CN1373810A

CN1373810A - Reglation of carbon assimilation

Info

Publication number: CN1373810A
Application number: CN99816826A
Authority: CN
Inventors: P·J·洛伊帕蒂; C·M·克拉夫顿
Original assignee: Archer Daniels Midland Co
Current assignee: Archer Daniels Midland Co
Priority date: 1999-06-29
Filing date: 1999-06-29
Publication date: 2002-10-09
Also published as: MXPA01013445A; JP2003503064A; KR20020015708A; EP1196611A1; CA2377488A1; WO2001000852A1; AU4720999A

Abstract

The present invention provides a method of increasing the productivity of a microorganism by improving the assimilation of carbon dioxide. Specifically, the invention provides a polypeptide having phosphoenolpyruvate carboxylase activity which does not require acetyl coenzyme a for activation and is desensitized to feedback inhibition by aspartic acid, and to genes coding for this polypeptide. A gene encoding a PEP carboxylase that is not regulated by acetyl-CoA or aspartic acid can improve carbon flow from the three carbon intermediate PEP to the four carbon intermediate OAA, contribute to compounds derived from OAA, and increase amino acid biosynthesis. The invention further provides recombinant DNA molecules containing these genes, bacteria transformed with these genes, and a method of producing amino acids using the transformed bacteria.

Description

The adjusting of carbon assimilation

Background of invention

Invention field

The present invention relates to have the active polypeptide of phosphoric acid enol pyruvic acid carboxylase, this polypeptide does not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid; The invention still further relates to the gene of this polypeptide of coding.The present invention also relates to contain these genes recombinant DNA molecules, produce amino acid whose method with the bacterium of these gene transformation and with this transform bacteria.

Correlation technique

Phosphoenolpyruvic acid (PEP) carboxylase (EC 4.1.1.31) is a kind of enzyme that exists in nearly all bacterium and all plants.The glycolysis-intermediate PEP of PEP carboxylase catalysis three carbon and the condensation reaction between the carbonic acid gas cause forming the oxaloacetic acid (OAA) of four carbon, and a kind of is tricarboxylic acid (TCA) circulation and the common metabolic intermediate of L-aspartic acid biosynthesizing.TCA circulation needs constantly to replenish four carbon molecules, and substituting synthetic this intermediate that is consumed of amino acid, and vitamin H dependency PEP carboxylase is by in that circulation provides and plays the covering effect among the OAA and help finish this function to TCA.

OAA is the very important material that produces cell metabolite, the example of these metabolites has amino acid, particularly L-glutamic acid family (being L-glutamic acid, arginine and proline(Pro)) and aspartate family (being aspartic acid, Methionin, methionine(Met), Threonine and Isoleucine).Cause forming the reaction of OAA by catalysis, the PEP carboxylase plays an important role providing in the organic acid through metabolic process.For example, PEP carboxylase overexpression significantly increases intestinal bacteria (Escherichiacoli) from glucose fermentation generation succsinic acid.See Millard, C. etc., Appl.Environ.Microbiol.62:1808-1810 (1996).Therefore, the PEP carboxylase also plays an important role in producing the amino acid that is formed by L-glutamic acid and aspartic acid.

Amino acid be in the cell ubiquity as the compound of protein component.But for the economy of energy metabolism and substance metabolism, it produces by strict control.This control mainly is feedback control, and wherein the end product of pathways metabolism suppresses the activity of the enzyme of the previous step of this approach of catalysis.The PEP carboxylase is also accepted various adjustings in its activity expression.

For example, with regard to the PEP carboxylase of brevibacterium sp (Brevibacterium), Corynebacterium (Corynebacterium) or Escherichia (Escherichia) microorganism, the PEP carboxylase activity is suppressed by aspartic acid.See for example Mori, M. etc., J.Biochem.98:1621-1630 (1985); O ' Regan, M. etc., gene (Gene) 77:237-251 (1989).Therefore, the above-mentioned amino acid bio of PEP carboxylase participation is synthetic is also suppressed by aspartic acid.But, the Corynebacterium microorganism PEP carboxylase activity that aspartic acid susceptibility is reduced has been described.See Eikmanns, B.J. etc., Mol.Gen.Genet.218:330-339 (1989).

Except being suppressed by the aspartic acid allosteric, (acetyl-CoA) is the allosteric activation thing of brevibacterium flavum (Brevibacterium flavum) and colibacillary PEP carboxylase for example to acetyl-CoA.See Mori, M. etc., J.Biochem.98:1621-1630 (1985); Morikawa, M. etc., J.Biochem.81:1473-1485 (1977).Report is not arranged yet by other biological PEP carboxylase of aspartic acid or acetyl-CoA adjusting.See Valle, F. etc., J.Indus.Microbiol.17:458-462 (1996); O ' Regan, M. etc., gene 77:237-251 (1989); Vance, C. etc., Plant Physiol.75:261-264 (1984).

Because it is crucial that covering enzyme PEP carboxylase is kept for best OAA storehouse, and has determined the amino acid whose biosynthesizing level from OAA thus, be the corresponding ppc gene of operation so improve a method of amino acid production by ferment method.For example, the amplification of brevibacterium (Brevibacterium lactofermentum) ppc gene shows the production that improves proline(Pro) and Threonine.See Sano, K. etc., Agric.Biol.Chem.51:597-599 (1987).

Developed to use the insensitive mutant strain of feedback inhibition has been carried out the various technology that amino acid fermentation is effectively produced.But, also be not used to be integrated into the report that excellent bacillus ppc gene fermentative production identical family amino acid among the microbial staining body DNA and PEP carboxylase are not wherein regulated by acetyl-CoA or aspartic acid substantially from the PEP of plant carboxylase fermentative production aspartic acid or L-glutamic acid family amino acid or utilization.

United States Patent (USP) 4,757,009 (Sano etc.; Ajinomoto company) a kind of amino acid whose method of fermentative production of passing through is disclosed, this method is included in and cultivates Corynebacterium or the brevibacterium sp bacterial strain that carries the chromogene that contains following plasmid and have coded amino acid in the substratum, functionally inserted the gene of coding PEP carboxylase in the described plasmid, wherein this gene is from the Corynebacterium that carries the PEP carboxylase gene or the chromogene of brevibacterium sp strains separation; And from substratum, separate this amino acid.The Corynebacterium or the brevibacterium sp bacterial strain that therefrom separate PEP carboxylase encoding gene are the bacterial strains that shows the aspartic acid feedback inhibition that weakens.

European patent 358,940 (Bachmann etc.; Degussa Aktiengesellschaft) the pDM6 plasmid that imports among Corynebacterium glutamicum (Corynebacterium glutamicum) DM58-1 is disclosed, this bacterial strain is deposited in Germany microbial preservation center (DSM), preserving number is DSM 4697, and wherein this plasmid contains the genetic sequence of the information that comprises coding PEP carboxylase activity protein production.This ppc gene is isolating from Corynebacterium glutamicum ATCC 13032 genomic libraries, and this PEP carboxylase is not stimulated by acetyl-CoA.Also disclose the method for fermentation production of L-lysine, L-Threonine and L-Isoleucine, it is included in cultivates Corynebacterium or the brevibacterium sp host bacterium that contains plasmid pDM6 in the appropriate culture medium, and reclaims L-amino acid from this substratum.

United States Patent (USP) 5,876,983 (Sugimoto etc.; Ajinomoto company) the amino acid whose method of a kind of production is disclosed, comprise by (growth Escherichia microorganism belonging to genus selected to contain the Escherichia microorganism belonging to genus to the insensitive sudden change of aspartic acid feedback inhibition PEP carboxylase DNA sequences encoding when the aspartic acid-β-hydrazide) and the wild-type PEP carboxylase inhibitor of DL-threo form-beta-hydroxy aspartic acid existed being selected from the acid of 3-martonite, aspartic acid-beta-hydrazides; In appropriate culture medium, cultivate Escherichia or excellent bacillus microorganisms with the dna sequence dna conversion of this encoding mutant PEP carboxylase; And from substratum, separate the amino acid be selected from L-Methionin, L-Threonine, L-methionine(Met), L-Isoleucine, L-L-glutamic acid, L-arginine, L-proline(Pro).

Although many examples by recombinant DNA technology cultivation product amino acid bacterium are arranged, not necessarily realize high-caliber amino acid throughput.Therefore, still also need high titre and the amino acid whose method of high productivity fermentative production.The PEP carboxylase that not regulated by acetyl-CoA or aspartic acid may improve from three carbon intermediate PEP mobile to the carbon of four carbon intermediate OAA.The mobile compound and the amino acid whose biosynthesizing of increase that may help to form of this improvement from OAA.

Brief summary of the invention

Therefore, the present invention relates to contain the dna fragmentation of gene of the polypeptide of coding tool PEP carboxylase activity, wherein this gene can be expressed in host microorganism, and this polypeptide do not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

The invention still further relates to recombinant DNA molecules, this molecule comprises plasmid and the coding that wherein functionally inserts has the gene of the polypeptide of PEP carboxylase activity, wherein this recombinant DNA molecules can be bred in the host microorganism that comprises Escherichia, Corynebacterium and brevibacterium sp, this gene can be expressed, and it is wherein this polypeptide does not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

The invention still further relates to Escherichia, Corynebacterium and brevibacterium sp host microorganism with the dna fragmentation conversion of the gene that contains coding PEP carboxylase activity polypeptide, wherein this gene is from the plant of Monocotyledonae (Monocotyledonae) or Dicotyledoneae (Dicotyledonae) or the microorganism of Corynebacterium or brevibacterium sp, this polypeptide does not need acetyl-CoA to activate, and insensitive, and with this this gene of dna fragmentation host transformed microbial expression to the feedback inhibition of aspartic acid.

In the present invention on the other hand, provide fermentative production amino acid whose method.This method is included in and cultivates the host microorganism that belongs to Escherichia, Corynebacterium or brevibacterium sp in the appropriate culture medium, and from substratum amino acid separation, wherein this host microorganism is contained the dna fragmentation conversion of the gene of coding PEP carboxylase activity polypeptide, this gene of this microbial expression, and this polypeptide do not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

In addition, the present invention relates to select to contain the method for dna fragmentation of the gene of coding PEP carboxylase activity polypeptide, wherein this polypeptide does not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.The invention still further relates to increases the method that PEP is converted into the transformation efficiency of OAA, the method of recirculation carbon in zymotechnique, the method that does not need vitamin H and in zymotechnique, assimilate carbon, in zymotechnique, increase the method that organic acid is produced, and in zymotechnique, increase the method that amino acid is produced.

The accompanying drawing summary

Fig. 1 is the policy map of gene substitution.

DESCRIPTION OF THE PREFERRED

Before describing the present invention in detail, with the several terms that use in the first defined declaration book.

" activator " used herein comprises that described polypeptide at first becomes the necessary material of activity form, and the material of enhanced activity only.

" amino acid " used herein is meant naturally occurring L type amino acid (L-Ala, arginine, aspartic acid, l-asparagine, halfcystine, L-glutamic acid, glutamine, glycine, Histidine, Isoleucine, leucine, Methionin, methionine(Met), proline(Pro), phenylalanine, Serine, Threonine, tryptophane, tyrosine and Xie Ansuan).

" mosaic gene " is meant the gene that contains allos adjusting and encoding sequence.It is the heterozygous genes that produces by recombinant DNA technology.

" dna fragmentation " is meant the fragment of dna molecules.

" expression " used herein is used to refer to the generation of the protein product of genes encoding.

" gene " is meant the nucleic acid fragment of expressing specified protein, comprises before the coding region (5 ' the non-coding) and (3 ' the non-coding) adjusting sequence afterwards.It is to comprise to be responsible for the discontinuous chromosomal region that control is expressed the adjusting dna sequence dna of (promptly transcribe and translate) and transcribed and translate the encoding sequence that produces unique polypeptide.

" host microorganism " is meant the microorganism that has transformed the genetic material that imports.

" inhibition " comprises that the activity of described polypeptide reduces and activity lacks fully.

" isolating " used herein is meant that this material takes out from its primal environment (if naturally occurring words, for example natural surroundings).

" polypeptide " used herein or " protein " are meant the molecule of being made up of the monomer (amino acid) that couples together by amido linkage (also claiming peptide bond) linearity.It is meant amino acid whose molecular chain, rather than refers to the product of length-specific.Therefore, comprise peptide, oligopeptides and protein in the definition of polypeptide.This term is modified after also being used to refer to polypeptide expression, as glycosylation, acetylize, phosphorylation etc.The reorganization or the polypeptide of deriving are not necessarily from specified nucleotide sequence translation.It can also produce by any way, comprises the expression of chemosynthesis or recombinant expression system.

" adjusting sequence " be meant and be positioned at encoding sequence upstream (5 '), middle and/or downstream (3 '), and control the nucleotide sequence of transcribing and/or expressing of this encoding sequence, is associated with the protein biosynthesizing device of cell potentially.

" synthetic DNA " is meant all or part of nucleic acid molecule that produces by chemical synthesis process.

" conversion " is meant that at this foreign gene is transferred in the host cell as the part of host cell gene group DNA or as independent molecule, and stable heredity on this gene genetic.

On the one hand, the invention provides and containing the dna fragmentation of gene that coding has the polypeptide of PEP carboxylase activity, wherein this gene can be expressed in host microorganism, and this polypeptide do not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

The ppc gene of coding PEP carboxylase can be any gene, as long as it is the gene of the PEP carboxylase of coding Monocotyledonae or Dicotyledoneae plant or Corynebacterium or brevibacterium sp microorganism, and polypeptide expressed do not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.The ppc gene has preferably been determined its base sequence and has been cloned.When it by when clone, can use amplification such as PCR method and separate the dna fragmentation that contains this gene, then with suitable carrier realization cloning.The preferred donor of ppc gene is the bacterial strain that shows the aspartic acid feedback inhibition that weakens.So that the resistance of aspartic acid antagonism inhibitor is discerned this class bacterial strain.

The PEP carboxylase is C ₄A key enzyme of photosynthesis of plant.It is special to be positioned in the cytosol of mesophyll cell, is regulated by phosphorylation/dephosphorylation process.See Giglioli-Guivarc ' h, N. etc., Cytometry 23:241-249 (1996).In addition, the PEP carboxylase plays a crucial role in carton dioxide assimilation in beans root nodule symbiotic nitrogen fixation process.See Pathirana, S. etc., Plant are (1997) J.12:293-304.

In one embodiment, contain the dna fragmentation of gene of coding tool PEP carboxylase activity polypeptide from the plant of Monocotyledonae or Dicotyledoneae.In preferred embodiments, this dna fragmentation is from alfalfa plant.Most preferably, this dna fragmentation from alfalfa (Medicagosativa) strain is.

Show that the PEP carboxylase activity of alfalfa strain system is not suppressed by the L-aspartic acid substantially.See Vance, C.P. etc., Phant Physiol.75:261-264 (1984).And, the natural ppc nucleotide sequence of alfalfa is known (Pathirana, S. etc., molecular biology of plants (Plant Molecular Biology) 20:437-450 (1992)), be provided among the SEQ ID NO:1, the aminoacid sequence of Bian Ma natural PEP carboxylase is provided among the SEQID NO:2 thus.Because these sequences are known,, be that template obtains this gene by PCR then with the messenger RNA(mRNA) so can design and synthesize primer based on these nucleotide sequences.

Regulate after the translation of plant PEP carboxylase and realize by for example this proteinic phosphorylation.See Jiao, J.A. etc., Arch.Biochem.Biophys.269:526-535 (1989); Duff, S.M. etc., Eur.J.Biochem.228:92-95 (1995).Clover PEP carboxylase contains some conserved sequences, and one of them is considered to participate in phosphorylation (MASIDAQLR, 8-16 position residue).See Pathirana, S.M. etc., molecular biology of plants 20:437-450 (1992).

In a further preferred embodiment, modify the dna fragmentation of the gene that contains coding Monocotyledonae or Dicotyledoneae plant origin PEP carboxylase activity polypeptide by one or more nucleotide substitution, disappearance and/or insertion.Most preferably, this modification comprises the Nucleotide of the following aminoacid sequence of disappearance coding: Met-Ala-Ser-Ile-Asp-Ala-Gln-Leu-Arg.

In another embodiment, contain the dna fragmentation of gene of coding tool PEP carboxylase activity polypeptide from the microorganism of brevibacterium sp or Corynebacterium.In preferred embodiments, this dna fragmentation is from Corynebacterium glutamicum strain.The natural ppc nucleotide sequence of Corynebacterium glutamicum is presented among the SEQ ID NO:3.

Should be understood that the amino acid no in the active PEP carboxylase of the present invention molecule can change, and the present invention's imagination comprises all aminoacid sequences of the desired character that has the PEP carboxylase activity and do not regulated of alfalfa plant or Corynebacterium bacterium source.Also comprise only because of the conservative peptide sequence that differs from one another that substitutes.These conservative substituting comprise another amino acid that an amino acid replacement of given position is a same type in this sequence.Also comprise be positioned at do not change on this sequence location that the one or more non-conserved amino acid that this polypeptide reaches the degree of destroying this polypeptide biologic activity substitutes, disappearance and/or insert.

The sequence modification that the silence that also considering to produce does not influence gained PEP carboxylase protein molecule functional performance basically changes is as the disappearance in the sequence, insertion and/or alternative.For example, imagined in this gene order reflection genetic code degeneracy or cause producing the amino acid whose change that chemically is equal at given position.

Therefore be appreciated that the present invention does not just comprise described concrete exemplary sequence.The same with the maintenance of definite coded product biologic activity, the modification of all propositions is all in the scope of this area routine techniques.

In another embodiment, the dna fragmentation that contains the gene of coding tool PEP carboxylase activity polypeptide is a mosaic gene, and it comprises the microbe-derived imperfect PEP carboxylase nucleotide sequence of brevibacterium sp or Corynebacterium and the imperfect PEP carboxylase nucleotide sequence of Monocotyledonae or Dicotyledoneae plant origin.These two imperfect sequences form the complete chimeric ppc gene that can express the polypeptide with PEP carboxylase activity together, and wherein this polypeptide does not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

In a preferred embodiment, an imperfect PEP carboxylase nucleotide sequence is from the microorganism that belongs to Corynebacterium, and another imperfect PEP carboxylase nucleotide sequence is from alfalfa plant.Most preferably, an imperfect PEP carboxylase nucleotide sequence is from Corynebacterium glutamicum strain, and another imperfect PEP carboxylase nucleotide sequence from the alfalfa strain is.

In another embodiment, described dna fragmentation is complementary DNA (cDNA), genomic dna or synthetic DNA.The encode dna fragmentation of PEP carboxylase of the present invention can easily obtain in every way, and these modes include but not limited to the pcr amplification of chemosynthesis, cDNA or genomic library screening, expression library screening and/or cDNA.Be used to separate these methods of this DNA and other method and for example be set forth in (molecular clonings: laboratory manual (Molecular Cloning:A Laboratory Manual) such as Sambrook, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989)), Ausubel etc. compile (molecular biology modernism (Current Protocols in Molecular Biology), Current Protocols Press (1994)), Berger and Kimmel (Enzymology method: molecule clone technology guide (Methods in Enzymology:Guide to MolecularCloning Techniques), the 152nd volume, Academic Press, Inc., San Diego (1987)).

The separation of ppc gene can be undertaken by for example following method.Although following for the sake of simplicity example is appreciated that the bacterium that can use brevibacterium sp equally at Corynebacterium.At first, extract chromogene (adopting for example H.Saito and K.Miura, the method for Biochem.Biophys.Acta 72:619 (1963)) from the Corynebacterium bacterial strain that carries the ppc gene.Cut this gene with suitable Restriction Enzyme, subclone is to the plasmid shuttle vectors that can breed in excellent bacillus or intestinal bacteria then.In order to cut chromogene, can pass through the Cutting Control degree, for example the time of Cutting Control reaction, temperature etc. are used various Restriction Enzymes.DNA is that those of ordinary skills know with the Restriction Enzyme cutting, need not set forth in detail at this.

Transform excellent bacillus or colibacillary PEP carboxylase deficient mutants with the gained recombinant DNA.Thus obtained transformant can be selected by ordinary method and separates based on feature that carrier DNA and/or acceptor had.For example, separate and cause having the bacterial isolates of PEP carboxylase activity, and therefrom separate the ppc gene.

When cultivating the mentioned microorganism that transforms with dna fragmentation of the present invention, this dna sequence dna is expressed, do not need can obtain then acetyl-CoA activate, and basically to the insensitive enzyme of the inhibition of aspartic acid.Whether by for example measure the PEP carboxylase activity when having and/or not having acetyl-CoA and exist, can know this enzyme needs acetyl-CoA as activator.By for example in enzymatic reaction system, having and/or measuring the PEP carboxylase activity when not having aspartic acid and existing, can know thus obtained this enzyme and whether be suppressed by aspartic acid basically.

Can adopt spectrometry (Yoshinage, T. etc., J.Biochem.68:747-750 (1970)) etc. to measure the activity of this enzyme.For example, when reaction is carried out, carry out the enzyme analysis, then can should react with spectrophotometry according to the reduction (usually in 340 nanometers) of absorbancy with continuous or kinetics mode.

In another aspect of this invention, provide the method for the dna fragmentation of the gene of selecting to contain coding tool PEP carboxylase activity polypeptide, wherein this polypeptide does not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.This method comprises from the Corynebacterium bacterial strain that carries the ppc gene extracts chromogene, cut this chromogene with suitable Restriction Enzyme, this ppc gene is connected with the plasmid vector that can breed in corynebacterium genus bacteria, transform wherein ppc and the non-functional Corynebacterium bacterial strain of pyc gene, being separated in glucose is the bacterial strain that shows good growth on the minimum medium of sole carbon source, and from this bacterial strain the DNA isolation fragment.

Pyruvate carboxylase (EC 6.4.1.1) is important covering enzyme, and this enzyme replenishes the OAA that biosynthesizing consumes the process of growth from pyruvic acid, and this enzyme is used in the Methionin and L-glutamic acid production of industrial fermentation.Except the PEP carboxylase, the vitamin H dependency pyruvate carboxylase of recent findings pyc genes encoding is the covering enzyme in Corynebacterium glutamicum.Ppc and pyc gene inactivation cause this microorganism can not rely on the glucose growth in the Corynebacterium glutamicum.See Peters-Wendisch, P., etc., Microbiology 144:915-27 (1998).By inactivation ppc and two genes of pyc, can identify the dna fragmentation that contains ppc gene of the present invention that is cloned in the rf plasmid on the minimum medium of glucose as sole carbon source by the ability that bacterial strain is grown.

In another embodiment, the inhibitor that also in substratum, adds the PEP carboxylase activity.For example, can add the aspartic acid analogue.Similar compound shows that preferably the Corynebacterium microorganism to producing wild-type PEP carboxylase has the growth-inhibiting effect, above-mentioned growth-inhibiting effect can recover by the existence of L-L-glutamic acid or L-aspartic acid, and similar compound suppresses wild-type PEP carboxylase activity.If select the bacterial strain of anti-this similar compound, the then very possible host microorganism that obtains to produce to the insensitive PEP carboxylase of the feedback inhibition of aspartic acid from the microorganism of Corynebacterium.

In another embodiment, separate the bacterial strain that shows from the amino acid production increase of OAA.These amino acid comprise aspartic acid, Methionin, methionine(Met), Threonine and Isoleucine.In addition, can not have to cultivate bacterial strain on the minimum medium of acetyl-CoA, and can measure the PEP carboxylase activity.

In another aspect of this invention, following recombinant DNA molecules is provided, it comprises plasmid and the coding that wherein functionally inserts has the gene of the polypeptide of PEP carboxylase activity, wherein this recombinant DNA molecules can be bred in the host microorganism that comprises Escherichia, Corynebacterium and brevibacterium sp, this gene can be expressed, and it is wherein this polypeptide does not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

The used plasmid vector of the present invention can be any carrier, as long as it can be bred in the bacterial cell of Escherichia, Corynebacterium or brevibacterium sp.This carrier DNA is with the cutting of cutting same restrictions enzyme that chromogene made, or with have the oligonucleotide of chromosomal DNA cutting fragment and link to each other with the complementary base sequence that is cut the carrier DNA associated end.Then plasmid vector is connected with the fragment that contains chromogene.If gene by this method or any other method is pressed sense orientation and correct frame inserts, make and when this plasmid is transcribed and translated by the hereditary machine of the cell of its insertion, express the PEP carboxylase, then claim this gene " functionally to insert " in this plasmid vector.

In a preferred embodiment, coding has the gene of polypeptide of PEP carboxylase activity from alfalfa plant.Most preferably, this dna fragmentation from the alfalfa strain is.In a further preferred embodiment, this gene is modified by one or more nucleotide substitution, disappearance and/or insertion.Most preferably, this modification comprises the Nucleotide of the following aminoacid sequence of disappearance coding: Met-Ala-Ser-Ile-Asp-Ala-Gln-Leu-Arg.

In another aspect of the present invention, provide dna fragmentation host transformed microorganism of the present invention with the gene that contains coding PEP carboxylase activity polypeptide.As the host, can use to be used to produce the amino acid whose microorganism of L-, for example those belong to the microorganism of brevibacterium sp, Corynebacterium, bacillus (Bacillus), Escherichia, serratia (Seratia), Providencia (Providencia) and genus arthrobacter (Arthrobacter).

In a preferred embodiment, the dna fragmentation of the described ppc of containing gene is expressed in the host microorganism of Escherichia, brevibacterium sp or Corynebacterium.As the host, example can be the microorganism that belongs to Escherichia, intestinal bacteria for example, and the intestinal bacteria that preferably produce L-Methionin, excellent bacillus preferably produces the bacterial strain of L-Methionin, or the like.The alleged excellent bacillus of the present invention is one group of aerobic, Gram-positive, not acidproof, shaft-like, the microorganism that can not form spore, comprise the bacterium that belongs to Corynebacterium, belong to being classified as brevibacterium sp up to now but being unified into the bacterium of corynebacterium genus bacteria at present of brevibacterium sp, and belong to brevibacterium sp but the bacterium in close relations with the bacterium that belongs to Corynebacterium.

In one embodiment, when described dna fragmentation during from the plant of Monocotyledonae or Dicotyledoneae, the recombinant DNA molecules of the available dna fragmentation that contains plasmid and wherein functionally insert transforms this host microorganism.Perhaps, can transform this host microorganism in the host chromosome DNA by dna fragmentation of the present invention is integrated into.

Preferably, described dna fragmentation is from alfalfa plant.Most preferably, this dna fragmentation from the alfalfa strain is.In a further preferred embodiment, the dna fragmentation of this plant origin is modified by one or more nucleotide substitutions, disappearance and/or insertion.Most preferably, this modification comprises the Nucleotide of the following aminoacid sequence of disappearance coding: Met-Ala-Ser-Ile-Asp-Ala-Gln-Leu-Arg.

And just as described above, acceptable is dna sequence dna of the present invention to be inserted in the carrier DNA of energy self-replacation, and import among the host.As carrier DNA, the preferred plasmid carrier, most preferably can be in host cell those of self-replacation.Perhaps, also can use the phage DNA carrier.

When the plant of the dna fragmentation that contains gene from Monocotyledonae or Dicotyledoneae, or during the microorganism of brevibacterium sp or Corynebacterium, also can accept by adopting for example transposon (Berg, D.E. and Berg, C.M., Bio/Technol.1:417 (1983)), the Mu phage (the open text 2-109985 of Japanese Patent) or the method for homologous recombination (" molecular genetics experiment " (Experiments in Molecular Genetics), Cold Spring HarborLab. (1972)) are integrated into this dna fragmentation in the chromosomal DNA of host microorganism.In addition, for DNA of the present invention is integrated in the excellent bacillus, can utilize disclosed temperature sensitivity plasmid among the open text 5-7491 of Japanese Patent.

In a preferred embodiment, described dna fragmentation is from Corynebacterium glutamicum strain, and is integrated in the chromosomal DNA of host microorganism.The flank region of ppc gene is by order-checking (SEQ ID NO:3) in the Corynebacterium glutamicum karyomit(e).According to gene substitution strategy of the present invention, remove the ppc gene of chromosome copies, and replace with antibiotics resistance gene mark (Fig. 1).This mark is conversely again with modification ppc of the present invention gene substitution.

The unique design of this gene substitution strategy helps removing fully the karyomit(e) ppc dna sequence dna of host microorganism and replaces new ppc gene, and does not change two adjacent genes, tpi gene and secG gene, expression.Tpi genes encoding glycolytic ferment triose-phosphate isomerase, and secG genes encoding secG, a conformity membrane albumen that participates in the protein outward transport.

The design of this gene substitution strategy depends on complete tpi and the secG gene of rebuilding ppc gene flank.Can use the DNA zone of four oligonucleotide clone ppc flanks:

(1)5′GTTGG?TGAGC?CACTG?GAAAT?CCGTG?3′(SEQ?ID：NO?4)

(2)5′GATGT?CATCG?CGTAA?AAAAT?CAGTC?3′(SEQ?ID：NO?5)

(3)5′CACTG?CGCTG?CGCAA?CTCTA?GATAG?3′(SEQ?ID：NO?6)

(4)5′GACCA?CCACC?TTGCC?GAAAT?CTTGG?3′(SEQ?ID：NO?7)。

In another aspect of this invention, provide fermentative production amino acid whose method.This method is included in and cultivates the host microorganism that belongs to Escherichia, Corynebacterium or brevibacterium sp in the appropriate culture medium, and from substratum amino acid separation, wherein this host microorganism is contained the dna fragmentation conversion of the gene of coding PEP carboxylase activity polypeptide, this host microorganism is expressed this gene, and this polypeptide do not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

It is not different especially to produce the amino acid method for culturing microbes in the method for cultivating above-mentioned host and the prior art.That is to say, use carbonaceous sources, nitrogenous source, mineral ion, the material of compensation nutrition defective and the ordinary culture medium of optional organic trace nutrient such as amino acid, VITAMIN etc.

For carbon source, can use carbohydrate such as glucose, sucrose, lactose etc., and organic acid such as acetate.For nitrogenous source, can use ammonia, moisture ammonium (aqueous ammonium), ammonium salt etc.For mineral ion, can in substratum, suitably add potassium ion, sodium ion, magnesium ion, phosphate ion etc. on demand.

Suitably control the pH and the temperature of substratum, under aerobic conditions, cultivate, stop substantially up to amino acid whose generation and accumulation.In order to collect the amino acid that in substratum, accumulates thus, can adopt ordinary method.For example, after removing cell, reclaim amino acid by for example concentrating this cell-free solution and crystalline aminoacid (or its salt) by filtration, ultrafiltration, centrifugal or other currently known methods.Perhaps, this compound can reclaim by ion exchange chromatography.

In a preferred embodiment, amino acid is the amino acid from OAA, as L-aspartic acid, L-Methionin, L-methionine(Met), L-Threonine and L-Isoleucine.Most preferably amino acid is L-Methionin.

On the other hand, the invention provides the method that increase PEP is converted into the transformation efficiency of OAA.This method comprises with dna fragmentation conversion host microorganism of the present invention.In a preferred embodiment, this host microorganism is chosen from Escherichia, brevibacterium sp or Corynebacterium.

Condensation reaction between PEP carboxylase catalysis PEP and the carbonic acid gas causes forming OAA.Therefore, the PEP carboxylase of the present invention that not regulated by acetyl-CoA or aspartic acid increases the transformation efficiency that PEP is converted into OAA.

When described dna fragmentation during from the plant of Monocotyledonae or Dicotyledoneae, can be by integrating or for example utilizing that recombinant DNA molecules transforms.When described dna fragmentation during,, dna fragmentation of the present invention transforms host microorganism by being incorporated in the host microorganism chromosomal DNA from the microorganism of brevibacterium sp or Corynebacterium.

On the other hand, the invention provides the method for recirculation carbon in zymotechnique.This method comprises with dna fragmentation conversion host microorganism of the present invention.In a preferred embodiment, this host microorganism is chosen from Escherichia, brevibacterium sp or Corynebacterium.

The TCA circulation needs constantly to replenish C ₄Molecule is to substitute synthetic this intermediate that is consumed of amino acid.The PEP carboxylase helps to finish this function by play the covering effect in the supply four carbon OAA that circulate to TCA.Transform host microorganism by dna fragmentation of the present invention, the method for recirculation carbon is provided with the polypeptide of coding tool PEP carboxylase activity.

Up to now, can produce these amino acid whose excellent bacillus in L-Methionin and L-L-glutamic acid use brevibacterium sp or the Corynebacterium and carry out industrial production by fermentation process.In these methods, known growth needs vitamin H that should the rod bacillus.The biologic activity of this PEP carboxylase does not need vitamin H.In addition, major physiological effect of PEP carboxylase is by assimilation carbon covering TCA circulation.The PEP carboxylase of not regulated of the present invention has been improved the assimilation of carbonic acid gas.

Therefore, on the other hand, the invention provides the method that does not need vitamin H and in zymotechnique, assimilate carbon.This method comprises with dna fragmentation conversion host microorganism of the present invention.In a preferred embodiment, this host microorganism is chosen from Escherichia, brevibacterium sp or Corynebacterium.

Covering enzyme PEP carboxylase is that to keep best OAA storehouse crucial, has therefore determined the organic acid biosynthesizing level of being come by OAA.By transforming host microorganism, can increase the productivity of OAA with dna fragmentation of the present invention.Equally, also increase organic acid production from OAA.

Therefore, on the other hand, the invention provides increases the method that organic acid is produced in zymotechnique.In a preferred embodiment, this host microorganism is chosen from Escherichia, brevibacterium sp or Corynebacterium.

OAA produces cell metabolite such as amino acid whose important substance.By increasing the transformation efficiency that PEP is converted into OAA, ppc gene of the present invention can increase amino acid whose production thus.Therefore, on the other hand, the invention provides increases the method that amino acid is produced in zymotechnique.This method comprises with dna fragmentation conversion host microorganism of the present invention.

In a preferred embodiment, this host microorganism is chosen from Escherichia, brevibacterium sp or Corynebacterium.In a further preferred embodiment, this amino acid comprises L-aspartic acid, L-Methionin, L-methionine(Met), L-Threonine and L-Isoleucine.Most preferably this amino acid is L-Methionin.

All patents quoted in the disclosure and publication all are the state of the art of expression field, place of the present invention those of ordinary skill, and all intactly are incorporated herein by reference.

After below having described the present invention prevailingly, will be more readily understood the present invention with reference to the following example.These embodiment provide in illustrational mode, and are not intended to limit the present invention, unless spell out.

Embodiment 1

Plant ppc gene has function in intestinal bacteria

The cDNA clone (APPC) of clover (alfalfa) ppc gene is lacking functional PEP carboxylase and can not be among the intestinal bacteria mutant strain CGSC3594 that grows on the M9 substratum of sole carbon source function to be arranged with glucose.When having transformed APPC plasmid (pMS2), intestinal bacteria mutant strain CGSC3594 can grow on the M9 substratum that with glucose is sole carbon source.The DNA of this clover PEP carboxylase and aminoacid sequence are provided at respectively among SEQ ID NO:1 and the SEQ ID NO:2.

Embodiment 2

Clover ppc gene shows shaking in the bottle

Corynebacterium growth-stimulating is arranged

The ppc gene pairs of having measured clover (alfalfa) source produces the effect of Methionin Corynebacterium bacterial strain BF100 growth-stimulating.Growth measurement is the optical density(OD) at 660nm place, and titer determination is determined as (glucose of gram Methionin/gram consumption) * 100 for gram Methionin/rise substratum, productive rate.There is 30mg/L inductor sec.-propyl-β-D-galactoside (IPTG).The result is presented in the table 1:

Table 1

Bacterial strain	Growth	Titre	Productive rate
Bacterial strain	Growth	Titre	Productive rate	?BF100	????25	????25	????42
?BF100/pMS2	????34	????23	????40	?BF100	????25	????25	????42
?BF100/pMS2	????34	????23	????40	?BF100/pMS2/IPTG	????40	????25	????43

Embodiment 3

The ppc gene of wild-type Corynebacterium bacterium source

Improve the throughput of producing Methionin Corynebacterium bacterial strain

The cDNA clone (CPPC) of the ppc gene in Corynebacterium glutamicum ATCC 13032 source is inserted in the pCPPC plasmid.When in shaking bottle, having transformed product Methionin Corynebacterium glutamicum strain BF100, improved throughput with this pCPPC plasmid.

Growth measurement is the optical density(OD) at 660nm place, and titer determination is determined as (glucose of gram Methionin/gram consumption) * 100 for gram Methionin/rise substratum, productive rate.The result is presented in the table 2:

Table 2

Bacterial strain	Growth	Titre	Productive rate
Bacterial strain	Growth	Titre	Productive rate	?BF100	????39	????27	????44
?BF100/pCPPC	????32	????29	????48	?BF100	????39	????27	????44

Embodiment 4

Wild-type and product Methionin Corynebacterium bacterial strain

Susceptibility to acetyl-CoA and L-aspartic acid

Observing the different susceptibility to acetyl-CoA and L-aspartic acid from the extract of wild-type Corynebacterium glutamicum strain (ATCC 13032) and product Methionin Corynebacterium glutamicum strain (BF100), this determines by the PEP carboxylase activity.Use crude extract to pass through the change (340 nm/min) of spectrophotometry absorbancy as activity unit.The results are shown in Table 3:

Table 3

Bacterial strain	The PEP carboxylase activity
Bacterial strain	The PEP carboxylase activity				All	-acetyl-CoA	+ aspartic acid (5mM)
ATCC?13032	????100％	????56％	????100％		All	-acetyl-CoA	+ aspartic acid (5mM)
ATCC?13032	????100％	????56％	????100％	BF100	????100％	????15％	????17％

Embodiment 5

With the ppc gene substitution karyomit(e) ppc gene of modifying

The flank region of ppc gene is by order-checking (SEQ ID NO:3) in the Corynebacterium glutamicum karyomit(e).Remove the chromosome copies of ppc gene, and replace with antibiotics resistance gene mark (Fig. 1).This mark is replaced with modification ppc gene of the present invention again conversely.The unique design of this gene substitution strategy helps removing the karyomit(e) ppc dna sequence dna of host microorganism fully and replacing new gene, and does not change two adjacent expression of gene.

(1)5′GTTGG?TGAGC?CACTG?GAAAT?CCGTG?3′(SEQ?ID：NO?4)

(2)5′GATGT?CATCG?CGTAA?AAAAT?CAGTC?3′(SEQ?ID：NO?5)

(3)5′CACTG?CGCTG?CGCAA?CTCTA?GATAG?3′(SEQ?ID：NO?6)

(4)5′GACCA?CCACC?TIGCC?GAAAT?CTTGG?3′(SEQ?ID：NO?7)。

In view of the above stated specification that comprises embodiment, those skilled in the art can implement the present invention with various forms and embodiment, and not deviate from the spirit and scope of the invention that limits in the appended claims.

Sequence table＜110〉Rayapati, P.John

Crafton，Corey?M.

Archer-Daniels-Midland Company＜120〉adjusting＜130〉1533.093PC00＜140〉PCT/US99/14437＜141〉1999-06-29＜160〉7＜170〉patentIn Ver.2.0＜210 of carbon assimilation〉1＜211〉2901＜212〉DNA＜213〉alfalfa＜220〉＜221〉CDS＜222〉(1) .. (2901)＜400〉1atg gca aac aag atg gaa aaa atg gca tca att gat gca cag ctt aga 48Met Ala Asn Lys Met Glu Lys Met Ala Ser Ile Asp Ala Gln Leu Arg 15 10 15caa ttg gtt cct gca aaa gtg agt gaa gat gat aaa ctt att gag tat 96Gln Leu Val Pro Ala Lys Val Ser Glu Asp Asp Lys Leu Ile Glu Tyr

20??????????????????25??????????????????30gat?gct?ttg?ttg?ttg?gat?cgg?ttt?ctt?gat?att?ctt?caa?gat?tta?cat????144Asp?Ala?Leu?Leu?Leu?Asp?Arg?Phe?Leu?Asp?Ile?Leu?Gln?Asp?Leu?His

35??????????????????40??????????????????45gga?gag?gat?ctg?aag?gat?tct?gtt?caa?gaa?gtg?tat?gaa?ctg?tct?gct????192Gly?Glu?Asp?Leu?Lys?Asp?Ser?Val?Gln?Glu?Val?Tyr?Glu?Leu?Ser?Ala

50??????????????????55?????????????????60gaa?tat?gaa?aga?aag?cat?gat?cct?aag?aaa?ctt?gaa?gag?ctt?gga?aat????240Glu?Tyr?Glu?Arg?Lys?His?Asp?Pro?Lys?Lys?Leu?Glu?Glu?Leu?Gly?Asn?65??????????????????70??????????????????75??????????????????80ttg?atc?aca?agt?ttc?gat?gca?ggt?gac?tca?att?gtt?gtt?gcc?aag?tcc????288Leu?Ile?Thr?Ser?Phe?Asp?Ala?Gly?Asp?Ser?Ile?Val?Val?Ala?Lys?Ser

85??????????????????90??????????????????95ttt?tca?cac?atg?ctt?aac?ttg?gcc?aac?tta?gct?gaa?gag?gtt?caa?att????336Phe?Ser?His?Met?Leu?Asn?Leu?Ala?Asn?Leu?Ala?Glu?Glu?Val?Gln?Ile

100?????????????????105?????????????????110gcg?cac?cgc?cga?agg?aac?aag?ttg?aag?aaa?ggt?gat?ttt?agg?gat?gag????384Ala?His?Arg?Arg?Arg?Asn?Lys?Leu?Lys?Lys?Gly?Asp?Phe?Arg?Asp?Glu

115?????????????????120?????????????????125agc?aat?gca?acc?act?gaa?tct?gac?att?gag?gaa?act?ctc?aag?aaa?ctt????432Ser?Asn?Ala?Thr?Thr?Glu?Ser?Asp?Ile?Glu?Glu?Thr?Leu?Lys?Lys?Leu

130?????????????????135?????????????????140gtg?ttt?gac?atg?aag?aaa?tct?cct?caa?gag?gtt?ttt?gat?gca?ttg?aag????480Val?Phe?Asp?Met?Lys?Lys?Ser?Pro?Gln?Glu?Val?Phe?Asp?Ala?Leu?Lys145?????????????????150?????????????????155?????????????????160aac?cag?act?gtt?gat?ctt?gtt?ctt?act?gct?cat?cct?act?cag?tcg?gtt????528Asn?Gln?Thr?Val?Asp?Leu?Val?Leu?Thr?Ala?His?Pro?Thr?Gln?Ser?Val

165?????????????????170?????????????????175cgt?cga?tct?ttg?ctt?caa?aag?cac?gga?agg?gta?agg?aac?tgt?tta?tct????576Arg?Arg?Ser?Leu?Leu?Gln?Lys?His?Gly?Arg?Val?Arg?Asn?Cys?Leu?Ser

180?????????????????185?????????????????190caa?ttg?tat?gct?aaa?gac?atc?act?cct?gat?gat?aag?cag?gag?ctt?gat????624Gln?Leu?Tyr?Ala?Lys?Asp?Ile?Thr?Pro?Asp?Asp?Lys?Gln?Glu?Leu?Asp

195?????????????????200??????????????????205gaa?gct?ctc?cag?agg?gag?att?caa?gct?gca?ttc?cgt?act?gac?gaa?atc????672Glu?Ala?Leu?Gln?Arg?Glu?Ile?Gln?Ala?Ala?Phe?Arg?Thr?Asp?Glu?Ile

210?????????????????215?????????????????220aag?agg?act?cca?cca?act?ccc?caa?gat?gaa?atg?aga?gct?ggg?atg?agt????720Lys?Arg?Thr?Pro?Pro?Thr?Pro?Gln?Asp?Glu?Met?Arg?Ala?Gly?Met?Ser225?????????????????230?????????????????235?????????????????240tac?ttc?cat?gaa?aca?att?tgg?aag?ggt?gtc?cct?aaa?ttt?ctt?cgc?cgt????768Tyr?Phe?His?Glu?Thr?Ile?Trp?Lys?Gly?Val?Pro?Lys?Phe?Leu?Arg?Arg

245?????????????????250?????????????????255gtt?gat?acg?gca?ttg?aag?aac?ata?ggg?att?aac?gaa?cgt?gtt?ccc?tat????816Val?Asp?Thr?Ala?Leu?Lys?Asn?Ile?Gly?Ile?Asn?Glu?Arg?Val?Pro?Tyr

260?????????????????265?????????????????270aat?gct?cct?ctt?att?caa?ttt?tct?tct?tgg?atg?ggt?ggt?gat?cgt?gac????864Asn?Ala?Pro?Leu?Ile?Gln?Phe?Ser?Ser?Trp?Met?Gly?Gly?Asp?Arg?Asp

275?????????????????280?????????????????285ggt?aat?cca?aga?gtg?act?cct?gaa?gtg?aca?agg?gat?gtt?tgc?tta?cta????912Gly?Asn?Pro?Arg?Val?Thr?Pro?Glu?Val?Thr?Arg?Asp?Val?Cys?Leu?Leu

290?????????????????295?????????????????300gct?aga?atg?atg?gct?gct?aac?ttg?tat?tat?tca?cag?ata?gaa?gat?ctt????960Ala?Arg?Met?Met?Ala?Ala?Asn?Leu?Tyr?Tyr?Ser?Gln?Ile?Glu?Asp?Leu305?????????????????310?????????????????315?????????????????320atg?ttt?gaa?ctt?tct?atg?tgg?cgt?tgc?aat?gac?gag?cta?cgt?gtt?cgc????1008Met?Phe?Glu?Leu?Ser?Met?Trp?Arg?Cys?Asn?Asp?Glu?Leu?Arg?Val?Arg

325?????????????????330?????????????????335gca?gaa?gaa?ctt?cac?agg?aat?tcc?aag?aaa?gat?gaa?gtt?gca?aaa?cac????1056Ala?Glu?Glu?Leu?His?Arg?Asn?Ser?Lys?Lys?Asp?Glu?Val?Ala?Lys?His

340?????????????????345?????????????????350tat?ata?gag?ttt?tgg?aaa?aaa?att?cct?ttg?aat?gaa?cca?tac?cgt?gtt????1104Tyr?Ile?Glu?Phe?Trp?Lys?Lys?Ile?Pro?Leu?Asn?Glu?Pro?Tyr?Arg?Val

355?????????????????360?????????????????365gta?ctc?ggg?gag?gta?agg?gac?aag?ctc?tat?cgc?act?cgt?gag?cgt?tct????1152Val?Leu?Gly?Glu?Val?Arg?Asp?Lys?Leu?Tyr?Arg?Thr?Arg?Glu?Arg?Ser

370?????????????????375?????????????????380cgt?tat?ctc?cta?gct?cat?ggc?tac?tgt?gaa?att?cct?gaa?gaa?gcc?aca????1200Arg?Tyr?Leu?Leu?Ala?His?Gly?Tyr?Cys?Glu?Ile?Pro?Glu?Glu?Ala?Thr385?????????????????390?????????????????395?????????????????400ttc?acc?aat?gtc?gat?gag?ttt?ctg?gaa?cct?ctt?gaa?ctc?tgc?tac?aga????1248Phe?Thr?Asn?Val?Asp?Glu?Phe?Leu?Glu?Pro?Leu?Glu?Leu?Cys?Tyr?Arg

405?????????????????410?????????????????415tca?ctc?tgt?gct?tgt?ggt?gat?cgt?gca?att?gct?gat?gga?agc?ctt?ctt????1296Ser?Leu?Cys?Ala?Cys?Gly?Asp?Arg?Ala?Ile?Ala?Asp?Gly?Ser?Leu?Leu

420?????????????????425?????????????????430gat?ttc?ttg?agg?caa?gtt?tcc?act?ttt?gga?ctg?tca?ctt?gta?agg?ctt????1344Asp?Phe?Leu?Arg?Gln?Val?Ser?Thr?Phe?Gly?Leu?Ser?Leu?Val?Arg?Leu

435?????????????????440?????????????????445gat?ata?cgg?caa?gag?tct?gat?cgt?cac?act?gac?gtg?atg?gat?gcc?att????1392Asp?Ile?Arg?Gln?Glu?Ser?Asp?Arg?His?Thr?Asp?Val?Met?Asp?Ala?Ile

450?????????????????455?????????????????460acc?aaa?cat?ttg?gaa?att?gga?tcc?tac?caa?gaa?tgg?tct?gaa?gaa?aaa????1440Thr?Lys?His?Leu?Glu?Ile?Gly?Ser?Tyr?Gln?Glu?Trp?Ser?Glu?Glu?Lys465?????????????????470?????????????????475?????????????????480aga?cag?gaa?tgg?ctt?ttg?tcc?gag?ttg?att?ggc?aaa?agg?cca?ctc?ttt????1488Arg?Gln?Glu?Trp?Leu?Leu?Ser?Glu?Leu?Ile?Gly?Lys?Arg?Pro?Leu?Phe

485?????????????????490?????????????????495gga?cct?gac?cta?ccc?caa?acc?gat?gaa?att?aga?gat?gtt?tta?gac?acg????1536Gly?Pro?Asp?Leu?Pro?Gln?Thr?Asp?Glu?Ile?Arg?Asp?Val?Leu?Asp?Thr

500?????????????????505?????????????????510ttc?cgt?gtc?ata?gca?gaa?ctt?cca?tct?gac?aac?ttt?gga?gcc?tac?atc????1584Phe?Arg?Val?Ile?Ala?Glu?Leu?Pro?Ser?Asp?Asn?Phe?Gly?Ala?Tyr?Ile

515?????????????????520?????????????????525att?tcg?atg?gca?act?gca?ccg?tct?gat?gtg?ctg?gca?gtt?gag?ctt?ctt????1632Ile?Ser?Met?Ala?Thr?Ala?Pro?Ser?Asp?Val?Leu?Ala?Val?Glu?Leu?Leu

530?????????????????535?????????????????540caa?cgt?gaa?tgc?aaa?gtc?agg?aat?cca?tta?aga?gtc?gtt?ccg?ttg?ttt????1680Gln?Arg?Glu?Cys?Lys?Val?Arg?Asn?Pro?Leu?Arg?Val?Val?Pro?Leu?Phe545?????????????????550?????????????????555?????????????????560gaa?aag?ctt?gat?gat?ctt?gag?tct?gct?cct?gct?gca?ttg?gct?cgg?ttg????1728Glu?Lys?Leu?Asp?Asp?Leu?Glu?Ser?Ala?Pro?Ala?Ala?Leu?Ala?Arg?Leu

565?????????????????570?????????????????575ttc?tcc?ata?gac?tgg?tac?att?aac?cgg?atc?gat?ggg?aag?caa?gaa?gtt????1776Phe?Ser?Ile?Asp?Trp?Tyr?Ile?Asn?Arg?Ile?Asp?Gly?Lys?Gln?Glu?Val

580?????????????????585?????????????????590atg?att?gga?tat?tct?gat?tca?gga?aaa?gat?gct?gga?agg?ttt?tct?gca????1824Met?Ile?Gly?Tyr?Ser?Asp?Ser?Gly?Lys?Asp?Ala?Gly?Arg?Phe?Ser?Ala

595?????????????????600?????????????????605gca?tgg?cag?cta?tat?aag?gct?cag?gag?gac?ctc?atc?aaa?gtc?gca?cag????1872Ala?Trp?Gln?Leu?Tyr?Lys?Ala?Gln?Glu?Asp?Leu?Ile?Lys?Val?Ala?Gln

610?????????????????615?????????????????620aaa?ttt?ggt?gtt?aag?cta?acc?atg?ttc?cac?ggt?cgt?ggt?gga?act?gtt????1920Lys?Phe?Gly?Val?Lys?Leu?Thr?Met?Phe?His?Gly?Arg?Gly?Gly?Thr?Val625?????????????????630?????????????????635?????????????????640gga?aga?gga?ggt?gga?cct?acc?cat?ctt?gct?atc?ttg?tct?caa?cca?cca????1968Gly?Arg?Gly?Gly?Gly?Pro?Thr?His?Leu?Ala?Ile?Leu?Ser?Gln?Pro?Pro

645?????????????????650?????????????????655gaa?aca?att?cac?gga?tct?ctt?cgt?gtg?aca?gtt?caa?ggt?gaa?gtt?att????2016Glu?Thr?Ile?His?Gly?Ser?Leu?Arg?Val?Thr?Val?Gln?Gly?Glu?Val?Ile

660?????????????????665?????????????????670gaa?cag?tcg?ttc?ggt?gag?gaa?cac?ttg?tgc?ttt?agg?aca?ctg?caa?cgt????2064Glu?Gln?Ser?Phe?Gly?Glu?Glu?His?Leu?Cys?Phe?Arg?Thr?Leu?Gln?Arg

675?????????????????680?????????????????685ttc?act?gct?gct?act?cta?gaa?cat?gga?atg?cgt?ccc?cca?agc?tct?cca????2112Phe?Thr?Ala?Ala?Thr?Leu?Glu?His?Gly?Met?Arg?Pro?Pro?Ser?Ser?Pro

690?????????????????695?????????????????700aaa?cca?gaa?tgg?cgc?gcc?ttg?atg?gat?cag?atg?gct?gtc?att?gca?act????2160Lys?Pro?Glu?Trp?Arg?Ala?Leu?Met?Asp?Gln?Met?Ala?Val?Ile?Ala?Thr705?????????????????710?????????????????715?????????????????720gag?gaa?tac?cgt?tca?att?gtg?ttc?aag?gaa?cca?cgt?ttt?gtt?gag?tat????2208Glu?Glu?Tyr?Arg?Ser?Ile?Val?Phe?Lys?Glu?Pro?Arg?Phe?Val?Glu?Tyr

725?????????????????730?????????????????735ttc?cgt?ctg?gct?aca?cca?gag?atg?gag?tat?ggt?agg?atg?aac?att?gga????2256Phe?Arg?Leu?Ala?Thr?Pro?Glu?Met?Glu?Tyr?Gly?Arg?Met?Asn?Ile?Gly

740?????????????????745?????????????????750agt?cga?ccg?gca?aag?aga?agg?cct?agt?gga?ggc?att?gaa?aca?ctg?cgt????2304Ser?Arg?Pro?Ala?Lys?Arg?Arg?Pro?Ser?Gly?Gly?Ile?Glu?Thr?Leu?Arg

755?????????????????760?????????????????765gcg?ata?cca?tgg?atc?ttt?gcc?tgg?aca?cag?aca?agg?ttt?cat?ctt?cca????2352Ala?Ile?Pro?Trp?Ile?Phe?Ala?Trp?Thr?Gln?Thr?Arg?Phe?His?Leu?Pro

770?????????????????775?????????????????780gta?tgg?ctg?ggc?ttt?gga?gca?gca?ttt?aga?caa?gtt?gtt?cag?aag?gat????2400Val?Trp?Leu?Gly?Phe?Gly?Ala?Ala?Phe?Arg?Gln?Val?Val?Gln?Lys?Asp785?????????????????790?????????????????795?????????????????800gtt?aag?aat?ctc?cat?atg?ctg?caa?gag?atg?tac?aat?caa?tgg?cct?ttc????2448Val?Lys?Asn?Leu?His?Met?Leu?Gln?Glu?Met?Tyr?Asn?Gln?Trp?Pro?Phe

805?????????????????810?????????????????815ttt?agg?gtt?aca?att?gat?tta?gtt?gaa?atg?gtg?ttt?gcc?aag?ggt?gac????2496Phe?Arg?Val?Thr?Ile?Asp?Leu?Val?Glu?Met?Val?Phe?Ala?Lys?Gly?Asp

820?????????????????825?????????????????830cct?ggt?att?gca?gca?ctg?aat?gat?agg?ctc?cta?gtt?tca?aag?gat?ctg????2544Pro?Gly?Ile?Ala?Ala?Leu?Asn?Asp?Arg?Leu?Leu?Val?Set?Lys?Asp?Leu

835?????????????????840?????????????????845tgg?cca?ttt?ggg?gaa?caa?ttg?aga?agc?aaa?tat?gaa?gaa?act?aag?aaa????2592Trp?Pro?Phe?Gly?Glu?Gln?Leu?Arg?Set?Lys?Tyr?Glu?Glu?Thr?Lys?Lys

850?????????????????855?????????????????860ctc?cta?ctt?cag?gtg?gct?gca?cac?aag?gaa?gtt?ctt?gaa?ggt?gac?ccc????2640Leu?Leu?Leu?Gln?Val?Ala?Ala?His?Lys?Glu?Val?Leu?Glu?Gly?Asp?Pro865?????????????????870?????????????????875?????????????????880tac?ttg?aag?caa?aga?ctc?aga?ctc?cgt?gat?tcg?tac?att?aca?acc?ctt????2688Tyr?Leu?Lys?Gln?Arg?Leu?Arg?Leu?Arg?Asp?Ser?Tyr?Ile?Thr?Thr?Leu

885?????????????????890?????????????????895aat?gtt?ttc?caa?gcc?tac?aca?ttg?aaa?cgg?atc?cgc?gat?cca?aac?tac????2736Asn?Val?Phe?Gln?Ala?Tyr?Thr?Leu?Lys?Arg?Ile?Arg?Asp?Pro?Asn?Tyr

900?????????????????905?????????????????910aag?gtg?gag?gtg?cgc?ccc?cca?ata?tcg?aaa?gag?tct?gct?gaa?aca?agt????2784Lys?Val?Glu?Val?Arg?Pro?Pro?Ile?Ser?Lys?Glu?Ser?Ala?Glu?Thr?Ser

915?????????????????920?????????????????925aaa?cca?gct?gat?gaa?ctt?gta?aca?ttg?aat?cca?aca?agt?gaa?tat?gct????2832Lys?Pro?Ala?Asp?Glu?Leu?Val?Thr?Leu?Asn?Pro?Thr?Ser?Glu?Tyr?Ala

930?????????????????935?????????????????940cct?ggt?ttg?gaa?gac?aca?ctc?att?ctt?acc?atg?aag?ggt?att?gct?gct????2880Pro?Gly?Leu?Glu?Asp?Thr?Leu?Ile?Leu?Thr?Met?Lys?Gly?Ile?Ala?Ala945?????????????????950?????????????????955?????????????????960ggc?atg?cag?aac?act?ggt?taa????????????????????????????????????????2901Gly?Met?Gln?Asn?Thr?Gly

965＜210〉2＜211〉966＜212〉PRT＜213〉alfalfa＜400〉2Met Ala Asn Lys Met Glu Lys Met Ala Ser Ile Asp Ala Gln Leu Arg, 15 10 15Gln Leu Val Pro Ala Lys Val Ser Glu Asp Asp Lys Leu Ile Glu Tyr

20??????????????????25??????????????????????30Asp?Ala?Leu?Leu?Leu?Asp?Arg?Phe?Leu?Asp?Ile?Leu?Gln?Asp?Leu?His

35??????????????????40??????????????????????45Gly?Glu?Asp?Leu?Lys?Asp?Ser?Val?Gln?Glu?Val?Tyr?Glu?Leu?Ser?Ala

50??????????????????55??????????????????60Glu?Tyr?Glu?Arg?Lys?His?Asp?Pro?Lys?Lys?Leu?Glu?Glu?Leu?Gly?Asn?65??????????????????70??????????????????75??????????????????80Leu?Ile?Thr?Ser?Phe?Asp?Ala?Gly?Asp?Ser?Ile?Val?Val?Ala?Lys?Ser

85??????????????????90??????????????????95Phe?Ser?His?Met?Leu?Asn?Leu?Ala?Asn?Leu?Ala?Glu?Glu?Val?Gln?Ile

100?????????????????105?????????????????110Ala?His?Arg?Arg?Arg?Asn?Lys?Leu?Lys?Lys?Gly?Asp?Phe?Arg?Asp?Glu

115?????????????????120?????????????????125Ser?Asn?Ala?Thr?Thr?Glu?Ser?Asp?Ile?Glu?Glu?Thr?Leu?Lys?Lys?Leu

130?????????????????135?????????????????140Val?Phe?Asp?Met?Lys?Lys?Ser?Pro?Gln?Glu?Val?Phe?Asp?Ala?Leu?Lys145?????????????????150?????????????????155?????????????????160Asn?Gln?Thr?Val?Asp?Leu?Val?Leu?Thr?Ala?His?Pro?Thr?Gln?Ser?Val

165?????????????????170?????????????????175Arg?Arg?Ser?Leu?Leu?Gln?Lys?His?Gly?Arg?Val?Arg?Asn?Cys?Leu?Ser

180?????????????????185?????????????????190Gln?Leu?Tyr?Ala?Lys?Asp?Ile?Thr?Pro?Asp?Asp?Lys?Gln?Glu?Leu?Asp

195?????????????????200?????????????????205Glu?Ala?Leu?Gln?Arg?Glu?Ile?Gln?Ala?Ala?Phe?Arg?Thr?Asp?Glu?Ile

210?????????????????215?????????????????220Lys?Arg?Thr?Pro?Pro?Thr?Pro?Gln?Asp?Glu?Met?Arg?Ala?Gly?Met?Ser225?????????????????230?????????????????235?????????????????240Tyr?Phe?His?Glu?Thr?Ile?Trp?Lys?Gly?Val?Pro?Lys?Phe?Leu?Arg?Arg

245?????????????????250?????????????????255Val?Asp?Thr?Ala?Leu?Lys?Asn?Ile?Gly?Ile?Asn?Glu?Arg?Val?Pro?Tyr

260?????????????????265?????????????????270Asn?Ala?Pro?Leu?Ile?Gln?Phe?Ser?Ser?Trp?Met?Gly?Gly?Asp?Arg?Asp

275?????????????????280?????????????????285Gly?Asn?Pro?Arg?Val?Thr?Pro?Glu?Val?Thr?Arg?Asp?Val?Cys?Leu?Leu

290?????????????????295?????????????????300Ala?Arg?Met?Met?Ala?Ala?Asn?Leu?Tyr?Tyr?Ser?Gln?Ile?Glu?Asp?Leu305?????????????????310?????????????????315?????????????????320Met?Phe?Glu?Leu?Ser?Met?Trp?Arg?Cys?Asn?Asp?Glu?Leu?Arg?Val?Arg

325?????????????????330?????????????????335Ala?Glu?Glu?Leu?His?Arg?Asn?Ser?Lys?Lys?Asp?Glu?Val?Ala?Lys?His

340?????????????????345?????????????????350Tyr?Ile?Glu?Phe?Trp?Lys?Lys?Ile?Pro?Leu?Asn?Glu?Pro?Tyr?Arg?Val

355?????????????????360?????????????????365Val?Leu?Gly?Glu?Val?Arg?Asp?Lys?Leu?Tyr?Arg?Thr?Arg?Glu?Arg?Ser

370?????????????????375?????????????????380Arg?Tyr?Leu?Leu?Ala?His?Gly?Tyr?Cys?Glu?Ile?Pro?Glu?Glu?Ala?Thr385?????????????????390?????????????????395?????????????????400Phe?Thr?Asn?Val?Asp?Glu?Phe?Leu?Glu?Pro?Leu?Glu?Leu?Cys?Tyr?Arg

405?????????????????410?????????????????415Ser?Leu?Cys?Ala?Cys?Gly?Asp?Arg?Ala?Ile?Ala?Asp?Gly?Ser?Leu?Leu

420?????????????????425?????????????????430Asp?Phe?Leu?Arg?Gln?Val?Ser?Thr?Phe?Gly?Leu?Ser?Leu?Val?Arg?Leu

435?????????????????440?????????????????445Asp?Ile?Arg?Gln?Glu?Ser?Asp?Arg?His?Thr?Asp?Val?Met?Asp?Ala?Ile

450?????????????????455?????????????????460Thr?Lys?His?Leu?Glu?Ile?Gly?Ser?Tyr?Gln?Glu?Trp?Ser?Glu?Glu?Lys465?????????????????470?????????????????475?????????????????480Arg?Gln?Glu?Trp?Leu?Leu?Ser?Glu?Leu?Ile?Gly?Lys?Arg?Pro?Leu?Phe

485?????????????????490?????????????????495Gly?Pro?Asp?Leu?Pro?Gln?Thr?Asp?Glu?Ile?Arg?Asp?Val?Leu?Asp?Thr

500?????????????????505?????????????????510Phe?Arg?Val?Ile?Ala?Glu?Leu?Pro?Ser?Asp?Asn?Phe?Gly?Ala?Tyr?Ile

515?????????????????520?????????????????525Ile?Ser?Met?Ala?Thr?Ala?Pro?Ser?Asp?Val?Leu?Ala?Val?Glu?Leu?Leu

530?????????????????535?????????????????540Gln?Arg?Glu?Cys?Lys?Val?Arg?Asn?Pro?Leu?Arg?Val?Val?Pro?Leu?Phe545?????????????????550?????????????????555?????????????????560Glu?Lys?Leu?Asp?Asp?Leu?Glu?Ser?Ala?Pro?Ala?Ala?Leu?Ala?Arg?Leu

565?????????????????570?????????????????575Phe?Ser?Ile?Asp?Trp?Tyr?Ile?Asn?Arg?Ile?Asp?Gly?Lys?Gln?Glu?Val

580?????????????????585?????????????????590Met?Ile?Gly?Tyr?Ser?Asp?Ser?Gly?Lys?Asp?Ala?Gly?Arg?Phe?Ser?Ala

595?????????????????600?????????????????605Ala?Trp?Gln?Leu?Tyr?Lys?Ala?Gln?Glu?Asp?Leu?Ile?Lys?Val?Ala?Gln

610?????????????????615?????????????????620Lys?Phe?Gly?Val?Lys?Leu?Thr?Met?Phe?His?Gly?Arg?Gly?Gly?Thr?Val625?????????????????630?????????????????635?????????????????640Gly?Arg?Gly?Gly?Gly?Pro?Thr?His?Leu?Ala?Ile?Leu?Ser?Gln?Pro?Pro

645?????????????????650?????????????????655Glu?Thr?Ile?His?Gly?Ser?Leu?Arg?Val?Thr?Val?Gln?Gly?Glu?Val?Ile

660?????????????????665?????????????????670Glu?Gln?Ser?Phe?Gly?Glu?Glu?His?Leu?Cys?Phe?Arg?Thr?Leu?Gln?Arg

675?????????????????680?????????????????685Phe?Thr?Ala?Ala?Thr?Leu?Glu?His?Gly?Met?Arg?Pro?Pro?Ser?Ser?Pro

690?????????????????695?????????????????700Lys?Pro?Glu?Trp?Arg?Ala?Leu?Met?Asp?Gln?Met?Ala?Val?Ile?Ala?Thr705?????????????????710?????????????????715?????????????????720Glu?Glu?Tyr?Arg?Ser?Ile?Val?Phe?Lys?Glu?Pro?Arg?Phe?Val?Glu?Tyr

725?????????????????730?????????????????735Phe?Arg?Leu?Ala?Thr?Pro?Glu?Met?Glu?Tyr?Gly?Arg?Met?Asn?Ile?Gly

740?????????????????745?????????????????750Ser?Arg?Pro?Ala?Lys?Arg?Arg?Pro?Ser?Gly?Gly?Ile?Glu?Thr?Leu?Arg

755?????????????????760?????????????????765Ala?Ile?Pro?Trp?Ile?Phe?Ala?Trp?Thr?Gln?Thr?Arg?Phe?His?Leu?Pro

770?????????????????775?????????????????780Val?Trp?Leu?Gly?Phe?Gly?Ala?Ala?Phe?Arg?Gln?Val?Val?Gln?Lys?Asp785?????????????????790?????????????????795?????????????????800Val?Lys?Asn?Leu?His?Met?Leu?Gln?Glu?Met?Tyr?Asn?Gln?Trp?Pro?Phe

805?????????????????810?????????????815Phe?Arg?Val?Thr?Ile?Asp?Leu?Val?Glu?Met?Val?Phe?Ala?Lys?Gly?Asp

820?????????????????825?????????????830Pro?Gly?Ile?Ala?Ala?Leu?Asn?Asp?Arg?Leu?Leu?Val?Ser?Lys?Asp?Leu

835?????????????????840?????????????845Trp?Pro?Phe?Gly?Glu?Gln?Leu?Arg?Ser?Lys?Tyr?Glu?Glu?Thr?Lys?Lys

850?????????????????855?????????????860Leu?Leu?Leu?Gln?Val?Ala?Ala?His?Lys?Glu?Val?Leu?Glu?Gly?Asp?Pro865?????????????????870?????????????875?????????????????????880Tyr?Leu?Lys?Gln?Arg?Leu?Arg?Leu?Arg?Asp?Ser?Tyr?Ile?Thr?Thr?Leu

885?????????????890??????????????????????895Asn?Val?Phe?Gln?Ala?Tyr?Thr?Leu?Lys?Arg?Ile?Arg?Asp?Pro?Asn?Tyr

900?????????????905?????????????????????910Lys?Val?Glu?Val?Arg?Pro?Pro?Ile?Ser?Lys?Glu?Ser?Ala?Glu?Thr?Ser

915?????????????920?????????????????????925Lys?Pro?Ala?Asp?Glu?Leu?Val?Thr?Leu?Asn?Pro?Thr?Ser?Glu?Tyr?Ala

930?????????????????935?????????????????940Pro?Gly?Leu?Glu?Asp?Thr?Leu?Ile?Leu?Thr?Met?Lys?Gly?Ile?Ala?Ala945?????????????????950?????????????????955?????????????????960Gly?Met?Gln?Asn?Thr?Gly

965＜210〉3＜211〉3372＜212〉DNA＜213〉＜400〉3cagacccgca agtcccttgc tggcctggat gctgctgagc tggccaacac cgttatcgcg 60tatgagccag tgtgggctat cggcactggc aaggttgctt ccgcggctga cgctcaggaa 120gtgtgcaagg ctatccgcgg tctgatcgtg gagcttgcag gcgacgaggt cgctgagggc 180ctgcgtattc tttacggtgg ttctgttaag gcagaaaccg tcgcagagat cgtcggtcag 240cctgacgtcg acggcggact tgtcggtggc gcttccctcg acggtgaagc attcgccaag 300ctggctgcca acgctgcgag cgttgcttaa agtacagagc tttaaagcac agccttaaag 360cacagcctta aagcacaagc actgtagaag tgcggttttg atgagcccat gaaagccatc 420gaaatcaatc gcccagctaa acacctgttt tgctgggtga ttttttatct catgcacgcc 480aacaccccca atgtgaaaga gtgtttaaag tagttatgac tgatttttta cgcgatgaca 540tcaggttcct cggtcaaatc ctcggtgagg taattgcgga acaagaaggc caggaggttt 600atgaactggt cgaacaagcg cgcctgactt cttttgatat cgccaagggc aacgccgaaa 660tggatagcct ggttcaggtt ttcgacggca ttactccagc caaggcaaca ccgattgctc 720gcgcattttc ccacttcgct ctgctggcta acctggcgga agacctctac gatgaagagc 780ttcgtgaaca ggctctcgat gcaggcgaca cccctccgga cagcactctt gatgccacct 840ggctgaaact caatgagggc aatgttggcg cagaagctgt ggccgatgtg ctgcgcaatg 900ctgaggtggc gccggttctg actgcgcacc caactgagac tcgccgccgc actgtttttg 960atgcgcaaaa gtggatcacc acccacatgc gtgaacgcca cgctttgcag tctgcggagc 1020ctaccgctcg tacgcaaagc aagttggatg agatcgaaaa gaacatccgc cgtcgcatca 1080ccattttgtg gcagaccgcg ttgattcgtg tggcccgccc acgtatcgag gacgagatcg 1140aagtagggct gcgctactac aagctgagcc ttttggaaga gattccacgt atcaaccgtg 1200atgtggctgt tgagcttcgt gagcgtttcg gcgaggatgt tcctttgaag cccgtggtca 1260agccaggttc ctggattggt ggagaccacg acggtaaccc ttatgtcacc gcggaaacag 1320ttgagtattc cactcaccgc gctgcggaaa ccgtgctcaa gtactatgca cgccagctgc 1380attccctcga gcatgagctc agcctgtcgg accgcatgaa taaggtcacc ccgcagctgc 1440ttgcgctggc agatgcaggg cacaacgacg tgccaagccg cgtggatgag ccttatcgac 1500gcgccgtcca tggcgttcgc ggacgtatcc tcgcgacgac ggccgagctg atcggcgagg 1560acgccgttga gggcgtgtgg ttcaaggtct ttactccata cgcatctccg gaagaattct 1620taaacgatgc gttgaccatt gatcattctc tgcgtgaatc caaggacgtt ctcattgccg 1680atgatcgttt gtctgtgctg atttctgcca tcgagagctt tggattcaac ctttacgcac 1740tggatctgcg ccaaaactcc gaaagctacg aggacgttct caccgagctt tttgagcgcg 1800cccaagtcac cgcaaactac cgcgagctgt ctgaagcaga gaagcttgag gtgctgctga 1860aggaactgcg cagccctcgt ccgctgatcc cgcacggttc agatgaatac agcgaggtca 1920ccgaccgcga gctcggcatc ttccgcaccg catctgaagc tgttaagaaa tttgggccac 1980ggatggtgcc tcactgcatc atctccatgg catcatcggt caccgatgtg ctggagccaa 2040tggtgttgct caaggaattc ggactcatcg cagccaacgg cgacaaccca cgcggcaccg 2100tcgatgtcat cccactgttc gaaaccatcg aagatctcca ggccggcgcc ggaatcctcg 2160acgaactgtg gaaaattgat ctctaccgca actacctcct gcagcgcgac aacgtccagg 2220aagtcatgct cggttactcc gattccaaca aggatggcgg atatttctcc gcaaactggg 2280cgctttacga cgcggaactg cagctcgtcg aactatgccg atcagccggg gtcaagcttc 2340gcctgttcca cggccgtggt ggcaccgtcg gccgcggtgg cggaccttcc tacgacgcga 2400ttcttgccca gcccaggggg gctgtccaag gttccgtgcg catcaccgag cagggcgaga 2460tcatctccgc taagtacggc aaccccgaaa ccgcgcgccg aaacctcgaa gctctggtct 2520cagccacgct tgaggcatcg cttctcgacg tctccgaact caccgatcac caacgcgcgt 2580acgacatcat gagtgagatc tctgagctca gcttgaagaa gtacgcctcc ttggtgcacg 2640aggatcaagg cttcatcgat tacttcaccc agtccacgcc gctgcaggag attggatccc 2700tcaacatcgg atccaggcct tcctcacgca agcagacctc ctcggtggaa gatttgcgag 2760ccatcccatg ggtgctcagc tggtcacagt ctcgtgtcat gctgccaggc tggtttggtg 2820tcggaaccgc attagagcag tggattggcg aaggggagca ggccacccaa cgcattgccg 2880agctacaaac actcaatgag tcctggccat ttttcacctc agtgttggat aacatggctc 2940aggtgatgtc caaggcagag ctgcgtttgg caaagctcta cgccgacctc atcccagata 3000gggaagtagc cgagcgcgtc tattccgtca tccgcgagga atacttcctg accaagaaga 3060tgttctgcgt aatcaccggt tctgatgatc tgcttgatga caacccactt ctcgcacgct 3120ctgtccagcg ccgttaccct tacctgcttc cactcaacgt gatccaggta gagatgatgc 3180gacgctaccg aaaaggcgac caaagcgagc aagtatcccg caacatccag ctgaccatga 3240acggtctttc cactgcgctg cgcaactccg gctagtccag ccggctgggt agtactcgtg 3300tatactgtct aaagttattc gaaatcaggt gggcataagg ttcacctggg ttctcaaacg 3360gcaaaggaac at 3372＜210〉4＜211〉25＜212〉DNA＜213〉＜220〉＜223〉：＜400〉4gttggtgagc cactggaaat ccgtg 25＜210〉5＜211〉25＜212〉DNA＜213〉＜220〉＜223〉：＜400〉5gatgtcatcg cgtaaaaaat cagtc 25＜210〉6＜211〉25＜212〉DNA＜213〉＜220〉＜223〉：＜400〉6cactgcgctg cgcaactcta gatag 25＜210〉7＜211〉25＜212〉DNA＜213〉＜220〉＜223〉：＜400〉7gaccaccacc ttgccgaaat cttgg 25

Claims

1.DNA fragment, it comprises the gene that coding has the active polypeptide of phosphoric acid enol pyruvic acid carboxylase, wherein said gene can be expressed in host microorganism, and described polypeptide do not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

2. the dna fragmentation of claim 1, wherein said dna fragmentation is from the plant that belongs to Monocotyledonae or Dicotyledoneae.

3. the dna fragmentation of claim 2, wherein said dna fragmentation is from alfalfa plant.

4. the dna fragmentation of claim 3, wherein said dna fragmentation is from the alfalfa strain.

5. the dna fragmentation of claim 2, wherein said dna fragmentation is modified by one or more nucleotide substitutions, disappearance or insertion.

6. the dna fragmentation of claim 5, wherein said modification comprise the Nucleotide of the following aminoacid sequence of disappearance coding: Met-Ala-Ser-Ile-Asp-Ala-Gln-Leu-Arg.

7. the dna fragmentation of claim 1, wherein said dna fragmentation is from the microorganism that belongs to brevibacterium sp or Corynebacterium.

8. the dna fragmentation of claim 7, wherein said dna fragmentation is from Corynebacterium glutamicum strain.

9. the dna fragmentation of claim 7, wherein said dna fragmentation is integrated in the chromosomal DNA of host microorganism.

10. the dna fragmentation of claim 1, wherein said dna fragmentation is expressed in the host microorganism that comprises Escherichia, Corynebacterium and brevibacterium sp.

11. the dna fragmentation of claim 1, wherein said dna fragmentation is a mosaic gene, and this mosaic gene comprises the microbe-derived imperfect phosphoric acid enol pyruvic acid carboxylase nucleotide sequence of brevibacterium sp or Corynebacterium and the imperfect phosphoric acid enol pyruvic acid carboxylase nucleotide sequence of Monocotyledonae or Dicotyledoneae plant origin.

12. the dna fragmentation of claim 1, wherein said dna fragmentation are cDNA, genomic dna or synthetic DNA.

13. dna fragmentation from Monocotyledonae or Dicotyledoneae plant, it comprises the gene that coding has the active polypeptide of phosphoric acid enol pyruvic acid carboxylase, wherein said gene can be expressed in the host microorganism that comprises Escherichia, Corynebacterium and brevibacterium sp, and described polypeptide do not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

14. the dna fragmentation of claim 13, wherein said dna fragmentation is from alfalfa plant.

15. the dna fragmentation of claim 14, wherein said dna fragmentation from the alfalfa strain is.

16. the dna fragmentation of claim 13, wherein said dna fragmentation is modified by one or more nucleotide substitutions, disappearance or insertion.

17. the dna fragmentation of claim 16, wherein said modification comprise the Nucleotide of the following aminoacid sequence of disappearance coding: Met-Ala-Ser-Ile-Asp-Ala-Gln-Leu-Arg.

18. the dna fragmentation of claim 13, wherein said dna fragmentation are cDNA, genomic dna or synthetic DNA.

19. dna fragmentation from brevibacterium sp or Corynebacterium microorganism, it comprises the gene that coding has the active polypeptide of phosphoric acid enol pyruvic acid carboxylase, wherein said gene can be expressed in the host microorganism that comprises Escherichia, Corynebacterium and brevibacterium sp, described gene is integrated in the chromosomal DNA of described host microorganism, and described polypeptide do not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

20. the dna fragmentation of claim 19, wherein said dna fragmentation is from Corynebacterium glutamicum strain.

21. the dna fragmentation of claim 19, wherein said gene is integrated in the following manner: the karyomit(e) ppc gene of removing host microorganism, and insert the gene that described coding has the active polypeptide of phosphoric acid enol pyruvic acid carboxylase, and do not change two expression of gene of host microorganism karyomit(e) ppc gene flank.

22. have the active isolated polypeptide of phosphoric acid enol pyruvic acid carboxylase, wherein said polypeptide does not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid, and described polypeptide is by claim 1,13 and 19 each dna fragmentation coding.

23. recombinant DNA molecules, this molecule comprises plasmid and the coding that wherein functionally inserts has the gene of the active polypeptide of phosphoric acid enol pyruvic acid carboxylase, wherein can breed at recombinant DNA molecules described in the host microorganism that comprises Escherichia, Corynebacterium and brevibacterium sp, described gene can be expressed, described gene is from monocotyledons or dicotyledons, and wherein said polypeptide do not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

24. the recombinant DNA molecules of claim 23, wherein said coding have the gene of the active polypeptide of phosphoric acid enol pyruvic acid carboxylase from alfalfa plant.

25. the recombinant DNA molecules of claim 24, wherein said coding have the gene of the active polypeptide of phosphoric acid enol pyruvic acid carboxylase.

26. having the gene of the active polypeptide of phosphoric acid enol pyruvic acid carboxylase, the recombinant DNA molecules of claim 23, wherein said coding modified by one or more nucleotide substitutions, disappearance or insertion.

27. the recombinant DNA molecules of claim 26, wherein said modification comprise the Nucleotide of the following aminoacid sequence of disappearance coding: Met-Ala-Ser-Ile-Asp-Ala-Gln-Leu-Arg.

28. have the active isolated polypeptide of phosphoric acid enol pyruvic acid carboxylase, wherein said polypeptide does not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid, and described polypeptide is by the dna molecule encode of claim 23.

29. the host microorganism of the Escherichia, Corynebacterium or the brevibacterium sp that transform with dna fragmentation, described dna fragmentation comprises the gene that coding has the active polypeptide of phosphoric acid enol pyruvic acid carboxylase, wherein said gene is from the plant of Monocotyledonae or Dicotyledoneae, described polypeptide does not need acetyl-CoA to activate, and, and the described host microorganism expressing said gene that with described dna fragmentation transform insensitive to the feedback inhibition of aspartic acid.

30. the host microorganism of claim 29, wherein said host microorganism is by in the chromosomal DNA that described dna fragmentation is incorporated into described host microorganism and by being transformed, or has transformed and contain the plasmid and the recombinant DNA molecules of the described dna fragmentation of insertion functionally wherein.

31. the host microorganism of claim 29, wherein said coding have the gene of the active polypeptide of phosphoric acid enol pyruvic acid carboxylase from alfalfa plant.

32. the host microorganism of claim 31, wherein said coding have the gene of the active polypeptide of phosphoric acid enol pyruvic acid carboxylase.

33. having the gene of the active polypeptide of phosphoric acid enol pyruvic acid carboxylase, the host microorganism of claim 29, wherein said coding modified by one or more nucleotide substitutions, disappearance or insertion.

34. the host microorganism of claim 33, wherein said modification comprise the Nucleotide of the following aminoacid sequence of disappearance coding: Met-Ala-Ser-Ile-Asp-Ala-Gln-Leu-Arg.

35. the host microorganism of Escherichia, Corynebacterium or brevibacterium sp, integrated the dna fragmentation of the gene that comprises coding tool phosphoric acid enol pyruvic acid carboxylase active polypeptide in the chromosomal DNA of wherein said host microorganism, described dna fragmentation is from the microorganism of Corynebacterium or brevibacterium sp, described polypeptide does not need acetyl-CoA to activate, and, and described host microorganism expressing said gene insensitive to the feedback inhibition of aspartic acid.

36. the host microorganism of claim 35, wherein said dna fragmentation is from Corynebacterium glutamicum strain.

37. the host microorganism of claim 35, wherein said gene is integrated in the following manner: the karyomit(e) ppc gene of removing host microorganism, and insert the gene that described coding has the active polypeptide of phosphoric acid enol pyruvic acid carboxylase, and do not change two expression of gene of host microorganism karyomit(e) ppc gene flank.

38. by the amino acid whose method of fermentative production, it comprises:

(a) in appropriate culture medium, cultivate the host microorganism of Escherichia, Corynebacterium or brevibacterium sp; With

(b) amino acid separation from this substratum, transformed the dna fragmentation of the gene that comprises coding tool phosphoric acid enol pyruvic acid carboxylase active polypeptide in the wherein said host microorganism (a), described host microorganism (a) expressing said gene, and described polypeptide do not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

39. the method for claim 38 wherein comprises L-aspartic acid, L-Methionin, L-methionine(Met), L-Threonine and L-Isoleucine at amino acid described in the step (b).

40. the method for claim 39 is a L-Methionin at amino acid described in the step (b) wherein.

41. the method for claim 38, wherein said dna fragmentation is from the plant that belongs to Monocotyledonae or Dicotyledoneae.

42. the method for claim 41, wherein said dna fragmentation is from alfalfa plant.

43. the method for claim 42, wherein said dna fragmentation from the alfalfa strain is.

44. the method for claim 38, wherein said dna fragmentation is modified by one or more nucleotide substitutions, disappearance or insertion.

45. the method for claim 44, wherein said modification comprise the Nucleotide of the following aminoacid sequence of disappearance coding: Met-Ala-Ser-Ile-Asp-Ala-Gln-Leu-Arg.

46. the method for claim 38, wherein said dna fragmentation is from the microorganism that belongs to brevibacterium sp or Corynebacterium.

47. the method for claim 46, wherein said dna fragmentation is from Corynebacterium glutamicum strain.

48. the method for claim 38, the gene that wherein said coding has the active polypeptide of phosphoric acid enol pyruvic acid carboxylase is a mosaic gene, and this mosaic gene comprises the microbe-derived imperfect phosphoric acid enol pyruvic acid carboxylase nucleotide sequence of brevibacterium sp or Corynebacterium and the imperfect phosphoric acid enol pyruvic acid carboxylase nucleotide sequence of Monocotyledonae or Dicotyledoneae plant origin.

49. the method for claim 38, wherein said dna fragmentation are cDNA, genomic dna or synthetic DNA.

50. by the amino acid whose method of fermentative production, it comprises:

(b) amino acid separation from this substratum, the dna fragmentation of the gene of wherein said host microorganism (a) by will comprising coding tool phosphoric acid enol pyruvic acid carboxylase active polypeptide is incorporated in the chromosomal DNA of described host microorganism (a) and is transformed, perhaps transformed the recombinant DNA molecules of the described dna fragmentation that contains plasmid and wherein functionally insert, described host microorganism (a) expressing said gene, described dna fragmentation is from monocotyledons or dicotyledons, and described polypeptide do not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

51. the method for claim 50 wherein comprises L-aspartic acid, L-Methionin, L-methionine(Met), L-Threonine and L-Isoleucine at amino acid described in the step (b).

52. the method for claim 51 is a L-Methionin at amino acid described in the step (b) wherein.

53. the method for claim 50, wherein said dna fragmentation is from alfalfa plant.

54. the method for claim 53, wherein said dna fragmentation from the alfalfa strain is.

55. the method for claim 50, wherein said dna fragmentation is modified by one or more nucleotide substitutions, disappearance or insertion.

56. the method for claim 55, wherein said modification comprise the Nucleotide of the following aminoacid sequence of disappearance coding: Met-Ala-Ser-Ile-Asp-Ala-Gln-Leu-Arg.

57. by the amino acid whose method of fermentative production, it comprises:

(b) amino acid separation from this substratum, wherein said host microorganism (a) is by in the chromosomal DNA that dna fragmentation is incorporated into described host microorganism and transformed, described dna fragmentation comprises the gene that coding has the active polypeptide of phosphoric acid enol pyruvic acid carboxylase, described host microorganism (a) expressing said gene, described dna fragmentation is from the microorganism of Corynebacterium or brevibacterium sp, and described polypeptide do not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

58. the method for claim 57, wherein integration method is a karyomit(e) ppc gene of removing host microorganism, and inserts described dna fragmentation, and does not change two expression of gene of host microorganism karyomit(e) ppc gene flank.

59. the method for claim 57 wherein comprises L-aspartic acid, L-Methionin, L-methionine(Met), L-Threonine and L-Isoleucine at amino acid described in the step (b).

60. the method for claim 59 is a L-Methionin at amino acid described in the step (b) wherein.

61. the method for claim 57, wherein said dna fragmentation is from Corynebacterium glutamicum strain.

62. select to contain the method for dna fragmentation of the gene of coding tool phosphoric acid enol pyruvic acid carboxylase active polypeptide, wherein said polypeptide does not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid, this method comprises:

(a) extract chromogene from the Corynebacterium bacterial strain that carries the ppc gene;

(b) cut described chromogene (a) with suitable Restriction Enzyme;

(c) described ppc gene (a) is connected with the plasmid vector that can breed in Corynebacterium;

(d) transform wherein ppc and pyc gene by the Corynebacterium bacterial strain of inactivation with described plasmid vector (c);

(e) being separated in glucose is the bacterial strain that shows good growth on the minimum medium of sole carbon source; With

(f) separate the dna fragmentation of the gene that contains coding tool phosphoric acid enol pyruvic acid carboxylase active polypeptide and from described bacterial strain (e), wherein said polypeptide does not need acetyl-CoA to activate, and insensitive to the feedback inhibition of aspartic acid.

63. the method for claim 62 wherein adds the active inhibitor of phosphoric acid enol pyruvic acid carboxylase in substratum in step (e).

64. the method for claim 62 is wherein separated the bacterial strain that shows from the amino acid production increase of oxaloacetic acid in step (e).

65. the method for claim 62, wherein strain growth is not having on the minimum medium of acetyl-CoA in step (e).

66. increase the method that phosphoenolpyruvic acid is converted into the transformation efficiency of oxaloacetic acid, this method comprises with claim 1,13 and 19 each dna fragmentation and transforms host microorganism.

67. comprising with claim 1,13 and 19 each dna fragmentation, the method for recirculation carbon in zymotechnique, this method transform host microorganism.

68. assimilation carbon in zymotechnique and do not need the method for vitamin H, this method to comprise to transform host microorganism with claim 1,13 and 19 each dna fragmentation.

69. increase the method that organic acid is produced in zymotechnique, this method comprises with claim 1,13 and 19 each dna fragmentation and transforms host microorganism.

70. increase the method that amino acid is produced in zymotechnique, this method comprises with claim 1,13 and 19 each dna fragmentation and transforms host microorganism.