CN101824392A - Method for producing L-lysine - Google Patents

Abstract

The invention relates to a method for producing L-lysine, which comprises the following concrete steps: culturing a bacterium of the invention in an appropriate culture medium to generate and accumulate the L-lysine in the culture medium, and then collecting the lysine from the culture material, wherein the bacterium of the invention belongs to Escherichia, and is characterized in that (1) dihydrodipicolinate synthetase the L-lysine feedback inhibition of which is desensitized and aspartokinase the L-lysine feedback inhibition of which is desensitized are contained; (2) the intra-cellular activity of dihydrodipicolinate reductase is enhanced; and (3) diaminopimelic acid dehydrogenase genes are introduced, or the intra-cellular activities of tetrahydro dipicolinic acid succinyl enzyme and succinyl diaminopimelic acid deacylase are enhanced, wherein the intra-cellular activity of aspartate-semialdehyde dehydrogenase or phosphoenolpyruvate carboxylase is enhanced.

Description

Produce the method for L-Methionin

The application is to be the dividing an application of Chinese patent application 00818456.9 method of Methionin " produce L-" on January 21st, 2000 applying date.

Technical field

The present invention relates to microbiological industry.More particularly, the present invention relates to method by fermentation production of L-lysine, and the microorganism that is used for this production method.

Background technology

By fermentation production of L-lysine the time, use strain separated from natural or artificial mutation bacterial strain usually, so that improve its output.It is well-known that many artificial mutation bacterial strains that can produce L-Methionin are arranged, wherein much be S-2-aminoethylcysteine (AEC) resistant strain, and belong to brevibacterium sp, Corynebacterium, bacillus or Escherichia.In addition, disclose the whole bag of tricks that is used to improve amino acid output already, for example, used the transformant that obtains by recombinant DNA.

For example, for the Escherichia bacterium, the method that the bacterial strain that had disclosed already in following document with wherein dihydrodipicolinic acid synthase (DDPS) increased activity is produced L-Methionin: Japanese Patent Application Publication (Kokai) No.56-18596, US4,346,170 and ' applied microbiology and biotechnology ', 15, pp.227-231 (1982).In addition, disclosed the method for producing L-Methionin to the Escherichia bacterium that has wherein imported the DDPS that comes from corynebacterium genus bacteria among the korean patent application NO.92-8382 already by using.In addition, in the open NO.WO95/16042 in the world, disclosed the method for producing L-Methionin by the bacterial strain that transformed with a kind of plasmid, described plasmid contains the coding dihydrodipicolinic acid synthase's who comes from the Escherichia bacterium DNA, and it has the sudden change of the feedback inhibition desensitization that makes L-Methionin; The DNA of the E.C. 2.7.2.4. that the feedback inhibition of its L-Methionin of encoding has desensitized; Coding dihydrodipicolinate reductase's DNA; And coding comes from the DNA of coryneform diaminopimelate dehydrogenase.

For the brevibacterium sp bacterium, open NO.WO95/11985 discloses according to the world, by strengthening the activity of Reduced nicotinamide-adenine dinucleotide transhydrogenase in the cell, can improve the L-lysine production.In addition, in Japanese Patent Application Publication NO.60-87788 and open (Kokoku) NO.6-102028 of Japanese Patent, disclosed respectively with the method that has only strengthened the active bacterial strain production of its Phosphoenolpyruvate carboxylase L-Methionin with only having strengthened the method that the active bacterial strain of aspartate-semialdehyde dehydrogenase is produced L-Methionin.

In the amino acid industrial production of being undertaken by fermentation, production is carried out with extensive form.Therefore,, can produce significant industrial value even the output of several percentage points improves, and, therefore need output to improve, and regardless of the raising of output.

Summary of the invention

An object of the present invention is, a kind of method of having improved of passing through fermentation production of L-lysine of comparing with ordinary method is provided.

To achieve these goals, the inventor has carried out research untiringly.The result, the inventor finds, if have the aspartate-semialdehyde dehydrogenase of Escherichia bacterium of particular characteristics or the increased activity of Phosphoenolpyruvate carboxylase, and, if in above-mentioned Escherichia bacterium, the increased activity of one or more specific enzymes except above-mentioned enzyme, the L-Methionin productivity of described bacterium can improve, and they have finished the present invention according to these discoveries.

In other words, the invention provides:

First aspect, a kind of Escherichia bacterium, wherein (1) dihydrodipicolinic acid synthase, E.C. 2.7.2.4. and dihydrodipicolinate reductase's intracellular reactive strengthens, (2) intracellular reactive of the intracellular reactive of diaminopimelate dehydrogenase or tetrahydropyridine dicarboxylic acid succinyl enzyme and succinyl-diaminopimelic acid deacylase strengthens, wherein, the intracellular reactive of aspartate-semialdehyde dehydrogenase or Phosphoenolpyruvate carboxylase strengthens;

Second aspect, a kind of Escherichia bacterium, wherein (1) dihydrodipicolinic acid synthase, E.C. 2.7.2.4. and dihydrodipicolinate reductase's intracellular reactive strengthens, (2) intracellular reactive of the intracellular reactive of diaminopimelate dehydrogenase or tetrahydropyridine dicarboxylic acid succinyl enzyme and succinyl-diaminopimelic acid deacylase strengthens, wherein, the intracellular reactive of the intracellular reactive of Phosphoenolpyruvate carboxylase and Reduced nicotinamide-adenine dinucleotide transhydrogenase or aspartate-semialdehyde dehydrogenase strengthens;

The third aspect, a kind of Escherichia bacterium, (1) dihydrodipicolinic acid synthase wherein, E.C. 2.7.2.4. and dihydrodipicolinate reductase's intracellular reactive strengthens, (2) intracellular reactive of the intracellular reactive of diaminopimelate dehydrogenase or tetrahydropyridine dicarboxylic acid succinyl enzyme and succinyl-diaminopimelic acid deacylase strengthens, wherein, the intracellular reactive of the intracellular reactive of Phosphoenolpyruvate carboxylase and Reduced nicotinamide-adenine dinucleotide transhydrogenase and aspartate-semialdehyde dehydrogenase or L-Aspartase strengthens (hereinafter, the bacterium of above-mentioned three aspects is collectively referred to as " bacterium of the present invention ").

In bacterium of the present invention, the intracellular reactive of preferred aspartate-semialdehyde dehydrogenase and L-Aspartase strengthens.

In addition, in bacterium of the present invention, E.C. 2.7.2.4., the dihydrodipicolinate reductase, tetrahydropyridine dicarboxylic acid succinyl enzyme, succinyl-diaminopimelic acid deacylase, Phosphoenolpyruvate carboxylase and aspartate-semialdehyde dehydrogenase preferably all come from the Escherichia bacterium, Reduced nicotinamide-adenine dinucleotide transhydrogenase and L-Aspartase (if any) preferably come from the Escherichia bacterium respectively, the dihydrodipicolinic acid synthase preferably comes from Escherichia bacterium or brevibacterium sp bacterium, and diaminopimelate dehydrogenase preferably comes from the brevibacterium sp bacterium.

In bacterium of the present invention, preferably its intracellular reactive is an enhanced, is by following in every one or its arbitrary combination and enhanced.

(1) importing has the plasmid of the gene of described enzyme.

(2) the gene copy quantity of the above enzyme of increase karyomit(e).

(3) promoter sequence of the gene of the above enzyme of change karyomit(e).

In addition, in bacterium of the present invention, preferably the feedback inhibition by obtaining its L-Methionin by the dihydro pimelic acid synthetic enzyme of desensitization with and the feedback inhibition of L-Methionin by the E.C. 2.7.2.4. of desensitization, strengthen the intracellular reactive of dihydrodipicolinic acid synthase and E.C. 2.7.2.4., and, by importing the diaminopimelate dehydrogenase gene, strengthen the intracellular reactive of diaminopimelate dehydrogenase.

The present invention also provides the method for producing L-Methionin, this method is included in and cultivates any bacterium of the present invention in the suitable medium, so that in substratum, produce and accumulation L-Methionin, and from culture, collect Methionin (following be referred to as again " production method of the present invention ").

Description of drawings

Fig. 1 represents to come from the preparation process of the plasmid RSF24P of RSF1010, and it contains dapA ^*24.

Fig. 2 represents to contain dapA ^*24 and lysC ^*The preparation process of 80 plasmid RSFD80.

Fig. 3 represents to contain dapA ^*24, lysC ^*80 and the preparation process of the plasmid pCAB1 of dapB.

Fig. 4 represents to contain dapA ^*24, lysC ^*80, the preparation process of the plasmid pCABD2 of dapB and ddh.

Fig. 5 represents to contain dapA ^*24, lysC ^*80, dapB, the preparation process of the plasmid pCABDE1 of dapD and dapE.

Fig. 6 represents to contain the structure of the plasmid pppc of ppc.

Fig. 7 represents to contain the structure of the plasmid pasd of asd.

Fig. 8 represents to contain the preparation process of the plasmid pMW::THY of pntAB (pntA and pntB).

Fig. 9 represents to contain the preparation process of the plasmid pMW118::aspA of aspA.

Figure 10 represents to contain the preparation process of the plasmid ppcd of ppc and asd.

Figure 11 represents to contain the preparation process of the plasmid pPTS of ppc and pntAB.

Figure 12 represents to contain the preparation process of the plasmid pAPW of ppc and aspA.

Figure 13 represents to contain ppc, the preparation process of the plasmid pPTd of pntAB and asd.

Figure 14 represents to contain ppc, the preparation process of the plasmid pAPT of pntAB and aspA.

Figure 15 represents to contain ppc, the preparation process of the plasmid pAPTK of pntAB and aspA.

Figure 16 represents to contain the preparation process of the plasmid pSd of asd.

Figure 17 represents to contain ppc, pntAB, the preparation process of the plasmid pKD of aspA and asd.

Figure 18 represents to contain tet ^ProThe preparation process of plasmid pUTES.

Figure 19 represents to contain tet ^ProBe positioned at tet ^ProThe preparation process of the plasmid pUEBL3 of the dapA downstream, that come from brevibacterium.

Figure 20 represents to contain the preparation process of the plasmid pCABD (B) of the dapA (Brev.dapA), lysC, dapB and the ddh that come from brevibacterium.

Embodiment

＜1〉bacterium of the present invention

Bacterium of the present invention is the Escherichia bacterium, wherein (1) dihydrodipicolinic acid synthase, E.C. 2.7.2.4. and dihydrodipicolinate reductase's intracellular reactive strengthens, (2) intracellular reactive of the intracellular reactive of diaminopimelate dehydrogenase or tetrahydropyridine dicarboxylic acid succinyl enzyme and succinyl-diaminopimelic acid deacylase strengthens, and the intracellular reactive of following enzyme further strengthens:

(1) aspartate-semialdehyde dehydrogenase or Phosphoenolpyruvate carboxylase,

(2) Phosphoenolpyruvate carboxylase and Reduced nicotinamide-adenine dinucleotide transhydrogenase (following be referred to as again " transhydrogenase ") or aspartate-semialdehyde dehydrogenase, or

(3) Phosphoenolpyruvate carboxylase, and transhydrogenase and aspartate-semialdehyde dehydrogenase or L-Aspartase.

Bacterium of the present invention is the Escherichia bacterium preferably, wherein, Phosphoenolpyruvate carboxylase, transhydrogenase, the intracellular reactive of aspartate-semialdehyde dehydrogenase and L-Aspartase further strengthens.

Bacterium of the present invention preferably belongs to intestinal bacteria.

In this manual, phrase " intracellular reactive enhancing " expression and wild type strain are (for example, intestinal bacteria W3110 bacterial strain) or parent strain (in the combination that the present invention refered in particular in this bacterial strain the intracellular reactive of all included enzymes all do not have strengthen) compare, the desmo enzyme increased activity represents that also a kind of bacterium has a kind of wild type strain or the unexistent enzymic activity of parent strain.The method that is used to measure above-mentioned enzymic activity is known, and those skilled in the art can confirm the enhancing of its intracellular reactive easily.

The method that strengthens described intracellular reactive comprises following method and combination thereof, but is not limited to these.

At first, mention the method for the expression amount that improves described enzyme.

Specifically, the method for raising expression of enzymes amount comprises following method.

(1) imports the plasmid that contains the enzyme gene

As plasmid, can use can be in the Escherichia bacterial cell carrier of self-replicating.Can it be imported with known method.In other words, interested gene can be inserted described carrier, and can transform the Escherichia bacterium with this carrier.This carrier is multiple copied type plasmid preferably.

Described gene can be carried by identical plasmid or different plasmid.Some gene can be carried by identical plasmid.When using two or more plasmid, the preferred plasmid that uses with stable distribution system, this system makes the coexistence that these plasmids are can be in cell stable.The order that imports described gene is not particularly limited.

(2) number of copies of enzyme gene on the increase karyomit(e)

By with the DNA on the amplification chromosomal DNAs such as Mu phage, increase described number of copies.

DNA on the chromosomal DNA can be that the Escherichia bacterium just had originally, or by using transduction, transposon (Berg, D.E. with Berg C.M., biotechnology, 1,417 (1983)), Mu phage (Japanese Patent Application Publication NO.2-109985), or the method for homologous recombination (molecular genetics experiment, cold spring harbor laboratory (1972)) is inserted in the host microorganism karyomit(e).

(3) promoter sequence of change enzyme gene

Can change promoter sequence, so that improve the gene transcription amount, and and then raising expression amount.For example, can import promotor by suddenling change and strengthen this promotor, so that improve the gene transcription amount that is positioned at this promotor downstream.Import except suddenling change the described promotor, can newly import the promotor that can in the Escherichia bacterium, work, as lac, trp, tac, trc, and PL.In addition, can improve the genetic transcription amount by the new enhanser that imports.Although described promoter sequence can be the promotor of enzyme gene on the promotor of enzyme gene on the karyomit(e) or the plasmid, the promotor of enzyme gene on the preferred karyomit(e).For example, Japanese Patent Application Publication NO.1-215280 has disclosed and will import chromosomal DNA such as the gene of promotor.

The source of above-mentioned enzyme gene is not particularly limited, therefore, can the using of various sources, as long as this gene can express in the Escherichia bacterium, and its gene product can work just in the Escherichia bacterium.

Colibacillary L-Methionin biosynthesis system gene and the dihydrodipicolinic acid synthase of transhydrogenase gene and brevibacterium and the method for diaminopimelate dehydrogenase gene of obtaining will be described below.

Can obtain this gene from plasmid pS2 (Sabe, H. etc., gene, 31,279 (1984)) or the pT2 that contains Phosphoenolpyruvate carboxylase gene (ppc).Can obtain to contain the dna fragmentation of ppc by digesting pS2 with AatII and AflII.In addition, can also obtain to contain the dna fragmentation of ppc by digesting pT2 with SmaI and ScaI.Intestinal bacteria F15 bacterial strain (AJ12873) with pT2 is deposited in (the postcode 305-8566 of life engineering Industrial Technology Research Institute of Govement Industrial Research Inst., Ministry of Commerce on July 15th, 1993,1-3Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japanese), and the preserving number that obtains is FERM P-13752.Then, according on July 11st, 1994 that is specified in of budapest treaty transferring international preservation to, the preserving number that is obtained is FERM BP-4732.

Can obtain aspartokinase gene (lysC) by pcr amplification, described amplification uses e. coli chromosomal dna to do template, and uses two types Oligonucleolide primers (Cassan according to the known nucleotide sequence preparation of lysC, M., Parsot, C., Cohen, G.N., and Patte, J.C., journal of biological chemistry 261,1052 (1986)) (for example, disclosed in the international publication number WO95/16042, SEQ ID NO:5 and 6).

Aspartate-semialdehyde dehydrogenase gene (asd) can obtain (Haziza, C. etc., EMBO, 1,379,1982) from the plasmid pAD20 that contains this gene.If with AseI and ClaI digestion pAD20, just can obtain to contain the dna fragmentation of asd.

Can obtain dihydrodipicolinic acid synthase's gene (dapA) by pcr amplification, described amplification uses e. coli chromosomal dna to do template, and use is according to the known nucleotide sequence (Richaud of dapA, F. etc., the bacteriology magazine, 297,1986) Zhi Bei two types Oligonucleolide primers (for example, at the SEQ ID NO:1 and 2 disclosed in the international publication number WO95/16042).

Can obtain dihydrodipicolinate reductase's gene (dapB) by pcr amplification, described amplification uses e. coli chromosomal dna to do template, and use is according to the known nucleotide sequence (Bouvier of dapB, J. etc., journal of biological chemistry, 259,14829,1984) Zhi Bei two types Oligonucleolide primers (for example, at the SEQID NO:9 and 10 disclosed in the international publication number WO95/16042).

Can obtain tetrahydropyridine dicarboxylic acid succinyl enzyme gene (dapD) by pcr amplification, described amplification uses e. coli chromosomal dna to do template, and use is according to the known nucleotide sequence (Richaud of dapD, C. etc., journal of biological chemistry, 259,14829,1984) Zhi Bei two types Oligonucleolide primers (for example, at the SEQ ID NO:15 and 16 disclosed in the international publication number WO95/16042).

Can obtain succinyl-diaminopimelic acid deacylase gene (dapE) by pcr amplification, described amplification uses e. coli chromosomal dna to do template, and use is according to the known nucleotide sequence (Bouvier of dapE, J. etc., the bacteriology magazine, 174,5265,1992) Zhi Bei two types Oligonucleolide primers (for example, at the SEQID NO:17 and 18 disclosed in the international publication number WO95/16042).

Can obtain L-Aspartase gene (aspA) by pcr amplification, described amplification uses e. coli chromosomal dna to do template, and use is according to the known nucleotide sequence (Woods of aspA, S.A. etc., biological chemistry 237, (2), 547-557,1986) Zhi Bei two types Oligonucleolide primers (for example, at the SEQ ID NO:5 and 6 disclosed in the sequence table of this specification sheets).

Can be according to known nucleotide sequence (D.M.Clark etc., european journal of biological chemistry, 158,647-653,1986) the preparation transhydrogenase gene (pntAB) of transhydrogenase gene.In intestinal bacteria, transhydrogenase is made up of two subunits, and these two subunits are respectively by pntA and pntB coding (D.M.Clark etc., the same).Therefore, these two genes (for example, referring to international publication number WO95/11985) have been prepared.

Can also from the plasmid that contains pntAB, obtain it.As the plasmid that contains pntAB, can be included in the plasmid pMW::THY that discloses among the international publication number WO95/11958.This plasmid is by connecting the 3.0kbDNA fragment that contains pntA and pntB and the BamHI of plasmid vector pMW118 and the recombinant plasmid that the HindIII fragment obtains of e. coli k-12 MC1061 bacterial strain.E. coli jm109 bacterial strain with pMW::THY is named as the AJ12929 bacterial strain, and be deposited in (the postcode 305-0046 of life engineering Industrial Technology Research Institute of Govement Industrial Research Inst., Ministry of Commerce on October 4th, 1993,1-3Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japanese), and the preserving number that obtains is FERM P-13890.Then, according on September 14th, 1994 that is specified in of budapest treaty transferring international preservation to, the preserving number that is obtained is FERMBP-4798.

Can obtain dihydrodipicolinic acid synthase's gene (dapA) of brevibacterium by pcr amplification, described amplification uses the brevibacterium chromosomal DNA to do template, and use is according to the known nucleotide sequence (Bonassie of dapA, S. etc., N.A.R., 18 (21), 6421 (1990)) Zhi Bei two types Oligonucleolide primers (for example, at the SEQ ID NO:3 and 4 disclosed in the sequence table of this specification sheets).

Can obtain the diaminopimelate dehydrogenase gene (ddh) of brevibacterium by pcr amplification, described amplification uses the brevibacterium chromosomal DNA to do template, and use is according to the known nucleotide sequence (Ishino of Corynebacterium glutamicum ddh, S. etc., nucleic acids research, 15,3917 (1987)) Zhi Bei two types Oligonucleolide primers (for example, at the SEQ ID NO:11 and 12 disclosed in the international publication number WO95/16042).

Method as strengthening described intracellular reactive also comprises the method that improves described specific enzyme activity.This method can make up with the method that improves the expression of enzymes amount.

As the method that improves specific enzyme activity, comprise importing enzyme with the sudden change that strengthens specific activity, comprise the enzyme of its feedback inhibition desensitizationization when described enzyme has been subjected to feedback inhibition etc.

The example of the enzyme of its feedback inhibition desensitizationization comprises that L-Methionin is to the dihydrodipicolinic acid synthase (DDPS) of its feedback inhibition desensitizationization and the L-Methionin E.C. 2.7.2.4. (AK) to its feedback inhibition desensitizationization.

It is just enough that phrase " L-Methionin is to its feedback inhibition desensitizationization " expression is significantly gone up desensitization to restraining effect, and do not need restraining effect is desensitized fully.In addition, L-Methionin also comprises the enzyme that comes from the biology except the Escherichia bacterium to the enzyme of its feedback inhibition desensitizationization, as long as L-Methionin is lower than the degree of the wild-type enzyme that comes from the Escherichia bacterium to its feedback inhibition degree, and its biological wild-type or mutant enzyme that be this no matter.Therefore, also comprise the enzyme that does not have the feedback inhibition of L-Methionin such as the script of the DDPS that comes from the brevibacterium sp bacterium.

Can assess the degree of L-Methionin feedback inhibition with known method, as be disclosed in the method in international publication number WO95/16042, example 1 and the example 2.

As L-Methionin to the DDPS (desensitizationization DDPS) of its feedback inhibition desensitizationization and L-Methionin to the AK (desensitizationization AK) of its feedback inhibition desensitizationization, be included in this class material disclosed in international publication number WO95/16042 and the Japanese Patent Application Publication NO.10-113183.

In other words, the example of desensitizationization DDPS is included in to have on the wild-type DDPS and makes the DDPS of L-Methionin to the sudden change of its feedback inhibition desensitizationization.As wild-type DDPS, comprise the DDPS that comes from the Escherichia bacterium, particularly come from colibacillary DDPS.The example of sudden change of DDPS of the feedback inhibition desensitizationization of L-Methionin is comprised:

(1) be equivalent to the sudden change of the amino-acid residue of No. 81 locational alanine residues with another kind of amino-acid residue (preferred Xie Ansuan residue) replacement,

(2) be equivalent to the sudden change of the amino-acid residue of No. 118 locational histidine residues with another kind of amino-acid residue (preferred tyrosine residues) replacement,

(3) replace the amino-acid residue that is equivalent to No. 81 locational alanine residues of the DDPS aminoacid sequence shown in the SEQ ID NO:4 among the international publication number WO95/16042 with another kind of amino-acid residue (preferred Xie Ansuan residue), and replace the sudden change of the amino-acid residue that is equivalent to No. 118 locational histidine residues of above-mentioned DDPS aminoacid sequence with another kind of amino-acid residue (preferred tyrosine residues), wherein, described position is that N-end from DDPS begins to count.Well-knownly be do not influence its active aminoacid sequence difference may existing between the kind or between the bacterial strain, and those skilled in the art can discern the amino-acid residue that is equivalent to above-mentioned particular amino acid residue easily.

Other example of desensitizationization DDPS comprises and comes from bar shaped bacteria, for example, and the DDPS of brevibacterium (Cremer J. etc., hereditary JOURNAL OF MICROBIOLOGY, 134,3221-3229,1988).

For example, in order to prepare the desensitization DDPS that the Escherichia bacterium is had, can import the DNA of coding desensitizationization DDPS.

The example of the DNA of coding desensitizationization DDPS, those of the DNA of the wild-type DDPS that comprising is equivalent to encode has a kind of sudden change, described sudden change makes the feedback inhibition desensitizationization of L-Methionin to coded DDPS.

Hereinafter, will be example with the DDPS that comes from the Escherichia bacterium, the method for the DNA (desensitizationization DDPS gene) that obtains coding desensitizationization DDPS is described.For other biological DDPS, can obtain DNA with similar approach.In addition, be desensitizationization DDPS if come from another kind of biological wild-type DDPS, just can be used as it is its DNA of coding.

The DNA of encoding wild type DDPS is not particularly limited, and is just passable as long as its coding comes from the DDPS of Escherichia bacterium.Specifically, the DNA that comprises the DDPS aminoacid sequence shown in the SEQ ID NO:4 among the coding international publication number WO95/16042.More particularly, the sequence that comprises the 272-1147 Nucleotide representative of nucleotide sequence shown in the SEQ ID NO:3 in the document.In described sequence, have the sequence of the nucleotide sequence sudden change that causes replacing above-mentioned amino-acid residue, be the DNA of coding desensitizationization DDPS.In addition, the codon type of the amino-acid residue that is equivalent to replace is not particularly limited, this amino-acid residue as long as it is encoded.In addition, estimate that also there is little difference in the aminoacid sequence of the DDPS that is contained between bacterium kind and the bacterial strain.But, have the sequence of the difference that can cause that amino-acid residue replacement, disappearance take place or insert on the position that does not influence enzymic activity, also belong to the scope of desensitizationization DDPS gene.

For example, can obtain desensitizationization DDPS gene in order to following method.At first, the DNA that contains wild-type DDPS gene or have other DDPS gene of sudden change is carried out vitro mutagenesis handle, and the DNA that will carry out mutagenic treatment is connected with the carrier DNA compatible with the host, so that prepare recombinant DNA.Recombinant DNA is imported host microorganism, obtains transformant, and from these transformant, select can desensitizationization DDPS transformant.Such transformant has desensitizationization DDPS gene.Perhaps, the DNA that contains wild-type DDPS gene or have other DDPS gene of sudden change can be connected with the carrier DNA compatible with the host, so that the preparation recombinant DNA.Then, can carry out vitro mutagenesis to recombinant DNA and handle, and it is imported host microorganism, obtain transformant, and from these transformant, select to become to express the transformant of desensitizationization DDPS.Such transformant has desensitizationization DDPS gene equally.

In addition, can carry out mutagenic treatment,, then, can from this mutant strain, obtain desensitizationization DDPS gene so that preparation can produce the mutant strain of desensitizationization DDPS to the microorganism that can produce wild-type DDPS.In addition, if wherein having imported the transformant of the recombinant DNA that is connected with wild type gene, subtend carries out mutagenic treatment, so that preparation can produce the mutant strain of desensitizationization DDPS, then, from these mutant strains, collect recombinant DNA, just can on this DNA, prepare desensitizationization DDPS gene.

The example that is used for the reagent of DNA vitro mutagenesis processing comprises azanol etc.Azanol is can be by cytosine(Cyt) is become N ⁴The chemomorphosis treatment agent that-hydroxyl cytosine(Cyt) causes cytosine(Cyt) to be replaced by thymus pyrimidine.When microorganism itself was carried out mutagenic treatment, this processing was to carry out with ultraviolet radiation or the mutagenic compound such as N-methyl-N '-nitro-N-nitrosoguanidine (NTG) and nitrous acid that are usually used in induced mutations.

As the bacterium of the DNA that other DDPS gene that contains wild-type DDPS gene or have sudden change can be provided, can use any microorganism that belongs to Escherichia.Specifically, can use the microorganism of in following document, mentioning: Neidhardt etc. (Neidhardt, F.C. etc., intestinal bacteria and Salmonella typhimurium, U.S. microbiology association, Washington D.C., 1208, table 1).For example, can use e. coli jm109 bacterial strain and MC1061 bacterial strain.When wild type strain is used as the donor of the DNA that contains the DDPS gene, can obtain to contain the DNA of wild-type DDPS gene.

The example of desensitizationization AK comprises the AK with the sudden change that appears at the feedback inhibition desensitizationization that can make L-Methionin on the wild-type AK.Wild-type AK can come from the Escherichia bacterium, particularly comes from colibacillary E.C. 2.7.2.4. (AKIII).Can make L-Methionin that the example of the sudden change of the feedback inhibition desensitizationization of AKIII is comprised:

(a) be equivalent to the sudden change of the amino-acid residue of No. 323 locational glycine residues with another kind of amino-acid residue (preferred asparagicacid residue) replacement,

(b) replace the amino-acid residue that is equivalent to No. 323 locational glycine residues with another kind of amino-acid residue (preferred asparagicacid residue), and replace the sudden change of the amino-acid residue that is equivalent to No. 408 locational glycine residues with another kind of amino-acid residue (preferred asparagicacid residue)

(c) replace the amino-acid residue that is equivalent to No. 34 locational arginine residues with another kind of amino-acid residue (preferred cysteine residues), and replace the sudden change of the amino-acid residue that is equivalent to No. 323 locational glycine residues with another kind of amino-acid residue (preferred asparagicacid residue)

(d) be equivalent to the sudden change of the amino-acid residue of No. 325 locational leucine residues with another kind of amino-acid residue (preferred phenylalanine residue) replacement,

(e) be equivalent to the sudden change of the amino-acid residue of No. 318 locational methionine residues with another kind of amino-acid residue (preferred Isoleucine residue) replacement,

(f) replace the amino-acid residue that is equivalent to No. 318 locational methionine residues with another kind of amino-acid residue (preferred Isoleucine residue), and replace the sudden change of the amino-acid residue that is equivalent to No. 349 locational Xie Ansuan residues with another kind of amino-acid residue (preferred methionine residues)

(g) be equivalent to the sudden change of the amino-acid residue of No. 345 locational serine residues with another kind of amino-acid residue (preferred leucine residue) replacement,

(h) be equivalent to the sudden change of the amino-acid residue of No. 347 locational Xie Ansuan residues with another kind of amino-acid residue (preferred methionine residues) replacement,

(i) be equivalent to the sudden change of the amino-acid residue of No. 352 locational threonine residues with another kind of amino-acid residue (preferred Isoleucine residue) replacement,

(j) replace the amino-acid residue that is equivalent to No. 352 locational threonine residues with another kind of amino-acid residue (preferred Isoleucine residue), and replace the sudden change of the amino-acid residue that is equivalent to No. 369 locational serine residues with another kind of amino-acid residue (preferred phenylalanine residue)

(k) be equivalent to the sudden change of the amino-acid residue of No. 164 locational glutaminic acid residues with another kind of amino-acid residue (preferred lysine residue) replacement,

(l) replace the amino-acid residue that is equivalent to No. 417 locational methionine residues with another kind of amino-acid residue (preferred Isoleucine residue), and replace the sudden change of the amino-acid residue that is equivalent to No. 419 locational cysteine residues with another kind of amino-acid residue (preferred tyrosine residues), said mutation is all based on the AKIII aminoacid sequence shown in the SEQ ID NO:8 among the international publication number WO95/16042, wherein, described position begins to count from the N-end.In addition, also comprise with another kind of amino-acid residue (preferred asparagicacid residue) and replace the amino-acid residue that is equivalent to No. 323 locational glycine residues, and replace the sudden change (Japanese Patent Application Publication NO.10-113183) of the amino-acid residue that is equivalent to No. 318 locational methionine residues with another kind of amino-acid residue (preferred Isoleucine residue), well-knownly be, do not influence its active aminoacid sequence difference may existing between the kind or between the bacterial strain, and those skilled in the art can discern the amino-acid residue that is equivalent to above-mentioned particular amino acid residue easily.

Other example of desensitizationization AK comprises the mutant AK (Japanese Patent Application Publication NO.6-62866) that comes from bar shaped bacteria.

For example, in order to prepare the desensitization AK that the Escherichia bacterium is had, the DNA of coding desensitizationization AK can be imported the Escherichia bacterium.

The example of the DNA of coding desensitizationization AK, those of the DNA of the wild-type AK that comprising is equivalent to encode has a kind of sudden change, described sudden change makes the feedback inhibition desensitizationization of L-Methionin to coded AK.

Hereinafter, will be example with the AKIII that comes from the Escherichia bacterium, the method for the DNA (desensitizationization DDPS gene) that obtains coding desensitizationization AK is described.Equally, for other biological AK, can obtain DNA with similar approach.In addition, be desensitizationization AK if come from another kind of biological wild-type AK, just can be used as it is its DNA of coding.

The DNA of encoding wild type AKIII is not particularly limited.For example, can be the DNA that coding comes from Escherichia bacterium, particularly colibacillary AKIII.Specifically, comprise the DNA of aminoacid sequence shown in the SEQ ID NO:8 among the coding international publication number WO95/16042, and the sequence of the 584-1930 Nucleotide representative of nucleotide sequence shown in the SEQ ID NO:7 in the document.Colibacillary AKIII is by the lysC genes encoding.

In described sequence, have the sequence of the sudden change that causes replacing above-mentioned amino-acid residue, be the DNA of encoding mutant type AKIII.In addition, the codon type of the amino-acid residue that is equivalent to replace is not particularly limited, this amino-acid residue as long as it is encoded.In addition, estimate that also there is little difference in the aminoacid sequence of the AKIII that is contained between bacterium kind and the bacterial strain.But, have the sequence of the difference that can cause that amino-acid residue replacement, disappearance take place or insert on the position that does not influence enzymic activity, also belong to the scope of mutant AKIII gene.For example, nucleotide sequence (the international publication number WO95/16042 of the wild-type lysC gene that obtains in the example that will mention 2 below, SEQ ID NO:7) on No. 6 positions, there are differences (Cassan M. with the corresponding position of the lysC nucleotide sequence of disclosed e. coli k-12 JC411 bacterial strain already, Parsot, C., Cohen, G.N., and Patte, J.C., journal of biological chemistry, 261,1052,1986), two species diversity wherein can provide different amino acids coding residue (lysC of JC411 bacterial strain provides with cysteine residues and replaced No. 58 locational glycine residues of lysC amino acid sequence coded shown in the SEQ ID NO:8 among the international publication number WO95/16042, and replaces the sudden change of No. 401 locational glycine residues with alanine residue, wherein, described position begins to count from the N-end).Estimate, even have the lysC of the sequence identical with the lysC of e. coli k-12 JC411 bacterial strain, the lysC of the sudden change with the feedback inhibition desensitizationization that can make L-Methionin also can be provided, prerequisite is to have imported any one sudden change of mentioning in above-mentioned (a)-(1), or replace the amino-acid residue that is equivalent to No. 323 locational glycine residues, and be equivalent to the sudden change of the amino-acid residue of No. 318 locational methionine residues with another kind of amino-acid residue replacement with another kind of amino-acid residue (preferred asparagicacid residue).

For example, can obtain the DNA of mutant AKIII of the feedback inhibition desensitizationization of its L-Methionin of coding in order to following method.At first, the DNA that contains wild-type AKIII gene or have other AKIII gene of sudden change is carried out vitro mutagenesis handle, and the DNA that will carry out mutagenic treatment is connected with the carrier DNA compatible with the host, so that prepare recombinant DNA.Recombinant DNA is imported host microorganism, obtain transformant, and from these transformant, select to become to express the transformant of mutant AKIII.Such transformant has mutant AKIII gene.Perhaps, the DNA that contains wild-type AKIII gene or have other AKIII gene of sudden change can be connected with the carrier DNA compatible with the host, so that the preparation recombinant DNA.Then, can carry out vitro mutagenesis to recombinant DNA and handle, and it is imported host microorganism, obtain transformant, and from these transformant, select to become to express the transformant of mutant AKIII.Such transformant has this mutated genes equally.

In addition, can carry out mutagenic treatment,, then, can from this mutant strain, obtain mutated genes so that preparation can produce the mutant strain of mutant enzyme to the microorganism that can produce wild-type enzyme.The example that is used for dna direct is carried out the reagent of mutagenic treatment comprises azanol etc.Azanol is can be by cytosine(Cyt) is become N ⁴The chemomorphosis treatment agent that-hydroxyl cytosine(Cyt) causes cytosine(Cyt) to be replaced by thymus pyrimidine.When microorganism itself was carried out mutagenic treatment, this processing was to carry out with ultraviolet radiation or the mutagenic compound such as N-methyl-N '-nitro-N-nitrosoguanidine (NTG) that are usually used in induced mutations.

As the bacterium of the DNA that other AKIII gene that contains wild-type AKIII gene or have sudden change can be provided, can use any microorganism that belongs to Escherichia.Specifically, can use the microorganism of in following document, mentioning: Neidhardt etc. (Neidhardt, F.C. etc., intestinal bacteria and Salmonella typhimurium, U.S. microbiology association, Washington D.C., 1208, table 1).For example, can use e. coli jm109 bacterial strain and MC1061 bacterial strain etc.

In bacterium of the present invention, E.C. 2.7.2.4., the dihydrodipicolinate reductase, tetrahydropyridine dicarboxylic acid succinyl enzyme, succinyl-diaminopimelic acid deacylase, Phosphoenolpyruvate carboxylase and aspartate-semialdehyde dehydrogenase preferably come from the Escherichia bacterium respectively, Reduced nicotinamide-adenine dinucleotide transhydrogenase and L-Aspartase (if any) preferably come from the Escherichia bacterium respectively, the dihydrodipicolinic acid synthase preferably comes from Escherichia bacterium or brevibacterium sp bacterium, and diaminopimelate dehydrogenase preferably comes from the brevibacterium sp bacterium.

The example of brevibacterium sp bacterium also comprises brevibacterium flavum, Brevibacterium divaricatum, Corynebacterium glutamicum and lily hedysarum scoparium bacillus etc. except brevibacterium.

In addition, in bacterium of the present invention, preferably the feedback inhibition by introducing its L-Methionin by the dihydro pimelic acid synthetic enzyme of desensitization with and the feedback inhibition of L-Methionin by the E.C. 2.7.2.4. of desensitization, strengthen the intracellular reactive of dihydrodipicolinic acid synthase and E.C. 2.7.2.4., and, by importing the intracellular reactive of diaminopimelate dehydrogenase gene enhancing diaminopimelate dehydrogenase.Above-mentioned preferred bacterium of the present invention can obtain by plasmid pCABD2 or the pCABDE1 importing Escherichia bacterium that will mention among the international publication number WO95/16042.

＜2〉production method of the present invention

Can effectively produce L-Methionin by the following method: in suitable culture medium, cultivate the bacterium of the present invention that obtains as stated above,, and from culture, reclaim L-Methionin so that in culture, produce and accumulation L-Methionin.

Being used to cultivate the substratum of bacterium of the present invention, can be the substratum commonly used that contains carbon source, nitrogenous source, mineral ion and other required organic trace nutrient.

As carbon source, can use carbohydrate such as glucose, lactose, semi-lactosi, fructose and starch hydrolysate; Alcohols such as glycerine and Sorbitol Powder; Or such as the acids of fumaric acid, citric acid and succsinic acid.

As nitrogenous source, can use inorganic ammonium salt such as ammonium sulfate, ammonium chloride or ammonium phosphate; Organonitrogen such as soya hydrolysate; Ammonia; Or ammoniacal liquor.

As organic trace nutrient, preferably add appropriate amount such as vitamins B ₁Necessary material with L-Isoleucine, yeast extract etc.In addition, also to add small amounts of phosphoric acid potassium, sal epsom, iron ion and mn ion etc.

Cultivate and preferably under aerobic conditions, carried out 16-72 hour.Between incubation period, culture temperature can be controlled at 20-45 ℃, pH can be controlled at 5-8.Can be with inorganic or organic, acidity or alkaline matter, and ammonia is used for pH regulator.

Collection L-Methionin is normally undertaken by the combination of ion exchange resin method, intermediate processing and other known technology from fermented liquid.

Embodiment

Below in conjunction with following examples the present invention is further described in detail.

Example 1

＜1〉prepares Escherichia bacterium with various features

Following plasmid is imported intestinal bacteria W3110 (tyrA).

The gene that the plasmid title is comprised

RSF24P??????????dapA ^*

RSFD80??????????dapA ^*，lysC ^*

pCAB1???????????dapA ^*，lysC ^*，dapB

pCABD2??????????dapA ^*，lysC ^*，dapB，ddh

pCABD(B)????????Brev.dapA，lysC ^*，dapB，ddh

pCABDE1?????????dapA ^*，lysC ^*，dapB，dapD，dapE

The shortenings that is used for gene has following implication.

Ppc: Phosphoenolpyruvate carboxylase

LysC: E.C. 2.7.2.4. III

LysC ^*: to the E.C. 2.7.2.4. III of restraining effect desensitizationization

Asd: aspartate-semialdehyde dehydrogenase

DapA: dihydrodipicolinic acid synthase

DapA ^*: to the dihydrodipicolinic acid synthase of restraining effect desensitizationization

Brev.dapA: to dihydrodipicolinic acid synthase's (coming from brevibacterium) of restraining effect desensitizationization

DapB: dihydrodipicolinate reductase

DapD: tetrahydropyridine dicarboxylic acid succinyl enzyme

DapE: succinyl-diaminopimelic acid deacylase

Ddh: diaminopimelate dehydrogenase (coming from brevibacterium)

Plasmid RSF24P, RSFD80, pCAB1, pCABD2 and pCABDE1 are disclosed among the international publication number WO95/16042.The structure of these plasmids is disclosed among the international publication number WO95/16042 equally, and is as mentioned below.

(1)RSF24P

According to known intestinal bacteria dapA nucleotide structure (bacteriology magazine, 166,297,1986), contain the SD sequence of dapA and the fragment of open reading-frame (ORF) (ORF) by pcr amplification.The fragment of amplification is connected on the cloning vector pCR1000, obtains plasmid pdapA2, wherein, connected dapA, make the transcriptional orientation of dapA and pCR1000 go up the transcriptional orientation opposite that the lacZ promotor instructs.With azanol plasmid pdapA2 is carried out mutagenic treatment, and will carry out the pdapA2 importing intestinal bacteria W3110 of mutagenic treatment.From transformant, select to have the transformant of AEC resistance.In addition, measured the inhibition degree of L-Methionin, and selected bacterial strain the restraining effect desensitizationization of L-Methionin to the plasmid-encoded DDPS that resistant strain had of selection.Plasmid pdapA24 (confirming that by order-checking No. 597 locational C become T) is connected on the pVIC40, is positioned at tetracycline resistance gene promotor downstream, so that obtain RSF24P (Fig. 1).

The e. coli jm109 bacterial strain that has imported RSF24P is named as AJ12395, and be deposited in (the postcode 305-8566 of life engineering Industrial Technology Research Institute of Govement Industrial Research Inst., Ministry of Commerce on October 28th, 1993,1-3Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan), the preserving number of acquisition is FERM P-13935.Then, according on November 1st, 1994 that is specified in of budapest treaty transferring international preservation to, the preserving number that is obtained is FERM BP-4858.

(2)RSFD80

According to known intestinal bacteria lysC nucleotide sequence (bacteriology magazine, 261,1052,1986), contain the SD sequence of lysC and the fragment of open reading-frame (ORF) (ORF) by pcr amplification.The fragment of amplification is connected on the multi-copy vector pUC18, obtains plasmid pLYSC1, wherein, connected lysC, make the transcriptional orientation opposite of lacZ on transcriptional orientation and the pUC18 of lysC.With azanol plasmid pLYSC1 is carried out mutagenic treatment, and will carry out the pLYSC1 importing intestinal bacteria GT3 of mutagenic treatment.From transformant, select to have the transformant of AEC resistance.In addition, pLYSC1 is imported intestinal bacteria MC1061, then, carry out mutagenic treatment, select to have the transformant of AEC resistance and L-Methionin resistance with the azanol pair cell.In addition, the L-Methionin of having measured the plasmid-encoded AK that resistant strain had that selects suppresses degree and thermostability, and selects the restraining effect desensitizationization of L-Methionin, the bacterial strain with remarkable stability.With plasmid pLYSC1 ^*80 (confirming that by order-checking No. 352 locational C become T) are connected on the pHSG399, are positioned at lacZ promotor downstream, so that obtain pLLC ^*80.As shown in Figure 2, by pLLC ^*80 and RSF24P make up RSFD80.

The e. coli jm109 bacterial strain that has imported RSFD80 is named as AJ12396, and be deposited in (the postcode 305-8566 of life engineering Industrial Technology Research Institute of Govement Industrial Research Inst., Ministry of Commerce on October 28th, 1993,1-3Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan), the preserving number of acquisition is FERM P-13936.Then, according on November 1st, 1994 that is specified in of budapest treaty transferring international preservation to, the preserving number that is obtained is FERM BP-4859.

(3)pCAB1

According to known dapB nucleotide sequence (Bouvier, J. etc., journal of biological chemistry, 259,14829,1984), by PCR by intestinal bacteria W3110 strain chromosome DNA cloning dapB.With the amplification of DNA fragments of AseI and DraI digestion acquisition, with the fragment end-filling that obtains, and the SmaI site of inserting pMW119, so that obtain plasmid pdapB.Then, as shown in Figure 3, dapB is imported RSFD80, so that obtain pCAB1.

(4)pCABD2

According to known Corynebacterium glutamicum ddh nucleotide sequence (Ishino, S. etc., nucleic acids research, 15,3917,1987), by PCR by brevibacterium ATCC13869 chromosomal DNA amplification ddh.With the amplification of DNA fragments of EcoT22I and AvaI digestion acquisition, with the fragment end-filling that obtains, and the SmaI site of inserting pMW119, so that obtain plasmid pddh.Then, as shown in Figure 4, ddh is imported pCAB1, so that obtain pCABD2.

(5)pCABDE1

According to known dapD nucleotide sequence (Richaud, C. etc., journal of biological chemistry, 259,14824,1984), by PCR by intestinal bacteria W3110 strain chromosome DNA cloning dapD.With the amplification of DNA fragments of EcoO109I and SacI digestion acquisition, with the fragment end-filling that obtains, and the SmaI site of inserting pMW118, so that obtain plasmid pdapD.In addition, according to known dapE nucleotide sequence (Bouvier, J. etc., bacteriology magazine, 174,5265,1992), by PCR by intestinal bacteria W3110 strain chromosome DNA cloning dapE.With the amplification of DNA fragments of MunI and BglII digestion acquisition, with the fragment end-filling that obtains, and the SmaI site of inserting pMW118, so that obtain plasmid pdapE.In addition, dapE is excised from pdapE, and be inserted among the pdapD, so that obtain to contain the plasmid pMWdapDE1 of dapE and dapD.As shown in Figure 5, the fragment that will contain dapE and dapD is excised from pMWdapDE1, and inserts among the pCAB1, so that obtain pCABDE1.

Plasmid pCABD (B) makes up in accordance with the following methods.

At first, with primer, contain the dna fragmentation in the promotor site of tetracycline resistance gene by the pBR322 amplification with following sequence.

5’-TCAAGAATCTTCATGTTTGA-3’(SEQ?ID?NO：1)

5’-GTTAGATTTGGTACCCGGTGCCTGACTGCGTTAGC-3’(SEQ?IDNO：2)

With the dna fragmentation of KpnI and EcoRI digest amplification, and insert between the KpnI and EcoRI cracking site of pUC18, obtain pUTES (Figure 18).

Then, use primer, make template, amplification Brev.dapA gene with the chromosomal DNA of brevibacterium Ysr bacterial strain with following sequence.

5’-GGTTGTGGTACCCCCAAATGAGGGAAGAAG-3’(SEQ?ID?NO：3)

5’-TGGAACCTCTGTTGCTGCAG-3’(SEQ?ID?NO：4)

With the Brev.dapA gene of KpnI and BamHI digest amplification, and insert between the KpnI and BamHI cracking site of pUTES, obtain pUEBL3 (Figure 19).

Then, with EcoRI and XbaI digestion pUEBL3, and the two ends benefit is flat, acquisition contains the fragment of Brev.dapA.Then, digest the pCABD2 that in international publication number WO95/16042, discloses, and two ends are mended flat with NheI and SpeI.Collection contains lysC, and the fragment of dpaB and ddh is inserted the fragment that contains Brev.dapA that obtains previously wherein again, obtains pCABD (B) (Figure 20).

Although in European Patent Publication No 488424, intestinal bacteria W3110 (tyrA) is had been described in detail, will explain the main points briefly to its preparation method below.Intestinal bacteria W3110 bacterial strain is that (Shizuoka-ken Mshima-shi) obtains from national genetic institute.This inoculation to the LB flat board that contains spectinomycin, and is selected to form the bacterial strain of bacterium colony, obtain the spectinomycin resistant strain.The spectinomycin resistant strain of selecting is mixed with e. coli k-12 ME8424 bacterial strain, and (the L-nutrient solution: 1% bacterium is used tryptone at perfect medium with static culture form, 0.5% yeast extract, 0.5% sodium-chlor) in, cultivated 15 minutes down at 37 ℃, so that induce joint.E. coli k-12 ME8424 bacterial strain has HfrPO45, thi, and relA1, tyrA::Tn10, the proterties of ung-1 and nadB, and can obtain from national genetic institute.

Then, culture is inoculated in the perfect medium (the L-nutrient solution: 1% bacterium is used tryptone, 0.5% yeast extract, 0.5% sodium-chlor, 1.5% agar) that contains spectinomycin, tsiklomitsin and L-tyrosine, and selects to form the bacterial strain of bacterium colony.This bacterial strain is named as intestinal bacteria W3110 (tyrA) bacterial strain.

In European Patent Publication No 488424, disclosed by plasmid being imported W3110 (tyrA) a lot of bacterial strains that bacterial strain produced.For example, the bacterial strain that is obtained by importing plasmid pHATerm is named as intestinal bacteria W3110 (tyrA)/pHATerm bacterial strain (intestinal bacteria AJ12662 bacterial strain), and be deposited in (the postcode 305-8566 of life engineering Industrial Technology Research Institute of Govement Industrial Research Inst., Ministry of Commerce as international preservation on November 16th, 1991,1-3Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan), the preserving number that is obtained is FERM BP-3653.Also can obtain W3110 (tyrA) bacterial strain by from intestinal bacteria W3110 (tyrA)/pHATerm bacterial strain, eliminating plasmid pHATerm.The elimination of plasmid can be finished with ordinary method.

＜2〉import aspartate-semialdehyde dehydrogenase gene (asd), Phosphoenolpyruvate carboxylase gene (ppc), or L-Aspartase gene (aspA), and assessment L-Methionin productivity

As plasmid that contains asd and the plasmid that contains ppc, can use the ppc that is disclosed among the international publication number WO95/16042, pasd, and pppc.In international publication number WO95/16042, disclosed the structure of these plasmids in detail.Be summarized as follows.

(1)pasd

Plasmid asd obtains from the plasmid pAD20 that contains described gene (Haziza, C. etc., EMBO, 1,379,1982).With AseI and ClaI digested plasmid pAD20, with end-filling, and the SmaI site of inserting pMW118, obtain plasmid pasd (Fig. 8).

(2)pppc

Plasmid pppc obtains from the plasmid pT2 that contains described gene.With SmaI and ScaI digested plasmid pT2, with end-filling, and the SmaI site of inserting pMW118, obtain plasmid pppc (Fig. 9).Intestinal bacteria F15 bacterial strain (AJ12873) with pT2 is deposited in (the postcode 305-8566 of life engineering Industrial Technology Research Institute of Govement Industrial Research Inst., Ministry of Commerce on July 15th, 1993,1-3Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan), the preserving number of acquisition is FERM P-13752.Then, according on July 11st, 1994 that is specified in of budapest treaty transferring international preservation to, the preserving number that is obtained is FERM BP-4732.

Make up the plasmid that contains aspA by the following method.

Use has the primer amplification intestinal bacteria aspA gene of following sequence.

5’-TGATCAGCGAAACACTTTTA-3’(SEQ?ID?NO：5)

5’-CAGCAAACTATGATGAGAA-3’(SEQ?ID?NO：6)

Then, the amplified fragments that obtains is inserted the SmaI cracking site of pMW118 (Nippon Gene), obtain pMW118::aspA (Fig. 9).

Among pMW118 (control plasmid), pasd, pppc and the pMW118::aspA (relatively plasmid) each imported intestinal bacteria W3110 (tyrA) respectively and in above-mentioned＜1 in the transformant that obtained.Except by pMW118, pasd, pppc or pMW118::aspA being imported the transformant that intestinal bacteria W3110 (tyrA) are obtained, the transformant that is obtained all contains two class plasmids, promptly, one of pMW118, pasd, pppc and pMW118::aspA, and one of RSF24P, RSFD80, pCAB1, pCABD2, pCABD (B) and pCABDE1.By being disclosed in the method among the international publication number WO95/16042, measure the L-Methionin productivity of described transformant.Concrete grammar is as follows.

Under 37 ℃, culturing cell is 30 hours in 500 milliliters of Sakaguchi flasks that 20 milliliters of substratum with following composition are housed, and stirs with the speed of 114-116rpm.

(substratum composition)

Glucose 40g/l

7 water magnesium sulfate 1g/l

Ammonium sulfate 16g/l

Potassium primary phosphate 1g/l

7 water ferric sulfate 0.01g/l

5 water manganous sulfate 0.01g/l

Yeast extract (Difco) 2g/l

L-tyrosine 0.1g/l

Adjust to pH7.0 with KOH, and at 115 ℃ of following autoclavings 10 minutes (glucose and 7 water magnesium sulfates are sterilized respectively).

Lime carbonate 25g/l

(according to Japanese Pharmacopoeia, 118 ℃ of following dry sterilizations 2 days)

Microbiotic (adding 20mg/l spectinomycin, 50mg/l penbritin or 25mg/l kantlex) according to the type that will import plasmid

With Asahi Chemical Industry Co., the Biotech AnalyzerAS210 that Ltd produces carries out quantitative assay to the L-Methionin in the nutrient solution (substratum after the cultivation).

The result is as shown in table 1.In table, the amount of L-Methionin is shown with the milligram numerical table in every dl substratum.

Table 1

N.d.: undetermined

From table 1, can be clear that, when in intestinal bacteria, using dapA (RSF24P), dapA+lysC (RSFD80), or dapA+lysC+dapB (pCAB1) is when strengthening asd or ppc individually or simultaneously, the output of L-Methionin (accumulation volume) does not change or has only little change, and, for asd, with asd or ppc wild phase ratio not, it might reduce (asd is-180-20mg/dl, and that ppc is 10-30mg/dl).On the contrary, if strengthen them simultaneously with dapA+lysC+dapB+ddh (pCABD2) or dapA+lysC+dapB+dapD+dapE (pCABDE1), with asd or ppc wild phase ratio not, find that the output of L-Methionin significantly improves (asd is 70mg/dl, and that ppc is 90mg/dl).But, when strengthening aspA, do not find that the L-lysine production significantly improves with dapA+lysC+dapB+ddh (pCABD2).In addition, promptly use the dapA come from brevibacterium to replace and come from colibacillary dapA (pCABD (B)), also to come from colibacillary dapA (pCABD2) identical with use for the effect that is obtained.Therefore, it is important that the source of this gene is not considered to, and still, their combination is important.

Example 2

＜1〉structure contains Phosphoenolpyruvate carboxylase gene (ppc) and aspartate-semialdehyde dehydrogenase gene (asd), the plasmid of transhydrogenase gene (pntAB) or L-Aspartase gene (aspA)

Make up the plasmid that contains ppc and asd in accordance with the following methods, contain the plasmid of ppc and pntAB and contain ppc and the plasmid of aspA.

(1) contains the plasmid (ppcd) of ppc and asd

Digest the plasmid pasd that is disclosed among the international publication number WO95/16042 with KpnI and SphI, will contain two end-fillings of the dna fragmentation of asd.Then, digest the plasmid pppc that is disclosed among the international publication number WO95/16042 with XbaI, with two end-fillings, and the asd fragment that will obtain in advance insertion wherein, so that obtain ppcd (Figure 10).

(2) contain the plasmid (pPTS) of asd and pntAB

Digest the plasmid pMW::THY that is disclosed among the international publication number WO95/11985 with SmaI and HindIII, and collect the dna fragmentation that contains pntAB.Then, be disclosed in pppc among the international publication number WO95/16042,, and further digest, the pntAB fragment that obtains is previously inserted cracking site, so that obtain pPTS (Figure 11) with HindIII with two end-fillings with XbaI digestion.

The structure of plasmid pMW::THY is disclosed among the international publication number WO95/11985 in detail.Be summarized as follows.

According to known intestinal bacteria pntA and pntB nucleotide sequence (D.M.Clarke etc., european journal of biological chemistry, 158,647-653,1986), contain the fragment that comprises these two genes by pcr amplification with promoter activity.With the dna fragmentation of BamHI and HindIII digest amplification, and the dna fragmentation that is obtained is connected on the plasmid vector pMW118 (Nippon Gene) that digest with BamHI and HindIII, so that acquisition pMW::THY (Fig. 8)

The e. coli jm109 bacterial strain that has imported pMW::THY is named as AJ12929, and be deposited in (the postcode 305-8566 of life engineering Industrial Technology Research Institute of Govement Industrial Research Inst., Ministry of Commerce on October 4th, 1993,1-3Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan), the preserving number of acquisition is FERM P-13890.Then, according on September 14th, 1994 that is specified in of budapest treaty transferring international preservation to, the preserving number that is obtained is FERM BP-4798.

(3) contain the plasmid (pAPW) of ppc and aspA

With SacI digestion be disclosed in the above-mentioned example 1 in plasmid pMW118::aspA, with two end-fillings, and further digest, so that obtain to contain the dna fragmentation of aspA with HindIII.Then, be disclosed in pppc among the international publication number WO95/16042,, and further digest, the aspA fragment that obtains is previously inserted the HindIII cracking site, so that obtain pAPW (Figure 12) with HindIII with two end-fillings with XbaI digestion.

＜2〉import two types gene, and assess L-Methionin productivity

Respectively the importing that is obtained to above-mentioned example 1 import pppc (with reference to plasmid) in the transformant of pCABD2, ppcd, each among pPTS and the pAPW (relatively plasmid).The transformant that is obtained contains two class plasmids, i.e. pppc, ppcd, pPTS, one of pAPW and pCABD2.With with example 1＜2 identical method measures the L-Methionin productivity of described transformant.

The result is as shown in table 2.

Table 2

	Methionin accumulation volume (mg/dl)
		??pCABD2+pppc	??1380
??pCABD2+ppcd	??1460
		??pCABD2+pPTS	??1450
??pCABD2+pAPW	??1390

From table 2, can be clear that, when strengthening asd or pntAB simultaneously, find that the output of L-Methionin significantly improves (that asd is 80mg/dl, and that pntAB is 70mg/dl) with dapA+lysC+dapB+ddh+ppc (ppcd or pPTS).But,, promptly use dapA+lysC+dapB+ppc (pAPW) to strengthen aspA simultaneously, do not find that also the L-lysine production significantly improves for aspA.

Example 3

＜1〉structure contains Phosphoenolpyruvate carboxylase gene (ppc), the plasmid of transhydrogenase gene (pntAB) and aspartate-semialdehyde dehydrogenase gene (asd) or L-Aspartase gene (aspA)

Make up in accordance with the following methods and contain ppc, the plasmid of pntAB and asd gene and contain ppc, the plasmid of pntAB and aspA.

(1) contains ppc, the plasmid of pntAB and asd (pPTd)

Digest the plasmid pasd that is disclosed among the international publication number WO95/16042 with KpnI and SphI, will contain two end-fillings of the dna fragmentation of asd.Then, digest the plasmid pPTS that is disclosed in the above-mentioned example 2 with HindIII, with two end-fillings, and the asd fragment that will obtain in advance insertion HindIII cracking site, so that obtain pPTd (Figure 13).

(2) contain ppc, the plasmid of pntAB and aspA (pAPT)

Digest the plasmid pMW::THY that is disclosed among the international publication number WO95/11985 with SmaI and HindIII, to obtain to contain the dna fragmentation of pntAB.Then, be disclosed in plasmid pAPW in the above-mentioned example 2,, and further digest with HindIII with two end-fillings with XbaI digestion.The fragment that contains pntAB that obtains is previously inserted the HindIII cracking site, so that obtain pAPT (Figure 14).

＜2〉import three types gene, and assess L-Methionin productivity

Respectively the importing that is obtained to above-mentioned example 1 import pPTS (with reference to plasmid), pPTd or pAPT in the transformant of pCABD2.The transformant that is obtained contains two class plasmids, i.e. pPTS, one of pPTd and pAPT, and pCABD2.With with example 1＜2 identical method measures the L-Methionin productivity of described transformant.

The result is as shown in table 3.

Table 3

	Methionin accumulation volume (mg/dl)
		??pCABD2+pPTS	??1450
??pCABD2+pPTd	??1510
		??pCABD2+pAPT	??1500

From table 3, can be clear that, when strengthening asd or aspA simultaneously, find that the output of L-Methionin significantly improves (that asd is 60mg/dl, and that aspA is 50mg/dl) with dapA+lysC+dapB+ddh+ppc+pntAB (pPTd or pAPT).

Example 4

＜1〉structure contains Phosphoenolpyruvate carboxylase gene (ppc), transhydrogenase gene (pntAB), the plasmid of aspartate-semialdehyde dehydrogenase gene (asd) and L-Aspartase gene (aspA)

Make up in accordance with the following methods and contain ppc, pntAB, the plasmid of asd and aspA.

Digest the plasmid pAPT that is disclosed in the above-mentioned example 3 with HindIII,, further digest, so that obtain to contain ppc, the dna fragmentation of pntAB and aspA with SmaI with two end-fillings.Then, digest pMW218 (Nippon Gene) with EcoRI, with two end-fillings, and will obtain in advance contain ppc, the dna fragmentation of aspA and pntAB inserts the EcoRI cracking site of end-filling, so that obtain pAPTK (Figure 15).

Then, digest the plasmid pasd that is disclosed among the international publication number WO95/16042,, further digest, so that obtain to have the dna fragmentation of asd with BamHI with two end-fillings with KpnI.Then, digest pSTV29 with PstI, with two end-fillings, and the asd fragment that will obtain in advance insertion BamHI cracking site, so that obtain pSd (Figure 16).

Then, digest pSd,, further digest, so that obtain to contain the dna fragmentation of asd once more with XbaI with two end-fillings with SphI.Then, digest pAPTK with SacI, with two end-fillings, and the asd fragment that will obtain in advance insertion XbaI cracking site, so that obtain pKD (Figure 17).

＜2〉import four types gene, and assess L-Methionin productivity

Respectively to importing that above-mentioned example 1 is obtained import pAPT (with reference to plasmid 1), pAPTK (with reference to plasmid 2) or pKD in the transformant of pCABD2, pCABD (B) or pCABDE1.The transformant that is obtained contains two class plasmids, i.e. pAPT, one of pAPTK and pKD, and pCABD2, one of pCABD (B) and pCABDE1.With with example 1＜2 identical method measures the L-Methionin productivity of described transformant.

The result is as shown in table 4.

Table 4

	Methionin accumulation volume (mg/dl)
		??pCABD2+pAPT	??1500
??pCABD2+pAPTK	??1500
		??pCABD2+pKD	??1600
??pCABD(B)+pKD	??1590
		??pCABDE1+pKD	??1580

From table 4, can be clear that, when strengthening asd simultaneously, find that the output of L-Methionin significantly improves with dapA+lysC+dapB+ddh+ppc+pntAB+aspA.When substituting pCABD2, obtained similar results with pCABD (B) or pCABDE1.

Plasmid pAPTK in the table 4 is equivalent to the drug resistant gene of anti-penbritin is changed over the plasmid pAPT (because this carrier has become pMW218 from pMW118) of anti-kantlex drug resistant gene.Because pKD is prepared into by asd is inserted pAPTK, think that therefore pAPTK is more suitable for contrast as pKD than pAPT.Therefore, the data of pAPTK are provided simultaneously.Even also confirmed to change the output that drug resistant gene also can not influence L-Methionin.

Industrial applicibility

The invention provides the Escherichia bacterium with high 1B productivity, and, the 1B of high yield can be obtained by this bacterium.

Claims (24)

1. Escherichia bacterium, wherein (1) dihydrodipicolinic acid synthase, E.C. 2.7.2.4. and dihydrodipicolinate reductase's intracellular reactive strengthens, (2) intracellular reactive of the intracellular reactive of diaminopimelate dehydrogenase or tetrahydropyridine dicarboxylic acid succinyl enzyme and succinyl-diaminopimelic acid deacylase strengthens, wherein, the intracellular reactive of aspartate-semialdehyde dehydrogenase or Phosphoenolpyruvate carboxylase strengthens.

2. Escherichia bacterium, wherein (1) dihydrodipicolinic acid synthase, E.C. 2.7.2.4. and dihydrodipicolinate reductase's intracellular reactive strengthens, (2) intracellular reactive of the intracellular reactive of diaminopimelate dehydrogenase or tetrahydropyridine dicarboxylic acid succinyl enzyme and succinyl-diaminopimelic acid deacylase strengthens, wherein, the intracellular reactive of the intracellular reactive of Phosphoenolpyruvate carboxylase and Reduced nicotinamide-adenine dinucleotide transhydrogenase or aspartate-semialdehyde dehydrogenase strengthens.

3. Escherichia bacterium, wherein (1) dihydrodipicolinic acid synthase, E.C. 2.7.2.4. and dihydrodipicolinate reductase's intracellular reactive strengthens, (2) intracellular reactive of the intracellular reactive of diaminopimelate dehydrogenase or tetrahydropyridine dicarboxylic acid succinyl enzyme and succinyl-diaminopimelic acid deacylase strengthens, wherein, the intracellular reactive of the intracellular reactive of Phosphoenolpyruvate carboxylase and Reduced nicotinamide-adenine dinucleotide transhydrogenase and aspartate-semialdehyde dehydrogenase or L-Aspartase strengthens.

4. the bacterium of claim 3, wherein, the intracellular reactive of described aspartate-semialdehyde dehydrogenase and L-Aspartase strengthens.

5. claim 3 or 4 bacterium, wherein, E.C. 2.7.2.4., dihydrodipicolinate reductase, tetrahydropyridine dicarboxylic acid succinyl enzyme, succinyl-diaminopimelic acid deacylase, Phosphoenolpyruvate carboxylase and aspartate-semialdehyde dehydrogenase come from the Escherichia bacterium respectively, if exist, Reduced nicotinamide-adenine dinucleotide transhydrogenase and L-Aspartase come from the Escherichia bacterium respectively, the dihydrodipicolinic acid synthase comes from Escherichia bacterium or brevibacterium sp bacterium, and diaminopimelate dehydrogenase comes from the brevibacterium sp bacterium.

6. each bacterium among the claim 1-5, wherein, the intracellular reactive of intracellular reactive enhanced enzyme is to have the plasmid of gene of described enzyme and enhanced by importing.

7. each bacterium among the claim 1-5, wherein, the intracellular reactive of intracellular reactive enhanced enzyme is enhanced by the gene copy quantity that increases the above enzyme of karyomit(e).

8. each bacterium among the claim 1-5, wherein, the intracellular reactive of intracellular reactive enhanced enzyme is enhanced by the promoter sequence of the gene that changes the above enzyme of karyomit(e).

9. each bacterium among the claim 1-8, wherein, feedback inhibition by obtaining its L-Methionin by the dihydrodipicolinic acid synthase of desensitization with and the feedback inhibition of L-Methionin by the E.C. 2.7.2.4. of desensitization, strengthen the intracellular reactive of dihydrodipicolinic acid synthase and E.C. 2.7.2.4., and, by importing the diaminopimelate dehydrogenase gene, strengthen the intracellular reactive of diaminopimelate dehydrogenase.

10. each bacterium among the claim 1-8, wherein said Escherichia bacterium is intestinal bacteria (Escherichia coli).

11. a method of producing L-Methionin, this method are included in the suitable medium bacterium of cultivating among the claim 1-10 each, so that produce in culture and accumulation L-Methionin, and collect L-Methionin from culture.

12. method of producing L-Methionin; this method is included in and cultivates the intestinal bacteria bacterium in the suitable medium; so that in culture, produce and accumulation L-Methionin; and from culture, collect L-Methionin, wherein said intestinal bacteria bacterium is carried the gene of coding dihydrodipicolinic acid synthase, E.C. 2.7.2.4., dihydrodipicolinate reductase, diaminopimelate dehydrogenase and Phosphoenolpyruvate carboxylase in identical plasmid or different plasmid.

13. the method for claim 12, the gene of the aspartate-semialdehyde dehydrogenase of wherein encoding is further introduced in the described bacterium by plasmid.

14. the method for claim 12, the gene of the Reduced nicotinamide-adenine dinucleotide transhydrogenase of wherein encoding is further introduced in the described bacterium by plasmid.

15. the method for claim 12, the gene of the gene of the aspartate-semialdehyde dehydrogenase of wherein encoding and coding Reduced nicotinamide-adenine dinucleotide transhydrogenase is further introduced in the described bacterium by plasmid.

16. the method for claim 14, the gene of the L-Aspartase of wherein encoding is further introduced in the described bacterium by plasmid.

17. the method for claim 16, the gene of the aspartate-semialdehyde dehydrogenase of wherein encoding is further introduced in the described bacterium by plasmid.

18. method of producing L-Methionin; this method is included in and cultivates the intestinal bacteria bacterium in the suitable medium; so that in culture, produce and accumulation L-Methionin; and from culture, collect L-Methionin, wherein said intestinal bacteria bacterium is carried the gene of coding dihydrodipicolinic acid synthase, E.C. 2.7.2.4., dihydrodipicolinate reductase, diaminopimelate dehydrogenase and aspartate-semialdehyde dehydrogenase in identical plasmid or different plasmid.

19. method of producing L-Methionin; this method is included in and cultivates the intestinal bacteria bacterium in the suitable medium; so that in culture, produce and accumulation L-Methionin; and from culture, collect L-Methionin, wherein said intestinal bacteria bacterium is carried the gene of coding dihydrodipicolinic acid synthase, E.C. 2.7.2.4., dihydrodipicolinate reductase, tetrahydropyridine dicarboxylic acid succinyl enzyme, succinyl-diaminopimelic acid deacylase and aspartate-semialdehyde dehydrogenase in identical plasmid or different plasmid.

20. method of producing L-Methionin; this method is included in and cultivates the intestinal bacteria bacterium in the suitable medium; so that in culture, produce and accumulation L-Methionin; and from culture, collect L-Methionin, wherein said intestinal bacteria bacterium is carried the gene of coding dihydrodipicolinic acid synthase, E.C. 2.7.2.4., dihydrodipicolinate reductase, tetrahydropyridine dicarboxylic acid succinyl enzyme, succinyl-diaminopimelic acid deacylase and Phosphoenolpyruvate carboxylase in identical plasmid or different plasmid.

21. each method among the claim 12-20, wherein said dihydrodipicolinic acid synthase, E.C. 2.7.2.4., dihydrodipicolinate reductase, Phosphoenolpyruvate carboxylase, Reduced nicotinamide-adenine dinucleotide transhydrogenase, L-Aspartase and aspartate-semialdehyde dehydrogenase come from intestinal bacteria separately, and described diaminopimelate dehydrogenase comes from brevibacterium (Brevibacterium lactofermentum).

22. each method among the claim 12-20, wherein said dihydrodipicolinic acid synthase and E.C. 2.7.2.4. come from the intestinal bacteria bacterium separately, and comprise and make the feedback inhibition of L-Methionin by the sudden change of desensitization.

23. the method for claim 22, wherein said dihydrodipicolinic acid synthase comes from intestinal bacteria, and comprises by Xie Ansuan and replace 81 L-Ala and replaced the sudden change of 118 hyte propylhomoserins by tyrosine.

24. the method for claim 22, wherein said E.C. 2.7.2.4. comes from intestinal bacteria, and comprises the sudden change that is selected from the group that is made of following member:

A) replace 323 glycine by aspartic acid;

B) replace 323 glycine and replace 408 glycine by aspartic acid by aspartic acid;

C) replace 34 arginine and replace 323 glycine by halfcystine by aspartic acid;

D) replace 325 leucines by phenylalanine;

E) replace 318 methionine(Met)s by Isoleucine;

F) replace 318 methionine(Met)s and replace 349 Xie Ansuans by Isoleucine by methionine(Met);

G) replace 345 Serines by leucine;

H) replace 347 Xie Ansuans by methionine(Met);

I) replace 352 Threonines by Isoleucine;

J) replace 352 Threonines and replace 369 Serines by Isoleucine by phenylalanine;

K) replace 164 L-glutamic acid by Methionin;

L) replace 417 methionine(Met)s and replace 419 halfcystines by Isoleucine by tyrosine;

M) replace 323 glycine and replace 318 methionine(Met)s by aspartic acid by Isoleucine; And

N) above-mentioned two or more combination arbitrarily.

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