MXPA97007921A - Procedure for the production of l-aminoacidomediantefermentac - Google Patents

Abstract

A procedure is provided to produce an L-amino acid with good efficiency by fermentation, a DNA encoding aspartakinase III derived from bacteria of the genus Escherichia and suffering mutation by which the display is released by feedback with L-lysine, is introduced into cells to form transforming bacteria of the genus Escherichia, and these bacteria are incubated in an appropriate culture medium, an L-amino acid is produced and accumulated in the culture, and is collected from this culture.

Description

PROCEDURE FOR THE PRODUCTION OF L-AMINOACIDO BY FERMENTATION FIELD OF THE INVENTION The present invention is related to the microorganism industry. Very specifically, the present invention relates to a process for producing an acidic L-arnino through fermentation, and a flDN and a microorganism that are used in this process.

PREVIOUS TECHNIQUE When L-lysine is produced through fermentation, strains separated from the natural field or synthetic strains thereof are used to improve productivity. A large number of synthetic mutants producing L-lysine are known, and many of them are resistant to the inoethylcycysteine (AEC) mutants belonging to the genus Brevibacteriurn, Corynebacterium, Bacillus or Escherichia. In addition, transformants obtained using recombinant DNA are also employed (U.S. Patent No. 4,278,765). Therefore, a variety of technologies are described for increp > entar the productivity of amino acids. For example, with respect to the genus Escherichia, a procedure to produce L-lysine by increasing an acid dihydrodipicolinic mtetase (hereinafter abbreviated in some instances as "DDPS") is disclosed in Japanese Patent Laid-open (Kokai) No. 10,595 / 1901, U.S. Pat. No. 4,346,170 and Applied Microbiology and Biotechnology 15, 227 (1982). The dihydrodipicolmico acid smtetasa (DDPS) in an enzyme that catalyzes the dehydro-condensation of aspartosernialdehyde and pyruvic acid to form dihydrodipicoliic acid. This reaction is an entry for an L-lysos biosthesis system in the biosynthesis of an amino acid of the aspartic acid type. i, e knows that this enzyme has an important regulatory site for the biosynthesis of Lysane in bacteria of the Escherich genus together with asp rtocmasa. DDP5 is encoded in a gene called dapA in Escherichia coll (E. coll). This dapA has already been cloned and its base sequence has been determined CRichaud, F. and others, 3. Bactenol. 29? (1986)]. Meanwhile, aspartakinase (hereinafter sometimes abbreviated as "AK") is an enzyme that catalyzes a conversion reaction from aspartic acid to (3-phosphoaspartic acid, and is a major regulatory enzyme in the biosynthesis system of a amino acid of the aspartic acid type AK of E. col includes three types (AKI, AKII, AKIII), and both of these are enzymes conjugated with hornoserine dehydrogenase (hereinafter abbreviated sometimes as "HD"). One of these enzyme conjugates is AKI-HDI encoded in the thrfl gene, and the other is AKII-HDII encoded in the metLM gene. AKI undergoes concentrated suppression with treomna and isoleucine and inhibition with treo ina, and AKIT undergoes suppression with inetionma. Meanwhile, AKIII alone is a single-function enzyme, and is a product of a gene called LysC. He knows that he suffers suppression and inhibition by realization with L-sine.

The relation of activities of the same in cells in AKI: AKIII: flKIII approximately 5: 1: 4. This lysC from E. coll has already been cloned, and its base sequence has been determined [Cassan, M., Parsot, C, Cohen, G. N., and Patte., 3. C. 3. Biol. Chem. 261, 1052 (1986) 3. A method for producing L-lysine using E. col containing dapfl having mutation whereby inhibition is released by feedback with lysine and lysC having mutation by which inhibition is released by lysine feedback is described in the Open document to the International Public UO95 / 16042. When L-threonma is produced through fermentation, strains separated from the natural field or synthetic mutants thereof are used as microorganisms. A large number of synthetic L-threonine producing mutants have been known, and many of them are resistant to α-arnino-β-hydroxyvalenic acid, which belongs to the genus Escherich a. Serra ia, Brevibacteriurn or Corynebacteriurn. With respect to the genus Lscherichia, a method for producing L-threonma using a transfored strain with a recornbinating plasmid containing a treoru operon is disclosed in Japanese Patents open to the public (Kokai) Nos. 131, 397/1980, 31,691 / 1984 and 15,696 / 1981 and Japanese Patent Publication No. 501,682 / 1991. In addition, a process for producing L-threonine using E. col containing lysC having mutation by which inhibition is released by realization with Usin is described in International Open Document U094 / U517.

PROBLEMS THAT MUST BE DETERMINED BY THE INVENTION The present invention is directed to obtain AKIIJ derived from bacteria of the genus Escherichia in which the inhibition by realirnentación with L-lysine is well released, and to provide "a procedure to produce an L-arninoacid through fermentation, which is better than before .

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the preparation procedure of pMLYSC2. Figure 2 shows the procedure for preparation of plasmids of the RSFD series. Figure 3 shows the preparation procedure of pl_LC * Y6.

MEANS TO RESOLVE THE PROBLEMS The inventors of the present have conducted assiduously investigations to solve the aforementioned problems, and consequently have succeeded in obtaining a DNA encoding AKII derived from bacteria of the genus Escherichia in which inhibition by feedback with L-lysine is well released. A DNA encoding AKIII derived from bacteria of the genus Escherichia in which inhibition by feedback with L-lysine in well released is sometimes referred to as the mutant lysC gene or AKIII gene of the present specification. A DNA encoding DDPS derived from E. coli in which inhibition by feedback with L-lysine is well released is sometimes referred to as the mutant dapA gene or DDPS gene of the present specification. In addition, the inventors of the present invention have produced bacteria of the genus Escherichia which contain lysC mutant in cells, and have found that a considerable amount of L-lysine or L-threonine can be produced and can accumulate in the culture. That is, the present invention relates to DNA encoding aspartokinase III of bacteria belonging to the genus Escherichia and having in the coding region mutation by which they release inhibition by realirnentacion with lysm of said aspar + ocmasa III, said mutation being mutation by which the residue of rne-t omna 318 of aspartokinase III is replaced by another residue of amino acid and the residue of glycine 323 by another acid residue, mutation whereby the residue of leucma 325 is replaced by another amino acid and the residue of valma 347 by another amino acid residue, mutation by which the residue of glycine 323 is replaced by another amino acid residue and the valine residue 347 by another amino acid residue, mutation whereby the leucine residue 325 is replaced by another amino acid residue and the serine residue 345 by another amino acid residue, mutation whereby the glycine 323 residue is substituted by another amino acid residue and the serine 358 residue by another amino acid residue, mutation whereby the threo ina 344 residue is replaced by another amino residue acid, mutation by which the glutarnic acid residue 250 is replaced by another amino acid residue, mutation by which the glutarnic acid residue 346 is replaced by another amino acid residue and leucine residue 347 by another amino acid residue, mutation by which the glutarnic acid residue 250 is replaced by another amino acid residue and the residue of threonine 364 by another amino acid residue, mutation by which the acid residue aspar, ico 202 is replaced by another amino acid residue and the senna 321 residue by another amino acid residue, mutation by which the residue of argin na 283 is replaced by another amino acid residue, the alanine residue 333 by another amino acid residue, the sepna residue 338 by another amino acid residue, the glutarnic acid residue 346 by another amino acid residue and the aspargin residue of 414 for another amino acid residue, or mutation whereby the rne + ionin residue 318 is replaced by another amino acid residue, the sepna residue 321 for another amino acid residue, the valine 320 residue for another amino acid residue, the valm residue 349 by another amino acid residue and glutarnic acid residue 405 by another amino acid residue. Preferably, the present invention relates to a DNA encoding aspartocmase III of bacteria belonging to the genus Esche ichia and having in the coding region mutation by which inhibition is released by lysine feedback of said aspartokinase III, said mutation being mutation by wherein the residue of rnetionma 318 of aspartocmase III is replaced by a residue of isoleucine and the residue of glycine 323 by a residue of aspartic acid, mutation whereby the residue of leucma 325 is substituted by phenylalanine residue and the valine residue 347 by a residue of rnetionma, mutation by which the glycine residue 323 is replaced by aspartic acid residue and the valine residue 347 by a rnet omna residue, mutation by which the residue of leucma 325 is replaced by phenylalanine residue and the residue of serine 345 for a leucma residue, a mutation whereby the residue of glycine 323 is replaced by aspartic acid residue and the septa residue 358 by a residue of leucma, a mutation by which the residue of threo ina 344 is replaced by a residue of rnethionine, a mutation by which the glutarnic acid residue 250 is replaced by a lysine residue, a mutation by which the glutamic acid residue 346 is replaced by a lysine residue and the leucine residue 347 by a phenylalanine residue, a mutation whereby the residue of glutamic acid 250 is replaced by a lysine residue and the threonine residue 364 by a residue of rnetiomna, mutation whereby the aspartic acid residue 202 is replaced by a glycine residue and the serine residue 321 by a prolyma residue, mutation whereby the arginine residue 283 is replaced by a serine residue, the alanine residue 333 by a threonine residue, the serine residue 330 by a threonine residue, the glutaric acid residue 346 by a residue of aspartic acid and the asparagine residue 414 by a residue of serine, or mutation by which the residue of methionine 318 is replaced by a lysine residue, the serine residue 321 by a proline residue, the valine residue 328 by a phenylalanine residue, the valine residue 349 by a glycine residue and the glutamic acid residue 405 for a residue of valine. The present invention relates to the aforementioned recornbinant DNA which is produced by ligating the DNA of claim 1 with the vector DNA capable of undergoing autonomous replication in cells of bacteria of the genus Escherichia, as well as a microorganism of the genus Escherichia having this DNA . The present invention relates to a process for producing an L-amino acid comprising incubating the aforementioned microorganism having a capacity to produce an L-amino acid in a fermentation medium, producing and accumulating an L-arnino acid in the culture, and collecting the L-amino acid of the culture. In the present specification, a DNA encoding DDPS or AKIII or a DNA containing the same and a promoter is sometimes referred to as the "DDP gene". or "AKIII gene" in addition, a mutant enzyme in which the inhibition by Realinentacion with L-Lysine is released is sometimes called simply "a enzyme utante", and a DNA that encodes the same or a DNA containing the same and a promoter as a "mutant gene". Furthermore, "releasing the method by realization with L-lysma" means that the inhibition is substantially liberated and is not necessarily released completely. The present invention is described in detail below. < 1 > DNA encoding the aspartosumase mutant (AKIII) of the present invention. The DNA encoding mutant aspartocmase (AKIII) of the present invention is a DNA encoding wild-type AKIII that has mutation by which it releases inhibition by realization with L-lysine of AKIII to be encoded. As AKIII, AKIII is mentioned as derived from bacteria of the genus Escherichia, especially AKIII derived from E. Coll. The mutation by which inhibition is released by realization with L-lysine of AKIII includes, in the amino acid sequence of AKIII represented by sequence No. 1 of the sequence table, as it is counted from the N-terminus of AKIII, (a) A mutation whereby the residue of rnetiomna 318 of aspartosinase III is replaced by another amino acid residue, preferably by a residue of isoleucine and the residue of glycine 323 by another amino acid residue, preferably by residue of aspartic acid, Ll (b) Mutation by which the residue of leucma 325 is replaced by another amino acid, preferably by a residue of phenylalanine, and the residue of valine 347 by another amino acid residue, preferably by a residue of ineionin, (c) Mutation by the which the glycine residue 323 is replaced by another amino acid residue, preferably by an aspartic acid residue, and the valine residue 347 by another amino acid residue, preferably by an inetiomna residue, (d) Mutation by which the leucma residue 325 is replaced by another amino acid residue, preferably by a phenylalanine residue, and sepna residue 345 by another amino acid residue, preferably by a leucine residue, (e) Mutation by which the glycine 323 residue is replaced by another amino acid residue, preferably by an aspartic acid residue, and the serine residue 358 by another amino acid residue, preferably by a leucma residue, ( ) A mutation by which the threonine residue 344 is replaced by another amino acid residue, preferably by a methionine residue, (g) A mutation whereby the residue of glutamic acid 250 is replaced by another amino acid residue, preferably by a residue of lysine, (h) Mutation by which the acid residue L2 Lutarnic 346 is replaced by another amino acid residue, preferably by a lysine residue, and the residue of Itema 347 by another residue, preferably by a tenilalamine residue, (i) Mutation by which the glutaric acid residue 250 is replaced by another amino acid residue, preferably by a lysine residue, and the threo in residue 364 by another amino acid residue, preferably by a methylene residue, (j) A mutation by which the aspartic acid residue 202 is replaced by another amino acid residue, preferably by a glycine residue, and the serine 321 residue by another amino acid residue, preferably by a proline residue, (k) Mutation by which the argimne residue 283 is replaced by another residue of amino acid, preferably by a serine residue, the residue of alanm 333 by another amino acid residue, preferably by a residue of treomna, the residue of septa 338 by another amino residue acid, preferably, by a threoon residue, the glutamic acid residue 346 by another amino acid residue, preferably by a residue of aspartic acid, and the asparagine residue 414 by another amino acid residue, preferably by a residue of serine, or (1) Mutation by which the rnethionine residue 318 is replaced by another amino acid residue, preferably for a residue of .lysine, the serine residue 321 for another amino acid residue, preferably for a proline residue, the valine residue 328 for another amino acid residue, preferably for a phenylalanine residue, the valine residue 349 for another amino acid residue, preferably by a glycine residue, and the glutamic acid residue 405 by another amino acid residue, preferably by a valine residue. The DNA encoding wild-type AKIII is not particularly limited. DNA encoding AKIII derived from bacteria of the genus Escherichia, for example E. coli, is mentioned. Specifically, a DNA containing the amino acid sequence represented by the sequence No 1 is mentioned, as well as the sequence represented by the base numbers 584 to 1930 in the base sequence represented by the sequence No. 1. AKIII of E. Coli is encoded in lysC gene. Of the aforementioned sequences, the sequence having the mutation of the base sequence by which it causes the substitution of the amino acid residue is the DNA encoding AKIII mutant of the present invention. A type of a codon corresponding to the substituted amino acid residue is not particularly limited as long as it encodes that amino acid residue. The amino acid sequence of wild-type AKIII varies slightly depending on the bacteria or strains. The sequence that has substitution, deletion or insertion of the amino acid residue in the position that does not L4 participates in enzynnat ca \ amblen activity is included in the non-mutant AKIII gene of the present invention. For example, the sequence of bases (sequence No. 1) of the lysC gene of the wild type obtained in example 1 is different from the sequence of bases of lysC of strain K-12 JC4U of E. Coll that has already been published CCassan, M., Parsot. C, Cohen, G.N. and Patte, J.C. , J.Biol. Chern. 261, 1052 (1986)] by 6 sites. The amino acid residues to be encoded are different at two sites between 6 sites (in lyeC of strain JC411, the residue of glycine 58, as it is counted from the N-terrninal, is replaced by a residue of cysteine and the glycine residue 401 by an alanine residue in the amino acid sequence of lysC represented by the sequence No.l. If any of the aforementioned mutations a) to 1) is introduced into lysC having the same sequence of lysC of strain K-12 JC411 of E. Coll, it is expected to obtain lysC having a mutation by which inhibition is released by realirnentacion with L-lisma. The DNA encoding AKIII mutant in which the inhibition by feedback with lysine is released is obtained in the following manner. First, a DNA containing wild-type AKIII gene or AKIII gene which has another mutation is cross-linked, and the mutagenized DNA is ligated with a vector DNA adaptable to a host to form a recornbinating DNA. The recornbmante DNA is introduced to a host microorganism to obtain a transformant. When 1. 5 the transformant that arrives to express AKIII mutant is selected, this transformant retains the mutant gene. In addition, a DNA containing wild-type AKIII gene or AKIII gene having another mutation is ligated with a vector DNA adaptable to a host to form a recombinant DNA. The recombinant DNA is then rnutagenized ij vitro, and the mutated DNA is introduced to host microorganisms to obtain a transformant. When the transformant arriving to express AKIII mutant is selected, the transformant also retains the mutant gene. Alternatively, it is also possible that a microorganism that produces a wild-type enzyme is mutagenized to form a mutant strain that produces a mutant enzyme, and a mutant gene is then obtained from this mutant strain. As an agent for directly mutating a DNA, similar hydroxylamine is mentioned. Hydroxylanine is a chemical mutagenization agent that produces rnutagenization from cytosine to thymine by changing the cytosine to N * -hydroxycytosine. In addition, when a microorganism is itself rnutagenized, irradiation with ultraviolet light or treatment with a mutagenizing agent ordinarily used in artificial mutation, such as N-rnethyl-N'-nitroeoguanidine (NTG) is conducted. As DNA donating bacteria containing AKIII gene or AKIII gene having another mutation, any microorganisms belonging to the genus can be used Eschepchia Specifically, those described in the documents of F. C. Neidhardt et al. (Neidhardt, F. C. et al., Escherichia coll and Salmonella typhimupurn, American Socie + and for Microbiology, Washington D. C, p.2020, Table 1) are available. Examples thereof are strain JM109 from E. coli and strain MC1061 from E. coll. (1) obtaining wild-type AKIII gene. An example for producing a DNA containing AKIII gene is described below. First, for example, an MC1061 strain of wild-type E. coll having LysC is incubated to obtain a culture. The aforementioned microorganism can be incubated through usual sun culture. In view of an efficiency of cell collection, it is preferable to employ a liquid culture. In this case, the culture medium is, for example, one obtained by adding one or more 4% of inorganic salts selected from potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate., sodium chloride, magnesium chloride, ferrous chloride, ferrous sulfate and manganese sulfate to one or more carbon sources selected from yeast extract, peptone, meat extract, macerated corn liquor and soybean or wheat affluent, and adding thereto suitable amounts of a starting material of extracting, vitamin and the like as required. It is appropriate to adjust the initial pH of the culture medium to between 6 and 8. The incubation is conducted at a temperature of 30 to 42 ° C, preferably at about 37 ° C for 4 to 24 hours at through a culture of submerged aerial agitation, shaking culture or stationary culture. The culture thus obtained is separated, for example, at 3,000 rprn for 5 minutes to obtain an MC1061 strain of E. coli. A chromosomal DNA can be obtained from this strain, for example, by the method of Saito and Miura CBiochen. Biophys. Acta. 7_2, 619, (1963)] or the method of K. S. Kirby CBiochern. J. 64, 405, (1956)]. In order to isolate the AKIII gene from the resulting chromornic DNA, the crornomic DNA library is prepared. First, the crsenoric DNA is partially cut with an appropriate restriction endonuclease to obtain several fragmented mixtures. If the degree of cut is controlled by cut-off time control or the like, a wide variety of fragmented mixes are formed. For example, the chromosomal DNA is reacted with Sau 3AI at an enzyme concentration of 1 to 10 units / nl at a temperature of 30 ° C or higher, preferably at 37 ° C for a period of 1 minute to 2 hours to digest the same. Then, the cut chromosomal DNA fragment is ligated with a vector DNA capable of undergoing autonomous replication within the cellulae of bacteria of the genus Escherichia to form a recombinant DNA. Specifically, a vector DNA is reacted with a restriction endonuclease that allows the formation of the same terminal base sequence as the restriction endonuclease Sau 3AI used in cutting of the crysomeric ODN, for example, Bam Hl at an enzyme concentration of 1 to 100 units / rnl at a temperature of 30 ° C or higher for L hour or more, preferably 1 to 3 hours to completely digest it, and short. Subsequently, the fragmented mixture of previously obtained chromosomal DNA is mixed with the vector DNA cor + ado. The mixture is reacted with a DNA ligase, preferably T4 DNA ligase at an enzyme concentration of 1 to 100 units / minute at a temperature of 4 to 166 ° C for 1 hour or more, preferably 6 to 24 hours to form a Recombinant DNA. A microorganism of the genus Eschepchia, for example a strain K-12 of E. coli, preferably a strain completely deficient in AKI, AKII and AKIII, for example, strain GT3 of E. coll [which can be obtained from a center of Genetic supply of E. coll (Connecticut, USA)] is transformed to prepare the chromosomal DNA library. The transformation can be conducted by the method of DM Morrison CMethods m Enzymology 6, 326, (1979)] or a method in which the permeability of a DNA is increased by treating cells of recipient bacteria with calcium chloride CMandel, M. and Higa, A., J. Mol. BlOl. 51,159 (1970)]. A strain having a recombinant DNA of AKIII gene is obtained from a strain having an increased AKIII activity or a strain in which the auxotrophy quashed by the AKIII gene deletion has been out of phase in the library L9 of resulting chromosomal DNA, for example, when a completely deficient AK is used as a host, a recombinant strain that can be grown in L-lysine-free medium, L-teronin, L-methionine and diaryninopyrinic acid or medium. Homoserin-free culture and diarmnopyrnepic acid, and the reclosing DNA is recovered from this strain, making it possible to obtain a DNA fragment containing the AKIII gene. A cell extract is prepared from a candidate strain, and a crude enzyme solution is formed from the cell extract. Then, the activity of AKIII is identified. The enzymatic activity of AKIII can be determined by the ER Stadtrnan method (Stadtman, ER, Cohen, GN, LeBras, G., and Robichon-Szulrnajster, H., J. Biol. Chem. 236, 2033 (1961)]. A recornbinant DNA obtained by inserting the DNA containing the AKIII gene into the vector DNA can be isolated, for example, by the method of P. Guerry et al. (J. Bactenol. F! 16, 1064, (1973)] or the DB Cleuell method (J. Bactepol. 110, 667, (1972)]. The wild type AKIII gene is also obtained by preparing a chromosoric DNA from a strain having AKII1 gene on a chromosome by the Saito and Miura method, and by amplifying the AKIII gene through the CPCR polymerase chain reaction, Uhite, TJ and other, Trends Genet. 5_, 185 (1989)]. A DNA primer in the amplification is complementary to the 3'-terminus of a double strand of DNA that contains the global region of the AKIII gene or a part of the rnisrna. When only part of the AKIII gene region is amplified, it is necessary to sift a DNA fragment containing the global region from the chromosomal DNA library using the DNA fragment having the region part < le AKIII as an initiator. When the entire AKIII gene region is amplified, the PCR solution containing the DNA fragment having the amplified AKIII gene is subjected to agarose gel electrophoresis, and a desired DNA fragment is then extracted, so that it can be recover the DNA fragment containing AKIII gene. The DNA primer can be appropriately formed, for example, on the basis of the known sequence of E. coli CCassan, M., Parsot, C., Cohe, GN and Patte, J. C, J. Biol. Chem. 261 , 1052 (1986)]. An initiator capable of expanding a region composed of 1347 bases encoding a lysC gene is appropriate. For example, two types of primers represented by sequences Nos. 2 and 3 are suitable. An initiator DNA can be synthesized by the usual method, for example, a phosphoarnidide method CTetrahedron Letters 2, 1859 (1981)] using a synthesizer from Commercially available DNA (eg, a DNA synthesizer model 380B manufactured by Applied Biosysterns). In addition, PCR can be carried out by a method indicated by a supplier using a commercially available PCR device (PJ2000 model DNA thermal cycler manufactured by Takara Shuzo Co., Ltd.) and a TaqDNA polyrnerase (provided by Ta ara Shuzo Co., Ltd.). The AKIII gene amplified by PCR is ligated with the DNA of a vector capable of undergoing autonomous replication in cells of bacteria belonging to the genus Eschenchia, and introduced into the cells of bacteria of the genus Escherichia, by means of which the introduction of the mutation in the AKIII gene can be carried out easily. The vector DNA, the transformation and the identification of the presence of the AKIII gene, are the same as those described above. (2) Introduction of the mutation in the AKIII gene The AKIII gene obtained above is subjected to mutation such as substitution, insertion or deletion of the amino acid residue by the recombinant PCR method CHiguchi, R. 61, in PCR Technology (Erlich, HA Eds, Stockton Press (1989)], a site-specific mutation method HKrarner, U. and Frits, HJ, Methods in Enzymology 154, 350 (1987), Kun el, TA et al, Methods m Enzimology 154, 367 (1987) )], or similar.These methods can cause the desired mutation at the desired site.The random mutation or mutation can be introduced at the desired site through a method of chemical synthesis of a desired gene.Furthermore, there is a method in which the AKIII gene on a chromosome or a plasmid is treated directly with hydroxylanine CHashirnoto, T. and Sekiguchi, M., 3. Bactenol 159, 1039 (1984).] Still further, a method can be used in which 7 0 bacteria of the genus Escherichia containing the AKIII gene are irradiated with ultraviolet light or a method using a chemical agent such as N-methyl-N'-nitrosoguanidma or nitrous acid. The utation can be introduced at random by these methods. A method for selecting the mutant AKIII gene is described below. That is, first, a recornbinant DNA containing the mutated AKIII gene is transformed into a strain completely deficient in AK, for example, the GT3 strain of E. coli. Then, the transformant is incubated in a minimal medium, for example, M9, containing a considerable amount of L-lysine. Since AK is only inhibited with L-lysine in a strain that has a recombinant DNA that contains the wild-type AKIII gene, L-threonine, L-isoleucm, L-rnetionma and diaminopyrimic acid (DAP) can not synthesized, controlling the growth of it. Meanwhile, a strain having a recornbinant DNA containing the mutant AKIII gene in which the inhibition with L-lysine is released, should be cultured in a minimal medium containing a considerable amount of L-lysine. During the use of this phenomenon, it is possible to select a strain resistant to L-liein or S-2-arn? Noet? L-cistern (AEC) analogous to the L-lysine in growth, namely a strain having a recombinant DNA containing the mutant AKIII gene in which the inhibition is released. The recombinant gene thus obtained as a recornbinant DNA is introduced into a microorganism (host) appropriate, and expressed to be able to obtain a microorganism containing AKIII in which the inhibition by realirnentación be released. As a host, microorganisms belonging to the genus Escherichia are preferred. For example, it is mentioned Escherichia coli (E. coli). In addition, a substance obtained by turning a fragment of the nt AKIII gene from a recombinant DNA can be used, and inserting it into the other vector. As vector DNA that can be used in the present invention, a plasmid vector DNA is preferred. . Examples thereof include pUC19, pUC.18, pBR322, pHSG299, pHSG298, pHSG399, PHSG398, RSF1010, pMUU9, pMIHB, pMU219 and pMU218. There is also a phage DNA vector and a transposon vector. In order to efficiently express the nt AKIII gene, another promoter acting within microorganisms, such as lac, trp and PL, can be ligated with a region towards the 5 'end of a DNA encoding the nt AKIII. Either a promoter contained in the AKIII gene that is being amplified is used. As previously mentioned, the substance obtained by inserting the nt gene into the vector DNA capable of undergoing autonomous replication can be introduced into a host, and be retained in the same way or an extrachromosoric DNA as a plasmid. Alternatively, the nt gene can be inserted into a chromosome of a host microorganism by transduction, with a transposon (Berg, D.E. and Berg, C.M., Bio / Technol., 1, 417 (1983)], with phage Mu [Japanese patent open to the public (Kokai) No. 1.09,985 / 1990] or by complementary recornbination [Experiments in Molecular Genetics, Cold Spring Harbor Lab. (1972)]. < 3 > Production of L-arninoacid according to the present invention An L-arnino acid with good efficiency can be produced by incubating in an appropriate culture medium bacteria of the genus Escherichia that have been transformed after introducing the nt AKIII gene obtained above, producing and accumulating an L -arninoacid in the culture, and collecting the L-arninoacid from the culture. The bacteria of the genus Escherichia that contain AK, in which the inhibition by realirnentación with L-lysine is released, include bacteria of the genus Escherichia that ee transformed inserting a DNA encoding AKIII undergoing tion by which inhibition is released by feedback with L -latin in a chromosomal DNA, and bacteria of the genus Escherichia that are transformed by introducing into the cells a recombinant DNA that is formed after ligating the aforementioned DNA with the DNA of a vector capable of undergoing autonomous replication in cells of bacteria of the Eecherichia genus. In addition, there are also bacteria nts of the genus Escherichia that allow the production of AKIII nt by rnutagenizing cells of bacteria of the genus Escherichia. With respect to the bacteria of the genus Escherichia which are used with host for transformation, any bacterium can be used if a promoter of the nt AKIII gene or another promoter expressing this gene acts within the cells of the rniema. 0, when the nt AKIII gene is introduced into a plasmid as an extrachlorasomal DNA, any bacterium can be used if an origin of replication of the vector DNA used for introduction purposes can act within the cells thereof and can replicate. The culture medium which is used to incubate the microorganism having the nt gene according to the present invention, is a common culture medium containing a carbon source, a nitrogen source, inorganic ions and other organic substances as requires Examples of the carbon source include sacapdoe such as glucose, lactose, galactose, fructose and a starch hydrolyzate; alcohols such as glycerol and sorbitol; and organic acids such as furnaric acid, citric acid and succinic acid. Examples of the carbon source include inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate; a soy hydrolyzate, gaseous ammonia and aqueous ammonia. As a source of organic germs, it is advisable to contain an appropriate amount of a necessary substance such as vitamin Bi or L-isoleucma, or a yeast extract. In addition, potassium phosphate is added in small amounts, magnesium sulfate, iron ions and manganese ions, as required. The incubation is carried out aerobically from 16 to 72 hours. The incubation temperature is between 25 ° C and 45 ° C. The pH is adjusted to between 5 and 8 during incubation. To adjust the pH, an organic or inorganic subetancy or an acid or alkaline eubstance can be used, as well as gaseous ammonia. Often an L-amino acid can be harvested from the fermentation liquid by the method of an ion exchange resin method, precipitation methods and other known methods.

EXAMPLES The present invention is described more specifically in relation to the following examples.

EXAMPLE 1 Obtaining mutant AKIII gene (1) < 1 > Cloning of the wild type AKIII gene The base sequence of the AKIII gene (lysC) of E. coli has already been reported [Caesan, M., Parsot, C., Cohen, G.N. and Patte, J.C., J. Biol. Chern. 261, 1052 (1986)]. It is known that an open reading frame (ORF) is composed of 1,347 base pairs, encoding a composite protein of 449 residues. amino acids. This gene has an operator, and suffers suppression with L-lysine. Therefore, to remove its region from the operator-, the SD sequence and the region containing SOI ORF were amplified by PCR, and cloned. The DNA of the total genome of strain K-12 MC1061 of E. coli was produced by the method of Saito and Miura [Biochem. Biophys. Acta. 72, 619 (1963)], and formed two types of primers having sequences represented by sequences numbers 2 and 3. The PCR was carried out by the method of H.A. Erlich et al. (PCR Technology, Stockton Press (1989)] using the same technique to amplify the AKIII gene, the resulting DNA was digested with Bam Hl and Asa I, then shaved at its ends and inserted into a Sma I site of a pMU119 low copy number vector to construct pMLYSC2 This SMa I site is located towards the 3 'end of a lacZ promoter present within the vector When a recombinant DNA obtained by meerning a DNA fragment into the eitium is introduced into E. coll. Sma I of? MU119, the inserted DNA fragment is tranecnbe by transcription controlling the lacZ promoter (Figure 1). <2> Cloning of the mutant AKIII gene Plasmids having the mutant AKIII gene, a eaber, pLYSCl * 8Q, pLYSCl * 117 and pLYSCl * 126, as described in the international brochures open to the public UQ94 / 11517 and UO95 / 16042, were digested with Eco Rl and Hind III to cut-off DNA fragments containing the mutant AKIII gene. fragments were inserted into an Eco RI-Hind III site of pMU119 pair-to construct plasmid designated as pMLYSC2 * 80,? MLY C2 * 117 and pMLYSC2 * 126, respectively. The plasmid pLYSC * 8Q can be produced from pLL2C * 8 in accordance with the description in the international brochure open to the public U094 / L1517. A strain obtained by introducing pLLC * 80 into strain E. coli HB1Q1 was designated as 12750. It was deposited at the National Institute of Bio-science and Human Technology of the Agency of Industrial Science and Technology (No. 1-3, Higashi 1-chorne, Tsuk? Ba-shi, Ibaragiken, 305) under deposit No. FERM P-13136 on September 1, 1992. This stock was transferred to the international warehouse under the Budapest Treaty on November 4, 1993, and the deposit No. FERM BP-446 was again assigned to the Ministry. pLYSC2 * 117 and pLYSC2 * 126 are not yet deposited. However, since the mutation points thereof are described in the international brochure open to the public W094 / 11517, they can be easily prepared using pLYSCl * B0 as starting material. < 3 > Study of the conditions for selecting the novel AKIII gene A transformant that was obtained by introducing pMLYSCl into a completely AK deficient strain, E. coli GTS (thrA1016b, rnetLM1005, lysC1004) was designated as GT3 / pMLYSC2. Also, GT3 / pMLYSC2 * 80, GT3 / pMLYSC2 * 117 and GT3 / pMLYSC2 * 126 were obtained. The strain GT3 /? MLYSC2 has a plasmid that contains wild-type lysC, and AKIII encoded in lysC of this plasmid is AK alone. Since wild-type AKTIE that is AK only in the presence of a considerable amount of L-lysine in the immediate medium is inhibited with L-lysine, L-treomna, L-isoleucine, L-rnethionine and diarynopyrimetic acid (DAP), it can be synthesized, suppressing the growth of the same. With the hope that a strain containing the plasmid and having a significant amount of L-C, in which the inhibition with L-Lysine is released, can be developed in the minimal medium containing a considerable amount of L-lysine, I selected a strain that exhibited resistance to L-lysine in growth, and thus a strain was selected that has a plasmid containing mutant lysC, in which the inhibition with L-liein was released. Strain GT3 / pMLYSC2, strain GT3 / pMLYSC2 * 80, strain GT3 / P LY C2 *! .7 and strain GT3 / pMLYSC2 * 126 were incubated on the minimal agar plate medium containing L-lysine at vains concentrations to examine the growth inhibitory concentration and the selection conditions of the strains containing the plasmid. The growth of the transformants in the minimum agar plate medium containing L-lysine at various concentrations, is shown in Table 1. In Table 1, + indicates that a transformant was developed, ± indicates that a traneformant was slightly developed, and - indicates that there is no trans-orman development.

TABLE 1 Concentration of L-lysine and growth The growth of strain GT3 / pMLYSC2 having lysC of type eilveetr-e was completely inhibited in the addition area containing lysine at 0.2M. In addition, the growth of strain GT3 / pMLYSC2 * 8Q, strain GT3 / pMLYSC2 * 117 and strain GT3 / pMLY? C2 * 126 which have significant lysC and which are well known, was also completely inhibited in the area of addition containing L-lysine at 0.4M. Accordingly, it was suggested that a mutant which has an even greater degree of inhibition release than the mutant lysC known hitherto, probably appeared to be obtained by selection in the addition area containing L-lysine at 0.4M. It was identified that this inhibition of growth was eliminated by the simultaneous addition of hornoserin and diaminopimerinic acid. In the mutation introduction test, ee I use a minimum medium of agar-M9 containing L-lysine at 0.4M to select a strain that contains the plasmid and that has the lysC rnutante. In example 1, this means is called selective medium. < 4 > Mutagenization of the AKIII gene and obtaining the gene AKIII mutant Two methods, namely, a method of metallurgical rnutagenization in which a plasmid is treated directly with hydroxylamine, and a PCR mutagenization method to provide several mutations in the hope of obtaining mutations other than the mutation of cytosma to thymine with hydroxylamine, were used in the introduction of the mutation in the piasrnido pMLYSCl. Micrograms of? MLYSC2 were treated in hydroxylamine at 0.4M [a solution containing 100 μl of a mixture of KH2PO-; and EDTA at lmM (pH 6.0) and a mixture of hydroxylanine at 1M and EDTA at lmM (pH 6.0) and 2-μg of DNA were adjusted to a total of 200 μl with the addition of water] at 75 ° C during the 4 hours. The DNA thus treated was purified with a glass powder, and was then introduced into a GT3 strain of E. coli completely deficient in AK. The traneformant was extended in a complete medium (containing L-broth at 1%, yeast extract at 0.5%, NaCl at 0.5%, 50 mg / liter ampicillin and 1.5% agar) to form colonies. The colonye were replicated in the selective medium, and the strains capable of being cultured in the selective medium were They selected as candidate strains. Random mutagenization by PCR was carried out by the method of C. Cad? Ell et al. (Cadwell, C. and GF Joyce, PCR methods Applic., 2, 28, (1902)]. fragment of lysC by the aforementioned method using pMLYSC2 and universal primer M13.This fragment was digested with Eco Rl and Hind III, and then inserted in an Eco RI-Hind III site of? M) 119 to introduce the same in strain GT3 of E. coli completely deficient in AK. The transformant was extended in half complete (containing L-broth to 1%, bacto trypton to 0.5%, yeast extract to 0.5%, NaCl to 0.5%, 50 rng / liter of ampicillin and agar to 1.5%) to form colonies The colonies were replicated in the selective medium, and strains capable of being cultured in the selective medium were selected as candidate strains. The plaeemids were recovered from a total of 47 candidate strains obtained previously, namely 44 strains resulting from the hydroxylamine rnutagenization, 3 strains resulting from random mutagenization by PCR. We determined the base sequence of the remaining lysC, and identified the mutation points. The base sequence determination was carried out by a usual method using an ABI DNA sequence determinant model 373A (provided by ABI). As a result, mutant AKIII strains could be obtained having 4 types (Nos. 1, 6, 14 and 21) of mutation sites from strains resulting from mutagenization with hydroxylamine, and it was also possible to obtain mutant AKI.II strains having 3 types (Nos. 28, 29 and 30) of mutation sites from strains resulting from randomization by randomization by PCR (Table 2).

TABLE 2 These seven plasmids (pMLYSC2 * Yl, pMLYSC2 * Y6, pMLYSC2 * Y14, pMLYSC2 * Y21, pMLYSC2 * Y28, pMLYSC2 * Y29 and? MLYSC2 * Y30) were introduced into strain GT3 completely deficient in AK, and free extracts were prepared from cells from the transformants The enzymatic activity of AKIII was measured using them The production of the cell-free extracts and the measurement of the enzymatic activity of AKIII were carried out by the method of ER Stadtman and others (Stadtnan , ER, Cohen, GN, LeBras, G. and Robichon-Szulrnajeter, H., 3. Biol. Chem. 236., 2033 (1961).] In addition, during the measurement of the enzymatic activity of AKIII, L was added. -Line at various concentrations to the enzymatic reaction solution to examine the degree of release of the inhibition with L-lysine.The results are plotted in the quadr-o 3. The degree of release of the inhibition refers to a ratio of the residual activity of AK in the presence of L-liema at 0.4M resp ecto to the activity of AK in the absence of L-liein.

TABLE 3 As is clear from the above referenced references, it could have been found that it had the highest degree of inhibition release than the conventional AKIII mutant (AKIII mutant encoded in? MLYSC2 * 8Q and? MLYSC2 * 117) by the method of selection used this time. A specific activity based on the total protein content is often influenced by the growth conditions of the cell or the preparation of the samples. The specific activity of them was equal to those of the wild type and of the conventional mutants, and almost no decrease was observed in the int activity of the mutation. Therefore, it is expected that the activity center of AKIII and the control site with L-lysine thereof would be independent of each other.

EXAMPLE 2 Obtaining mutant AKIII gene (2) The lysC gene was prepared in which Ser 358 was replaced with Leu by introducing the site-specific mutation using an in vitro PCR rnutagenesis kit from LA (provided by Takara Sh? Zo Co., Ltd.). On this occasion, pLYSCl described in the international brochure open to public U094 / 11517 and UO95 / 16042 was used as a template, and the primer described in sequence table No. 4 as the initiator, to introduce the mutation, respectively. Both terminals of a PCR amplification fragment were cut with Eco Rl and Hind III, and the cut fragment was ligated with a fragment obtained by cutting pUC19 with Eco Rl and Hmd III from pUC19 to form? LYSC358L. The AKIII mutant gene in which Ser 354 was rnutagenized in Lie or Val was prepared in the above-mentioned manner, except that the primer described in sequence table No. 5 or 6 was used as an initiator. This gene was ligated with a fragment obtained by cutting? UC19 with Eco Rl and Hmd III. Thus, they were formed? LYSC345I and? LYSC354V. Fragments containing lysC that were obtained cutting pLY? Cl * 48, pLYSCl * 117, pLYSCl * l2b, pLYSCl * 150 and? LYSCl * 158 with Eco I and Hmd III were inserted into an Eco Hmd III cleavage site of pUC19, and the resulting fragments designated pLYSC2 * 48, pLYSC2 * 117, pLYSC2 * 126, pLYSC2 * 15Q and? LYSC2 * 158, respectively. International brochures open to the public I094 / 11517 and IO95 / 16042 describe the structure of pLYSCl and the sites of the mutation sites of pLYSCl * 48, pLYSC2 * 117, pLYSCl * 126, pLYSC2 * 15Q and pLYSC2 * 158. Therefore, these plasmids can be prepared from the above-described pLYSCl * 80. Subsequently, a mutant lysC gene was prepared having two types of mutations using a Sep I cleavage site present in the vicinity of the center of the mutation sites of the obtained monobasic lysC monomer obtained, and a Sepl cut site located towards the 3 'end. of lysC in? UC19. That is, pU2547M was prepared by ligating an Ssp I fragment containing a lysC gene towards the 5 'end of pLYSC2 * 48 with an Ssp I fragment containing a lyeC gene towards the 3' end of pLYSC2 * 158 pU2347M was prepared by ligating an Ssp I fragment containing a lysC gene towards the 5 'end of? LYSC2 * 126 with an Ssp I fragment containing a lysC gene towards the 3' extrusion of pLYSC2 * 158; Prepared pU2545L by ligating a Sep I fragment containing a lysC gene towards the 5 'end of pLYSC2 * 48 with an Ssp I fragment containing a lysC gene towards the 3' end of? LYSC2 * 117, pU2358L ligand was prepared in a Ssp I fragment containing a lyeC gene towards the 5 'end of pLYSC2 * 126 with a sp L fragment containing a lysC gene towards the 3 'end of? LYSC358L; pU2345V was prepared by ligating a Sep I fragment containing a lysC gene towards the 5' end of pLYSC2 * 126 with an Ssp I fragment containing a lysC gene towards the end 3 'of pLYSC345V; and U2345I was prepared by ligating an Ssp I fragment containing a lysC gene towards the 5 'end of? LYSC2 * 126 with a Sep I fragment containing a lyeC gene towards the 3' end of pLYSC345I. Since the mutation sites of pLYSC2 * 150 and? LYSC2 * 126 are located more towards the 5 'end than the Sep I cleavage sites, the dibasic quantum lysC having two mutation sites was prepared as follows. First, a DNA fragment having two mutation sites was amplified using? LYSC2 * 126 as a template, a cleotide oligon described in the text box No. 7 as an initiator, and the aforementioned equipment. Subsequently, ambae terrninalee of the resulting DNA fragment were cut with Eco Rl and Hmd III, and ligated with a fragment obtained by cutting pUC19 with Eco Rl and Hmd III.

The product thus obtained was designated pU1823D. The degree of release of the inhibition of the lyeC gene product (AKIII) mutant dibasic thus obtained with respect to Lys was determined as described in Example 1. Consequently, the degree of release of the inhibition of any dibasic mutant AKIII was greater than that of the monobaemic AKIII (Table 4). By the way, the mutant lysC gene in pLYSC582L, pLYSC345I and? LYSC345V is a novel mutant gene. In each of these genetic products, inhibition by r -feeding with Usin is released, and the binding with the other mutation improves the degree of inhibition release. The product is also useful as an intermediary to build the dynastic AKIII.

TABLE 4 EXAMPLE 3 Production of L-lysine by fermentation using a strain having the mutant DDPS gene and mutant AKIII gene introduced therein The effect to produce L-lysine with respect to the DDPS gene and the mutant AKIH gene is described in FIG. international brochure open to the public UO95 / 16042. To improve this effect, the mutant AKIII gene obtained from Example 1 was made to coexist with the mutant DDPS gene. A strain obtained by introducing the RSF24P strain having the DDPS gene described in the international leaflet open to the public UO95 / 16042 in strain E. coli DM109 was designated A312395. It was deposited at the National Institute of Bioscience and Human Technology of the Industrial Science and Technology Agency (No. 1-3, Higashi 1-chorne, Tsukuba-shi, Ibaragi-ken, 305) under deposit No. FERM P-13935 on October 28, 1993. Eeta cepa ee transferred to the international deposit under the Budapest Treaty on November 1, 1994, and deposit No. FERM BP-4858 was recently assigned to it. The plasmids RSFDY1, R? FDY6, RSFDY14, RSFDY21, RSFDY28, RSFDY29, RSFDY30, R? FD2547M, RSFD2347M, RSFD2545L, R? FD2358L, RSFD2345V, RSFD2345I and RSFD1823D were prepared as shown in FIG. 2 from a selected type of plasmids containing the mutant AKIII gene, pMLYSC2 * Yl , pMLYSC2 * Y6, pMLYSC2 * Y14, pMLYSC2 * Y21, pMLYSC2 * Y28, pMLYSC2 * Y29, pMLYSC2 * Y30, pU2547M, pU2347M, pU2545L, pU2358L, pU2545V, PU2345I and pU1823D. The plaemido RSFD80 described in the international brochure open to the public U095 / 16042 was used as control. A strain that was obtained by introducing the plasmid RSFD80 into strain JM109 of E. coli was designated as AJ12396. HE deposit in the National Institute of Bioscience and Human Technology of the Agency of Science and Industrial Technology (No. 1-3, Higashi 1-chorne, Tsu? ba-shi, Ibaragi-ken, 305) under the deposit Mo. FERM P- 13936 on October 28, 1993. This stock was transferred to the international warehouse under the Budapest Treaty on November 1, 1994, and deposit No. FERM BP-4859 was recently assigned to the country. The plasmids obtained previously, RSFDY1, RSFDY6, RSFDY14, RSFDY21, RSFDY28, RSFDY29, RSFDY30, RSFD2547M, RSFD2347M, R5FD2545L, RSFD2358L, RSFD2345V, RSFD2345I and RSFD1823D were inoculated into strain B-399 in a usual manner to form L-lysine producing strains. The lysine productivity of the aforementioned strains was evaluated. The evaluation of the productivity of lisma was also carried out with respect to B-399 / RSFD80 as control. A method to obtain strain B-399 is described in the international brochure open to the public UO95 / 16042. Incubation was carried out in the L-lysine producing culture medium at 37 ° C for an incubation time of 48 hours by agitation at 114 to 116 rpm in accordance with the method described in the international brochure open to the public UO95 / 16042. The results are shown in Table 5.

TABLE 5 EXAMPLE 4 Production of L-lysine by fermentation using a strain having the mutant DDPS gene and the mutant AKIII gene introduced therein (2) It was identified in Example 3 that the productivity of L-lysine could be improved by making the bacteria of the genus Escherichia have the mutant DDPS gene and the mutant AKIII gene. The test was carried out to see if this effect could be maintained even when a guest was changed. Strain U3110 (tyr-A) from E. coli was used as Guest. Strain U3110 (tyrA) is described in detail in European Patent No. 488,424 / 1992. European Patent Laid-Open No. 488,424 / 1992 describes a large number of strains obtained by introducing a plasmid in strain U3110 (tyrA). For example, a strain obtained by introducing the plasmid pHATerrn was designated as strain U311Q (tyrA) / pHATerrn from E. coli. It was deposited internationally in the National Institute of Bioscience and Human Technology of the Agency of Industrial Science and Technology (No. 1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi-Ken, 305) on November 18, 1991 under the Treaty of Budapeet, and deposit No. FERM BP-3653 was assigned to it. Strain U3110 (tyrA) can also be obtained by dropping the plasmid pHATerrn dropwise from this strain U3110 (tyrA) ZpHATerm from E. coll. The trickle of the plasmid can be performed in a usual manner. Two containing the DDPS gene and the mutant AKIII gene obtained in Example 3, namely, RSFDY1, RSFDY6, RSFDY14, RSFDY21, RSFDY28, RSFDY29, RSFDY30, RSFD2547M, RSFD2347M, RSFD2545L, RSFD2358L, RSFD2345V, RSFD2345I and RSFD1823D. were introduced into strain U3110 (tyrA) previously obtained, and the productivity of L-lysine was evaluated in Example 3. As a control, U3110 (tyrA) / RSFD80 was produced by introducing RSFD80 into strain U3110 (tyrA), and The L-lysine productivity of this strain was also evaluated. The results are shown in Table 6.

TABLE 6 EXAMPLE 5 Production of L-threonine by fermentation using a strain that has the mutant AKIII gene introduced therein As a strain of threonine-producing E. coli, strain B-3996 has high productivity among those that are ee They know in the present. Therefore, during the evaluation of the mutant AKIII gene, strain B-3996 was used as a host. This strain B-3996 is described in the patent of E.U.A. 5,175,107, and is inscribed in the list as it was deposited in the Institute of Research in Genetic and Industrial Genetic Improvement of Microorganisms under deposit No. BKIIM B-3996. In addition, as a long-running AKIII gene for evaluation, three types of mutant genes (AKIII genes of RSFDY6, RSFE254M and RSFD1823D) were selected from among mutant genes that have a high productivity of L-lysine in Example 4, and were subjected to the test . First, to increase the degree of expression of the mutant AKIII gene, the AKHI gene present in? MLYSC2 * Y6 bound to the 3 'end of the lacZ promoter of pUC19 (provided by Takara Shuzo Co., Ltd). The novel plasmid thus formed was designated pLLC * Y6 (Figure 3). Since in pU2547M and pU1823D, the AKIII mutant gene ee originally located towards the 3 'end of the lacZ promoter of pUC19 (provided by Takara Shuzo Co., Ltd), was used as such. These plasmids were introduced into strain B-3996 in a usual manner, and the evaluation was carried out. Incubation was carried out by the method described in the international brochure open to the public U094 / H517. pLLC * Y6, pU2547M and pU1823D were transformed into strain B-3996, and the transforrnantee were incubated in the presence or absence of 1 g / liter of lysine. Host strain B-3996 alone and B-3996 /? LLC * 80 described in the f-international brochure open to the public U094 / 1151? 1. The results are shown in table 7. The sensitivity to lysine in table 7 refers to a ratio of sugar yield consumed in the presence of lysine to the yield of sugar consumed in the absence of lysine. In strain B-3996, the decrease in the yield of sugar consumed in the incubation in the presence of lysine is approximately 0.74 with respect to that in the incubation area in the absence of lysine; and in B-3996 /? LLC * 80, the decrease in the yield of confectionary sugar in the incubation in lieu of ee of approximately 0.90 relative to that in the incubation area in lieutenancy. The AKIII mutant genes newly obtained this time are slightly superior in treomna productivity and lysine sensitivity.

TABLE 7 EFFECTS OF THE INVENTION The present invention has made it possible to produce the mutant AKIII gene derived from bacteria of the genus Eecherichia in which inhibition by feedback with L-lysine is well released. A L-arnino acid producing strain that is more improved than the previous one can be formed by introducing the above-mentioned gene into bacteria of the genus Escherichia. A process for producing an L-arnino acid by fermentation can be provided, which is superior to the conventional procedure using this L-arnino acid producing strain.

Feature key: signal -10 Position; 265.-273 Determination method: S Property key: initiator link Position: 536.-555 Determination method: E Feature key: initiator link Position: 2128..2147 Determination method: E Feature key: RBS Poeition: 575..578 Determination method: S Property key: CDS Position: 584..1933 Determination method: S Property key: terminator Poetry: 1941..1968 Determination method:? DESCRIPTION OF THE SEQUENCE TCGAAGTGTT TCTGTAGTGC CTGCCAGGCA GCGGTCTGCG TTGGATTGAT GTTTTTCATT " AGCAATACTC TTCTGATTTT GAGAATTGTG ACTTTGGAAG ATTGTAGCGC CAGTCACAGA 1 AAAATGTGAT GGTTTTAGTG CCGTTAGCGT AATGTTGAGT GTAAACCCTT AGCGCAGTGA 1 AGCATTTATT AGCTGAACTA CTGACCGCCA GGAGTGGATG AAAAATCCGC ATGACCCCAT 24 CGTTGACAAC CGCCCCGCTC ACCCTTTATT TATAAATGTA CTACCTGCGC TAGCGCAGGC 30 CAGAAGAGGC GCGTTGCCCA AGTAACGGTG TTGGAGGAGC CAGTCCTGTG ATAACACCTG 36 AGGGGGTGCA TCGCCGAGGT GATTGAACGG CTGGCCACGT TCATCATCGG CTAAGGGGGC 42 TGAATCCCCT GGGTTGTCAC CAGAAGCGTT CGCAGTCGGG CGTTTCGCAA GTGGTGGAGC 48 ACTTCTGGGT GAAAATAGTA GCGAAGTATC GCTCTGCGCC CACCCGTCTT CCGCTCTTCC 5 CTTGTGCCAA GGCTGAAAAT GGATCCCCTG ACACGAGGTA GTT ATG TCT GAA ATT 5 Met Ser Glu He 1 GTT GTC TCC AAA TTT GGC GGT ACC AGC GTA GCT GAT TTT GAC GCC ATG 6 Val Val Ser Lys Phe Gly Gly Thr Ser Val Wing Asp Phe Asp Wing Met 5 10 15 20 AAC CGC AGC GCT GAT ATT GTG CTT TCT GAT GCC AAC GTG CGT TTA GTT 6 Asn Arg Ser Wing Asp He Val Leu Ser Asp Wing Asn Val Arg Leu Val 25 '30 35 GTC CTC TCG GCT TCT GCT GGT ATC ACT AAT CTG CTG GTC GCT TTA GCT 7 Val Leu Being Wing Being Wing Gly He Thr Asn Leu Leu Val Wing Leu Wing 40 45 50 GAA GGA CTG GAA CCT GGC GAG CGA TTC GAA AAA CTC GAC GCT ATC CGC 7 Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu Asp Wing He Arg 55 60 65 AAC ATC CAG TTT GCC ATT CTG GAA CGT CTG CGT TAC CCG AAC GTT ATC 8 Asn He Gln Phe Wing He Leu Glu Arg Leu Arg Tyr Pro Asn Val He 70 75 80 CGT GAA GAG ATT GAA CTG CTG GAG AAC ATT ACT GTT CTG GCA GAA Arg Glu Glu He Glu Arg Leu Leu Glu Asn He Thr Val Leu Ala Glu 85 90 95 100 GCG GCG GCG CTG GCA ACG TCT CCG GCG CTG ACA GAT GAG CTG GTC AGC Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp Glu Leu Val Ser 105 110 115 CAC GGC GAG CTG ATG TCG ACC CTG CTG TTT GTT GAG ATC CTG CGC GAA His Gly Glu Leu Met Ser Thr Leu Leu Phe Val Glu He Leu Arg Glu 120 125 130 CGC GAT GTT CAG GCA CAG TGG TTT GAT GTA CGT AAA GTG ATG CGT ACC 1 Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys Val Met Arg Thr 135 - 140 '145 AAC GAC CGA TTT GGT CGT GCA GAG CCA GAT ATA GCC GCG CTG GCG GAA 1 Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp He Ala Ala Ala Glu 150 155 160 CTG GCC GCG CTG CAG CTG CTC CTC CGT CTC AAT GAA GGC TTA GTG ATC 1 Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu Gly Leu Val He 165 170 175 180 ACC CAG GGA TTT ATC GGT AGC GAA AAT AAA GGT CGT AC ACG ACG CTT 1 Thr Gln Gly Phe He Gly Ser Glu Asn Lys Gly Arg Thr Thr Thr Leu 185 190 195 GGC CGT GGA GGC AGC GAT TAT ACG GCC GCC TTG CTG GCG GCT GCT TTA 1219 Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu Ala Glu Ala Leu 200 205 210 CAC GCA TCT GTT GAT ATC TGG ACC GAC GTC CCG GGC ATC TAC ACC 1267 His Wing Ser Arg Val Asp He Trp Thr Asp Val Pro 'Gly He Tyr Thr 215 220 225 ACC GAT CCA CGC GTA GTT TCC GCA GCA AAA CGC ATT GAT GAA ATC GCG 1315 Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg He Asp Glu He Ala 230 235 240 TTT GCC GAA GCG GCA GAG ATG GCA ACT TTT GGT GCA AAA GTA CTG CAT 1363 Phe Wing Glu Wing Wing Glu Met Wing Thr Phe Gly Wing Lys Val Leu His 245 250 255 260 CCG GCA ACG TTG CTA CCC GCA GTA CGC AGC GAT ATC CCG GTC TTT GTC 1411 Pro Wing Thr Leu Leu Pro Wing Val Arg Ser Asp He Pro Val Phe Val 265 ^ 270 275 GGC TCC AGC AAA GAC CCA CGC GCA GGT GGT ACG CTG GTG TGC AAT AAA 1459 Gly Ser Ser Lys Asp Pro Arg Wing Gly Gly Thr Leu Val Cys Asn Lys 280 285 290 ACT GAA AAT CCG CCG CTG TTC CGC GCT CTG GCG CTT CGT CGC AAT CAG 1507 fhr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu Arg Arg Asn Gln 295 300 305 ACT CTG CTC ACT TTG CAC AGC CTG AAT ATG CTG CAT TCT CGC GGT TTC 1555 Thr Leu Leu Thr Leu Kis Ser Leu Asn Met Leu His Ser Arg Gly Phe 310 315 320 CTC GCG GAA GTT TTC GGC ATC CTC GCG CGG CAT AAT ATT TCG GTA GAC 1603 Leu Wing Glu Val Phe Gly He Leu Wing Arg His Asn He Ser Val Asp 325 330 335 - • 340 TTA ATC ACC ACG TCA GAA GTG AGC GTG GCA TTA ACC CTT GAT ACC ACC 1651 Leu He Thr Thr Ser Glu Val Ser Val Ala Leu Thr Leu Asp Thr Thr 345 350 355 GGT TCA ACC TCC ACT GGC GAT ACG TTG CTG ACG CAA TCT CTG CTG ATG 1699 Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln Ser Leu Leu Met 360 365 370 GAG CTT TCC GCA CTG TGT CGG GTG GAG GTG GAA GAA GGT CTG GCG CTG 1747 Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu Glu Leu Ala Leu 375 380 '«385 GTC GCG TTG ATT GGC AAT GAC CTG TCA AAA GCC TGC GGC GTT GGC AAA 1795 Val Ala Leu He Gly Asn Asp Leu Ser Lys Wing Cys Gly Val Gly Lys 390 395 400 GAG GTA TTC GGC GTA CTG GAA CCG TTC AAC ATT CGC ATG ATT TGT TAT 1843 Glu Val Phe Gly Val Leu Glu Pro Phe Asn H e Arg Met He Cys Tyr 405 410 415 420 GGC GCA TCC AGC CAT AAC CTG TGC TTC CTG GTG CCC GGC GAA GAT GCC 1891 Gly Ala Being Ser His Asn Leu Cys Phe Leu Val Pro Gly Glu Asp Wing 425 430 435 GAG CAG GTG GTG CAA AAA CTG CAT AGT AAT TTG TTT GAG TAAATACTGT 194 Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe Glu 440 445 ATGGCCTGGA AGCTATATTT CGGGCCGTAT TGATTTTCTT GTCACTATGC TCATCAATAA 200 ACGAGCCTGT ACTCTGTTAA CCAGCGTCTT TATCGGAGAA TAATTGCCTT TAATTTTTTT 206 ATCTGCATCT CTAATTAATT ATCGAAAGAG ATAAATAGTT AAGAGAAGGC AAAATGAATA 212 TTATCAGTTC TGCTCGCAAA GGAATTC 214 ID. SEC. NO: 2 LENGTH OF THE SEQUENCE: 20 TYPE OF SEQUENCE: nucleic acid TYPE OF CHAIN: euphoria TOPOLOGY: linear TYPE MOLECULAR: other DNA DESCRIPTION OF THE SEQUENCE CTTCCCTTGT GCCAAGGCTG 20 ID. SEC. NO: 3 LENGTH OF SEQUENCE: 18 TYPE OF SEQUENCE: nucleic acid TYPE OF CHAIN: simple TOPOLOGY: linear MOLECULAR TYPE: other DNA DESCRIPTION OF THE SEQUENCE GAATTCCTTT GCGAGCAG 18 ID. SEC. NO: 4 LENGTH OF THE SEQUENCE: 32 TYPE OF SEQUENCE: nucleic acid TYPE OF CHAIN: einchor TOPOLOGY: linear TYPE MOLECULAR: other DNA DESCRIPTION OF THE SEQUENCE ID. SEC. NO: 5 LENGTH OF THE SEQUENCE: 51 TYPE OF SEQUENCE: nucleic acid TYPE OF CHAIN: simple TOPOLOGY: linear TYPE MOLECULAR: other DNA DESCRIPTION OF THE SEQUENCE ID. SEC. NO: 6 LENGTH OF THE SEQUENCE: 51 TYPE SEQUENCE: nucleic acid CHAIN TYPE: simple TOPOLOGY: linear MOLECULAR TYPE: Other DNA DESCRIPTION OF THE SEQUENCE ID. SEC. NO: 7 LENGTH OF THE SEQUENCE: 30 TYPE OF SEQUENCE: nucleic acid TYPE OF CHAIN: simple TOPOLOGY: linear TYPE MOLECULAR: other DNA DESCRIPTION OF THE SEQUENCE

Claims (6)

NOVELTY OF THE INVENTION CLAIMS

1. - A DNA that encodes asp rtocmaea III of bacteria belonging to the genus Eschepchia and that have in the coding region mutation by which they release inhibition by feedback with lysine of said aspartakinase III, said mutation being mutation by which the residue of Etionine 318 from aspartacinaea III ee is replaced by another amino acid residue and glycine 323 residue from another amino acid residue, mutation by which the leccine residue 325 is replaced by another amino acid and the reuse of value 347 by another residue of amino acid, mutation by which the glycine residue 323 is replaced by another amino acid residue and the valm residue 347 by another amino acid residue, mutation by which the leucma residue 325 is replaced by another amino acid residue and the serine 345 residue by another amino acid residue, mutation by which the glycine 323 residue is replaced by another amino acid residue and the residue of septen to 358 by another amino acid residue, a mutation by which the tremene residue 344 is replaced by another amino acid residue, a mutation by which the residue of glutamic acid 250 is replaced by another amino acid residue, a mutation whereby the residue of glutamic acid 346 is replaced by another amino acid residue and the residue of leucine 347 by another amino acid residue, mutation by which the residue of glutarnic acid 250 is replaced by another amino acid residue and the residue of threo in 364 by another amino acid residue, mutation by which the acid residue aspartic 202 is replaced by another amino acid residue and the serine residue 321 by another amino acid residue, mutation whereby the arginine residue 283 is replaced by another amino acid residue, the alanine residue 333 by another amino acid residue, the residue of septa 338 by another amino acid residue, the residue of glutamic acid 346 by another amino acid residue, and the residue of aepargma 414 by another amino acid residue, or mutation by which the residue of rnetiomna 318 is substitued by another residue of amino acid, the residue of serine 321 by another amino acid residue, the valine residue 328 by another amino acid residue, the residue of valma 349 by another amino acid residue and the amino acid residue. 405 glutarnic acid residue for another amino acid residue.

2. The DNA according to claim 1, further characterized in that the mutation is a mutation whereby the residue of rnethionine 318 of aspartakinase III is replaced by a residue of isoleucine and the re-residue of glycine 323 by a residue of acid of aspar. -tico, mutation by which the leucine residue 325 is replaced by phenylalanine residue and the valine residue 347 by a methionine residue, mutation by which the glycine 323 residue is replaced by residue of aspherical acid and the residue of valma 347 by a rnetiomna residue, mutation by which the leucine residue 325 is replaced by residue of f- "in? lalan? na and the residue of serine 345 for a leucine residue, a mutation whereby the glycine residue 323 is replaced by aspartic acid residue and the serine 358 residue is a leucine residue, a mutation whereby the residue of treomna 344 is replaced by a residue of rnethionine, a mutation by which the glutarnic acid residue 250 is replaced by a lysine residue, a mutation whereby the glutaric acid residue 346 is replaced by a lysine residue and the leucine residue 347 is replaced by a phenylalanine residue, mutation whereby the glyceric acid residue 250 is replaced by a lysine residue and the threonine residue by a rnethionine residue, a mutation by which the aspartic acid redox 202 is replaced by a residue of glycine and the senna 321 residue by a prolma residue, mutation whereby the arginine residue 283 is replaced by a sepna residue, the alanine residue 333 by a treomna residue, the sepna residue 338 by a threonine residue, the re-residue of glutamic acid 346 by a residue of aspartic acid and the residue of asparagma 414 by a sepna residue, or mutation whereby the residue of methiomna 318 is replaced by a lysine residue, the residue of serine 321 by a residue of prolma, the residue of valine 328 by a residue of phenylamine, the residue of valine 349 by a residue of glycine and the residue of 405 giutarnic acid for a residue of valine.

3. A recornbinante DNA that is produced by ligating the DNA of claim 1 with a vector DNA capable of undergoing autonomous replication in cells of bacteria of the genus Eschenchia.

4. A microorganism of the genus Eschenchia carrying the DNA of claim 1.

5. A process for producing an L-ammo acid, which comprises incubating the microorganism of claim 4 that has a capacity to produce an L-amino acid in a fermentation medium, producing and accumulating an L-arninoacid in said culture, and collecting the L-arninoacid from the culture.

6. The process according to claim 5, further characterized in that the L-arninoacid is L-lysine or L-treomna.