

VI. illustration

Following embodiment has further explained the present invention, and they not should be understood to restrictive.In an embodiment, use following material and method, except as otherwise noted.

Material and method

Bacterial isolates

Use e. coli k-12 bacterial strain W3110 (ATCC 27325) and derivative strain AH242 in this research, it is the preserved material representative at the farming research culture collection center of NRRL B-30967 (Δ ldhA and Δ (focA-pflB)) by specifying preserving number.Bacterial strain SE2378 is the producing and ethanol mutant of strains A H242.According to people such as Dastenko, deletion gene pflB, adhE, mgsA and aceF.After transposon Tn10 is introduced ldhA, make up the ldhA deletion mycopremna, subsequently fusaric acid substratum (people 1991 such as Klekner; People such as Maloy 1981) select in.The structure of other bacterial strain uses the heredity of standard and Protocols in Molecular Biology (people 1982 such as Maniatis; People such as Miller 1972).In the table 1 below, listed the genotype of bacterial strain used herein.

Table 1: bacterial isolates and genes involved type

Growth medium and fermentation

Rich medium (L-meat soup) contains (every liter), trypticase peptone (10g), yeast extract (5g) and NaCl (5g) (Lee, et al.1985).Mineral salts medium (Lee, et al.1985) as previously mentioned adds glucose or wood sugar as required.(Hasona, et al.2004) ferments at 37 ℃ as previously mentioned.By adding KOH, will cultivate pH and maintain 7.0.(people 2006 such as Patel) carries out batch fermentation at the 13x 100mm screw cap cuvette cartridge that is filled to the top as previously mentioned.

Carrier and conversion

According to the described standard operation in this area, clone and expression lpd and the gene in the pdh district.Except other general known carrier, the carrier that adopts in conversion can comprise pTrc99a (GE), pCR2.1-TOPO, pBR322, pUC19, pACYC184, pBAD24.According to the standard operation of people such as Maniatis (1989), use based on CaCl ₂Chemical conversion process.

Analytical procedure

By HPLC (Underwood, et al.2002), measure sugar and tunning.(Talarico, et al.2001) as previously mentioned measures the pyruvic carboxylase activity in the cell product of fragmentation.

Embodiment 1

The non-recombinant escherichia coli strain SE2377 of producing and ethanol, SE2378, SE2382, SE2383,

The separation of SE2384, SE2385

The separation of the non-reorganization producing and ethanol bacterial strain of bacteria Escherichia coli has been described in this embodiment.

The described high ethanol that initial strain AH242 is used for separating Escherichia coli generates mutant.Because the encode ldh of serum lactic dehydrogenase (LDH) and pyruvic acid formic acid lyase (PFL) and the sudden change in the pflB gene respectively, strains A H242 can not be in sacchariferous rich medium anaerobic growth people such as (, 1989) Mat-Jan.Although these sudden changes are arranged, the aerobic growth of AH242 is unaffected.The anaerobic growth defective of AH242 is that NADH is to NAD ⁺The result of the defective that reoxidizes, described NAD ⁺The necessary substrate that to be crucial glycolytic ferment glyceraldehyde-3-phosphate dehydrogenase produce with relevant ATP.By pyruvate salt is reduced into lactic acid salt, the disappearance of LDH can be got rid of the NADH oxidation.Under the situation of the acetyl-CoA that disappearance is produced by PFL usually, there are not enough acetyl-CoAs to be used for the effective NADH oxidation and the alcoholdehydrogenase activity of natural aldehyde.

In the present invention, described method (Datsenko and Wanner, 2000) before using, from the H242 bacterial strain, make up focA-and-pflB (pyruvic acid formic acid lyase) disappearance.Single deletion mutant AH240 ,-(focA-pflB) and AH241-(ldhA) be the parent strain of two AH242 bacterial strains that suddenly change.In the position of disappearance, insert the FRT-Km-FRT box, thereby provide kalamycin resistance for strains A H242.Because 2 sudden changes, strains A H242 is the anaerobic growth defective type in all substratum.Following table 2 has been listed the growth characteristics of the intestinal bacteria mutant of the sudden change that has in the aerobic fermentation approach.

Table 2: growth characteristics with intestinal bacteria mutant of the sudden change in the aerobic fermentation approach

LB, L-meat soup; Minimum-as not have to add or the glucose minimal medium of interpolation acetate and succinate (1mg/ml separately)

NG-does not grow

Value in the bracket is the growth velocity in the glucose minimal medium that contains acetate, succinate and glutaminate (1mg/ml)

In shaking table 200RPM, 37 ℃, under aerobic condition, in 5ml L-meat soup, cultivate the anaerobic growth deficient strain AH242 obtain.In mid-term exponential phase of growth, take out culture from shaking table, be coated on the L-agar that contains glucose or contain on the L-agar of glucose+toluylene red reduction-oxidation dyestuff.The Whatman paper filter is placed on the surface of each nutrient agar.In dish, add mutagenic compound ethyl methane sulfonates (EMS), flat board be transferred to anaerobic jar, this is canned have outer by the H of palladium catalyst ₂+ CO ₂Producer is to set up no O ₂Environment.Can adopt other standard reagent that is applicable to mutagenesis in the present invention.To dull and stereotyped anaerobic jar be housed 37 ℃ of incubations 5 days.

After 5 days, on specified substratum, do not detect visible growth.Subsequently, 2 of incubations coiled about 20 hours under aerobic condition.When this incubation finishes, in 2 kinds of substratum, in the All Ranges around the zone of the placement location of the paper disc that contains EMS, observe the bacterial cell lawn.By replica plating,, and placed anaerobic jar 5 days with the fresh culture of the cell transfer on every kind of media surface to same composition.After 5 days, each flat board has 100 bacterium colonies of surpassing at the All Ranges except the placement location of EMS.Select 31 bacterium colonies from each glucose (15 bacterium colonies) and glucose+toluylene red flat board (16 bacterium colonies), on fresh L-agar+glucose, rule.Culture plate under anaerobic.All colony growths under anaerobic.

31 mutant are inoculated into L-meat soup+glucose cultures, after growth, each is transferred to the surface of L-agar+kantlex flat board.All mutant are grown in the presence of kantlex, show that they carry the antibiotics resistance of parent strain AH242.

31 mutant are transferred in L-meat soup+glucose in the screw cap test tube, under not mixing situation at 37 ℃ of incubations.After detecting visible growth,, use tunning in the useless substratum of high-performance liquid chromatogram determination people such as (, 2002) Underwood with substratum and cellular segregation.Table 3 shows, produces ethanol as basic or main tunning (73%) for 30 in 31 mutant.Other product is the combination of succinate and acetate.

These results show, isolate non-recombinant mutant from the AH242 parent strain that can not grow anaerobic environment, and it can be grown in anaerobic condition, and produce ethanol as main tunning.

Table 3: the fermentation character of the producing and ethanol sudden change derivative of coli strain AH242

Embodiment 2

The growth velocity of the non-recombinant bacteria of producing and ethanol and fermentation character

The growth velocity and the fermentation character of the non-recombinant bacteria of producing and ethanol have been described in this embodiment.

Selection is used for further research from the mutant strain SE2378 that can under anaerobic grow and produce the main tunning of ethanol conduct of above-mentioned research.

Growth characteristics

Checked the growth characteristics of intestinal bacteria mutant with the sudden change of aerobic fermentation approach.As above when cultivating in the described rich medium of table 2, any in the aerobic growth of bacterial strain SE2378 and wild-type e. coli bacterial strain W3110 single or two (focA-pflB) or the ldhA mutant is suitable.In minimal medium, the aerobic growth speed of bacterial strain SE2378 be parent strain AH242 pact half.Add acetate and succinate to growth medium, growth velocity is returned near parent strain.Although bacterial strain SE2378 can aerobic growth, even the growth velocity in rich medium only is about 50% (referring to top table 2) of AH240 and AH241 single mutant.Bacterial strain SE2378 can not be in glucose-minimal medium aerobic growth, this is the phenotype relevant with pflB sudden change people such as (, 1989) Clark.Add the growth that acetate can be supported pflB mutant strain AH240 to minimal medium, but the derivative strain SE2378 of producing and ethanol can not.Except acetate, bacterial strain SE2378 also need L-glutamic acid could be in glucose-minimal medium anaerobic growth.Research in the past is verified, and producing and ethanol coli strain KO11 also needs the best xylose-fermenting of L-glutamic acid (people such as Underwood, 2004).This L-glutamic acid demand can overcome by add protectiveness penetrant trimethyl-glycine in substratum.But bacterial strain SE2378 L-glutamic acid demand of anaerobic growth in minimal medium can not be suppressed by trimethyl-glycine, and this is hinting that acetyl-CoA flows to the biosynthesizing defective of (flux to) 2-oxoglutaric acid salt (a kind of glutamate precursor), rather than the infiltration demand.Think that acetyl-CoA can be changed into ethanol fast by this ethanol source, and acetyl-CoA is biosynthetic speed limit.Utilize these additives, the growth velocity of bacterial strain SE2378 in minimal medium reaches the growth velocity of pflB parent strain AH240.For the growth of bacterial strain SE2378 in glucose-minimal medium, corn steep liquor (a kind of low-cost medium additives) can substitute L-glutamic acid.

Glucose fermentation

50gl in pH control ^-1In glucose fermentation people such as (, 2004) Hasona, bacterial strain SE2378 lagged behind the back with 0.46h at about 6 hours ^-1Specific growth rate growth, and produce ethanol as base product (Fig. 3 and following table 4).Because directly parent strain AH242 can not aerobic growth, and the fermentation of bacterial strain SE2378 and the fermentation of wild type strain W3110 are compared.W3110 finished 50gl in 24 hours ^-1Glucose fermentation, and mutant strain needs about 72 hours.This difference mainly owing to the difference (dry weight of wild-type is 2.5mg/ml, and the dry weight of mutant is 1.7mg/ml) of cell density, (wild-type and mutant are respectively 4.1-3.3g glucose h to the carbohydrate metabolism high specific speed of 2 kinds of bacterial strains ^-1The g cell ^-1Following table 5).Bacterial strain SE2378 produces about 480mmol l ^-1Ethanol (22gl ^-1), accounting for 88% of gross product, described gross product comprises acetate, lactic acid salt and succinate in a small amount.This is different from bacterial strain W3110 fermentation, and wherein ethanol only accounts for 27% of product, is 6.6gl ^-1(table 4).1.6gh with yeast batch fermentation report ^-1The g cell ^-1Value people such as (, 2000) Smits is compared, and is 1.34gh with the observed high specific productivity of bacterial strain SE2378 (specific productivity) ^-1The g cell ^-1(table 5).

Table 4: coli strain SE2378 and wild type strain W 3110 ^aFermenting characteristic

^aAdding 50gl ^-1Ferment at 37 ℃ in the L-meat soup of sugar, pH7.0.

^bAlcohol yied is as the mark of theoretical maximum (0.51g ethanol/g sugar).

^cEthanol is as the molar fraction of the gross product of the glucose of every mole of fermentation.

Table 5: growth and the alcohol production of the coli strain SE2378 that on glucose or wood sugar, grows

Abbreviation: μ _Max, specific growth rate, h ^-1Y _X/8, g cell (g substrate) ^-1

Q _s, the sugared L that g consumes ^-1h ^-1Q _P, g ethanol L ^-1h ^-1Y _P/8, g ethanol (g substrate) ^-1

q _s, the sugar (g dry cell weight) that g consumes ^-1h ^-1q _p, (g dry cell weight) ^-1h ^-1

Wood-sugar fermentation

At 50g l ^-1In the wood sugar aerobic fermentation process, wild-type W3110 and mutant SE2378 bacterial strain are with speed growth similarly, although bacterial strain SE2378 lags behind about 8 hours (Fig. 3 and top table 4 and 5).Specific growth rate on wood sugar is 80% of a glucose, this consistent with previously disclosed report (people 2002 such as Gonzalez).Mutant strain is than wild-type W3110 xylose-fermenting quickly.After 48 hours, the xylose utilization of bacterial strain SE2378 has surpassed glucose utilization.About 88% of the tunning that reclaims with bacterial strain SE2378 is an ethanol; 20gl ^-1From 50gl ^-1Wood sugar.It is 2.23gh that bacterial strain SE2378 utilizes the ethanol high specific productivity of wood sugar ^-1The g cell ^-1

W3110 and SE2378 utilize the ethanol specific production rate of wood sugar all to be higher than and utilize glucose, and be as shown in table 5.This can indicate the lower energy yield (people such as Hasona, 2004) from xylose metabolism.For wild-type, be only about 1.5/ wood sugar from the clean ATP productive rate of wood sugar, be different from 3.0/ glucose.This needs cell to use more wood sugar to produce the cell quality of same amount.But the wood sugar consumption rate speed of wild-type only slightly is higher than glucose (4.93 contrast 4.10gh ^-1The g cell ^-1), shown in top table 5, thereby explained and compared lower cell yield and longer fermentation time with glucose fermentation.On the contrary, bacterial strain SE2378 lacks pyruvic acid formic acid lyase, and this enzyme is for be crucial (people 2004 such as Hasona) for the wood-sugar fermentation in the minimal medium.Because this sudden change, be 0.67/ wood sugar only from the clean calculating ATP productive rate of wood-sugar fermentation among the bacterial strain SE2378.Obviously, this lower ATP productive rate drives the high wood sugar flow in this producing and ethanol mutant.2.23gh ^-1The g cell ^-1The ethanol specific production rate from wood sugar be higher than the 1.6gh of yeast on glucose ^-1The g cell ^-1Value people 2000 such as () Smits and carry producing and ethanol coli strain KO11 (about 2gh on glucose and wood sugar of zymomonas mobilis pdc and adh gene ^-1The g cell ^-1).

These results show that non-reorganization SE2378 mutant is from glucose and the basic tunning of xylose production ethanol conduct.In addition, alcohol production speed and other producing and ethanol biophase are worked as.

Embodiment 3

Discriminating from the colibacillary mutant lpd gene of non-reorganization producing and ethanol

In this embodiment, discriminating from the mutant lpd gene of coli strain SE2377, SE2378, SE2382, SE2383, SE2384, SE2385 has been described.

By zac ∷ Tn10 cotransduction, describe the mutation map in the non-reorganization producing and ethanol intestinal bacteria mutant strain.When by cotransduction deletion aroP-pdhR-aceEF gene (Fig. 5), the ability that the transduttant forfeiture is under anaerobic grown in the LB that contains 1% glucose, and the identical disappearance under the wild-type background can not influence anaerobic growth.These results are hinting the effect of pyruvic oxidase in the anaerobic growth of bacterial strain SE2378.Cause that SE2377, the SE2378 of discriminating, the sudden change of the producing and ethanol phenotype in the SE2382 mutant strain are plotted in the pyruvic oxidase gene locus.

Pyruvate dehydrogenase complex (PDH) is made up of 3 enzymes: pyruvic oxidase/decarboxylase (enzyme 1, E1), lipoate acetyltransferase (enzyme 2, E2), and dihydrolipoamide dehydrogenase (enzyme 3, E3) subunit.Known pdhR promotor is the promotor of pdhR-aceEF-lpd genetic transcription, although there is independent startup (people such as Quail, 1995) of aceEF and lpdA gene.Because the expression of PDH operon is subjected to the proteic negative adjusting of pdhR people such as (, 1995) Quail, to the pdhR gene sequencing (Fig. 6 A) of SE2377, SE2378, SE2382.The sequential analysis of bacterial strain SE2378 has disclosed 2 sudden changes in the coding region of pdhR: 1 amino acid is replaced (S 12P) and 1 amino sour insertion as amino acid/11 18 of leucine.Found the another kind of Nucleotide replacement (Fig. 6 B) of G to A in the intergenic region between pdhR gene and aceE gene.Bacterial strain SE2377 and SE2382 are not carried at any sudden change in the pdhR-aceEF zone of genomic dna.But these bacterial strains and bacterial strain SE2383, SE2384 and SE2385 have the single sudden change (Fig. 5 A) in the lpd gene.PdhR albumen is the pyruvate salt responsiveness instrumentality of pdhR-lpd operon, thereby the sudden change in this albumen is paid no attention to outside the material.Except the initiation site that the pdhR that transcribes at pdhR-lpd begins to locate, aceEF can contain its aceEF-lpd transcription initiation site.Thereby the sudden change in the intergenic region can support that also high-caliber aceEF-lpd expresses in the anaerobic cell.The level that was reported that the pyruvic oxidase/decarboxylase of PDH mixture in the intestinal bacteria of aerobic growth in the past is than high about 5 times of the cell of anaerobic growth people such as (, 1999) deGraef.

These results provide the position of the sudden change in the producing and ethanol coli strain that identifies of the present invention.

Embodiment 4

The producing and ethanol phenotype is responsible in sudden change in the lpd gene

In this embodiment, confirmed the PDH mixture, particularly the sudden change in the lpd gene is the origin cause of formation of producing and ethanol phenotype.

The preliminary genetic analysis of bacterial strain SE2378 discloses, cause the sudden change of anaerobic growth and high alcohol production be positioned at the gene of coding PDH mixture (pdh locus: pdhR, aceF, lpd) locate or its near.For the producing and ethanol phenotype that confirms bacterial strain SE2378 needs PDH, with aceF gene (Thioctic acid, dihydro-Transacetylase; The E2 enzyme of PDH) the into bacterial strain YK1 that transduces of the sudden change in promptly lacks the derivative of the bacterial strain SE2378 of kalamycin resistance gene.Transduttant bacterial strain YK93 has lost the anaerobic growth ability, shown in following table 6.

Table 6: growth characteristics with producing and ethanol coli strain SE2378 of the sudden change in the pdh locus (aceF)

??YK152	??YK29，aceF	??0.83	??NG	??0.50	??NG	??NG
??YK152	??YK29，aceF	??0.83	??NG	??0.50	??NG	??NG	??YK1	??pflB，ldhA，Ana ⁺	??1.14	??0.51	??0.83	??0.41	??NG ^*
??YK93	??YK1，aceF	??0.68	??NG	??0.46	??NG	??NG	??YK1	??pflB，ldhA，Ana ⁺	??1.14	??0.51	??0.83	??0.41	??NG ^*
??YK93	??YK1，aceF	??0.68	??NG	??0.46	??NG	??NG	??YK157	??YK152，aceF ⁺(W3110)	??1.32	??0.96	??0.87	??NG	??NG
??YK158	??YK152，aceF ⁺(SE2378)	??1.17	??0.51	??0.80	??0.45	??NG ^*	??YK157	??YK152，aceF ⁺(W3110)	??1.32	??0.96	??0.87	??NG	??NG

Minimum-as not have to add or the glucose minimal medium of interpolation acetate and succinate (1mg/ml separately).

NG-does not grow

^*2 kinds of producing and ethanol derivatives need acetate and succinate to be used for the anaerobic growth of bacterial strain SE2378 in minimal medium.

The bacterial strain YK93 of the negative phenotype of this anaerobism is similar to strains A H242, i.e. the parent of bacterial strain SE2378.Although the aceF mutant is aerobic minus in minimal medium, this is because this cell can not be produced acetyl-CoA and be used for biosynthesizing, this function is by PFL catalysis under the anaerobic growth condition, thereby, the aceF sudden change can not influence intestinal bacteria (bacterial strain YK153, have the W3110 of aceF sudden change, as shown in table 6) anaerobic growth.The anaerobic growth of bacterial strain YK93 is defective in all substratum of test.By containing phage P1, aceF is advanced in the sudden change of the aceF among bacterial strain YK152 transduction from the gene of W3110 (wild-type) or SE2378 (ethanol source) ⁺, select transduttant growth in minimal medium under aerobic condition.Also test the anaerobic growth and the tunning of transduttant.Acceptance is from the aceF of wild type strain W3110 ⁺The transduttant of gene aerobic growth in minimal medium, but can not be in the substratum of arbitrarily test anaerobic growth, this is owing to have ldhA and pflB sudden change.Acceptance is from the aceF of bacterial strain SE2378 ⁺All transduttants of gene are anaerobic growth all, and the transduttant of all tests is produced ethanol as main tunning.These results show, the producing and ethanol phenotype of bacterial strain SE2378 needs complete pdh locus and PDH activity, and with the dependent alcohol production approach of PDH-consistent (referring to Fig. 8 C).In this high alcohol production approach, pyruvate salt is become acetyl-CoA by PDH oxidative decarboxylation, and further is reduced into acetaldehyde and ethanol (Fig. 8 C) by alcoholdehydrogenase.Acetyl-CoA is produced required aceF (PDH-minus; Bacterial strain YK93) or required adhE (the ADH minus of alcohol production; Bacterial strain YK91) disappearance can cause anaerobic growth to bear phenotype, and this has supported this approach in the high alcohol production of the bacterial strain SE2378 that lacks lactic acid-fermenting desaturase and pyruvic acid formic acid lyase and the effect in the reduction-oxidation balance.

In next group experiment, confirmed that the lpd gene is the origin cause of formation of producing and ethanol phenotype.To advance expression vector from wild type strain W3110 with from the lpd gene clone of producing and ethanol mutant strain SE2378, and be used for producing Lpd albumen from the trc promotor, with IPTG as inductor.These plasmids are transformed the bacterial strain YK100:ldhA that into has following 3 disappearances, (focA-pflB), and lpd.Except 3 sudden changes, bacterial strain YK100 is similar to the W3110 bacterial strain.Because these 3 disappearances, bacterial strain YK100 is the anaerobic growth defective in all substratum of test, is the aerobic growth defective in minimal medium.As previously discussed, pyruvate dehydrogenase complex (PDH) is made up of 3 enzymes: pyruvic oxidase/decarboxylase (enzyme 1), lipoate acetyltransferase (enzyme 2), and lipoamide dehydrogenase (enzyme 3).Colibacillary aerobic growth is subjected to any the damage of sudden change in 3 components of PDH mixture.

To contain from the plasmid pKY32 (Fig. 7 A) of the lpd gene (lpd+) of bacterial strain W3110 or contain mutant lpd gene (lpd from bacterial strain SE2378 ^*) plasmid pKY33 (Fig. 7 B) transform bacterial strain YK100, and select the transformant of Ampicillin Trihydrate resistance.These transformant are PDH-male, as viewed by the aerobic growth in minimal medium; The enzyme 1 of PDH mixture and enzyme 2 are from karyomit(e), and lpd is from plasmid.Only having the conversion physical efficiency of carrying from the plasmid pKY33 of the lpd gene of producing and ethanol SE2378 bacterial strain under anaerobic grows.Ethanol is from the main tunning in the useless substratum of bacterial strain YK100/pKY33 (being called YK129).On the contrary, having the bacterial strain YK100 that carries from the plasmid pKY32 of the natural lpd gene of W3110 can not under anaerobic grow.

In a word, these results show that LPD albumen has caused the activity of observed pyruvate dehydrogenase complex under the anaerobic growth condition, and further shows, the mutant form of Lpd is enough to support colibacillary high alcohol production.The reason of colibacillary lpd mutant producing and ethanol is the ability of its production 4NADH/ glucose.

Embodiment 5

The NADH insensitivity is responsible in sudden change in the lpd gene

In this embodiment, confirmed that the sudden change in the lpd gene can cause the NADH insensitivity.More specifically, dihydrolipoamide dehydrogenase (LPD) activity of discovery NADH sensitivity in wild-type (natural) enzyme becomes NADH-in mutant insensitive, as shown in Figure 10 and Figure 11.Because LPD is a kind of component of pyruvate dehydrogenase complex (PDH), this NADH insensitivity of LPD is carried into PDH from the producing and ethanol mutant.

In glucose-mineral salts medium, cultivate intestinal bacteria wild type strain W3110 or producing and ethanol mutant strain SE2378 to mid-term exponential phase of growth.The extract of harvested cell, and preparation then.As substrate,, measure the enzymic activity in the cell extract with pyruvate salt and NAD with the NADH of different concns inhibitor as enzymic activity.Figure 10 has shown that NADH is to the active inhibition of PDH.In last figure, for wild type strain W3110 and producing and ethanol mutant strain SE2378, NAD concentration is 2mM NAD.In figure below, for the natural enzyme from bacterial strain W3110, NAD concentration is 2mM NAD; For enzyme, be 1mM from the mutant form of bacterial strain SE2378.

By the lpd gene of pcr amplification from intestinal bacteria wild type strain W3110 and producing and ethanol mutant strain SE2378, and the clone advances protein expression vector pET15b.By to inserting dna sequencing, verify the dna sequence dna of lpd gene in the plasmid of selecting.Induce lpd expression of gene in the plasmid, and purifying protein.Measure enzymic activity in reversed reaction, wherein 2 kinds of substrates are Thioctamide (3mM) and NADH (0.1mM), in containing the 0.1M potassium phosphate buffer pH 8.0 of 1.5mM EDTA.Figure 11 has shown the inhibition of NADH to LPD.Under these conditions, natural enzyme does not have detectable activity, shown in the figure among Figure 11.NAD (being reaction product) is the essential activator of enzymic activity, and activity increases along with the increase of NAD concentration.Be the influence of the enzymatic determination NADH/NAD comparison enzymic activity of natural and mutant form, the result as shown in figure 11.

PDH is generated by all aerobes (from the bacterium to the mankind).This enzyme becomes acetyl-CoA, CO with pyruvate salt oxidative decarboxylation ₂And NADH, acetyl-CoA enters tricarboxylic acid cycle then, is used for further oxidation and energy generation subsequently.In intestinal bacteria, PDH generates under aerobic and anaerobic condition.But under anaerobic, owing to the inhibition of NADH to PDH, this enzyme is a non-activity.NADH is present in the anaerobic cell with higher concentration usually, thereby prevention can not be lacked the generation of NADH of the cellular oxidation of external electrical acceptor.As a result, cell is only produced 2NADH/ glucose, and second group of reductive agent discharges as hydrogen.Need 2 NADH because an acetyl-CoA is reduced into ethanol, wild-type cell can not generate 2 ethanol/glucose.

In producing and ethanol mutant strain of the present invention, PDH is more insensitive to NADH.The susceptibility of this reduction allows enzyme even under anaerobic works under higher NADH pond.Because this biochemistry changes, cell can be produced 4 NADH molecule/glucose (2 from glycolysis-, 2 from the PDH reaction).All 4 NADH are used for 2 acetyl-CoAs are reduced into ethanol, make mutant become high ethanol producer.On the biochemistry and on the physiology, described cell is high ethanol producer, this be because PDH to the reduction of the susceptibility of NADH and it in addition high NADH/NAD than under the ability that works.

At last, in another experiment (data not shown), the sudden change (E354K) of the LPD that will find in bacterial strain SE2378 imports among the LPD of anaerobe subtilis in similar position.The anaerobic growth of mutant (MR1) is supported in the E356K sudden change.

Embodiment 6

Contrast comparison from other biological LPD sequence

In this embodiment, carried out contrast comparison from the aminoacid sequence of different biological dihydrolipoamide dehydrogenases (LPD).

Pyruvic oxidase (PDH) is present in all aerobes from the bacterium to the mankind.LPD is the basal component of PDH enzyme complex, and it is present in PDH mixture and the 2-oxoglutaric acid dehydrogenase complex.In intestinal bacteria, lpd is total by these 2 kinds of enzyme complexs, because this demand, except the promotor that is positioned at the pdhR upstream region of gene, the lpd gene is transcribed from independent startup.

The Lpd homologue is present in all life fields.In bacterial isolates, Lpd albumen is from 458 to 581 amino acid, and no molecular weight water is 49 000 to 62 000 Da.The amino acid sequence identity that has shown 20 kinds of Lpd homologues of the bacterium of classifying in the table 7 below from system not of the same race generation.

Use the Blast of NCBI server or European biotechnology research ClustalW, will compare from the aminoacid sequence and the proteic aminoacid sequence of intestinal bacteria LPD of different biological dihydrolipoamide dehydrogenases.From 2 databases any obtains the identity per-cent of particular sequence and intestinal bacteria sequence.Value in the bracket is represented total similarity of specific protein and intestinal bacteria Lpd, and comprises identical amino acid position and conservative position of replacing takes place.For subtilis, in contrast, comprise 2 kinds of dihydrolipoic acid dehydrogenases, a kind of from the PDH mixture, another kind of from the 3-acetoin dehydrogenase.

Sequence identity from the low value 24% of Pasteur's sarcina methanica (a kind of archeobacteria (archaeon)) to 98% of salmonella typhimurium strain LT2 (a kind of gram negative bacterium).Salmonella typhimurium LT2 LPD albumen and intestinal bacteria LPD are the most closely related, and this is that gram negative bacterium in the same section enterobacteriaceae is consistent with two kinds of division bacterias.With coli strain W3110 or MG1655Lpd aminoacid sequence with from acinetobacter calcoaceticus ADP1, bacillus cereus ATCC 10987, bacillus subtilis strain 168, clostridium tetani bacterial strain Massachusetts/E88, corynebacterium glutamicum strains A TCC13032, metallic reducing ground bacillus GS-15, oxidizing glucose acidfast bacilli 621H, lactobacterium casei ATCC334, lactococcus lactis subsp SK11, plant lactobacillus WCFS1, Pasteur's sarcina methanica bacterial strain Fusaro, drinks wine coccus MCW PSU-1, Pseudomonas aeruginosa PAO1 (ATCC15692), Spherical red antibacterial 2.4.1, Salmonella typhimurium LT2, Shiva Salmonella ANA-3, streptococcus mutans ATCC 700610, streptomyces coelicolor M145, the thermophilic anaerobic ethanol bacillus, the known Lpd sequence of Fei Xiershi vibrios strains A TCC700601 compares.The homology comparison is [this figure will renumber] as shown in Figure 9.As if when the total identity per-cent of contrast, intestinal bacteria LPD is the most similar to other Gram-negative LPD; But when calculating identity per-cent based on conservative replacement, table 4 has reflected higher percent homology between the different biological LPD albumen of checking.For example, the bacillus subtilis strain 168LPD albumen amino acid sequence identity of comparing with intestinal bacteria LPD albumen is 34%.But, consider conservative the replacement, the identity scoring increases to 57%.From comparison chart as can be seen, several amino acid is a high conservative in 20 kinds of LPD homologue groups from very various biotic population.Residue total between the biology highlights with asterisk.The target area indicates underscore.In the different biological sequences of analyzing, sequence identity is the highest in the N-stub area.Between amino acid 40 to 55 (intestinal bacteria LPD numbering), can see sequence identity, its possible flavine site of representative.Another similarity zone is at amino acid/11 80 to 190.Have several position in sequence, the amino-acid residue is here guarded in from different all the biological 20 kinds of LPD that analyze.It should be noted that amino acid position 322 encoding histidines (H), in 3 bacterial strains of the present invention (SE2377, SE2383 and SE2382), exist in of the sudden change of the Histidine of position 322 to tyrosine (Y).Histidine in position 322 is being guarded to all 20 kinds of LPD of archeobacteria since Gram-positive, gram negative bacterium.Other conservative residue is included in the proline(Pro) (18/20LPD) of position 355 and 356 the L-glutamic acid (17/20LPD) in the position in this different range.

Table 7: intestinal bacteria LPD albumen with from the amino acid sequence identity of other biological LPD homologue

Biological	Amino acid number	% identity (gauged %) ^a
Biological	Amino acid number	% identity (gauged %) ^a	Acinetobacter calcoaceticus strains A DP1	??468	??35(55)
Bacillus cereus strains A TCC 10987	??470	??44(62)	Acinetobacter calcoaceticus strains A DP1	??468	??35(55)
Bacillus cereus strains A TCC 10987	??470	??44(62)	The E3 albumen of bacillus subtilis strain 168 pyruvic oxidases	??470	??47(64)

The E3 albumen of bacillus subtilis strain 1683-acetoin dehydrogenase	??458	??35(57)
	??458	??35(57)	Clostridium tetani bacterial strain Massachusetts/E88	??589	??35(58)
Corynebacterium glutamicum strains A TCC 13032	??469	??34(53)	Clostridium tetani bacterial strain Massachusetts/E88	??589	??35(58)
Corynebacterium glutamicum strains A TCC 13032	??469	??34(53)	Coli strain W3110 or MG1655	??474	??100
Metallic reducing ground bacillus GS-15	??476	??35(57)	Coli strain W3110 or MG1655	??474	??100
Metallic reducing ground bacillus GS-15	??476	??35(57)	Oxidizing glucose acidfast bacilli bacterial strain 621H	??468	??32(51)
Lactobacillus casei bacterial strain ATCC 334	??471	??30(52)	Oxidizing glucose acidfast bacilli bacterial strain 621H	??468	??32(51)
Lactobacillus casei bacterial strain ATCC 334	??471	??30(52)	Lactococcus lactis subsp bacterial strain SK11	??472	??40(59)
Lactobacterium plantarum strain WCFS1 (NCIMB 8826)	??470	??39(58)	Lactococcus lactis subsp bacterial strain SK11	??472	??40(59)
Lactobacterium plantarum strain WCFS1 (NCIMB 8826)	??470	??39(58)	Pasteur's sarcina methanica bacterial strain Fusaro	??476	??24(49)
Drinks wine coccus bacterial strain MCW PSU-1	??473	??39(59)	Pasteur's sarcina methanica bacterial strain Fusaro	??476	??24(49)
Drinks wine coccus bacterial strain MCW PSU-1	??473	??39(59)	Pseudomonas aeruginosa bacterial strain PAO1 ATCC 15692	??467	??37(57)
Spherical red antibacterial bacterial strain 2.4.1	??462	??40(58)	Pseudomonas aeruginosa bacterial strain PAO1 ATCC 15692	??467	??37(57)
Spherical red antibacterial bacterial strain 2.4.1	??462	??40(58)	Salmonella typhimurium LT2 ATCC 700720	??474	??98(99)
Shiva Salmonella strains A NA-3	??475	??85(94)	Salmonella typhimurium LT2 ATCC 700720	??474	??98(99)
Shiva Salmonella strains A NA-3	??475	??85(94)	Streptococcus mutans ATCC 700610	??581	??36(56)
Streptomyces coelicolor M145	??486	??36(55)	Streptococcus mutans ATCC 700610	??581	??36(56)
Streptomyces coelicolor M145	??486	??36(55)	The thermophilic anaerobic ethanol bacillus	??479	??40(58)
Fei Xiershi vibrios strains A TCC 700601	??475	??86(94)	The thermophilic anaerobic ethanol bacillus	??479	??40(58)

^aRepresentative changes with monoamino-acid and conserved amino acid

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Equivalent

Those skilled in the art only use routine test, will appreciate that the many equivalents that maybe can determine particular of the present invention described herein.Such equivalent is intended to comprise in the present invention.

The introducing of reference

All publications, patent application and the patent that this paper mentions is complete especially to be incorporated herein as a reference.

Sequence table

SEQ?ID?NO：1

ATGACCGCCGGAGATAAATATATAGAGGTCATGATGAGTACTGAAATCAAAA

CTCAGGTCGTGGTACTTGGGGCAGGCCCCGCAGGTTACTCCGCTGCCTTCCGT

TGCGCTGATTTAGGTCTGGAAACCGTAATCGTAGAACGTTACAACACCCTTG

GCGGTGTTTGCCTGAACGTCGGCTGTATCCCTTCTAAAGCACTGCTGCACGTA

GCAAAAGTTATCGAAGAAGCCAAAGCGCTG\GCTGAACACGGTATCGTCTTC

GGCGAACCGAAAACCGATATCGACAAGATTCGTACCTGGAAAGAGAAAGTG

ATCAATCAGCTGACCGGTGGTCTGGCTGGTATGGCGAAAGGCCGCAAAGTCA

AAGTGGTCAACGGTCTGGGTAAATTCACCGGGGCTAACACCCTGGAAGTTGA

AGGTGAGAACGGCAAAACCGTGATCAACTTCGACAACGCGATCATTGCAGCG

GGTTCTCGCCCGATCCAACTGCCGTTTATTCCGCATGAAGATCCGCGTATCTG

GGACTCCACTGACGCGCTGGAACTGAAAGAAGTACCAGAACGCCTGCTGGTA

ATGGGTGGCGGTATCATCGGTCTGGAAATGGGCACCGTTTACCACGCGCTGG

GTTCACAGATTGACGTGGTTGAAATGTTCGACCAGGTTATCCCGGCAGCTGA

CAAAGACATCGTTAAAGTCTTCACCAAGC?GTATCAGCAAGAAATTCAACCTG

ATGCTGGAAACCAAAGTTACCGCCGTTGAAGCGAAAGAAGACGGCATTTATG

TGACGATGGAAGGCAAAAAAGCACCCGCTGAACCGCAGCGTTACGACGCCG

TGCTGGTAGCGATTGGTCGTGTGCCGAACGGTAAAAACCTCGACGCAGGCAA

AGCAGGCGTGGAAGTTGACGACCGTGGTTTCATCCGCGTTGACAAACAGCTG

CGTACCAACGTACCGCACATCTTTGCTATCGGCGATATCGTCGGTCAACCGAT

GCTGGCATACAAAGGTGTTCACGAAGGTCACGTTGCCGCTGAAGTTATCGCC

GGTAAGAAACACTACTTCGATCCGAAAGTTATCCCGTCCATCGCCTATACCG

AACCAGAAGTTGCATGGGTGGGTCTGACTGAGAAAGAAGCGAAAGAGAAAG

GCATCAGCTATGAAACCGCCACCTTCCCGTGGGCTGCTTCTGGTCGTGCTATC

GCTTCCGACTGCGCAGACGGTATGACCAAGCTGATTTTCGACAAAGAATCTC

ACCGTGTGATCGGTGGTGCGATTGTCGGTACTAACGGCGGCGAGCTGCTGGG

TGAAATCGGCCTGGCAATCGAAATGGGTTGTGATGCTGAAGACATCGCACTG

ACCATCCACGCGCACCC?GACTCTGCACGAGTCTGTGGGCCTGGCGGCAGAAG

TGTTCGAAGGTAGCATTACCGACCTGCCGAACCCG

SEQ?ID?NO：2

MSTEIKTQVV?VLGAGPAGYS?AAFRCADLGL?ETVIVERYNT?LGGVCLNVGC

IPSKALLHVA?KVIEEAKALA?EHGIVFGEPK?TDIDKIRTWK?EKVINQLTGG

LAGMAKGRKV?KVVNGLGKFT?GANTLEVEGE?NGKTVINFDN?AIIAAGSRPI

QLPFIPHEDP?RIWDSTDALE?LKEVPERLLV?MGGGIIGLEM?GTVYHALGSQ

IDVVEMFDQV?IPAADKDIVK?VFTKRISKKF?NLMLETKVTA?VEAKEDGIYV

TMEGKKAPAE?PQRYDAVLVA?IGRVPNGKNL?DAGKAGVEVD?DRGFIRVDKQ

LRTNVPHIFA?IGDIVGQPML?AYKGVHEGHV?AAEVIAGKKH?YFDPKVIPSI

AYTEPEVAWV?GLTEKEAKEK?GISYETATFP?WAASGRAIAS?DCADGMTKLI

FDKESHRVIG?GAIVGTNGGE?LLGEIGLAIE?MGCDAEDIAL?TIHAHPTLHE

SVGLAAEVFE?GSITDLPNPK?AKKK

SEQ?ID?NO：3

ATGACCGCCGGAGATAAATATATAGAGGTCATGATGAGTACTGAAATCAAAA

CTCAGGTCTGGTACTTGGGGCAGGCCCCGCAGGTTACTCCGCTGCCTTCCGTT

GCGCTGATTTAGGTCTGGAAACCGTAATCGTAGAACGTTACAACACCCTTGG

CGGTGTTTGCCTGAACGTCGGCTGTATCCCTTCTAAAGCACTGCTGCACGTAG

CAAAAGTTATCGAAGAAGCCAAAGCGCTGGCTGAACACGGTATCGTCTTCGG

CGAACCGAAAACCGATATCGACAAGATTCGTACCTGGAAAGAGAAAGTGAT

CAATCAGCTGACCGGTGGTCTGGCTGGTATGGCGAAAGGCCGCAAAGTCAAA

GTGGTCAACGGTCTGGGTAAATTCACCGGGGCTAACACCCTGGAAGTTGAAG

GTGAGAAC?GGCAAAACCGTGATCAACTTCGACAACGCGATCATTGCAGCGGG

TTCTCGCCCGATCCAACTGCCGTTTATTCCGCATGAAGATCCGCGTATCTGGG

ACTCCACTGACGCGCTGGAACTGAAAGAAGTACCAGAACGCCTGCTGGTAAT

GGGTGGCGGTATCATCGGTCTGGAAATGGGCACCGTTTACCACGCGCTGGGT

TCACAGATTGACGTGGTTGAAATGTTCGACCAGGTTATCCCGGCAGCTGACA

AAGACATCGTTAAAGTCTTCACCAAGCGTATCAGCAAGAAATTCAACCTGAT

GCTGGAAACCAAAGTTACCGCCGTTGAAGCGAAAGAAGACGGCATTTATGTG

ACGATGGAAGGCAAAAAAGCACCCGCTGAACCGCAGCGTTACGACGCCGTG

CTGGTAGCGATTGGTCGTGTGCCGAACGGTAAAAACCTCGACGCAGGCAAAG

CAGGCGTGGAAGTTGACGACCGTGGTTTCATCCGCGTTGACAAACAGCTGCG

TACCAACGTACCGCACATCTTTGCTATCGGCGATATCGTCGGTCAACCGATGC

TGGCACACAAAGGTGTTCACGAAGGTCACGTTGCCGCTGAAGTTATCGCCGG

TAAGAAACACTACTTCGATCCGAAAGTTATCCCGTCCATCGCCTATACCAAA

CCAGAAGTTGCATGGGTGGGTCTGACTGAGAAAGAAGCGAAAGAGAAAGGC

ATCAGCTATGAAACCGCCACCTTCCCGTGGGCTGCTTCTGGTCGTGCTATCGC

TTCCGACTGCGCAGACGGTATGACCAAGCTGATTTTCGACAAAGAATCTCAC

CGTGTGATCGGTGGTGCGATTGTCGGTACTAACGGCGGCGAGCTGCTGGGTG

AAATCGGCCTGGCAATCGAAATGGGTTGTGATGCTGAAGACATCGCACTGAC

CATCCACGCGCACCCGACTCTGCACGAGTCTGTGGGCCTGGCGGCAGAAGTG

TTCGAAGGTAGCATTACCGACCTGCCGAACCCGAAAGCGAAGAAGAAGTAA

SEQ?ID?NO：4

MSTEIKTQVV?VLGAGPAGYS?AAFRCADLGL?ETVIVERYNT?LGGVCLNVGC

IPSKALLHVA?KVIEEAKALA?EHGIVFGEPK?TDIDKIRTWK?EKVINQLTGG

LAGMAKGRKV?KVVNGLGKFT?GANTLEVEGE?NGKTVINFDN?AIIAAGSRPI

QLPFIPHEDP?RIWDSTDALE?LKEVPERLLV?MGGGIIGLEM?GTVYHALGSQ

IDVVEMFDQV?IPAADKDIVK?VFTKRISKKF?NLMLETKVTA?VEAKEDGIYV

TMEGKKAPAE?PQRYDAVLVA?IGRVPNGKNL?DAGKAGVEVD

DRGFIRVDKQ?LRTNVPHIFA?IGDIVGQPML?AHKGVHEGHV?AAEVIAGKKH

YFDPKVIPSI?AYTKPEVAWV?GLTEKEAKEK?GISYETATFP?WAASGRAIAS

DCADGMTKLI?FDKESHRVIG?GAIVGTNGGE?LLGEIGLAIE?MGCDAEDIAL

TIHAHPTLHE?SVGLAAEVEE?GSITDLPNPK?AKKK

SEQ?ID?NO：5

ATGACCGCCGGAGATAAATATATAGAGGTCATGATGAGTACTGAAATCAAAA

CTCAGGTCGTGGTACTTGGGGCAGGCCCCGCAGGTTACTCCGCTGCCTTCCGT

TGCGCTGATTTAGGTCTGGAAACCGTAATCGTAGAACGTTACAACACCCTTG

GCGGTGTTTGCCTGAACGTCGGCTGTATCCCTTCTAAAGCACTGCTGCACGTA

GCAAAAGTTATCGAAGAAGCCAAAGCGCTGGCTGAACACGGTATCGTCTTCG

GCGAACCGAAAACCGATATCGACAAGATTCGTACCTGGAAAGAGAAAGTGA

TCAATCAGCTGACCGGTGGTCTGGCTGGTATGGCGAAAGGCCGCAAAGTCAA

AGTGGTCAACGGTCTGGGTAAATTCACCGGGGCTAACACCCTGGAAGTTGAA

GGTGAGAACGGCAAAACCGTGATCAACTTCGACAACGCGATCATTGCAGCGG

GTTCTCGCCCGATCCAACTGCCGTTTATTCCGCATGAAGATCCGCGTATCTGG

GACTCCACTGACGCGCTGGAACTGAAAGAAGTACCAGAACGCCTGCTGGTAA

TGGGTGGCGGTATCATCGGTCTGGAAATGGGCACCGTTTACCACGCGCTGGG

TTCACAGATTGACGTGGTTGAAATGTTCGACCAGGTTATCCCGGCAGCTGAC

AAAGACATCGTTAAAGTCTTCACCAAGCGTATCAGCAAGAAATTCAACCTGA

TGCTGGAAACCAAAGTTACCGCCGTTGAAGCGAAAGAAGACGGCATTTATGT

GACGATGGAAGGCAAAAAAGCACCCGCTGAACCGCAGCGTTACGACGCCGT

GCTGGTAGCGATTGGTCGTGTGCCGAACGGTAAAAACCTCGACGCAGGCAAA

GCAGGCGTGGAAGTTGACGACCGTGGTTTCATCCGCGTTGACAAACAGCTGC

GTACCAACGTACCGCACATCTTTGCTATCGGCGATATCGTCGGTCAACCGATG

CTGGCACACAAAGGTGTTCACGAAGGTCACGTTGCCGCTGAAGTTATCGCCG

GTAAGAAACACTACTTCGATCCGAAAGTTATCCCGTCCATCGCCTATACCGA

ACCAGAAGTTGCATGGGTGGGTCTGACTGAGAAAGAAGCGAAAGAGAAAGG

CATCAGCTATGAAACCGCCACCTTCCCGTGGGCTGCTTCTGGTCGTGCTATCG

CTTCCGACTGCGCAGACGGTATGACCAAGCTGATTTTCGACAAAGAATCTCA

CCGTGTGATCGGTGGTGCGATTGTCGGTACTAACGGCGGCGAGCTGCTGGGT

GAAATCGGCCTGGCAATCGAAATGGGTTGTGATGCTGAAGACATCGCACTGA

CCATCCACGCGCACCCGACTCTGCACGAGTCTGTGGGCCTGGCGGCAGAAGT

GTTCGAAGGTAGCATTACCGACCTGCCGAACCCGAAAGCGAAGAAGAAGTA

A

SEQ?ID?NO：6

MSTEIKTQVV?VLGAGPAGYS?AAFRCADLGL?ETVIVERYNT?LGGVCLNVGC

IPSKALLHVA?KVIEEAKALA?EHGIVFGEPK?TDIDKIRTWK?EKVINQLTGG

LAGMAKGRKV?KVVNGLGKFT?GANTLEVEGE?NGKTVINFDN?AIIAAGSRPI

QLPFIPHEDP?RIWDSTDALE?LKEVPERLLV?MGGGIIGLEM?GTVYHALGSQ

IDVVEMFDQV?IPAADKDIVK?VFTKRISKKF?NLMLETKVTA?VEAKEDGIYV

TMEGKKAPAE?PQRYDAVLVA?IGRVPNGKNL?DAGKAGVEVD?DRGFIRVDKQ

IGDIVGQPML?AHKGVHEGHV?AAEVLAGKKH?YFDPKVIPSI?AYTEPEVAWV

GLTEKEAKEK?GISYETATFP?WAASGRAIAS?DCADGMTKLI?FDKESHRVIG

GAIVGTNGGE?LLGEIGLAIE?MGCDAEDIAL?TIHAHPTLHE?SVGLAAEVFE

GSITDLPNPK?AKKK

Claims

1. the isolating non-recombinant bacteria that comprises sudden change, wherein said sudden change make this non-recombinant bacteria can under anaerobic produce 4 moles of NADH/ mole sugar.

2. the isolating non-recombinant bacteria of claim 1, wherein said sudden change is arranged in the pdh operon.

3. the isolating non-recombinant bacteria of claim 2, wherein said pdh operon comprises pdhR, aceEF and lpd gene.

4. the isolating non-recombinant bacteria of claim 3, wherein said sudden change is in the lpd gene.

5. each isolating non-recombinant bacteria among the claim 1-4, wherein the production of 4 moles of NADH/ mole sugar causes the alcoholic acid production as basic tunning.

6. each isolating non-recombinant bacteria among the claim 1-5, wherein said sugar is selected from: glucose, wood sugar, pectinose, seminose, semi-lactosi, sucrose and lactose.

7. the isolating non-recombinant bacteria that comprises the lpd gene with sudden change, wherein said sudden change make this non-recombinant bacteria can under anaerobic produce ethanol as basic tunning.

8. each isolating non-recombinant bacteria in the claim 5 or 7, wherein the ethanol of Sheng Chaning under anaerobic comprises the total non-gas tunning greater than 50%.

9. each isolating non-recombinant bacteria among the claim 1-8, wherein said bacterium producing and ethanol not when not suddenling change.

10. the isolating non-recombinant bacteria of claim 9, wherein ethanol is less important tunning, and comprises the total non-gas tunning less than 40%.

11. each isolating non-recombinant bacteria among the claim 1-8, wherein said sudden change provides high ethanol fermentation approach.

12. each isolating non-recombinant bacteria among the claim 1-8, wherein the one or more alternative fermentation approach in the bacterium is inactivated.

13. the isolating non-recombinant bacteria of claim 12, wherein said alternative fermentation approach comprises the lactic acid-producing of serum lactic dehydrogenase (ldhA), from acetate, ethanol, formic acid, the H of pyruvic acid formic acid lyase (pfl) beginning ₂And CO ₂, and succsinic acid.

14. the isolating non-recombinant bacteria of claim 13, wherein said alternative fermentation approach is by the deactivation that suddenlys change.

15. the isolating non-recombinant bacteria of claim 13, wherein said sudden change are in the ldhA gene.

16. the isolating non-recombinant bacteria of claim 13, wherein said sudden change are in the pfl gene.

17. the isolating non-recombinant bacteria of claim 15 or claim 16, wherein said sudden change are in ldhA or pflB gene.

18. be selected from following isolated nucleic acid molecule:

19. the isolated nucleic acid molecule of claim 18, wherein the ethanol of Sheng Chaning under anaerobic comprises the total non-gas tunning greater than 50%.

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

21. the isolated nucleic acid molecule of claim 20, wherein bacterial cell producing and ethanol not when not expressing described nucleic acid molecule.

22. the isolated nucleic acid molecule of claim 21, wherein ethanol is less important tunning, and comprises the total non-gas tunning less than 40%.

23. the isolated nucleic acid molecule of claim 19, wherein said bacterial cell are under anaerobic produced ethanol as basic tunning.

24. the isolated nucleic acid molecule of claim 19, wherein said nucleic acid molecule being expressed in bacterial cell provides high ethanol fermentation approach in the described bacterial cell.

25. isolated nucleic acid molecule according to claim 18, wherein said nucleic acid molecule comprises the fragment of SEQID NO:1, the length of wherein said nucleic acid molecule is at least 100 Nucleotide, and contain with the T of the 997 corresponding positions, position of SEQ ID NO:1.

26. isolated nucleic acid molecule according to claim 18, wherein said nucleic acid molecule comprises the fragment of SEQID NO:3, the length of wherein said nucleic acid molecule is at least 100 Nucleotide, and contain with the G of the 1023 corresponding positions, position of SEQ ID NO:1.

27. be selected from following isolated polypeptide:

B) comprise the naturally occurring allele variant of polypeptide of the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4, wherein said polypeptide by under rigorous condition with the nucleic acid molecule encoding of the complementary sequence hybridization of the nucleic acid molecule that comprises SEQ ID NO:1 or SEQ ID NO:3;

E) comprise the isolated polypeptide of the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4;

When wherein said polypeptide is expressed in cell, make described cell can produce ethanol as basic tunning.

28. the isolated polypeptide of claim 27, wherein the ethanol of Sheng Chaning under anaerobic comprises the total non-gas tunning greater than 50%.

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

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

31. the isolated polypeptide of claim 30, wherein bacterial cell producing and ethanol not when not expressing this polypeptide.

32. the isolated polypeptide of claim 30, wherein ethanol is less important tunning, and comprises the total non-gas tunning less than 40%.

33. the isolated polypeptide of claim 27, wherein said bacterial cell are under anaerobic produced ethanol as basic tunning.

34. the isolated polypeptide of claim 30, wherein the ethanol of Sheng Chaning under anaerobic comprises the total non-gas tunning greater than 50%.

35. the isolated polypeptide of claim 30, wherein said polypeptide being expressed in bacterial cell provides high ethanol fermentation approach in the described bacterial cell.

36. comprise the bacterial host cell of each nucleic acid molecule among the claim 18-26.

37. the bacterial host cell of claim 36, it comprises nucleotide sequence or its segmental carrier that contains SEQ ID NO:1 or SEQ IDNO:3.

38. the bacterial host cell of claim 30, wherein said carrier is pKY33.

39. the bacterial host cell of claim 36, it is genetically engineered to express described nucleic acid molecule.

40. comprise the bacterial host cell of each polypeptide among the claim 27-35.

41. produce the method that is selected from following polypeptide:

A) comprise the polypeptide of aminoacid sequence SEQ ID NO:2 or SEQ ID NO:4;

This method comprises, under the condition of expressing described nucleic acid molecule, cultivates the host cell of claim 30.

42. each isolating non-recombinant bacteria in claim 1-5 and 7, it comprises the nucleic acid molecule of claim 18.

43. each isolating non-recombinant bacteria among the claim 1-4, wherein ethanol generates from sugar.

44. the isolating non-recombinant bacteria of claim 42 or 43, wherein said sugar is selected from: glucose, wood sugar, pectinose, seminose, semi-lactosi, sucrose and lactose.

45. the isolated polypeptide of the isolated nucleic acid molecule of each isolating non-recombinant bacteria, claim 18, claim 27 or the bacterial host cell of claim 36 among the claim 1-7, wherein said bacterium is selected from: gram negative bacterium and gram positive bacterium.

46. the isolated polypeptide of the isolated nucleic acid molecule of each isolating non-recombinant bacteria, claim 18, claim 27 or the bacterial host cell of claim 36 among the claim 1-7, wherein said bacterium is a gram negative bacterium.

47. the isolated polypeptide of the isolated nucleic acid molecule of each isolating non-recombinant bacteria, claim 18, claim 27 or the bacterial host cell of claim 36 among the claim 1-7, wherein said gram negative bacterium is selected from: acinetobacter calcoaceticus, gluconobacter suboxydans, escherich's bacillus, bacillus, Xie Wanala bacterium, Salmonellas, intestinal bacteria and klebsiella bacillus.

48. the isolated polypeptide of the isolated nucleic acid molecule of each isolating non-recombinant bacteria, claim 18, claim 27 or the bacterial host cell of claim 36 among the claim 1-7, wherein said bacterium is a gram positive bacterium.

49. the isolated polypeptide of the isolated nucleic acid molecule of each isolating non-recombinant bacteria, claim 18, claim 27 or the bacterial host cell of claim 36 among the claim 1-7, wherein said gram positive bacterium is selected from: genus bacillus, clostridium, coryneform bacteria, lactobacillus, galactococcus, wine coccus, suis and eubacterium.

50. the isolated polypeptide of the isolated nucleic acid molecule of each isolating non-recombinant bacteria, claim 18, claim 27 or the bacterial host cell of claim 36 among the claim 1-7, wherein said bacterium is intestinal bacteria.

51. the isolating non-recombinant bacteria of claim 4 or 7, wherein said sudden change comprise with an amino acid in the polypeptide of the lpd genetic expression of another amino acid replacement sudden change, wherein said replacement changes the pK of described polypeptide.

52. the isolating non-recombinant bacteria of claim 51, wherein said polypeptide comprise SEQ ID NO:6, and described sudden change is included in following position and replaces wild-type amino acid with another amino acid:

53. the isolating non-recombinant bacteria of claim 51 or 52, wherein said another amino acid is to be selected from following neutral amino acids: L-Ala, halfcystine, glycine, Isoleucine, leucine, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine and Xie Ansuan.

54. the isolating non-recombinant bacteria of claim 51 or 52, wherein said another amino acid is to be selected from following basic aminoacids: arginine, l-asparagine, glutamine, Histidine and Methionin.

55. the isolating non-recombinant bacteria of claim 52, wherein said sudden change comprise the H that replaces 322 places in the position with arbitrary amino acid, make this amino acid replace the acidity of increase by the polypeptide of the lpd genetic expression of sudden change.

56. the H that the isolating non-recombinant bacteria of claim 55, wherein said sudden change comprise 322 places, position among the SEQ IDNO:6 replaces with Y.

57. the isolating non-recombinant bacteria of claim 52, wherein said sudden change comprise the E that replaces 354 places in the position with arbitrary amino acid, make this amino acid replace the acidity that reduces by the polypeptide of the lpd genetic expression that suddenlys change.

58. the E that the isolating non-recombinant bacteria of claim 57, wherein said sudden change comprise 354 places, position among the SEQ IDNO:6 replaces with K.

59. the isolating non-recombinant bacteria of claim 56, wherein said bacterium are coli strain SE2377.

60. the isolating non-recombinant bacteria of claim 59, wherein said bacterium comprise SEQ ID NO:1 or its fragment.

61. the isolating non-recombinant bacteria of claim 58, wherein said bacterium are coli strain SE2378.

62. the isolating non-recombinant bacteria of claim 61, wherein said bacterium comprise SEQ ID NO:3 or its fragment.

63. the isolating non-recombinant bacteria of claim 58, wherein said bacterium are coli strain SE2382.

64. the isolating non-recombinant bacteria of claim 63, wherein said bacterium comprise SEQ ID NO:3 or its fragment.

65. the isolating non-recombinant bacteria of claim 56, wherein said bacterium are coli strain SE2383.

66. the isolating non-recombinant bacteria of claim 65, wherein said bacterium comprise SEQ ID NO:1 or its fragment.

67. the isolating non-recombinant bacteria of claim 56, wherein said bacterium are coli strain SE2384.

68. the isolating non-recombinant bacteria of claim 67, wherein said bacterium comprise SEQ ID NO:1 or its fragment.

69. the isolating non-recombinant bacteria of claim 58, wherein said bacterium are coli strain SE2385.

70. the isolating non-recombinant bacteria of claim 69, wherein said bacterium comprise SEQ ID NO:3 or its fragment.

71. being applicable to from sugar, each isolating non-recombinant bacteria among the claim 56-70, wherein said bacterium produce ethanol.

72. isolating non-recombinant bacteria, it comprises the lpd gene with one or more sudden changes, wherein said sudden change makes this non-recombinant bacteria can under anaerobic produce ethanol as basic tunning, and wherein said bacterium prepares by the method that comprises the steps:

A) under the anaerobic growth condition in the sacchariferous substratum of richness the candidate mutant strain of culturing bacterium; With

B) select to produce the mutant of ethanol as main tunning.

73. the method for claim 72, wherein the ethanol of Sheng Chaning under anaerobic comprises the total non-gas tunning greater than 50%.

74. the method for each non-recombinant bacteria among the production claim 1-8, it comprises the steps:

B) select to produce the mutant of ethanol as main tunning.

75. the method for claim 72, wherein the ethanol of Sheng Chaning under anaerobic comprises the total non-gas tunning greater than 50%.

76. the isolating non-recombinant bacteria of claim 72 or the method for claim 74, wherein said mutant is derived from spontaneous mutation.

77. the isolating non-recombinant bacteria of claim 72 or the method for claim 74, wherein said bacterial exposure is in mutagenic compound.

78. the isolating non-recombinant bacteria of claim 72 or the method for claim 74, wherein said mutagenic compound are selected from: ethyl methane sulfonate, 2-aminopurine, ICR-191, methylmethanesulfonate, N-methyl-N '-nitro-N-nitrosoguanidine.

79. the isolating non-recombinant bacteria or the method for claim 78, wherein said mutagenic compound are ethyl methane sulfonates.

80. the isolating non-recombinant bacteria of claim 72 or the method for claim 74, the sugar in the wherein rich sacchariferous substratum is selected from: glucose, wood sugar, pectinose, seminose, semi-lactosi, sucrose and lactose.

81. the isolating non-recombinant bacteria of claim 72 or the method for claim 74 also comprise the step of the alternative fermentation approach in the deactivation bacterium.

82. the isolating non-recombinant bacteria of claim 72 or the method for claim 74, wherein said alternative fermentation approach is come deactivation by import deletion mutantion in bacterium.

83. the isolating non-recombinant bacteria of claim 72, wherein said bacterium producing and ethanol not when not suddenling change.

84. the isolating non-recombinant bacteria of claim 83, wherein ethanol is less important tunning, and comprises the total non-gas tunning less than 40%.

85. the isolating non-recombinant bacteria of claim 72, wherein bacterium under anaerobic produces ethanol as basic tunning.

86. the isolating non-recombinant bacteria of claim 85, wherein the ethanol of Sheng Chaning under anaerobic comprises the total non-gas tunning greater than 50%.

87. the isolating non-recombinant bacteria of claim 72, wherein said sudden change provide high ethanol fermentation approach.

88. the isolating non-recombinant bacteria of claim 87, wherein the one or more alternative fermentation approach in the bacterium is inactivated.

89. the isolating non-recombinant bacteria of claim 88, wherein said alternative fermentation approach is by the deactivation that suddenlys change.

90. the isolating non-recombinant bacteria of claim 88, wherein said alternative fermentation approach comprises the lactic acid-producing of serum lactic dehydrogenase (ldh), from acetate, ethanol, formic acid, the H of pyruvic acid formic acid lyase (pfl) beginning ₂And CO ₂, and succsinic acid.

91. produce the alcoholic acid method from the oligosaccharides source, it comprises, makes among the described oligosaccharides contact claim 1-8 each the isolating non-recombinant bacteria or the bacterial host cell of claim 36, thereby produces ethanol from the oligosaccharides source.

92. the method for claim 91, wherein said oligosaccharides is selected from: lignocellulose, hemicellulose, Mierocrystalline cellulose, pectin and its arbitrary combination.

93. test kit, it comprises among the claim 1-8 each isolating non-recombinant bacteria and is used to produce the alcoholic acid specification sheets.

94. the test kit of claim 93, it also comprises sugared source.

95. be the coli strain AH218 of preserved material representative at the farming research culture collection center of NRRL B-30967 by preserving number.

96. be the coli strain AH241 of preserved material representative at the farming research culture collection center of NRRL B-30968 by preserving number.

97. be the coli strain AH242 of preserved material representative at the farming research culture collection center of NRRL B-30969 by preserving number.

98. be the coli strain SE2377 of preserved material representative at the farming research culture collection center of NRRL B-30970 by preserving number.

99. be the coli strain SE2378 of preserved material representative at the farming research culture collection center of NRRL B-30971 by preserving number.

100. be the coli strain SE2382 of preserved material representative at the farming research culture collection center of NRRL B-30972 by preserving number.

101. be the coli strain SE2383 of preserved material representative at the farming research culture collection center of NRRL B-30973 by preserving number.

102. be the coli strain SE2384 of preserved material representative at the farming research culture collection center of NRRL B-30974 by preserving number.

103. be the coli strain SE2385 of preserved material representative at the farming research culture collection center of NRRL B-30975 by preserving number.

104. claim 4,7 or 72 isolating non-recombinant bacteria, wherein the sudden change in the lpd gene causes the NADH insensitivity.

105. claim 72,74 or 91 method or the test kit of claim 93, wherein said mutant is derived from the sudden change in the lpd gene.

106. the method for claim 105 or test kit, wherein the sudden change in the lpd gene causes the NADH insensitivity.