JP2007068437A - Variant aspartokinase and method for utilizing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To create a variant aspartokinase in which concerted feedback-inhibiting activities by L-lysine and L-threonine are reduced by gene recombination to enable the L-lysine or ε-poly-L-lysine to be mass-produced. <P>SOLUTION: An expression vector encoding the variant aspartokinase comprising an amino acid sequence having variation of 68th methionine residue to another amino acid residue in an amino acid sequence possessed by the aspartokinase in a bacterium of the genus Streptomyces or the genus Corynebacterium is created, and expressed by introducing the expression vector into an ε-poly-L-lysine-producing bacterium. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a mutant aspartokinase and a method for using the same.

  Currently, ε-poly-L-lysine, which is widely used as a food preservative, is produced by a fermentation method using ε-poly-L-lysine-producing bacteria belonging to the genus Streptomyces. In order to increase productivity in substance production by fermentation, it is common to obtain mutant strains by applying chemical and physical mutation treatments such as NTG and ultraviolet rays. Even in the case of ε-poly-L-lysine, such a mutation treatment is performed, and S- (2-aminoethyl) -cysteine (hereinafter abbreviated as AEC) resistant mutant strain (for example, see Patent Document 1), AEC And glycine-resistant mutant strains (for example, see Patent Document 2) and valine, isoleucine-requiring mutant strains (for example, see Patent Document 3), thereby obtaining a strain having higher productivity than the wild strain. It is done.

  In bacteria, L-lysine is mainly biosynthesized by the diaminopimelate pathway, and aspartokinase is an important enzyme that regulates the amount of L-lysine synthesized in a series of enzymes in this biosynthetic pathway. is there. In the ε-poly-L-lysine producing strain, the productivity is increased by using the AEC resistant mutant. Therefore, L-lysine is a precursor of ε-poly-L-lysine. It is thought that the amount of L-lysine accumulated increased due to the decreased sensitivity of Aspartokinase to feedback inhibition of L-lysine, resulting in an increase in ε-poly-L-lysine production (for example, Non-patent document 1).

Thus, it was suggested that aspartokinase is an important enzyme in the production of ε-poly-L-lysine, but the structure of the aspartokinase gene of ε-poly-L-lysine producing bacteria has been elucidated. Therefore, it was not possible to introduce mutations directly using genetic recombination techniques. Further, it has not been known at all what part of aspartokinase can introduce a mutation to reduce the feedback inhibitory activity by L-lysine.
Japanese Patent Publication No. 3525190 Japanese Patent No. 1713630 Japanese Patent Laid-Open No. 10-290688 Hiraki et al., Japanese Society for Biotechnology, Volume 76, 1998

  In view of the above-mentioned problems, the present invention produces a mutant aspartokinase with reduced concerted feedback inhibition activity by L-lysine and L-threonine by gene recombination, and produces L-lysine or ε-poly-L. -The purpose is to enable mass synthesis of lysine.

  The present inventors firstly synthesized L-lysine and ε-poly-L-lysine from Streptomyces albulus subsp. Lysinopolymerus, which is an ε-poly-L-lysine-producing bacterium. We cloned an aspartokinase gene that plays an important role in the regulation of. Mutations were randomly introduced into the aspartokinase gene and the activity of the mutant enzyme was examined. Aspartokin having SEQ ID NO: 1 replaced the 68th methionine residue with a valine residue. It was found that aspartokinase with reduced concerted feedback inhibition activity by L-lysine and L-threonine can be obtained. Then, when a transformant of an ε-poly-L-lysine producing bacterium having a gene encoding this mutant aspartokinase as a foreign gene was prepared, ε-poly-L-lysine was more productive than the wild type As a result, the present invention has been completed. In the present specification, “feedback inhibitory activity” refers to “sensitivity to feedback inhibition”.

The present invention is constituted by the following items.
(1) The following protein (a) or (b).
(A) A protein comprising the amino acid sequence set forth in SEQ ID NO: 1.
(B) A protein having an aspartokinase activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1.

(2) The following protein (a) or (b).
(A) A protein comprising an amino acid sequence in which the 68th methionine residue is mutated to another amino acid residue in the amino acid sequence described in SEQ ID NO: 1.
(B) In the amino acid sequence set forth in SEQ ID NO: 1, the 68th methionine residue is mutated to another amino acid residue, and one or several amino acids other than the 68th methionine residue are A protein having an aspartokinase activity, comprising an amino acid sequence deleted, substituted or added and having reduced feedback inhibition activity by L-lysine and L-threonine.

(3) The following protein (a) or (b).
(A) From the amino acid sequence of the homologue of the protein having the amino acid sequence of SEQ ID NO: 1 in Streptomyces or Corynebacterium, the amino acid sequence in which the 68th methionine residue is mutated to another amino acid residue Protein.
(B) The amino acid sequence of the protein described in (a) consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or added, and the feedback inhibitory activity by L-lysine and L-threonine is reduced. A protein having aspartokinase activity.

(4) The following protein (a) or (b).
(A) an aspart comprising an amino acid sequence of a protein in which the 68th methionine residue in the amino acid sequence described in SEQ ID NO: 1 is conserved, wherein the 68th methionine residue is mutated to another amino acid residue; A protein having tokinase activity.
(B) The amino acid sequence of the protein described in (a) consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or added, and the feedback inhibitory activity by L-lysine and L-threonine is reduced. A protein having aspartokinase activity.

(5) The protein according to any one of Items 2 to 4, which comprises an amino acid sequence in which the 68th methionine residue is mutated to a valine residue.

(6) DNA encoding the protein according to any one of Items 1 to 5.

(7) The DNA according to item 6, which has the base sequence described in any one of SEQ ID NOs: 2 to 6.

(8) An expression vector containing the DNA according to item 6 or 7 and constructed so as to express the protein according to any one of items 1 to 5.

(9) A transformed cell comprising the vector according to item 8.

(10) A method for producing L-lysine, comprising culturing the transformed cell according to item 9 and isolating L-lysine produced by the transformed cell.

(11) A method for producing ε-poly-L-lysine, comprising culturing the transformed cell according to item 9 and isolating ε-poly-L-lysine produced by the transformed cell.

  According to the present invention, a mutant aspartokinase having reduced concerted feedback inhibition activity by L-lysine and L-threonine can be produced by genetic recombination. This enables large-scale synthesis of L-lysine or ε-poly-L-lysine.

== Synthesis of DNA for use in L-lysine or ε-poly-L-lysine synthesis ==
The wild type aspartokinase from which the DNA encoding the mutant aspartokinase according to the present invention is prepared may be an aspartokinase in which the 68th methionine residue in the amino acid sequence described in SEQ ID NO: 1 is conserved. The amino acid sequence around the 68th methionine residue is preferably conserved. Examples of such aspartokinase include homologs of aspartokinase described in SEQ ID NO: 1 in Streptomyces or Corynebacterium. FIG. 1 illustrates S. coelicolor A3 (2) (NP_627820), S. avermitilis MA-4680 (NP_825736), Streptomyces sp. NRRL 5331 (CAD24815) C. glutamicium (S15276). S. noursei (FERM P-9797), Streptomyces sp. Strain SP-72 (FERM P-16810) and the like. The aspartokinase described in SEQ ID NO: 1 is an enzyme inherently possessed by Streptomyces albulus subsp. Lysinopolymerus.

  DNA encoding these wild-type aspartokinase can be produced by determining the nucleotide sequence from the codon table and chemically synthesizing, using each gene sequence described in the database, from bacteria having each gene, Cloning is preferably performed using a conventional method such as RT-PCR using genomic DNA or cDNA, or screening of a genomic library or cDNA library. For example, in the case of the aspartokinase described in FIG. 1, cloning can be performed from each bacterium using the database information described in parentheses. In the case of the aspartokinase described in SEQ ID NO: 1 possessed by Streptomyces albulus subsp. Lysinopolymerus, it can be cloned using the base sequence described in SEQ ID NO: 2.

  In the amino acid sequences of these aspartokinases, DNA encoding mutant aspartokinase consisting of an amino acid sequence in which the 68th methionine residue is mutated to another amino acid residue is templated with DNA encoding wild-type aspartokinase. For example, it can be prepared by introducing a mutation into the 68th methionine residue using in vitro mutagenesis. As described in Examples, the mutant aspartokinase in which the 68th methionine residue was mutated to a valine residue had reduced feedback inhibitory activity by L-lysine and L-threonine. The second methionine residue was found to have an important role in feedback inhibitory activity by L-lysine and L-threonine. Therefore, in the mutant aspartokinase used in the present invention, any mutation may be introduced as long as a mutation that reduces the feedback inhibitory activity is introduced into the 68th methionine residue.

  Furthermore, in the amino acid sequence of SEQ ID NO: 1, even if one or several amino acids other than the 68th methionine residue are deleted, substituted or added, it has aspartokinase activity, and L-lysine and L -Any mutant aspartokinase having reduced feedback inhibitory activity by threonine may be used.

  The base sequence of the DNA thus obtained can be confirmed using a DNA sequencer. The obtained base sequence is analyzed with base sequence analysis software such as DNASIS (manufactured by Hitachi Software Engineering Co., Ltd.) or GENEX (manufactured by Software Development Co., Ltd.) to identify the coding region of the aspartokinase gene in the DNA. You can also.

== Intracellular expression of aspartokinase ==
The genes encoding wild-type aspartokinase and mutant aspartokinase obtained as described above can be incorporated into expression vectors by a conventional method, introduced into cells and expressed. In this case, the expression vector is not particularly limited, such as a plasmid or a viral vector, and is appropriately selected in relation to the host cell. The host cell is not limited to Streptomyces bacteria, but is not particularly limited to Escherichia coli and mammalian cells.

  Conventionally known methods such as an ion exchange resin method and a crystallization method are used for culturing the transformed cells thus prepared and then producing L-lysine or ε-poly-L-lysine produced by the transformed cells. And the combination thereof.

<< Example >>
(1) Cloning of Aspartokinase Gene from ε-Poly-L-Lysine-Producing Bacteria Streptomyces albulus IFO14147 strain, an ε-poly-L-lysine-producing bacterium, Tryptone 10 g / L, yeast extract The cells were inoculated into 100 ml of a medium consisting of 5 g / L and NaCl 5 g / L, and cultured with shaking at 30 ° C. for 24 hours. Bacterial cells were collected from this culture broth by centrifugation, and chromosomal DNA was prepared according to a conventional method. Using this chromosomal DNA as a template, based on the amino acid sequence highly conserved in Aspartokinase derived from Streptomyces genus, the following two types of primers were designed, and a Takara Bio PCR Kit (TaKaRa LA Taq with GC Buffer) and a PCR reaction solution was prepared according to the protocol (primer concentration was 100 μM). The reaction conditions are step 1 (94 ° C., 2 minutes), step 2 (94 ° C., 1 minute), step 3 (50 ° C., 1 minute), step 4 (72 ° C., 1 minute), step 5 (steps 2 to 2). (4, 30 cycles) and step 6 (72 ° C., 10 minutes) for a total of 6 steps.

ASK-1: 5′-CTGTSGTGSCAGAAGTACGGGSGG-3 ′ (SEQ ID NO: 7)
ASK-2: 5′-GATCTSTSSCAYCTGGTCCGTCTY-3 ′ (SEQ ID NO: 8)

  From the obtained amplified fragment of about 1 kb, a labeled probe was prepared using an ECL labeling kit (Amersham Bioscience) and screened against a cosmid library prepared from the chromosomal DNA of this strain. A 4.5 kb BamHI fragment containing the full length of the aspartokinase gene was obtained. The base sequence of this fragment was determined, and the ORF (SEQ ID NO: 2) of the aspartokinase gene was determined.

(2) Expression of recombinant aspartokinase in Escherichia coli Based on this base sequence, the following two types of primers were prepared and PCR was performed according to the protocol using a Takara Bio PCR kit (TaKaRa LA Taq with GC Buffer). A reaction solution was prepared (primer concentration was 10 μM). The reaction conditions are step 1 (94 ° C., 2 minutes), step 2 (94 ° C., 1 minute), step 3 (65 ° C., 1 minute), step 4 (72 ° C., 1 minute), step 5 (step 2 to 4 and 30 cycles) and step 6 (72 ° C., 10 minutes) for a total of 6 steps.

ASK-N4: 5'-GGGGGATCCGGCCTTGTCTGCAGAAGTAC-3 '(SEQ ID NO: 9)
ASK-C4: 5′-ACCAAGCTTTCATCGCCCCGGTGCCGCCGTA-3 ′ (SEQ ID NO: 10)

  This amplified fragment was treated with restriction enzymes BamHI and HindIII, and then inserted into pQE30 (Qiagen) treated with restriction enzymes BamHI and HindIII to obtain a fusion protein with a histidine tag added to the N-terminal side of aspartokinase. The encoding plasmid ask / pQE30 was constructed. The prepared plasmid was introduced into Escherichia coli (M15 pREP4), inoculated into 50 mL of a medium consisting of tryptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, and cultured with shaking at 37 ° C. for 3 hours. After cooling the culture in ice, IPTG was added to a final concentration of 0.1 mM, and the culture was further performed at a low temperature (18 ° C.) for 16 hours. The cells were collected, suspended in a buffer for disrupting cells (50 mM potassium phosphate buffer pH 7.5, 300 mM sodium chloride, 5 mM β mercaptoethanol, 10 mM imidazole), and then disrupted ultrasonically. After centrifugation, the soluble fraction is passed through a Ni-NTA agarose column (Qiagen), and the column is washed with a washing buffer (50 mM potassium phosphate buffer pH 7.5, 300 mM sodium chloride, 5 mM β mercaptoethanol, 20 mM imidazole). did. Subsequently, elution buffer (50 mM potassium phosphate buffer pH 7.5, 300 mM sodium chloride, 5 mM β mercaptoethanol, 250 mM imidazole) was passed through the column to elute aspartokinase to obtain purified aspartokinase.

  Aspartokinase activity of this purified aspartokinase is measured as aspartate hydroxamic acid by the black method (Methods in Enzymology, Vol. 5, p820-827, Academic Press Inc., New York (1962)). Thus, it was confirmed that the obtained gene encoded aspartokinase. That is, 100 mM Tris-sulfate buffer (pH 7.5), 10 mM ATP, 10 mM magnesium sulfate, 600 mM hydroxylamine / hydrochloride, 600 mM ammonium sulfate, 50 mM L-aspartic acid, and purified aspartokinase are added to 1 mL of the final solution. After reacting at 30 ° C. for 15 minutes, 1.5 mL of 10% iron (II) chloride hexahydrate-3.3% trichloroacetic acid-0.7N hydrochloric acid solution was added, and the precipitate was removed by centrifugation. The enzyme activity was determined by measuring the absorbance at 540 nm.

  Next, the feedback mode was analyzed using the obtained aspartokinase. When the aspartokinase activity was measured by the above black method, various amounts of L-lysine and / or L-threonine were added to the reaction solution, and the relative activity of each sample was determined and plotted in FIG. is there. These results revealed that feedback inhibition of aspartokinase is a concerted feedback inhibition by L-lysine and L-threonine.

(3) Preparation of mutant aspartokinase gene The DNA fragment containing aspartokinase gene derived from ε-poly-L-lysine producing bacteria obtained as described above was randomly introduced by error-prone PCR. An amplified fragment was obtained. Error-prone PCR uses the above two types of primers (ASK-N4 and ASK-C4) and plasmid ask / pQE30 as template DNA, and the concentration balance of various deoxyribonucleotides in the PCR reaction solution is changed as shown in Table 1 4 A total of five types of PCR solutions, that is, a PCR solution (1 to 4) of a kind and a PCR solution (5) containing a low concentration of magnesium, were prepared and subjected to a PCR reaction. The reaction conditions are Step 1 (94 ° C., 2 minutes), Step 2 (94 ° C., 1 minute), Step 3 (65 ° C., 1 minute), Step 4 (72 ° C., 1 minute), Step 5 (Steps 2 to 2). 4, 30 cycles), step 6 (72 ° C., 10 minutes).

The DNA fragments amplified in each reaction were mixed, treated with restriction enzymes BamHI and HindIII, and then inserted into pQE30 treated with restriction enzymes BamHI and HindIII to construct a mutation library.

  The library was introduced into E. coli XL1-blue MRF 'and spread on M9 minimal medium containing 8 mM, 16 mM, 32 mM AEC and AHV. AEC and AHV are structural analogs of L-lysine and L-threonine, respectively, and inhibit the growth of many microorganisms. In fact, it was revealed in vitro that AEC and AHV act as structural analogs of L-lysine and L-threonine in S. albulus aspartokinase and show weak feedback inhibition in the presence of them (see FIG. (See FIG. 2). In E. coli as well, these analogs show growth inhibition and are unable to grow on M9 minimal medium containing 8 mM AEC and AHV, respectively. However, in a high-producing mutant of L-lysine or L-threonine with reduced feedback inhibitory activity of aspartokinase, the relative concentration of intracellular AEC or AHV to L-lysine or L-threonine decreases, and these structures It is known that it can grow even in a medium containing an analog. Therefore, if an aspartokinase gene with reduced feedback inhibitory activity is introduced into Escherichia coli in a mutation library, the transformant becomes a high-producing strain of L-lysine or L-threonine, and AEC and AHV are It was thought that it became possible to grow on the M9 minimal medium containing. Thus, cells having mutant aspartokinase with reduced feedback inhibitory activity can be identified using proliferation in the presence of AEC and AHV as an index, and actually grow in a medium containing 8 mM of AEC and AHV. A number of transformants were obtained. Of these transformants, 4 strains (ASK er2, 13, 16, 17) were able to grow on a medium containing 16 mM of AEC and AHV (FIG. 3). This indicates that these four strains are high-producing strains of L-lysine or L-threonine.

  Next, when plasmids were prepared from these transformants and the mutation sites were identified, it was found that all four strains had the same mutation pattern (I19V, M68V, T309A). Thus, the feedback inhibitory activity was analyzed using the mutant aspartokinase ASK er2 purified as described above. As a result, it was revealed that ASK er2 maintains about 80% of the activity of L-lysine and L-threonine even in the presence of 100 mM, and the feedback inhibitory activity is reduced (FIG. 4). . Thus, using the plasmid ask / pQE30 as a template, using the QuickCange XL Site-Directed Mutagenesis Kit (Stratagene) and the following primers, various site-specific mutagenesis were prepared by in vitro mutagetesis, and the feedback inhibitory activity was similarly determined. As a result of analysis, all mutant aspartokinases into which the M68V mutation was introduced (hereinafter referred to as M68V mutant aspartokinase), ie, ASK (M68V) (SEQ ID NO: 3) and ASK (I19V, M68V) in FIG. (SEQ ID NO: 4), ASK (M68V, T309A) (SEQ ID NO: 5), and ASK (I19V, M68V, T309A) (SEQ ID NO: 6) were detected to have reduced feedback inhibitory activity (FIG. 4). In addition, the specific activity of M68V mutant aspartokinase was observed to be more than doubled compared to wild-type aspartokinase (Table 2). That is, when the M68V mutation is introduced into aspartokinase, the feedback inhibitory activity can be reduced and the aspartokinase activity can be improved.

ASKt309a-sense; 5'-CCACCGGTCTGGCGGGACATCTCCTTCA-3 '(SEQ ID NO: 11)
ASKt309a-anti; 5'-TGAAGGAGAGTCCCGCCAGACCGGGTGG-3 (SEQ ID NO: 12) '
ASKm68v-sense; 5′-GCGAGTTCGACGTGCTGCTGACCGC-3 ′ (SEQ ID NO: 13)
ASKm68v-anti; 5′-GCGGTCAGCAGCACCGTCGAACTCGC-3 ′ (SEQ ID NO: 14)
ASKi19v-sense; 5′-TGCCGAGGGGCGCACACGCGTGCC-3 ′ (SEQ ID NO: 15)
ASKi19v-anti; 5'-GGCAACCGCGCTTGACGCCCCTCGCA-3 '(SEQ ID NO: 16)

(4) Expression of mutant aspartokinase gene in ε-poly-L-lysine-producing bacteria The gene encoding M68V mutant aspartokinase obtained in this study is expressed in ε-poly-L-lysine-producing bacteria Therefore, this gene was inserted into the conjugation plasmid pLAE003.
First, a 1.9 kb ApaI fragment containing the wild type aspartokinase gene was inserted into the ApaI site of plasmid pGEM 11Zf (+) to construct plasmid ask / pGEM 11Z. In addition, the 0.2 kb ClaI fragment present in the 1.9 kb ApaI fragment was replaced with the 0.2 kb ClaI fragment into which the M68V mutation was introduced to construct plasmid ask (M68V) / pGEM 11Z. Next, these plasmids were digested with BamHI and HindIII, and 1.9 kb fragments having a wild type aspartokinase gene or M68V mutant aspartokinase gene were obtained, respectively, and inserted into pLAE003 digested with BamHI and HindIII. By conjugating the thus constructed plasmid, ask / pLAE003 or ask (M68V) / pLAE003, with Escherichia coli according to the method of Kanno et al. (Journal of Bioscience and Bioenginering vol. 99, No. 6, 636-641 (2005)) And introduced into S. albulus CR1 (FIG. 5). In a 500 mL Sakaguchi flask, ε-poly-L-lysine production medium (50 g of glycerol, 5 g of yeast extract, 10 g of ammonium sulfate, 1.58 g of disodium hydrogen phosphate, 1.36 g of potassium dihydrogen phosphate, 7.36 g of magnesium sulfate 50 g of hydrate 0.5 g, zinc sulfate heptahydrate 0.04 g, iron sulfate heptahydrate 0.03 g) was added, and the resulting introduced strain was cultured with shaking at 30 ° C. for 6 days. As a result of measuring the amount of ε-poly-L-lysine production, about 10% improvement in ε-poly-L-lysine productivity was observed as compared with the vector alone or the strain into which the wild type aspartokinase gene was introduced. (FIG. 5).
The ε-poly-L-lysine accumulated in the culture broth was quantified by the Itzhaki method (Analitical Biochemistry vol. 50, 569-574 (1972)) using methyl orange. That is, 2 mL of a 1 mM methyl orange aqueous solution was mixed with a solution obtained by adding 1.9 mL of 0.1 M phosphate buffer (pH 6.9) to 0.1 mL of the culture supernatant and left at 30 ° C. for 30 minutes. The complex of poly-L-lysine and methyl orange was removed by centrifugation, and the absorbance of the supernatant was measured at 465 nm. On the other hand, the amount of ε-poly-L-lysine was calculated from a calibration curve prepared using a standard product of ε-poly-L-lysine.

Diagram showing amino acid sequences of Aspartokinase of Streptomyces albulus, S. coelicolor A3 (2) (NP_627820), S. avermitilis MA-4680 (NP_825736), Streptomyces sp. NRRL 5331 (CAD24815) C. glutamicium (S15276) It is. (A) is the figure which showed the characteristic of the concerted feedback inhibitory activity with respect to aspartokinase by L-lysine and L-threonine. (B) is a diagram showing that AEC and AHV do not have feedback inhibitory activity against aspartokinase. In one embodiment of the present invention, (A) shows the results of examining whether a transformant having wild type aspartokinase or various mutant aspartokinases can survive in the presence of AEC and AHV. FIG. (B) is a figure showing mutant amino acids in ASK er2. In one Example of this invention, it is a figure which shows the result by which the fall of feedback inhibitory activity was detected by all the mutant | variant aspartokinase which introduce | transduced the M68V mutation. In one Example of this invention, it is the figure which showed the result of having quantified ε-poly-L-lysine which wild type aspartokinase or M68V mutant type aspartokinase produces.

Claims (11)

The following protein (a) or (b).
(A) A protein comprising the amino acid sequence set forth in SEQ ID NO: 1.
(B) A protein having an aspartokinase activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1. The following protein (a) or (b).
(A) A protein comprising an amino acid sequence in which the 68th methionine residue is mutated to another amino acid residue in the amino acid sequence described in SEQ ID NO: 1.
(B) the amino acid sequence of the protein according to (a), comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in amino acid residues other than the 68th methionine residue; -A protein having aspartokinase activity in which feedback inhibitory activity by lysine and L-threonine is reduced. The following protein (a) or (b).
(A) From the amino acid sequence of the homologue of the protein having the amino acid sequence of SEQ ID NO: 1 in Streptomyces or Corynebacterium, the amino acid sequence in which the 68th methionine residue is mutated to another amino acid residue Protein.
(B) The amino acid sequence of the protein described in (a) consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or added, and the feedback inhibitory activity by L-lysine and L-threonine is reduced. A protein having aspartokinase activity. The following protein (a) or (b).
(A) A protein having aspartokinase activity, comprising an amino acid sequence in which the 68th methionine residue in the amino acid sequence described in SEQ ID NO: 1 is mutated to another amino acid residue.
(B) The amino acid sequence of the protein described in (a) consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or added, and the feedback inhibitory activity by L-lysine and L-threonine is reduced. A protein having aspartokinase activity.   The protein according to any one of claims 2 to 4, comprising an amino acid sequence in which the 68th methionine residue is mutated to a valine residue.   DNA encoding the protein according to any one of claims 1 to 5.   The DNA according to claim 6, which has the base sequence according to any one of SEQ ID NOs: 2 to 6.   An expression vector containing the DNA according to claim 6 or 7 and constructed so as to express the protein according to any one of claims 1 to 5.   A transformed cell comprising the vector according to claim 8.   A method for producing L-lysine, comprising culturing the transformed cell according to claim 9 and isolating L-lysine produced by the transformed cell.   A method for producing ε-poly-L-lysine, comprising culturing the transformed cell according to claim 9 and isolating ε-poly-L-lysine produced by the transformed cell.