CN115725537A - Aspartokinase mutant and application thereof in producing L-homoserine - Google Patents

Abstract

The invention belongs to the field of bioengineering, and particularly discloses an aspartokinase mutant with improved enzyme activity and application thereof in preparation of L-homoserine or a derivative thereof. Compared with wild-type aspartokinase derived from saccharomyces cerevisiae, the amino acid sequence of the mutant has one mutation type of V299A, N317S, N485S, Q107R/N317S, T114S/Q510R and T155A/K390E. In specific implementation, through enzyme activity determination analysis of induced expression purification, the enzyme activity of the mutant is improved by 1.7 to 3.8 times compared with that of a wild type. Therefore, the beneficial mutant provided by the invention can lay a good foundation for industrial high-efficiency production of L-homoserine and downstream metabolites.

Description

Aspartokinase mutant and application thereof in producing L-homoserine

Technical Field

The invention belongs to the field of bioengineering, and particularly discloses an aspartokinase mutant with improved enzyme activity and application thereof in preparation of L-homoserine or a derivative thereof.

Background

L-homoserine is an important non-protein amino acid, is also a precursor of methionine, lysine and threonine, participates in various physiological and biochemical reactions and various biological metabolic processes in vivo, and has important physiological functions and application values. Homoserine and derivatives thereof have abundant biological activity, can be used as antifungal drugs and can effectively inhibit sickle red blood cells, and the derivative homoserine methyl ester is an important intermediate of a compound Chinese bitersweet alkaloid with anticancer activity. Homoserine is also the main raw material for synthesizing a novel pesticide L-glufosinate-ammonium, and glufosinate-ammonium is used as a systemic conductive herbicide, and has high activity of broad-spectrum weeding, so that the homoserine is widely applied in foreign developed countries, and the demand is increased year by year. Due to the urgent need for these drugs, the large demand for the key intermediate, L-homoserine, has been stimulated. In addition, homoserine has potential application in crop fertilizer additives and animal husbandry feed additives, for example, in chicken diets, homoserine has similar biological activity as threonine, and in the absence of threonine, homoserine can replace threonine to improve the growth performance of young chicks. Meanwhile, homoserine is an intermediate which can be converted and synthesized into acrylic acid, 3-hydroxypropionyl-CoA, 3-hydroxypropionate, poly-3-hydroxypropionate and 1, 2-propanediol. In conclusion, homoserine can be used as an intermediate or a raw material to synthesize various chemical products, pesticides and feeds, can be applied to the fields of food, cosmetics, medicines, feeds and the like, and has a wide application prospect.

At present, the synthesis method of L-homoserine mainly comprises a chemical method and a biological enzyme method. The chemical method uses L-methionine or aspartic acid as a raw material, requires the use of iodide and a large amount of organic solvent, and produces sulfide, accompanied by serious environmental and safety problems. The biological enzyme method takes pyruvic acid and aldehyde compounds as raw materials, and obtains homoserine by the reaction under the common catalysis of aldolase, formate dehydrogenase and L-amino acid dehydrogenase complex enzyme, so that the cost is high, toxic raw materials of formaldehyde and formic acid are required to be used, and expensive coenzyme and the like are required to be used. In recent years, the microbial fermentation production of L-homoserine is receiving more and more attention, and the microbial fermentation method provides a safe, economic and environment-friendly alternative production strategy.

In microorganisms such as E.coli, C.glutamicum, saccharomyces cerevisiae, etc., L-homoserine biosynthesis is initiated via the aspartate pathway. Glucose generates oxaloacetate precursor through glycolysis pathway, pentose phosphate pathway, TCA cycle and the like, and enters aspartate branch pathway; then, L-Homoserine is finally produced from the Aspartate precursor under the sequential catalysis of Aspartokinase (EC: 2.7.2.4), aspartate-semialdehyde dehydrogenase (EC: 1.2.1.11), and Homoserine dehydrogenase (EC: 1.1.3). Wherein aspartokinase is the first common key enzyme in the synthetic pathway of amino acids in aspartate family such as homoserine, threonine, lysine, isoleucine, methionine and the like, can introduce central carbon metabolic flux into an aspartate branch, and the catalytic activity of aspartokinase is usually subjected to feedback inhibition or repression effect of end products or intermediate metabolites. Therefore, the key enzyme mutant with excellent catalytic performance is obtained by screening or molecular modification of aspartokinase, and has very important significance for the high-efficiency production of aspartate family amino acid.

Disclosure of Invention

In view of the above-mentioned needs, the primary object of the present invention is to provide aspartokinase mutants, so as to improve the catalytic performance thereof, and facilitate the production of metabolites such as L-homoserine by microbial fermentation.

The invention provides a polypeptide derived from wild typeSaccharomyces cerevisiaeAspartate kinase LysC mutant of S288C, characterized in that it is as defined in SEQ ID No.2In terms of amino acid sequences, only one site mutation of mutation from valine V to alanine A at position 299, mutation from asparagine N to serine S at position 317, or mutation from asparagine N to serine S at position 485 is adopted; there are only combinatorial mutations with a mutation from glutamine Q to arginine R at position 107 and asparagine N to serine S at position 317; there are only combined mutations with threonine T to serine S at position 114 and glutamine Q to arginine R at position 510; there are only combinatorial mutations with a mutation from threonine T to alanine a at position 155 and lysine K to glutamic acid E at position 390.

The present invention further provides a gene encoding the aspartokinase LysC mutant. In a preferred embodiment, the nucleotide sequence of the gene encoding aspartokinase LysC is obtained by mutation based on the nucleotide sequence shown in SEQ ID No. 1. The invention also provides an expression vector and a host cell containing the coding gene of the aspartokinase LysC mutant. In a preferred embodiment, the expression vector includes, but is not limited to, pACYC184 and pXMJ19, and is used for fermentative production of L-homoserine and downstream derivatives, etc., by constructing a recombinant plasmid containing a gene encoding the LysC mutant and introducing it into a microbial underpan cell. The derivatives generally refer to derivatives that are downstream metabolites of their metabolic pathways, such as threonine, leucine, isoleucine, etc. (see FIG. 1). Although lysine is not a derivative thereof, since LysC enzyme is located upstream of the metabolic pathway, it can also be obtained by the method of the present invention.

In a particular embodiment, the microbial underpan cells may be selected from the genus Corynebacterium, enterobacter or Saccharomyces, preferably E.coli ((E.coli))Escherichia coli) Corynebacterium glutamicum (C.) (Corynebacterium glutamicum) And Saccharomyces cerevisiae (Saccharomyces cerevisiae）。

The invention also provides application of the aspartokinase mutant in fermentation production of L-homoserine and derivatives thereof. Selecting chassis engineering bacteria with certain L-homoserine production capacity for application performance test, wherein in a specific embodiment, the engineering bacteria Ec-Hom is specially usedCharacterized by Escherichia coliEscherichia coli Deletion of autologous homoserine degradation pathway Gene in MG1655thrB(ProteinID: NP-414544) andmetA(Protein ID: NP-418437) while overexpressing the aspartokinase/homoserine dehydrogenase genethrA(Protein ID: NP-414543); in another specific embodiment, the engineered bacterium Cg-Hom is characterized by Corynebacterium glutamicumCorynebacterium glutamicum Deletion of the autologous homoserine degradation pathway Gene in ATCC 13032thrB(Protein ID: CAF 19888) while overexpressing the homoserine dehydrogenase genehom（Protein ID: CAF19887）。

The method is realized by the following technical thought, a saccharomyces cerevisiae genome is taken as a template, an error-prone PCR random mutation method is utilized to obtain a LysC coding gene mutation library, and a vector construction strategy based on a Golden Gate technology is adopted to subclone the mutation library onto an expression vector, so that a recombinant plasmid library containing an aspartate kinase LysC mutant coding gene is obtained. And then introducing the recombinant plasmid library into escherichia coli (containing a homoserine biosensor PhomBio) with a homoserine degradation pathway weakened, and finally screening mutants with improved enzyme activity through primary screening of a 96-well plate and secondary screening of a test tube and a shake flask, wherein mutation sites of the mutants are V299A, N317S, N485S, Q107R/N317S, T114S/Q510R and T155A/K390E respectively.

The invention has the beneficial effects that the aspartokinase mutant with improved catalytic activity in different degrees is obtained by mutating and screening the aspartokinase coding gene LysC from saccharomyces cerevisiae. Therefore, the aspartokinase mutants provided by the invention can lay a good foundation for efficiently fermenting and producing L-homoserine and other downstream aspartate family metabolites, and have better industrial application prospect compared with non-mutant aspartokinase.

Drawings

FIG. 1 is a schematic diagram of the biosynthetic pathway of microbial L-homoserine.

FIG. 2 determination of enzyme activity of aspartokinase mutants.

FIG. 3 analysis of the yield of L-homoserine engineering bacteria.

Detailed Description

The invention will be described in further detail with reference to specific embodiments and drawings for better understanding of the objects, technical solutions and advantages thereof, but the invention should not be construed as being limited thereto. The experimental procedures used in the examples are, unless otherwise specified, conventional procedures well known to those skilled in the art. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 construction of aspartokinase LysC variant library

The invention adopts EasyTaq DNA polymerase (Beijing TransGen Biotech, china) with low fidelity and wild Saccharomyces cerevisiaeSaccharomyces cerevisiaeThe S288C genome is used as a template, and the primers P1 (5-lysCEncoding a library of gene mutations. By adding magnesium ions and manganese ions with certain concentration into a PCR reaction system, the fidelity in the PCR amplification process is further reduced, and the obtained product is controlledlysCThe coding gene contains 2-3 point mutations. The error-prone PCR system adopted by the invention is as follows: 5. mu.L 10 × EasyTaq buffer, 0.2. Mu.M forward primer P1, 0.2. Mu.M forward primer P2, 200. Mu.M dNTPs, 0.8 mM MnCl ₂ 、6 mM MgSO ₄ 50 ng template DNA and 1. Mu.L EasyTaq DNA polymerase were added to make up to a 50. Mu.L system with sterile water. The PCR reaction program is: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s; annealing at 58 ℃ for 30s; extending for 2 min at 72 ℃ and circulating for 35 times; extension at 72 ℃ for 5min and storage at 4 ℃.

The pACYC plasmid skeleton was obtained by a general PCR method using Phusion High-Fidelity DNA polymerase and pACYC184 plasmid as a template and primers P3 (5-. 10. mu.L of 5 XPHUSION HF buffer, 0.5. Mu.M forward primer P1, 0.5. Mu.M forward primer P2, 200. Mu.M dNTPs, 3% DMSO, 50 ng template DNA, 0.5. Mu.L Phusion High-Fidelity DNA polymerase (Thermo Scientific, USA), sterile water was added to make up to the 50. Mu.L system. The PCR reaction program is: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 ℃ for 10 s; annealing at 62 ℃ for 20s; extending for 3min at 72 ℃ and circulating for 35 times; extending for 5min at 72 ℃, and storing at 4 ℃.

Obtained by PCR based on the golden gate methodlysCThe coding gene mutation library is connected with pACYC184 plasmid skeleton to obtain a plasmid containinglysCRecombinant expression plasmid pACYC184-lysC encoding different mutants of a gene ^mut . The golden gate linker system is: t4 DNA Ligase buffer 1.5. Mu.L, T4 DNA Ligase 1. Mu.L, bsaI 1. Mu.L, 10 × BSA 1.5. Mu.L, plasmid backbone, fragments (molar ratio 1, total 10. Mu.L), total volume 15. Mu.L. PCR ligation conditions: 3min at 37 ℃ and 4 min at 22 ℃ (40 cycles); 30min at 22 ℃; 5min at 80 ℃; 10 min at 4 ℃. Then, the above-mentioned ligation system was introduced into E.coli DH 5. Alpha. Competent cells according to a conventional E.coli heat shock transformation method to obtain a recombinant plasmid library.

Example 2 screening identification of aspartate kinase LysC variant libraries

The present invention established a screening method for screening aspartokinase LysC mutants by a homoserine biosensor, phomBio, which has been disclosed in the aforementioned patent application of the present applicant (application No. 202110059860.2), constructed based on the Corynebacterium glutamicum transcription regulator NCgl0581, and a genomic region containing the NCgl0581 open reading frame and the NCgl0580 promoter was cloned in front of GFP so that the expression of GFP was controlled by the NCgl0580 promoter. The biosensor can link the concentration of L-homoserine with the intensity of a fluorescence signal, so that the concentration level of L-homoserine is visualized, and the sensitive detection of the target product L-homoserine is realized.

The homoserine biosensor can respond to L-homoserine, and the fluorescence intensity is improved along with the increase of intracellular L-homoserine concentration, so that the high-throughput screening method is established on the basis of the L-homoserine biosensor. The recombinant plasmid mutation library and homoserine biosensor plasmid in example 1 were introduced into E.coli whose homoserine degradation pathway had been weakened, and the resulting strain was analyzed by flow cytometryScreening to obtain the beneficial mutant with high fluorescence intensity. The mutant with the improved aspartokinase activity has higher intracellular L-homoserine concentration and enhanced fluorescence intensity. The Escherichia coli with the homoserine degradation pathway weakened is shown inE.coliKnockout in MG1655metAAndthrBthe genotype of the strain is as follows:∆metA ∆thrB。

the specific screening steps are as follows: homoserine biosensor plasmid is first transferred intoE. coli MG1655∆metA∆ thrBIn the strain, then made competent, will be transferred into the embodiment 1 in the recombinant plasmid mutation library, coated on containing chloramphenicol and kanamycin selective antibiotic agar plate. Washing colonies on the plate with sterile PBS solution, inoculating into shake flasks containing fermentation medium, initial OD =0.5, overnight fermentation at 30 ℃,200 rpm; 1ml of the fermentation broth was centrifuged, washed 3 times with PBS solution, diluted to OD 0.1, sorted by flow cytometry, and the mutants with higher fluorescence intensity than the control (wild type) were collected on a plate and cultured overnight at 37 ℃. Picking single bacterial colony to a 96 shallow hole plate containing 200ul LB culture medium by using a sterilized toothpick, culturing for about 12h at 37 ℃, transferring to a 96 deep hole plate containing 600 mu L of LB culture medium until OD =0.6, adding 0.4mMIPTG, inducing overnight at 16 ℃ and 800rpm, centrifugally collecting bacteria, adding 200 mu L of lysis buffer containing 3mg/ml lysozyme for resuspension, reacting for 2h at 37 ℃, centrifugally taking supernatant, and measuring enzyme activity; connecting the mutants with improved enzyme activity obtained by primary screening to a test tube containing 5ml of LB culture medium, culturing at 37 ℃ until OD =0.6, adding 0.4mM IPTG, inducing overnight at 16 ℃, centrifuging to collect thalli, ultrasonically crushing, centrifuging to obtain supernatant to measure enzyme activity, sanger sequencing to determine mutation sites, and finally obtaining 6 different beneficial LysC mutants, namely V299A, N317S, N485S, Q107R/N317S, T114S/Q510R and T155A/K390E through three rounds of screening programs.

The LB culture medium comprises the following components: 1% yeast extract, 2% tryptone, 1% NaCl. The fermentation medium comprises the following components: 50 g/L glucose, 2 g/L yeast extract, 2 g/L MgSO ₄ ·7H ₂ O, 4 g/L KH ₂ PO ₄ , 14 g/L(NH ₄ ) ₂ SO ₄ , 0.5 g/L methionine, 0.5 g/L thReonine, 20 mL of an inorganic salt stock solution comprising 10 g/L FeSO ₄ ·7H ₂ O, 0.5 g/L MnSO ₄ ·4H ₂ O。

Example 3 determination of enzyme Activity of aspartate kinase LysC variants

Obtained by PCR based on the golden gate methodlysCThe coding gene mutation fragment is connected with pET21b plasmid skeleton to obtain the product containinglysCExpression plasmid pET21b-lysC encoding different mutants of a gene ^mut The plasmid was introduced into E.coli BL21 competent cells according to a conventional E.coli heat shock transformation method. The cells induced to express were collected, centrifuged to remove the medium, and the cells were resuspended in a 10 mL precooled lysis buffer to pellet (20 mM Na) ₂ HPO ₃ 200 mM NaCl, pH 7.5). Crushing cells by using an ultrasonic cell crusher, setting 200W power, performing ultrasonic treatment for 2 s and 1 s at intervals, and performing ultrasonic treatment for 10 min; followed by centrifugation at 8000 Xg for 10 min in a high speed refrigerated centrifuge and collection of the supernatant for subsequent protein purification and enzyme activity determination.

Since aspartokinase catalyzes aspartic acid to produce aspartic acid phosphate, and then consumes NADPH to reduce it to aspartic acid semialdehyde by aspartate semialdehyde dehydrogenase, the aspartokinase activity can be calculated by continuously measuring the absorbance decrease of NADPH at 340 nm. Reaction system 11.6mM aspartic acid, 70mMATP,5.8mM MgAc ₂ 0.60mM NADPH, 588mKCl, 140mM Tris-HCl (pH7.5), 3 to 5 units of aspartate semialdehyde dehydrogenase and a proper amount of aspartate kinase sample. Under the above conditions, the enzyme activity for catalytically oxidizing 1. Mu. Mol NADPH per minute is 1 enzyme activity unit.

The assay was performed by the aspartokinase activity assay, as shown in FIG. 2. The results show that most aspartokinase LysC mutants show obviously enhanced enzyme activity compared with the non-mutant aspartokinase, wherein the enzyme activity of the V299A and Q107R/N317S mutants can reach 12.49 +/-0.57 mu mol/min/mg and 13.08 +/-0.13 mu mol/min/mg, and is respectively improved by 3.6 and 3.8 times compared with a wild type control, thereby prompting that the mutant aspartokinase has more industrial application prospect.

Example 4 expression of aspartokinase LysC mutant to improve L-homoserine production by fermentation of engineering bacteria

Based on the aspartokinase mutant obtained in example 3, a microbial cell factory was constructed for fermentative production of L-homoserine and downstream derived products. The preferred plasmid for this example is the E.coli-C.glutamicum pXMJ19 shuttle-inducible expression vector, which itself contains sequences related to the strong promoter tac (including the operator lacO), which when an inducer such as IPTG or lactose is added, causes the repressor protein to leave the operator and thus initiate gene expression. Obtained by PCR based on the golden gate methodlysCThe mutant fragment of the coding gene is connected with the pXMJ19 plasmid skeleton to obtain a plasmid containinglysCExpression plasmid pXMJ19-lysC encoding different mutants of a gene ^mut . Corresponding expression plasmids are respectively transformed into chassis engineering bacteria with certain L-homoserine production capacity for fermentation performance test. In a specific embodiment, the engineered bacterium Ec-Hom is characterized by Escherichia coliEscherichia coli MG1655 with its homoserine degradation pathway gene deletedthrB(ProteinID: NP-414544) andmetA(Protein ID: NP-418437) while overexpressing the aspartokinase/homoserine dehydrogenase genethrA(Protein ID: NP-414543); in another specific embodiment, the engineered bacterium Cg-Hom is characterized by Corynebacterium glutamicumCorynebacterium glutamicum Deletion of the autologous homoserine degradation pathway Gene in ATCC 13032thrB(Protein ID: CAF 19888) while overexpressing the homoserine dehydrogenase genehom（Protein ID: CAF19887）。

The engineered strains obtained above were inoculated into triangular flasks containing 20 mL of LBHIS medium, respectively, and cultured for 16 h to the middle log phase. Cells were collected by centrifugation, suspended in fresh fermentation medium, inoculated into a 500 mL Erlenmeyer flask containing 25 mL of fermentation medium, and the starting OD was adjusted ₆₀₀ Is 1.0. The LBHIS culture medium is as follows: 5 g/L of yeast powder, 10 g/L of peptone, 10 g/L of sodium chloride, 18.5 g/L of brain-heart infusion and 91 g/L of sorbitol. The fermentation medium comprises: 60 g/L glucose, 0.5 g/L urea, 30 g/L corn steep liquor, 20 g/L ammonium sulfate and KH ₂ PO ₄ 5 g/L，MgSO ₄ ·7H ₂ O 5 g/L，FeSO ₄ ·7H ₂ O 0.2 g/L，MnCl ₂ ·4H ₂ 0.1 g/L of O, 0.2 mg/L of biotin, 10 mg/L of vitamin mixture, and CaCO ₃ 10 g/L buffer for the fermentation medium. The seed culture medium comprises 20 g/L glucose, 0.2 g/L urea, 15 g/L corn steep liquor and 20 g/L ammonium sulfate.

The engineering strains obtained are respectively inoculated into a 250 mL triangular flask containing 20 to 25 mL seed culture medium, and cultured for 12h to 16 h to the logarithmic phase. The cells were then harvested by centrifugation, suspended in fresh fermentation medium, inoculated into a 250 mL Erlenmeyer flask containing 50 mL of fermentation medium, and the OD was started ₆₀₀ 0.8 to 1.2, and 5 to 15 g/L CaCO is added ₃ . The fermentation condition is 30 ℃ and 150 to 200rpm, and the culture is carried out under constant temperature oscillation. The fermentation medium is as follows: 50-60 g/L of glucose, 0.1-0.2 g/L of urea, 5-10 g/L of corn steep liquor, 15-20 g/L of ammonium sulfate and KH ₂ PO ₄ 5~6 g/L，MgSO ₄ ·7H ₂ O 2 ~5 g/L，FeSO ₄ ·7H ₂ O 0.2~0.5 g/L，MnCl ₂ ·4H ₂ 0.2 to 0.4 g/L of O, 0.2 to 0.4 mg/L of biotin, 1 to 2 mg/L of vitamin B, and CaCO ₃ 5 to 15 g/L of buffer used for the fermentation medium. The seed culture medium comprises 10-20 g/L glucose, 0.1-0.2 g/L urea, 15-20 g/L corn steep liquor, 15-20 g/L ammonium sulfate and 0.2 mM IPTG (isopropyl thiogalactoside) addition concentration. Results of the study table 1 shows:

TABLE 1 influence of aspartokinase LysC mutant expression on L-homoserine production by fermentation of Chassis-engineering bacteria

After 72 h fermentation culture, after the aspartokinase LysC mutant is over-expressed in escherichia coli Ec-Hom engineering bacteria, the L-homoserine yield of the contrast spawn can be improved by 80-285%; after the aspartokinase LysC mutant is over-expressed in Corynebacterium glutamicum Cg-Hom engineering bacteria, the yield of L-homoserine of the contrast spawn can be improved by 5% -49%. Compared with the non-mutant aspartokinase, the expression of the aspartokinase LysC mutant in the chassis engineering bacteria can obviously improve the capability of producing L-homoserine by strain fermentation.

Claims (10)

1. An aspartokinase LysC mutant derived from saccharomyces cerevisiae, which is characterized in that only one site mutation of valine V to alanine A at position 299, asparagine N to serine S at position 317, or asparagine N to serine S at position 485 is adopted as a mutation relative to an amino acid sequence shown in SEQ ID No. 2; there are only combinatorial mutations with a mutation from glutamine Q to arginine R at position 107 and asparagine N to serine S at position 317; there are only combined mutations with threonine T to serine S at position 114 and glutamine Q to arginine R at position 510; there are only combined mutations with a mutation from threonine T to alanine a at position 155 and lysine K to glutamic acid E at position 390.

2. The aspartokinase LysC mutant gene according to claim 1.

3. The encoding gene of claim 2, wherein the nucleotide sequence is obtained by mutation based on the nucleotide sequence shown in SEQ ID No. 1.

4. An expression vector comprising the gene encoding the gene according to claim 2 or 3.

5. Host cell comprising a gene coding for the gene according to claim 2 or 3, preferably E.coli, C.glutamicum or s.cerevisiae, more preferably E.coli, C.glutamicum or s.cerevisiae.

6. Use of the aspartokinase mutant or its encoding gene according to claim 1 for producing L-homoserine or its derivative selected from the group consisting of L-homoserine and its derivativeO-acetylhomoserine or methionine.

7. A method for producing L-homoserine or its derivatives, or lysine, characterized in that L-homoserine or its derivatives are produced by fermentation of a recombinant microorganism containing the coding gene of claim 2 or 3, wherein the microorganism is a chassis-engineered bacterium having L-homoserine production ability; the derivatives are threonine, leucine and isoleucine.

8. The method according to claim 7, wherein the microorganism is Escherichia coli, corynebacterium glutamicum or Saccharomyces cerevisiae having L-homoserine producing ability, more preferably Escherichia coli, corynebacterium glutamicum or Saccharomyces cerevisiae having L-homoserine producing ability.

9. The method according to claim 8, wherein the self homoserine degradation pathway gene is deleted in E.colithrBSimultaneous overexpression of aspartokinase/homoserine dehydrogenase genethrA。

10. The method of claim 8, wherein the corynebacterium glutamicum has a knockout of a self-homoserine degradation pathway genethrBSimultaneous overexpression of the homoserine dehydrogenase genehom。

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