RU2133772C1 - Modified phosphoenolpyruvate carboxylase, dna fragment, strain escherichia coli, recombinant dna, method of preparing amino acid (variants), method of escherichia coli cells selection, method of preparing modified phosphoenolpyruvate carboxylase, method of preparing dna fragment and method of microorganism preparing - Google Patents

Abstract

FIELD: molecular biology, genetic engineering, biotechnology. SUBSTANCE: invention proposes phosphoenolpyruvate carboxylase where glutamic acid residue at position 625 is substituted with lysine and/or arginine residue at position 222 and/or glutamic acid residue at position 223 is substituted with histidine residue and lysine residue, respectively. Also, serine residue at position 288, glutamic acid residue at position 289, methionine residue at position 551 and glutamic acid residue at position 804 can be substituted with phenylalanine residue, lysine residue, isoleucine residue and lysine residue, respectively. Also, alanine residue at position 867 can be substituted with threonine residue and/or arginine residue at position 438 can be substituted with cysteine residue and/or lysine residue at position 620 can be substituted with serine residue. Phosphoenolpyruvate carboxylase is produced by Escherichia coli strain. Modified phosphoenolpyruvate carboxylase shows low sensitivity to inhibition effect of aspartic acid. EFFECT: improved method of preparing and selection. 18 cl, 13 dwg, 9 tbl, 6 ex

Description

 This invention relates to a mutant phosphoenolpyruvate carboxylase, a gene encoding it and a method for producing an amino acid, and in particular, to a gene having a mutation causing desensitization (decrease in sensitivity) to feedback inhibition by aspartic acid, and its use.

BACKGROUND OF THE INVENTION
Phosphoenolpyruvate carboxylase is an enzyme that is found in almost all bacteria and in all plants. This enzyme plays a role in the biosynthesis of aspartic acid and glutamic acid and the supply of C4-dicarboxylic acid to the citric acid cycle to maintain its turnover. However, in the field of enzymatic production of amino acids by microorganisms, there have been several reports of the action of this enzyme that have not clarified its role (Atsushi Yokota and Isamu Shiio, Agric. Biol. Chem., 52, 455-463 (1988), Josef Cremer et al ., Appl. Environ. Microbiol., 57, 1746-1752 (1991), Petra, G. Peters-Weintisch, FEMS Microbiol. Letters, 112, 269-274 (1993)).

 Incidentally, an amino acid is a compound that exists everywhere in the cells in the form of protein components, however, in order to economically metabolize energy and metabolize substances, its formation is strictly controlled. This control is in principle a feedback control in which the end product of the metabolic pathway inhibits the activity of the enzyme catalyzing an earlier stage of this pathway. Phosphoenolpyruvate carboxylase also undergoes various types of regulation in the manifestation of its activity.

 For example, in the case of phosphoenolpyruvate carboxylase of microorganisms belonging to the genus Corynebacterium or to the genus Escherichia, the activity of the enzyme is inhibited by aspartic acid. Thus, the aforementioned amino acid biosynthesis, in which phosphoenolpyruvate carboxylase is involved, is also inhibited by aspartic acid.

 In previous studies, various methods were developed for the efficient production of amino acids by fermentation, and leucine, isoleucine, tryptophan, phenylalanine, and the like were obtained enzymatically. using mutant strains that were made insensitive to control by type of feedback. However, neither the mutant phosphoenolpyruvate carboxylase converted into an enzyme insensitive to inhibition of aspartic acid was known, nor the attempts to use this enzyme to enzymatically produce amino acids from the aspartic acid family and glutamic acid family.

 On the other hand, the ppc gene has already been cloned, which is the gene encoding Escherichia coli phosphoenolpyruvate carboxylase, and its nucleotide sequence has also been determined (Fujita, N., Miwa, T., Ishijima, S., Izui, K. and Katsuki, H ., J. Biochem., 95, 909-916 (1984). However, there is no reported mutant in which sensitivity to inhibition of aspartic acid is reduced.

 This invention is carried out on the basis of this point of view and the purpose of this invention is the creation of mutant phosphoenolpyruvate carboxylase with a significantly reduced sensitivity to inhibition of aspartic acid by the type of feedback, providing a gene encoding the enzyme, and method of its use.

Description of the invention
As a result of a diligent study to achieve the above goal, the authors of the present invention found that the inhibition of aspartic acid is significantly desensitized by replacing the amino acid in the specific site of the phosphoenolpyruvate carboxylase of Escherichia coli with another amino acid, which led to the generation of a gene encoding a mutant enzyme, which was the completion of this invention.

 Specifically, the present invention provides a mutant phosphoenolpyruvate carboxylase derived from a microorganism of the genus Escherichia and having a mutation to reduce feedback feedback sensitivity and desensitization of aspartic acid, and a DNA sequence encoding a mutant phosphoenolpyruvate carboxylase.

 Further, this invention provides microorganisms belonging to the genus Escherichia or coryneform bacteria carrying this DNA fragment, and a method for producing an amino acid comprising culturing any of these microorganisms in a preferred medium and isolating amino acids selected from L-lysine, L-threonine, L-methionine, L-isoleucine, L-glutamic acid, L-arginine and L-proline, from this medium.

 As applied to this description of DNA, a sequence encoding a mutant phosphoenolpyruvate carboxylase, or a DNA sequence containing, in addition, a promoter, is sometimes referred to simply as the "DNA sequence of the present invention", "mutant gene" or "phosphoenolpyruvate carboxylase gene".

 The invention will now be explained in detail.

 <1> Mutant phosphoenolpyruvate carboxylase.

 The mutant phosphoenolpyruvate carboxylase of the present invention (hereinafter referred to as the "mutant enzyme") is based on the phosphoenolpyruvate carboxylase of a microorganism belonging to the genus Escherichia having a mutation that causes desensitization (decrease in sensitivity) to feedback inhibition of aspartic acid.

Such a mutation can be any mutation, provided that the feedback inhibition sensitivity is significantly reduced without loss of the enzymatic activity of phosphoenolpyruvate carboxylase. An example is the mutation, which in the case of the existence of a mutant phosphoenolpyruvate carboxylase with such a mutation in the cells of the Escherichia microorganism gives these cells resistance to a compound having the following properties:
the compound exhibits a growth inhibitory effect against a microorganism of the genus Escherichia producing non-mutant phosphoenolpyruvate carboxylase (wild type);
said growth inhibitory effect is removed by the presence of L-glutamic acid or L-aspartic acid; and
the compound inhibits the wild-type phosphoenolpyruvate carboxylase activity.

More specifically, as examples of such a mutation, the following mutations may be given (numbering from the N-terminus of the phosphoenolpyruvate carboxylase):
(1) a mutation to replace glutamic acid 625 with lysine;
(2) a mutation to replace 222 with histidine and glutamic acid 223 with lysine, respectively;
(3) a mutation to replace serine 288 with phenylalanine, glutamic acid 289 with lysine, methionine 551 with isoleucine, and glutamic acid 804 with lysine, respectively;
(4) a mutation to replace alanine 867 with threonine;
(5) a mutation to replace arginine 438 with cysteine; and
(6) mutation to replace lysine 620 with serine.

 For a phosphoenolpyruvate carboxylase of a microorganism belonging to the genus Escherichia, the amino acid sequence deduced from the phosphoenolpyruvate carboxylase gene of Escherichia coli (Fujita, N., Miwa, T., Ishijima, S., Izui, K. and Katsuki, H., J. Biochem., 95 , 909-916 (1984)), shown in SEQ ID N 2 in the List of sequences. In addition, the complete nucleotide sequence of the pT2 plasmid containing the Escherichia coli phosphoenolpyruvate carboxylase gene is shown in SEQ ID N 1 together with the amino acid sequence (see end of description).

 The above mutant enzymes are encoded by the DNA sequences of the invention described below, which are formed by expression of these DNA sequences in Escherichia coli and the like.

 <2> The DNA sequence of the present invention and the microorganisms containing it.

 The DNA sequence of the present invention includes DNA sequences encoding the aforementioned mutant enzymes and has a mutation causing desensitization to feedback inhibition of phosphoenolpyruvate carboxylase by aspartic acid in the coding regions in DNA fragments encoding the phosphoenolpyruvate carboxylase of a microorganism belonging to the genus Escherichia.

Specifically, an DNA sequence encoding a phosphoenolpyruvate carboxylase having any of the above mutations (1) to (6) can be carried out as an example. With respect to the nucleotide sequence of SEQ ID N 1, an example of a DNA sequence having any of the following mutations can be given:
i) a mutation for the conversion of GAA with base numbers 2109-2111 in AAA or AAG;
ii) a mutation to convert CGC with base numbers 900-902 to CAT or CAC and GAA 903-905 to AAA or AAG, respectively;
iii) a mutation to convert TCT with base numbers 1098-1100 to TTT or TTC, GAA 1101-1103 to AAA or AAC, ATG 1887-1889 to ATT, ATC or ATA and GAA 2646-2648 to AAA or AAG, respectively:
iv) a mutation to convert GCG 2835-2837 into any of the ACT, ACC, ACA, and ACG codons; and
v) a mutation to convert CGT 1548-1550 to TGT or TGC; and
vi) a mutation to convert AAA 2094-2096 to TCT, TCC, TCA or TCG.

 Such a mutant gene is prepared in such a way that a recombinant DNA obtained by ligating a phosphoenolpyruvate carboxylase gene as a non-mutant enzyme gene or having a different gene mutation, with a host adapted DNA vector, is subjected to a mutation treatment to screen for transformants using recombinant DNA. Alternatively, it is also acceptable to subject the non-mutant enzyme producing microorganism to a mutation treatment, which results in a mutant strain producing the mutant enzyme, and then the mutant gene is screened from the mutant strain. To obtain a mutation of recombinant DNA, hydroxylamine and the like are used. Further, when treating the microorganism itself with the conditions of mutagenesis, a drug or method commonly used for artificial mutagenesis can be used.

 Further, in accordance with methods such as a method of overlapping elongation (Ho, SN, Hunt, HD, Horton, RM, Publen, JK and Pease, LR, Gene, 77, 51-59 (1989)), a method for site-specific mutagenesis ( Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. In Enzymol., 154, 367 (1987)) and others, the indicated mutant gene may can also be obtained by introducing a mutation, such as an amino acid substitution, insertion, deletion and the like, into the phosphoenolpyruvate carboxylase gene as a non-mutant enzyme gene or as a gene having a different mutation. These methods are based on the principle that a non-mutated DNA gene is used as a template, and synthetic DNA containing erroneous base pairing at the mutation point is used as one of the primers for the synthesis of complementary chains of the indicated DNA gene, as a result of which the mutation is introduced. Using these methods, the putative mutation can be obtained at the target site.

 Alternatively, a sequence is synthesized that has restriction enzyme cleavage sites at both ends and includes both sides of the mutation point, and replaces the corresponding portion of the non-mutated gene with it, whereby a mutation can be introduced (cassette mutation method).

 A phosphoenolpyruvate carboxylase gene, representing a non-mutant gene of an enzyme or having a different mutation used to introduce a mutation, can be any gene of this enzyme provided that it is a gene encoding a phosphoenolpyruvate carboxylase of a microorganism belonging to the genus Escherichia, which preferably has a specific base sequence (i.e. sequenced) and cloned. If it has not been cloned, a DNA fragment containing this gene can be amplified and isolated using the PCR method, etc. followed by the use of a suitable vector to achieve cloning.

 An example of the gene described above is the Escherichia coli gene, which has been cloned and sequenced (Fujita, N., Miwa, T., Ishijima, S., Izui, K. and Katsuki, H., J. Biochem., 95, 909-916 (1984)). The sequence in the coding region of this gene is shown in SEQ ID N 1 (base N 237-2888).

 Screening of a host carrying this mutant gene can be performed using an aspartic acid analog compound. The analog compound preferably has the following properties. It exhibits a growth inhibitory effect against a microorganism belonging to the genus Escherichia producing non-mutant phosphoenolpyruvate carboxylase, this inhibitory effect is removed by the presence of L-glutamic acid or L-aspartic acid, and the analogue inhibits wild-type phosphoenolpyruvate carboxylase.

 If a mutant strain is selected from microorganisms of the genus Esherichia that is resistant to the analogue compound mentioned above, for example, Escherichia coli HB101 producing wild-type phosphoenolpyruvate carboxylase using growth inhibition of this microorganism as a criterion, there is a high probability of obtaining a host microorganism that produces phosphoenolpyruvate carboxylase with reduced sensitivity to feedback inhibition by aspartic acid.

 As a general structure of the phosphoenolpyruvate carboxylase inhibitor, the structure of C4 carboxylic acids is mainly proposed. From this point of view, the authors of the present invention screened various compounds. Escherichia coli HB101 was cultured in LB medium and transferred to M9 medium (containing 20 μg / ml thiamine and 3 μg / ml each of Leu and Pro), also containing one of the following compounds: DL-2-amino-4-phosphonobutyric acid, bromosuccinic acid, meso-2,3-dibromosuccinic acid, 2,2-difluorosuccinic acid, 2-bromopyruvic acid, α-ketobutyric acid, α-ketoadipic acid, DL-threo-β-hydroxyaspartic acid, L-aspartic acid β-methyl ester , α-methyl-DL-aspartic acid, 2,3-diamino-succinic acid or aspartic acid β-hydrazide, and The absorption of this medium was measured at 660 nm with increasing time, observing the growth.

 Further, when these compounds were present at growth inhibitory concentrations, it was investigated whether this inhibition was removed by the addition of nucleic acids (uridine, adenosine, each at a concentration of 10 mg / dl), glutamic acid or amino acids of the aspartic acid family (Asp: 0.025%, each of Met, Thr, Lys: 0.1%).

 As a result, three compounds were selected: 3-bromopyruvate (3BP) (1), aspartate-β-hydrazide (AHY) (2) and DL-tert-β-hydroxyaspartate (βHA) (3).

Ингибирование роста Escherichia coli этими соединениями-аналогами показано на фиг. 1-3. Далее восстановление роста Escherichia coli в случае добавления снимающих ингибирование веществ, отдельно или в виде смеси 2 или 3 типов веществ, показано на фиг. 4-6. Кроме того, в качестве контроля рост в случае добавления снимающих ингибирование веществ в отсутствие ингибирующего вещества показан на фиг. 7. На фиг. 4-7 добавки 1, 2 и 3 обозначают нуклеиновые кислоты, глутаминовую кислоту или аминокислоты семейства аспарагиновой кислоты соответственно.

Inhibition of Escherichia coli growth by these analog compounds is shown in FIG. 1-3. Further, the growth recovery of Escherichia coli in the case of adding inhibition-removing substances, separately or as a mixture of 2 or 3 types of substances, is shown in FIG. 4-6. In addition, as a control, growth in the case of adding inhibition-removing substances in the absence of an inhibitory substance is shown in FIG. 7. In FIG. 4-7, additives 1, 2 and 3 are nucleic acids, glutamic acid or amino acids of the aspartic acid family, respectively.

 Further, the inhibition of activity by an analog compound of phosphoenolpyruvate carboxylase was also investigated. The crude enzyme was obtained from Escherichia coli strain HB101 according to the method described in Journal of Biochemistry, vol. 67, No. 4 (1970) and enzyme activity was measured according to the method described in Eur. J. Biochem., 202, 797-803 (1991).

 Escherichia coli HB101 cells cultured in LB medium were disrupted, and the suspension was centrifuged to obtain a supernatant, which was used as a crude enzyme solution. The enzyme activity was measured by measuring the decrease in absorbance at 340 nm, adding acetyl coenzyme A, which is known to affect the enzyme activity, at a concentration of 0.1 mM in a measurement system containing 2 mM potassium phosphoenolpyruvate, 0.1 mM NADH, 0.1 M Tris Acetate (pH 8.5), 1.5 E malate dehydrogenase and crude enzyme. The results are shown in FIG. eight.

 In accordance with the above results, it is obvious that these three compounds inhibit the growth of Escherichia coli, this inhibition cannot be removed by the addition of nucleic acids only, but it can be removed by the addition of glutamic acid or amino acids of the aspartic acid family. Thus, these analog compounds are selective inhibitors of phosphoenolpyruvate carboxylase. As shown in the examples described below, using these compounds in the present invention, it was possible to select Escherichia coli, which produces mutant phosphoenolpyruvate carboxylase.

 When screening for a transformant having a target gene of a mutant enzyme using these compounds and isolating recombinant DNA, a mutant gene of the enzyme is obtained. Alternatively, in the case of a mutating treatment of the microorganism itself, when a mutant strain having the target mutant enzyme gene is screened using these compounds, a DNA fragment containing the target mutant enzyme gene is isolated from the strain, and it is ligated with a suitable vector and then the mutant enzyme gene is obtained.

 On the other hand, as a result of a thorough study of the authors of this invention, paying attention to the importance of the arginine residue in the aspartate binding protein Esherichia coli (Krikos, A., Mouth, N., Boyd, A. and Simon, MI Cell, 33, 615-622 ( 1983), Mowbray, SL and Koshland, DEJ Biol. Chem., 264, 15638-15643 (1990), Milburn, MV, Prive. GG, Milligan, DL, Scott, WG, Yeh, J., Jancarik, J., Koshland, DE and Kim, SH, Science, 254, 1342-1347 (1991)), it was found that inhibition of aspartic acid is substantially desensitized by the conversion of arginine 438 phosphoenolpyruvate carboxylase to cysteine. To convert arginine 438 to cysteine, the arginine codon 438 of the gene encoding phosphoenolpyruvate carboxylase can be converted to a cysteine codon. For example, in SEQ ID N 1 CGT, nucleotides 1548-1550 can be converted to TGT or TGC.

 Further, the authors of the present invention carried out chemical modification of lysine residues of phosphoenolpyruvate carboxylase with 2,4,6-trinitrobenzenesulfonic acid (TNBS), which is a compound that chemically modifies lysine residues in a protein. During the modification reaction, malic acid was also present, capable of acting as an inhibitor of phosphoenolpyruvate carboxylase. It was assumed that the lysine residue near the binding position of phosphoenolpyruvate carboxylase could be protected by binding of malic acid and not be chemically modified. As a result, it was suggested that the lysine 620 residue was important for the binding of malic acid to phosphoenolpyruvate carboxylase, and it was found that feedback inhibition by aspartic acid was desensitized while maintaining the enzymatic activity of phosphoenolpyruvate carboxylase by converting the lysine 620 residue to the serine residue. To convert a lysine residue to a serine residue, the lysine codon 620 of a gene encoding phosphoenolpyruvate carboxylase can be converted to a serine codon. For example, in SEQ ID N 1 AAA with nucleotide numbers 2094-2096, TCT, TCC, TCA, TCG, AGT or AGC can be replaced.

 In accordance with methods such as the overlapping extension method (Ho, SN, Hunt, HD, Horton, RM, Pullen, JK, and Pease, LR, Gene, 77, 51-59 (1989)), the site-specific mutation method ( Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. In Enzymol., 154, 367 (1987)) and the like, converting this the codon can be achieved by introducing a mutation such as amino acid substitution, insertion, deletion, etc., into the phosphoenolpyruvate carboxylase gene as a wild-type enzyme gene or as a gene having a different mutation. These methods are based on the principle that a non-mutated DNA gene is used as a template, and synthetic DNA containing mismatching at a mutation point is used as one of the primers for the synthesis of complementary chains of the aforementioned DNA gene, thereby introducing a mutation. Using these methods, the desired mutation can be obtained at the target site.

 Alternatively, a sequence is synthesized having restriction enzyme cleavage sites at both ends and containing both sides of the mutation point, and replaces with it the corresponding portion of the non-mutated gene, whereby a mutation can be introduced (cassette mutation method).

 A DNA fragment encoding a phosphoenolpyruvate carboxylase with the mutation introduced as described above is expressed using a suitable host-vector system by which a mutant enzyme can be obtained. Alternatively, even by performing a transformation by inserting a DNA fragment of the invention into the chromosomal DNA of the host, the desired mutant enzyme can be obtained.

 Microorganisms belonging to the genus Escherichia, for example Escherichia coli, coryneform bacteria, and the like, can be used as a host. Coryneform bacteria include bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus Brevibacterium, classified so far as belonging to the genus Brevibacterium, but currently combined as bacteria belonging to the genus Corynebacterium, and bacteria actually belonging to the genus Breviberium closely related to bacteria belonging to the genus Corynebacterium. Hosts preferred for the production of amino acids will be described below.

 On the other hand, a plasmid vector is preferred as a DNA vector, and plasmid vectors capable of self-replicating in a host cell are preferred. In the case of the Escherichia coli host, for example, pUC19, pUC18, pBR322, pHSG299, pHSG399, RSF1010 and the like are applicable. Alternatively, phage DNA can also be used as a vector.

 Further, in the case where the host is a coryneform bacterium, the vectors and host carriers thereof indicated below can be used. In these examples, deposit numbers in international repositories (depositories) are shown in parentheses.

pAj655 Escherichia coli Aj11882 (FERM BP-136) Corynebacterium glutamicum SR8201 (ATCC 39135)
pAj1844 Escherichia coli Aj11883 (FERM BP-137) Corynebacterium glutamicum 8202 (ATCC 39136)
pAj611 Escherichia coli Aj11884 (FERM BP-138)
pAj3148 Corynebacterium glutamicum 8203 (ATCC 39137)
pAj440 Bacillus subtilis Aj11901 (FERM BP-140)
These vectors can be obtained from deposited microorganisms as follows. Cells collected in the logarithmic growth phase are subjected to bacteriolysis using lysozyme and sodium dodecyl sulfate (SDS) and centrifuged at 30000g to obtain a supernatant solution from the lysate, to which polyethylene glycol is added to separate and purify the vectors by equilibrium centrifugation in a density gradient of cesium chloride- ethidium bromide.

 To transform Escherichia coli with a recombinant vector obtained by inserting the DNA sequence of the present invention into the above vector, a method commonly used to transform Escherichia coli can be used, for example, a method in which cells are treated with calcium chloride to enhance DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1977)) and the like.

 Further, as the method for transforming coryneform bacteria, the aforementioned method is also used in which the cells are treated with calcium chloride, or a method in which inclusion is performed during a specific growth period in which the cells may include DNA (message regarding Bacillus subtilis Duncan, CH et al.). Further incorporation into bacterial cells can be achieved by the formation of protoplasts or spheroplasts of DNA recipients that readily include plasmid DNA. They are known for Bacillus subtilis, Actinomyces, and yeast (Chang, S. et al., Molec. Gen. Genet., 168, 111 (1979), Bibb et al., Nature 274, 398 (1978), Hinnen, A. et al., Proc. Natl. Acad. Sci. USA, 75 1929 (1978)). Additionally, a method for transforming coryneform bacteria is described in JP Laid-open N 2-207791.

 For expression of the DNA sequence of the present invention in the above hosts, promoters such as lac, trp, PL and the like, which work efficiently in microorganisms, or when the sequence of the present invention contains a promoter of the phosphoenolpyruvate carboxylase gene, this promoter can be used as such. Alternatively, if a coryneform bacterium is used as a host, the known trp promoter from a bacterium belonging to the genus Brevibacterium (JP Laid-open N 62-244382) and others can also be used.

 Further, as described above, it is permissible that the DNA sequence of the present invention is inserted into a self-replicating DNA vector and introduced into the host so that the host retains it in the form of a plasmid. It is also permissible that the DNA sequence of the present invention be integrated into the chromosome of the microorganism using a transposon method (Berg, DE and Berg, CM, Bio / Technol, 1, 417 (1983)) of the Mu-- phage (Japanese Patent Laid-open N 2-109985) or homologous recombination (Experiments in Molecular Genetics, Cold Spring Harbor Lab. (1972)). In addition, the temperature-sensitive plasmid described in Japanese Patent Laid-open N 5-7491 can be used to integrate the DNA of the present invention into coryneform bacteria.

 By culturing a microorganism transformed with the DNA sequence of the invention described above and expressing this DNA sequence, a mutant enzyme is obtained. As a result of measuring activity by adding aspartic acid to the enzyme reaction system, it becomes clear whether the mutant enzyme thus obtained has a reduced sensitivity to feedback inhibition by aspartic acid. A spectrometric method (Yoshinage, T., Izui, K. and Katsuki, H., J. Biochem., 68, 747-750 (1970)) and the like can be used to measure enzyme activity.

 Further, the DNA sequence of the present invention encodes a mutant enzyme in which feedback inhibition by aspartic acid is desensitized, so that a microorganism carrying this DNA sequence can be used to efficiently enzymatically produce amino acids of the aspartic acid family and the glutamic acid family, as described below.

 Escherichia coli AJ12907, AJ12908, AJ12909 and AJ12910 carrying the mutant enzyme genes obtained in the examples described below were deposited with the National Institute of Bioscience and Human Technology of Agancy of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba- shi, Ibaraki-ken, Japan; zip code 305) August 3, 1993 under deposit numbers FERM P-13774, FERM P-13775, FERM P-13776 and FERM P-13777, transferred from the original depositories to the international depository based on Budapest Treaty July 11, 1994 and deposited under deposit numbers FERM BP-4734, FERM BP-4735, FERM BP-4736, FERM BP-4737, respectively, in this sequence.

 <3> A method for producing amino acids.

 Amino acids can be obtained by culturing a microorganism carrying the DNA sequence of the invention in a preferred medium and isolating the resulting amino acids. Such amino acids may be, for example, L-lysine, L-threonine, L-methionine, L-isoleucine, L-glutamic acid, L-arginine and L-proline.

 Examples of preferred hosts into which the DNA sequence of this invention is introduced for use in the preparation of each of these amino acids, and the cultivation method are given below.

 (1) Hosts preferred for the production of L-lysine.

 An example of a host that can be used to produce L-lysine according to this invention can be bacteria belonging to the genus Escherichia, preferably producing L-lysine of Escherichia coli. Specifically, a mutant strain resistant to a lysine analogue can be used. Such lysine analogs are those that inhibit the growth of microorganisms belonging to the genus Escherichia, however, this suppression is fully or partially desensitized, provided that L-lysine is also present in the medium. For example, there are analogues such as oxalysine, lysine hydroxamate-S- (2-aminoethyl) -cysteine (hereinafter abbreviated as "AEC"), γ-methyllysine, α-chlorocaprolactam, etc. Mutant strains that are resistant to these lysine analogues may be obtained by applying conventional artificial mutational processing to microorganisms of the genus Escherichia. Specifically, Escherichia coli AJ11442 (deposited as FERM P-5084, see lower left column on Japanese Patent Laid-open N 56-18596) can be used as a bacterial strain that can be used to produce L-lysine.

On the other hand, various artificial mutant strains of coryneform bacteria can be used for the present invention as lysine producing bacteria. Such artificial mutant strains are the following: AEC-resistant mutant strain; a mutant strain requiring amino acids for its growth, such as L-homoserin (Japanese Patent Publication N 48-28078 and N 56-6499); A mutant strain exhibiting resistance to AEC and requiring amino acids such as L-leucine, L-homoserin, L-proline, L-serine, L-arginine, L-alanine, L-valine, and the like. for its growth (US Patent N 3708395 and N 3825472); a L-lysine-producing mutant strain exhibiting resistance to DL α-amino ε-caprolactam, α-amino-laurillactam, quinoid and N-lauroylleucine; L-lysine-producing mutant strain resistant to an oxaloacetate decarboxylase inhibitor or respiratory system enzyme (Japanese Patent Laid-open N 50-53588, 52-102498, 53-86089, 55-9783, 55-9759, 56-32995, 56-39778 and Japanese Patent Publication N 53-43591 and 53-1833); L-lysine-producing mutant strain requiring inositol or acetic acid (Japanese Patent Laid-open N 55-9784 and 56-8692); mutant strain producing L-lysine, exhibiting sensitivity to fluoropyruvate or a temperature of at least 34 ^o C (Japanese Patent Laid-open N 55-9783 and 53-86090); and a mutant strain of Brevibacterium or Corynebacterium exhibiting resistance to ethylene glycol and producing L-lysine (see US Patent Application Serial N 333455).

The following are specific examples of coryneform bacteria useful for producing lysine:
Brevibacterium lactofermentum AJ12031 (FERM-BP277), see page 525 in Japanese Patent Laid-open N 60-62994;
Brevibacterium lactofermentum ATCC 39134, see lower right column on page 473 in Japanese Patent Laid-open N 60-62994;
Brevibacterium lactofermentum AJ3463 (FERM-P1987), see Japanese Patent Publication N 51-34477.

 In addition, wild strains of coryneform bacteria described below can also be used in the same manner for the present invention.

Corynebacterium acetoacidophilum ATCC 13870
Corynebacterium acetoglutamicum ATCC 15806
Corynebacterium callunae ATCC 14991
Corynebacterium glutamicum ATCC 13032
ATCC 13060
(Brevibacterium divaricatum) ATCC 14020
(Brevibacterium lactofermentum) ATCC 13869
(Corynebacterium lilium) ATCC 15990
Corynebacterium melassecola ATCC 17965
Brevibacterium saccharolyticum ATCC 14066
Brevibacterium immariophilum ATCC 14068
Brevibacterium roseum ATCC 13825
Brevibacterium flavum ATCC 13826
Brevibacterium thiogenitalis ATCC 19240
Microbacterium ammoniaphilum ATCC 15354
(ii) Hosts preferred for the production of L-threonine.

Escherichia coli B-3996 (RIA 1867), see Japanese Patent Laid-open N 3-501682 (PCT);
Escherichia coli AJ12349 (FERM-P9574), see upper left column on page 887 in Japanese Patent Laid-open N 2-458;
Escherichia coli AJ12351 (FERM-P9576), see lower right column on page 887 in Japanese Patent Laid-open N 2-458;
Escherichia coli AJ12352 (FERM P-9577), see upper left column on page 888 in Japanese Patent Laid-open N 2-458;
Escherichia coli AJ11332 (FERM P-4898), see upper left column on page 889 in Japanese Patent Laid-open N 2-458;
Escherichia coli AJ12350 (FERM P-9575), see upper left column on page 889 in Japanese Patent Laid-open N 2-458;
Escherichia coli AJ12353 (FERM P-9578), see upper right column on page 889 in Japanese Patent Laid-open N 2-458;
Escherichia coli AJ12358 (FERM P-9764), see upper left column on page 890 in Japanese Patent Laid-open N 2-458;
Escherichia coli AJ12359 (FERM P-9765), see upper left column on page 890 in Japanese Patent Laid-open N 2-458;
Escherichia coli AJ11334 (FERM P-4900), see column 6 on page 201 of Japanese Patent Publication N 1-29559;
Escherichia coli AJ11333 (FERM P-4899), see column 6 on page 201 of Japanese Patent Publication N 1-29559;
Escherichia coli AJ11335 (FERM P-4901), see column 7 on page 202 in Japanese Patent Publication N 1-29559.

The following bacterial strains are given as examples of coryneform bacteria:
Brevibacterium lactofermentum AJ11188 (FERM P-4190), see upper right column on page 473 in Japanese Patent Laid-open N 60-87788;
Corynebacterium glutamicum AJ11682 (FERM BP418), see column 8 on page 230 in Japanese Patent Publication N 2-31956;
Brevibacterium flavum AJ11683 (FERM BP-119), see column 10 on page 231 of Japanese Patent Publication N 2-31956.

 (iii) Hosts preferred for the production of L-methionine.

The following bacterial strains are given as examples of bacteria for producing L-methionine:
Escherichia coli AJ11457 (FERM P-5175), see upper right column on page 552 in Japanese Patent Laid-open N 56-35992;
Escherichia coli AJ11458 (FERM P-5176), see upper right column on page 552 in Japanese Patent Laid-open N 56-35992;
Escherichia coli AJ11459 (FERM P-5177), see upper right column on page 552 in Japanese Patent Laid-open N 56-35992;
Escherichia coli AJ11539 (FERM P-5479), see lower left column on page 435 in Japanese Patent Laid-open N 56-144092;
Escherichia coli AJ11540 (FERM P-5480), see lower left column on page 435 in Japanese Patent Laid-open N 56-144092;
Escherichia coli AJ11541 (FERM P25481), see lower left column on page 435 in Japanese Patent Laid-open N 56-144092;
Escherichia coli AJ11542 (FERM P-5482), see lower left column on page 435 in Japanese Patent Laid-open N 56-144092.

 (iv) Hosts preferred for the production of L-aspartic acid.

The following bacterial strains are given as examples of bacteria for obtaining L-aspartic acid:
Brevibacterium flavum AJ3859 (FERM P-2799), see upper left column on page 524 Japanese Patent Laid-open N 51-61689;
Brevibacterium lactofermentum AJ3860 (FERM P-2800), see upper left column on page 524 in Japanese Patent Laid-open N 51-61689;
Corynebacterium acetoacidophilum AJ3877 (FERM P-2803), see upper left column on page 524 in Japanese Patent Laid-open N 51-61689;
Corynebacterium glutamicum AJ3876 (FERM P-2802), see upper left column on page 524 in Japanese Patent Laid-open N 51-61689;
(v) Hosts preferred for the production of L-isoleucine.

 Escherichia coli KX141 (VKPM-B4781) (see paragraph 45 in Japanese Patent Laid-open N 4-33027) is listed as a bacterium belonging to the genus Escherichia, and Brevibacterium lactofermentum AJ12404 (FERM P-10141) (see bottom left column on page 603 in Japanese Patent Laid-open N 2-42988) and Brevibacterium flavum AJ12405 (FERM P-10142) (see lower left column on page 524 in Japanese Patent Laid-open N 2-42988) are given as examples coryneform bacteria.

 (vi) Hosts preferred for the production of L-glutamic acid.

The following bacterial strains are listed as bacteria belonging to the genus Escherichia:
Escherichia coli AJ12628 (FERM P-12389), see Franch Patent Publication No. 2,680 (1993);
Escherichia coli AJ12624 (FERM P-12379), see French Patent Publication No. 2,680,178 (1993).

The following bacterial strains are given as examples of coryneform bacteria:
Brevibacterium lactofermentum AJ12745 (FERM BP-2922), see lower right column on page 561 in Japanese Patent Laid-open N 3-49690;
Brevibacterium lactofermentum AJ12746 (FERM BP-2923), see upper left column on page 562 in Japanese Patent Laid-open N 3-49690;
Brevibacterium lactofermentum AJ12747 (FERM BP-2924), see upper left column on page 562 in Japanese Patent Laid-open N 3-49690;
Brevibacterium lactofermentum AJ12748 (FERM BP-2925), see upper left column on page 562 in Japanese Patent Laid-open N 3-49690;
Brevibacterium flavum ATCC 14067, see table 1 on page 3 in Japanese Patent Laid-open N 5-3793;
Corynebacterium glutamicum ATCC 21492, see table 1 on page 3 in Japanese Patent Laid-open N 5-3793.

 (vii) Preferred hosts for the production of L-arginine.

The following bacterial strains are listed as bacteria belonging to the genus Escherichia:
Escherichia coli AJ11593 (FERM P-5616), see upper left column on page 468 in Japanese Patent Laid-open N 57-5693;
Escherichia coli AJ11594 (FERM P-5617), see upper right column on page 468 in Japanese Patent Laid-open N 57-5693.

The following bacterial strains are given as examples of coryneform bacteria:
Brevibacterium flavum AJ12144 (FERM P-7642), see column 4 on page 174 in Japanese Patent Publication N 5-27388;
Corynebacterium glutamicum AJ12145 (FERM P-7643), see column 4 at Art. 174 in Japanese Patent Publication N 5-27388;
Brevibacterium flavum ATCC 21493, see table 1 on page 3 in Japanese Patent Laid-open M 5-3793;
Corynebacterium glutamicum ATCC 21659, see table 1 on page 3 in Japanese Patent Laid-open N 5-3793.

 (viii) Hosts preferred for the production of L-proline.

The following bacterial strains are listed as bacteria belonging to the genus Escherichia:
Escherichia coli AJ11543 (FERM P-5483), see lower left column on page 435 in Japanese Patent Laid-open N 56-144093;
Escherichia coli AJ11544 (FERM P-5484), see lower left column on page 435 in Japanese Patent Laid-open N 56-144093.

The following bacterial strains are given as examples of coryneform bacteria:
Brevibacterium lactofermentum AJ11225 (FERM P-4370), see upper left column on page 473 in Japanese Patent Laid-open N 60-87788;
Brevibacterium flavum AJ11512 (FERM P-5332), see column 2 on page 185 in Japanese Patent Publication N 62-36679;
Brevibacterium flavum AJ11513 (FERM P-5333), see column 2 on page 185 in Japanese Patent Publication N 62-36679;
Brevibacterium flavum AJ11514 (FERM P-5334), see column 2 on page 185 of Japanese Patent Publication N 62-36679;
Corynebacterium glutamicum AJ11522 (FERM P-5342), see column 2 on page 185 of Japanese Patent N 62-36679;
Corynebacterium glutamicum AJ11523 (FERM P-5343), see column 2 on page 185 of Japanese Patent Publication N 62-36679.

 (2) The cultivation method.

 The method of culturing the above hosts is not particularly different from the method of culturing amino acid-producing microorganisms in the preceding descriptions. Specifically, a standard medium containing a carbon source, a nitrogen source and inorganic ions, and sometimes organic nutrients in trace amounts, such as amino acids, vitamins, and the like, is used.

 As a carbon source, glucose, sucrose, lactose, etc., as well as starch hydrolyzate, whey, molasses, and other products containing these components can be used. As a nitrogen source, gaseous ammonia, aqueous ammonium, an ammonium salt, and the like can be used. In some cases, when a mutant strain is used as the host that has the need for amino acids or other substances, a nutrient such as amino acids or other necessary substances should be added to the medium. An example of a medium for producing lysine is given in table. 1 as a medium that can be used to produce amino acids. In some cases, calcium carbonate is added to the other components after separate sterilization.

 Cultivation is carried out until the generation and accumulation of amino acids essentially stops when controlling the pH and temperature of the medium under aerobic conditions. In order to collect the amino acids thus accumulated in the medium, the usual method can be used.

 A brief description of the drawings.

 FIG. 1 shows growth inhibition by 3-bromopyruvate.

 FIG. 2 shows growth inhibition by aspartate-β-hydrazide.

 FIG. 3 shows growth inhibition of DL-threo-β-hydroxyaspartate.

 FIG. 4 shows the effect of anti-inhibition agents on 3-bromopyruvate.

 FIG. 5 shows the effect of anti-inhibition agents on aspartate β-hydrazide.

 FIG. 6 shows the effect of anti-inhibition agents on DL-threo-β-hydroxyaspartate.

 FIG. 7 shows the growth effect of inhibition-releasing factors.

 FIG. 8 shows the inhibition of phosphoenolpyruvate carboxylase by growth inhibitory substances.

 FIG. 9 shows the inhibition of the phosphoenol carboxylase of the present invention with aspartic acid.

 FIG. 10 shows the inhibition of the phosphoenolpyruvate carboxylase of the present invention with aspartic acid.

 FIG. 11 shows a method for introducing a mutation into a phosphoenolpyruvate carboxylase gene.

 FIG. 12 shows the effect exerted by aspartic acid on the activity of wild-type phosphoenol carboxylase and mutant phosphoenol pyruvate carboxylase in which arginine 438 is replaced by cysteine (N-terminal numbering).

 FIG. 13 shows the effect exerted by aspartic acid on wild-type phosphoenolpyruvate carboxylase and mutant phosphoenolpyruvate carboxylase activity in which lysine 620 is replaced by serine (N-terminus numbering).

The best way to carry out the invention
The invention will be explained in more detail below by way of examples.

 Example 1: obtaining a mutant gene of phosphoenolpyruvate carboxylase.

 A mutant gene was obtained using the plasmid pS2 obtained by shaking the cloned and sequenced gene of phosphoenolpyruvate carboxylase at the Sall site of the vector plasmid pBR322. pS2 has an ampicillin resistance gene as a marker gene for drug resistance (Sabe, H. et al., Gene, 31, 279-283 (1984)). The nucleotide sequence of the phosphoenolpyruvate carboxylase gene contained in pS2 is the same as that contained in the above plasmid pT2.

pS2 DNA was treated at 75 ^° C for 2 hours with a hydroxylamine solution containing 20 μg / ml pS2 DNA, 0.05 M sodium phosphate (pH 6.0), 1 mM EDTA, 0.4 M hydroxylamine. Due to the effect of pH on hydroxylamine treatment, 80 μl of a solution of 1M hydroxylamine HCl and 1 mM EDTA having a pH adjusted to 6.0 with sodium hydroxide, 100 μl of a solution of 0.1 M sodium phosphate (pH 6.0) and 1 mM EDTA were mixed, and TE buffer (10 mM Tris-HCl, 1 mM EDTA) containing 2 μg pS2 DNA, bringing the final volume to 200 μl with water.

 Under these conditions, the transformants have a proportion of surviving organisms of 0.2% based on the state before treatment in the medium containing ampicillin during transformation with plasmid pS2 Escherichia coli HB101 after this treatment.

Cells of Escherichia coli HB101 transformed with hydroxylamine-treated plasmid pS2 were applied to solid Petri dish medium to give approximately 10,000 transformant colonies. They were suspended in a liquid medium and plated on solid Petri dish medium containing one of the analogues of aspartic acid (3-bromopyruvate (3BP), aspartate-β-hydroxamate (ANX), aspartate-β-hydrazide (ANU) and DL-threo β-hydroxyaspartate (βHA) at a concentration close to the minimum inhibitory concentration, receiving 10 ³ - 10 ⁵ cells per plate with medium, and growing colonies were selected.

 Of the 100 strains thus obtained which are resistant to the analogue compound, the phosphoenolpyruvate carboxylase produced by each of them was partially purified according to the method described in Journal of Biochem., Vol. 67, No. 4 (1970), and inhibition of enzyme activity by analog compounds was investigated. Measurement of enzyme activity was performed as described above.

 Next, plasmids were isolated from bacterial strains producing mutant enzymes with activities not inhibited by analog compounds, and these plasmids were introduced into Escherichia coli PCR1, which is defective in relation to phosphoenolpyruvate carboxylase strain (Sabe, H. et al., Gene 31, 279 283 (1984 )), to confirm the formation of mutant enzymes.

 Thus, 5 transformants carrying mutant enzyme genes were obtained. The determination of the base sequences of these genes showed that 2 strains had the same mutation, and 4 types of mutant genes were obtained. Transformants carrying these genes were named AJ12907, AJ12908, AJ12909 and AJ12910 and were deposited with the National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken , Japan; zip code 305) On August 3, 1993 under deposit numbers FERM P-137, FERM P-13775, FERM P-13776 and FERM P-13777, they were transferred from the original depository to the international depository (repository) based on Budapest Treaty 11 July 1994 and were deposited in the form of deposit numbers FERM BP-4734, FERM BP-4735, FERM BP-4736 and FERM BP-4737, respectively, in this sequence. Further, plasmids having these genes were designated as pBP5, pHA19, pBP122 and pR6, respectively. Mutations of the phosphoenolpyruvate carboxylase genes contained in each of these plasmids are shown in Table. 2. The numerical values in the table indicate the nucleotide numbers or amino acid numbers in SEQ ID N 1.

 Typically, selection for AJ12907 and AJ12909 was performed in a medium containing 500 μg / ml 3BP, for AJ12908 in a medium containing 1000 μg / ml βHA, and for AJ12910 in a medium containing 500 μg / ml AHY.

 Example 2: mutant phosphoenolpyruvate carboxylase.

 For phosphoenolpyruvate carboxylases produced by the above 4 transformants, sensitivity to aspartic acid was investigated. These bacterial strains are defective in the phosphoenolpyruvate carboxylase gene originating from the host, so that the phosphoenolpyruvate kinase of these strains is a phosphoenolpyruvate kinase plasmid.

 Sensitivity to aspartic acid was investigated in accordance with a known method (Yoshinaga, T., Izui, K. and Katsuki, H., J. Biochem. 68, 747-750 (1970)), namely by measuring the activity of the enzyme produced by each from transformers or Escherichia coli carrying pS2 in the presence of acetyl coenzyme A, which affects the activity in the enzyme activity measuring system, at a concentration of 0.1 or 1 mM, sensitivity to aspartic acid was studied, and these data are presented in FIG. 9 and FIG. ten.

 According to these data, it is seen that the wild-type enzyme loses its activity in the presence of a high concentration of aspartic acid, while the mutant phosphoenolpyruvate carboxylase of the present invention continues to essentially remain active.

 Example 3: Enzymatic production of Escherichia coli L-threonine with introduced mutant phosphoenolpyruvate carboxylase.

 As a threonine-producing strain of Escherichia coli B-3996 (Japanese Patent Laid-open N 3-501682 (PCT)) has the highest productivity among currently known bacteria producing threonine. Thus, in evaluating the mutant phosphoenolpyruvate carboxylase, B-3996 was used as the host. This strain B-3996 was deposited at the Research Institute for Genetics and Industrial Microorganism Breeding under registration number R1A 1867. In addition, the plasmid vector pBH5, which was experimentally selected, was selected to evaluate the mutant phosphoenolpyruvate carboxylase.

 Plasmid pBP5 having the mutant phosphoenolpyruvate carboxylase (gene) was introduced into Escherichia coli B-3996 according to the Hanahan method (J. Mol. Biol., Vol. 106, 577 (1983)) and the transformant was isolated. As a control, Escherichia coli B-3996 was transformed in the same manner using pS2 as a plasmid expressing the wild-type phosphoenolpyruvate carboxylase gene.

When inoculating Escherichia coli B-3996 and the transformants obtained from it, respectively, into 500 ml Sakaguchi flasks, filled with 20 ml of a medium having the composition shown in table. 3, and culturing at 37 ^o C for 40 h to study the production of L-threonine, the results were obtained, are presented in table. 4. Usually, the aforementioned medium was prepared in two parts: first, glucose and MgSO ₄ • 7H ₂ O and other components were mixed and adjusted to pH 7.0 with KOH, followed by autoclaving at 115 ^° C for 10 minutes, and then after mixing was separately sterilized with CaCO ₃ and up to 30 g / L was added.

 As these data showed, Escherichia coli B-3996 / pBP5, carrying the mutant enzyme expressing plasmid having the DNA sequence of the present invention, had an improved threonine producing ability compared to Escherichia coli B-3996 / pS2 carrying the wild-type expressing plasmid.

Example 4: Enzymatic Production of Escherichia coli L-Glutamic Acid with Mutant Phosphoenolpyruvate Carboxylase Introduced
As a bacterium producing glutamic acid, the strain Escherichia coli AJ-12628 described in Japanese Patent Laid-open N 4-11461 has the highest production capacity among currently known bacteria. Therefore, when evaluating the mutant phosphoenolpyruvate carboxylase, AJ-12628 was used as the host.

 Strain AJ-12628 was deposited with the National Institute of Bioscience and Human Tachnology of Agency of Industrial Science and Technology under registration number FERM BP-385. Next, the plasmid pBP5 was selected as the evaluated mutant phosphoenolpyruvate carboxylase, which was subjected to experiment.

 Plasmid pBP5 having a mutant phosphoenolpyruvate carboxylase was introduced into Escherichia coli AJ-12628 according to the Hanahan method (J. Mol. Biol., Vol. 106, 577 (1983)) and the transformant was isolated. The Escherichia coli transformant AJ-12628 with pS2 was isolated in the same way.

When inoculating Escherichia coli AJ-12628 and the transformants obtained from it, respectively, into 500 ml Sakaguchi flasks, filled with 20 ml of medium having the composition shown in table. 5, and culturing at 37 ^° C. for 36 hours to study the production of L-glutamic acid, the results are shown in the table. 6. The above medium was prepared in two parts: glucose and MgSO ₄ • 7H ₂ O and the other components were mixed, adjusted to pH 7.0 with KOH, followed by autoclaving at 115 ^° C for 10 minutes, and then added separately after stirring sterilized CaCO ₃ up to 30 g / l.

 As it was found from the obtained data, Escherichia coli AJ-126 / pBP5 carrying the mutant enzyme expression plasmid having the DNA sequence of the present invention had an improved ability to produce glutamic acid compared to Escherichia coli AJ-12628 / pS2 carrying the wild-type plasmid expressing enzyme .

Example 5: production of L-lysine by a coryneform bacterium with introduced mutant. phosphoenolpyruvate carboxylase
To introduce and express the mutant gene into the coryneform bacterium, a promoter derived from a bacterium belonging to the genus Brevibacterium was obtained and ligated to the mutant gene to obtain an expression type plasmid. Further, this construct was introduced into a bacterium of the genus Brevibacterium to produce L-lysine.

 <1> Obtaining the aspartokinase (AK) gene, originating from a bacterium belonging to the genus Brevibacterium.

 Chromosomal DNA was prepared according to a standard method from wild-type Brevibacterium lactofermentum (Corynebacterium glutamicum) (ATCC 13869). The AK gene was amplified from chromosomal DNA using PCR (polymerase chain reaction: see White, T. J. et al., Trends Genet., 5, 185 (1989)). For the DNA primers used in the amplification, a 23-membered oligonucleotide (SEQ ID N 3) and a 21-membered oligonucleotide (SEQ ID N 4) were synthesized to amplify a region of approximately 1643 bp encoding the AK gene based on the sequence known in Corynebacterium glutamicum (see Molecular Microbiology (1991) 5 (5), 1197-1204, Mol. Gen. Genet. (1990) 224, 317-324).

 DNA synthesis was carried out according to the standard phosphoamidite method (see Tetrahedron Letters (1981), 22, 1859) using a model 380B DNA synthesizer manufactured by Applied Biosystems Co. DNA Thermal Cycler PJ2000 manufactured by Takara Shuzo Co., Ltd. was used in the PCR reaction, and gene amplification was performed using Taq DNA polymerase according to the manufacturer's instructions.

 The amplified 1643 kb gene fragment confirmed by agarose gel electrophoresis and then this fragment was excised from the gel and purified in a standard manner, and then digested with restriction enzymes Nrul (manufactured by Takara Shuzo Co., Ltd.), and Eco RI (manufactured by Takara Shuzo Co., Ltd.). PHSG399 was used as a cloning vector for this gene fragment (see Takeshita S. et al.,; Gene (1987), 61, 63-74). pHSG399 was digested with Smal restriction enzymes (manufactured by Takara Shuzo Co., Ltd.) and EcoR1 and ligated with an amplified fragment of the AK gene.

 DNA ligation was carried out in a known manner using a DNA ligation kit (Takara Shuzo Co., Ltd.). Thus, a plasmid was made in which pHSG399 was ligated with a fragment of the AK gene amplified from the Bravibacterium chromosome. A plasmid having the AK gene derived from ATCC 13869 (wild-type strain) was named p399AKY.

 <2> Determination of the base sequence of the AK gene of Bravibacterium lactofermentum.

 After receiving the AK plasmid, p399AKY, the base sequence of the AK gene was determined. The determination of the sequence of the bases was carried out according to the method of Sanger et al. (F. Sanger et al .: Proc. Natl. Acad. Sci. US, 74, 5463 (1977), etc.). The results are shown in SEQ ID N 5 and SEQ ID N 7. These DNA fragments have two open reading frames that correspond to the α-subunit and the β-subunit of AK, respectively. In SEQ ID N 5 and SEQ ID N 7, the amino acid sequences corresponding to each of the open reading frames are shown together with the nucleotide sequences. Further, only amino acid sequences corresponding to each of the open reading frames are shown in SEQ ID N 6 and SEQ ID N 8.

 <3> Obtaining expressing plasmids phosphoenolpyruvate carboxylase.

 Sall fragments of approximately 4.4 kb containing phosphoenolpyruvate carboxylase genes were extracted from pS2 as a plasmid having the wild-type phosphoenolpyruvate carboxylase gene and pBP5 as a plasmid having the obtained mutant phosphoenolpyruvate carboxylase gene, and these fragments were inserted into S sites plasmid vector pHSG399, universally used for Escherichia coli. The prepared plasmids were named pHS2 for the wild type and pHBP5 for the mutant.

 To obtain pHS2 and pHPB5, plasmids capable of being expressed in Brevibacterium were introduced with a promoter and a replication seed (start of replication) functioning in Brevibacterium. As a promoter, a gene fragment containing the base sequence from the 1st site of Nru 1 to the 297th site of ApaL1, which is considered the promoter region of the cloned AK gene, was extracted from p399AKY, and this fragment was inserted into the Ava1 site, located approximately 60 bp ahead of the structural genes pHS2 and pHBP5 so that the direction of transcription is correct.

 Then, a gene fragment was introduced into the site located on the vector, making autonomous replication of this plasmid into Brevibacterium possible, namely, the beginning of replication of this plasmid. A gene fragment containing the start of replication of this plasmid was extracted from Brevibacterium pHC4 vector (see 10 in Japanese Patent Laid-open N 5-7491; Escherichia coli AJ12039 carrying such a plasmid was deposited at the National Institute of Bioscience and Human Technology of Agency Industrial Science and Technology under deposit number FERM P12215) and restriction enzyme sites at both ends were modified at the PSt1 sites by the introduction of linkers.

 This fragment was introduced into the PSt1 site in the vector part of the plasmid with the added promoter from Brevibacterium. The constructed plasmid expressing phosphoenolpyruvate carboxylase were named pHS2B for the pS2-derived plasmid with the wild-type phenolpyruvate carboxylase gene and pHBP5B for the pBP5-derived plasmid with the mutant phosphoenolpyruvate carboxylase gene, respectively.

 <4> Obtaining L-lysine using expressing phosphoenolpyruvate carboxylase plasmids.

The resulting plasmids pHS2B and pHBP5B were respectively introduced into strain AJ3463, which is an L-lysine producing strain, Brevibacterium lactofermentum (see Japanese Patent Publication N 51-24477). To introduce this gene, a transformation method using an electric pulse was used (see Japanese Patent Laid-open N 2-207791). The host strain and transformants were cultured with shaking for 72 hours at 31.5 ^o C in a medium for the production of lysine having the composition shown in table. 7. The above medium was prepared as follows: all of the components listed in the table, except for CaCO ₃ , were added to 1 L of water and adjusted to pH 8.0 with KOH, followed by autoclaving at 115 ^° C for 15 minutes, and then CaCO was added ₃ , subjected to heat sterilization. The amounts of L-lysine accumulated after cultivation in the medium are shown in Table. eight.

 As shown by these data, Brevibacterium lactofermentum AJ3463, carrying the mutant enzyme expression plasmid having the DNA sequence of the present invention, had an improved lysine production ability compared to Brevibacterium lactofermentum AJ3463 / pHS2B carrying the wild-type enzyme expression plasmid.

 Example 6: another example of the mutant phosphoenolpyruvate carboxylase of the present invention and its gene.

 <1> Generation of mutant phosphoenolpyruvate carboxylase gene.

 When obtaining DNA encoding a mutant phosphoenolpyruvate carboxylase, the phosphoenolpyruvate carboxylase gene cloned in plasmid pT2 was used as a material.

 The host that should carry the pT2 plasmid should preferably be defective in the phosphoenolpyruvate carboxylase gene so that only the activity of the phosphoenolpyruvate carboxylase originating from the plasmid can be detected. Escherichia coli F15 (Hfr, recAl, met, Δ (ppc-argECBH), Tn10) was used as such a gene-free strain of this enzyme. Escherichia coli AJ-12873, capable of retaining pT2 in the F15 strain, was deposited as FERM P-13752 at the National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology (1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki- ken, Japan; zip code 305) July 15, 1993, transferred from the original depository to the international depository based on the Budapest Treaty on July 11 and deposited as FERM PB-4732. In addition, the complete base sequence of pT2 is shown in SEQ ID N 1.

 To replace the codon of arginine 438 phosphoenolpyruvate carboxylase with a cysteine codon using pT2, an overlapping extension method was used (Ho, SN, Hunt, HD, Horton, RM, Pullen, JK and Pease, LR, Gene, 77, 51-59 (1989)) using PCR method (polymerase chain reaction).

 The PCR method is a method in which the amplification cycle is repeated, including thermal denaturation of double-stranded DNA, hybridization of oligonucleotide primers corresponding to the sequences at both ends of the target amplifiable site and thermally denatured DNA, and a polymerase reaction using these oligonucleotides as primers. As a result, the DNA sequence amplifies exponentially.

The region subjected to the site-specific PCR mutation and method is shown in FIG. 11. The primers used in this invention were 4 types: a primer with (SEQ ID N 11 corresponding to bases 1535-1554 in SEQ ID N 1) having a sequence near the arginine codon 438, a primer in (SEQ ID N 10) having a sequence complementary to primer c, primer a (SEQ ID N 9 corresponding to bases 1185-1200 in SEQ ID N 1) having a sequence to the left of it, and primer d (SEQ ID N 12 corresponding to bases 2317-2342 in SEQ ID N 1) having a sequence complementary to the sequence located in the direction 5′_ → 3 ′.
In primer b and primer c, the codon (CGT) of arginine 438 was replaced by the cysteine codon (TGT). Such a substitution may employ TGC, which is another cysteine codon. Then, the C codon (AAC) of asparagine 435 was replaced by T, and therefore, the EcoR1 site was introduced without amino acid substitution, so that a mutant plasmid could be selected using this site as an indicator. However, this mutation is not essential for the present invention.

By performing a PCR reaction using pT2 DNA as template and primer a and primer b as primers, a fragment was amplified from the position to the left of the mutation site to the mutation site (AB fragment in Fig. 11). Then, by performing a PCR reaction using primer c and primer d, the fragment to the right of the mutation site was amplified (CD fragment in Fig. 11). After repeated hybridization of each of the amplified products (AB, CD) after thermal denaturation, they were ligated to conduct the polymerase reaction to obtain the AD fragment (AD in FIG. 11). Typically, the PCR reaction was carried out by repeating 30 cycles, each of which involved heating at 94 ^° C for 1 min followed by denaturation (94 ^° C, 1.5 min), hybridization (50 ^° C, 2 min), and polymerase extension reaction (72 ^° C) 3.5 min). In addition, the reaction compositions are shown in table. nine.

 <2> Selection is not sensitive to inhibition of phosphoenolpyruvate carboxylase.

 Escherichia coli was transformed with the plasmid obtained as described above, and the transformed strain was cultured to extract the plasmid and select the EcoR1 plasmid. In the selected DNA, the base sequence of the region amplified by the described PCR method was determined by dideoxy sequencing to confirm that the base replacement that was scheduled was introduced. This plasmid was named pT2R438C. The strain (AJ12874) obtained by introducing this plasmid into Escherichia coli F15 was deposited as FERM P-1375 at the National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan; zip code 305) July 15, 1993, transferred from the original depository to the international depository based on the Budapest Treaty July 11, 1994 and was deposited as FERM BP-4733.

 The base sequence pT2R438C is a sequence in which the nucleotides 1541 and 1550 are replaced by the introduction of T instead of C, respectively, in SEQ ID N 1.

 <3> Confirmation of a decrease in sensitivity (desensitization) to inhibition of mutant phosphoenolpyruvate carboxylase with aspartic acid.

 Sensitivity to aspartic acid was studied for phosphoenolpyruvate carboxylase produced by Escherichia coli AJ 12874 bearing pT2 438C. Since, as described above, Escherichia coli F15 is insufficient for phosphoenolpyruvate carboxylase, the phosphoenolpyruvate carboxylase produced by strain AJ12874 originates from the plasmid.

 Sensitivity to aspartic acid was investigated in accordance with a known method (Yoshinaga, T., Izui, K., and Katsuki, H., J. Biochem. 68, 747-750 (1970)), namely by measuring the activity of the enzyme in the presence of acetyl coenzyme A, which affects, as you know, activity in the system for measuring activity, sensitivity to aspartic acid was measured at a concentration of 1 mM or 2 mM, and the results are presented in FIG. 12.

 It is obvious that the wild-type enzyme significantly reduces its activity at a high concentration of aspartic acid, while the mutant phosphoenolpyruvate carboxylase of the present invention continues to remain active.

 <4> Generation of mutant phosphoenolpyruvate carboxylase gene (11).

 To replace the lysine codon 620 with the serine codon in the phosphoenolpyruvate carboxylase gene contained in plasmid pT2, the overlapping extension method was used (Ho, SN, Hunt, HD, Horton, RM, Pullen, JK and Pease, LR, Gene, 77, 51-99 (1989 )) using the PCR method (polymerase chain reaction). Specific procedures are similar to those described in <1>. A plasmid carrying the mutant gene with the targeted substitution was called pT2K620S. The resulting mutant enzyme was called the mutant enzyme K620S.

 <5> Confirmation of reduced sensitivity to inhibition of aspartic acid for mutant phosphoenolpyruvate carboxylase.

 For phosphoenolpyruvate carboxylase produced by the transformant obtained by introducing the plasmid pT2K620S into the indicated Escherichia coli F15 strain, sensitivity to aspartic acid was investigated. Since, as described above, Escherichia coli F15 does not have phosphoenolpyruvate carboxylase, any phosphoenolpyruvate carboxylase produced by troformant originates from the plasmid.

 Sensitivity to aspartic acid was investigated in accordance with a known method (Yoshinaga, T., Izui, K. and Katsuki, H., J. Biochem. 68, 747-750 (1970)), namely by measuring the activity of the enzyme in the presence of acetyl- of coenzyme A, which is known to affect the activity of the enzyme in the system for measuring activity, sensitivity to aspartic acid was measured at a concentration of 1 mM or 2 mM, and the results are shown in FIG. 13.

 It is obvious that the wild-type enzyme significantly reduces its activity at a high concentration of aspartic acid, while the phosphoenolpyruvate carboxylase of the present invention continues to remain active.

 In FIG. 13 also shows sensitivity to aspartic acid for a mutant phosphoenolpyruvate carboxylase in which lysine 650 is replaced by serine (mutant enzyme K650A), and for a mutant phosphoenolpyruvate carboxylase in which lysine 491 is replaced by serine (mutant enzyme K491A). In the case of these mutant enzymes, inhibition of aspartic acid was not removed (there was no desensitization).

 Industrial applicability.

 The DNA sequence of the present invention encodes a mutant phosphoenolpyruvate carboxylase and the microorganism carrying this DNA sequence produces the aforementioned enzyme.

 The mutant phosphoenolpyruvate carboxylase of the present invention is not substantially inhibited by its activity with aspartic acid, so that it can be used to enzymatically produce amino acids whose biosynthesis is regulated by aspartic acid and the like.

Claims (18)

 1. Modified phosphoenolpyruvate carboxylase, originating from a microorganism belonging to the genus Escherichia, having a modification causing desensitization and feedback inhibition of phosphoenolpyruvate carboxylase with aspartic acid, said modification being at least one modification selected from the group consisting of residues of the modification at position 625 on the lysine residue, modification of the arginine residue at position 222 and the glutamic acid residue at position 223 at about histidine and lysine residue, respectively, modification of the serine residue at position 288, glutamic acid residue at position 289, methionine residue at position 551 and glutamic acid residue at position 804 to phenylalanine residue, lysine residue, isoleucine residue and lysine residue, respectively, modifications of alanine residue at position 867 to the threonine residue, modification of the arginine residue at position 438 to the cysteine residue and modification of the lysine residue at position 620 to the serine residue, wherein the indicated amino acid positions consider from the N-terminus of the modified phosphoenolpyruvate carboxylase.  2. Phosphoenolpyruvate carboxylase according to claim 1, characterized in that said mutation is a replacement of glutamic acid 625 with lysine, counting from the N-terminus of phosphoenolpyruvate carboxylase.  3. Phosphoenolpyruvate carboxylase according to claim 1, characterized in that said mutation is a replacement of arginine 222 with histidine and glutamic acid 223 with lysine, counting from the N-terminus of phosphoenolpyruvate carboxylase.  4. Phosphoenolpyruvate carboxylase according to claim 1, characterized in that said mutation is a substitution of serine 288 with phenylalanine, glutamic acid 289 with lysine, methionine 551 with isoleucine and glutamic acid 804 with lysine, counting from the N-terminus of phosphoenolpyruvate carboxylase.  5. The phosphoenolpyruvate carboxylase according to claim 1, characterized in that the mutation is a substitution of alanine 867 with threonine, counting from the N-terminus of the phosphoenolpyruvate carboxylase.  6. The phosphoenolpyruvate carboxylase according to claim 1, characterized in that the mutation is a replacement of arginine 438 with cysteine, counting from the N-terminus of the phosphoenolpyruvate carboxylase.  7. The phosphoenolpyruvate carboxylase according to claim 1, characterized in that it is a replacement of lysine 620 with serine from the N-terminus of the phosphoenolpyruvate carboxylase.  8. A DNA fragment encoding a modified phosphoenolpyruvate carboxylase according to any one of claims 1 to 7.  9. The strain Escherichia coli carrying the gene encoding the modified phosphoenolpyruvate carboxylase selected from the group consisting of Escherichia coli Aj 12873 (FERM BP-4732), Escherichia coli Aj 12874 (FERM BP-4733), Escherichia coli Aj 12907 (FERM BP-4734 ), Escherichia coli Aj 12908 (FERM BP-4735), Escherichia coli Aj 12909 (FERM BP-4736) and Escherichia coli Aj 12910 (FERM BP-4737).  10. Recombinant DNA formed by ligation of the DNA fragment of claim 8 with a vector DNA capable of autonomously replicating in bacterial cells belonging to the genus Escherichia or to coryneform bacteria.  11. A method for producing an amino acid, comprising culturing in a suitable medium a microorganism belonging to the genus Escherichia or coryneform bacteria, transformed by integrating into the chromosomal DNA a DNA fragment according to claim 8 or recombinant DNA according to claim 10, and isolating from this medium an amino acid selected from the group consisting of L-lysine, L-threonine, L-methionine, L-isoleucine, L-glutamic acid, L-arginine and L-proline.  12. A method for selecting E. coli cells producing a modified phosphoenolpyruvate carboxylase having a modification causing desensitization to feedback inhibition of phosphoenolpyruvate carboxylase with aspartic acid, comprising the step of culturing E. coli in the presence of a compound selected from 3-bromopyruvate, β-hydrazide and asparagin DL-threo-β-hydroxyaspartic acid.  13. The method according to p. 12, characterized in that the modified phosphoenolpyruvate carboxylase is a modified phosphoenolpyruvate carboxylase according to any one of claims 1 to 5.  14. A method for producing an amino acid, including culturing E. coli cells selected by the method according to item 12 in a suitable medium and isolating from this medium an amino acid selected from the group consisting of L-lysine, L-threonine, L-methionine, L- isoleucine, L-glutamic acid, L-arginine and L-proline.  15. A method of obtaining a modified phosphoenolpyruvate carboxylase, comprising the step of isolating phosphoenolpyruvate carboxylase from E. coli cells, selected by the method according to item 12.  16. A method of obtaining a DNA fragment encoding a modified phosphoenolpyruvate carboxylase, comprising the step of isolating said DNA fragment from E. coli cells selected by the method of claim 12.  17. A method of obtaining a microorganism producing a modified phosphoenolpyruvate carboxylase, comprising the step of introducing a DNA fragment obtained by the method according to clause 16 into a microorganism belonging to the genus Escherichia or coryneform bacteria.  18. A method for producing an amino acid, comprising culturing in a suitable medium E. coli cells obtained by the method according to 17, and isolating from this medium an amino acid selected from the group consisting of L-lysine, L-threonine, L-methionine, L- isoleucine, L-glutamic acid, L-arginine and L-proline.

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