Construction method and application of genetically engineered bacterium for efficiently producing O-acetyl-L-homoserine

Abstract

The invention relates to the technical field of microbial metabolism engineering, in particular to a construction method and application of genetically engineered bacteria for efficiently producing O-acetyl-L-homoserine. The construction method specifically comprises the following steps: (1) taking a strain Escherichia coli W3110 as a chassis, and knocking out a homoserine O-succinyl transferase coding gene metA; (2) knocking out a gamma-cystathionine synthetase encoding gene metB; (3) knocking out a cysteine lyase coding gene metC; (4) carrying out weakening expression on an O-acetyl-L-homoserine thiolase coding gene metYcm from the marine cyclobacterium; (5) overexpressing a ribulose diphosphate carboxylase coding gene rbcLSse from the synechococcus elongate; (6) overexpressing a ribulose phosphate kinase coding gene prkse from the synechococcus slender; and (7) weakening the expression of the citrate synthase coding gene gltA to obtain the genetically engineered bacterium for efficiently producing the O-acetyl-L-homoserine. The strain provided by the invention is used for producing O-acetyl-L-homoserine, and has the advantages of high yield, high yield and high sugar-acid conversion rate.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial metabolic engineering, and in particular to a method for constructing a genetically engineered bacterium capable of efficiently producing O-acetyl-L-homoserine and an application thereof. Background Art

[0002] O-acetyl-L-homoserine (OAH) is an important non-protein synthetic amino acid with the molecular formula of C6H 11 NO4, molecular weight is 161.16. O-acetyl-L-homoserine can hardly accumulate in microbial cells. O-acetyl-L-homoserine is an important platform compound that can be used to synthesize a variety of chemicals, among which methionine is an extremely important one.

[0003] There are two pathways for the synthesis of methionine, the transsulfuration pathway and the direct sulfurization pathway. In the process of transsulfuration of methionine, homoserine is converted to L-homocysteine ​​through three steps (catalyzed by MetA, MetB, and MetC, respectively). Inorganic sulfur is required to first reach cysteine, and then cysteine ​​is used as a sulfur donor. In the direct sulfurization pathway of methionine, homoserine only needs two steps to be converted into L-homocysteine, and MetY directly combines with inorganic sulfur. The advantages of the direct sulfurization pathway are obvious. It synthesizes amino acids used for survival in cells in a simpler way, which is conducive to more carbon flow for the synthesis of target products.

[0004] At present, there have been many studies on the production of O-acetyl-L-homoserine by fermentation of Escherichia coli as the chassis. Wei Liang et al. from the Tiangong Institute redistributed the metabolic flow and used protein engineering to transform the homoserine O-acetyltransferase MetX from Leptospira meyeri, and then regulated the cofactors including acetyl-CoA and NADPH, so that the shake flask yield of O-acetyl-L-homoserine of the engineered strain reached 12.1g / L. Li Bo et al. from Zhejiang University of Technology screened exogenous MetX, knocked out the degradation and competition pathways, and then redistributed the carbon flux of central carbon metabolism, which could accumulate 8.29g / L of O-acetyl-L-homoserine. CJ CheilJedang Co., Ltd. from South Korea achieved the extracellular accumulation of O-acetyl-L-homoserine by increasing the activity of MetX and strengthening the expression of the O-acetyl-L-homoserine extracellular transporter YjeH, and its shake flask yield reached 5.4g / L. These studies have achieved remarkable results, but there is still room for improvement in various indicators of the strain, and the yield can still be further improved. Therefore, improving the production performance of O-acetyl-L-homoserine engineering bacteria is a goal of those skilled in the art. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems of low yield and high fermentation cost of O-acetyl-L-homoserine production strains in the prior art in the fermentation production of O-acetyl-L-homoserine, and to provide a construction method of a genetically engineered bacterium for efficiently producing O-acetyl-L-homoserine and its application.

[0006] In order to achieve the above object, the present invention is implemented by the following technical solutions: In a first aspect, the present invention provides a method for constructing a genetically engineered bacterium for efficiently producing O-acetyl-L-homoserine, which specifically comprises the following steps: (1) Using the strain Escherichia coli W3110 as the chassis, the metA gene encoding homoserine O-succinyltransferase was knocked out; (2) knocking out the gene encoding γ-cystathionine synthase, metB; (3) knocking out metC, the gene encoding cysteine ​​cleavage enzyme; (4) Weakly expressing metYcm, the gene encoding O-acetyl-L-homoserine thiolase from Cyclobacter maritima; (5) Overexpression of the ribulose bisphosphate carboxylase encoding gene rbcLSse from Synechococcus elongatus; (6) Overexpression of the phospholipid kinase encoding gene prkse from Synechococcus elongatus; (7) Weakening the expression of the citrate synthase encoding gene gltA to obtain the genetically engineered bacteria that efficiently produces O-acetyl-L-homoserine.

[0007] The present invention enables the genetically engineered bacteria of O-acetyl-L-homoserine to increase its yield as much as possible, and can enhance the glycolysis flux and weaken the TCA pathway by knocking out the original methionine transsulfurization synthesis pathway, introducing a heterologous methionine direct sulfurization synthesis pathway, and weakening the TCA pathway. The present invention knocks out the homoserine O-succinyltransferase encoding gene metA, the γ-cystathionine synthetase encoding gene metB, and the cysteine ​​lyase encoding gene metC in the previously constructed Escherichia coli Escherichia coli W3110 (i.e., Escherichia coli OAH7-X, disclosed in patent application number CN202410775092.4, application publication number CN118531032A, and provided by the microbial strain collection of Zhejiang University of Technology) to block the intracellular methionine transsulfurization synthesis pathway; express the methionine direct sulfurization synthesis pathway O-acetyl-L-homoserine thiolase encoding gene metY from marine cyclobacillus cm, so that methionine can still be synthesized normally in the cell, and reducing its expression is to reduce the degradation of O-acetyl-L-homoserine. In addition, the methionine transsulfurization pathway requires additional consumption of succinyl-CoA, which can be produced in the TCA cycle and is closely related to cell growth. When replaced with the direct sulfurization pathway, its consumption can be reduced, laying the foundation for the weakening of the TCA cycle and reducing its impact; overexpression of the ribulose bisphosphate carboxylase encoding gene rbcLS from Synechococcus elongatus se and the phosphoribosyl kinase encoding gene prk se , in order to increase the cell's carbon fixation capacity and enhance the flux of glycolysis; the promoter of the citrate synthase encoding gene gltA was replaced with a dynamically regulated promoter, which greatly reduced its transcription in the middle and late stages of fermentation, thereby weakening the TCA cycle. This can not only reduce the additional consumption of acetyl-CoA, provide more acetyl-CoA for the synthesis of O-acetyl-L-homoserine, but also strengthen the L-homoserine synthesis pathway.

[0008] The genes encoding homoserine O-succinyltransferase, γ-cystathionine synthase, cysteine ​​lyase, O-acetyl-L-homoserine thiolase from Cyclobacter maritima, ribulose bisphosphate carboxylase and phospholipid kinase from Synechococcus elongatus, and citrate synthase are generally represented by metA, metB, metC, metY, and metY. cm rbcLS se 、prk se The gene sequences can be obtained from the public database National Center for Biotechnology Information (NCBI). For example, the GenBank ID No. 85676765 of metA, the GenBank ID No. 85676121 of metB, the GenBank ID No. 85675812 of metC, the GenBank ID No. 85675813 of metY cm GenBank: NLF91922.1, rbcLS se GenBank ID.No.56685098 and 56685097, prk se GenBank ID. No. 56685535, gltA GenBank ID. No. 85674748.

[0009] Preferably, in step 5, a copy of rbcLSse and prkse genes is added in the genome to enhance their expression; and the rbcLSse and prkse genes are expressed by a strong promoter.

[0010] Preferably, the strong promoter in step 5 is Ptrc, and its nucleotide sequence is SEQ ID NO.1.

[0011] Preferably, the method for constructing a genetically engineered bacterium for efficiently producing O-acetyl-L-homoserine is characterized in that the weakened expression of metYcm in step 4 is achieved by using a dynamically regulated promoter, and the weakened expression of gltA in step 7 is achieved by replacing the original promoter with a dynamically regulated promoter.

[0012] Preferably, the dynamic regulation promoter is PfliC, and its nucleotide sequence is SEQ ID NO.2.

[0013] Preferably, the metYcm is integrated into the metA gene locus, and the rbcLSse and prkse are integrated into the efeU gene locus.

[0014] Preferably, the metYcm is from Cyclobacter maritima, and the rbcLSse and prkse are from Synechococcus elongatus.

[0015] In a second aspect, a genetically engineered bacterium for efficiently producing O-acetyl-L-homoserine is prepared by any of the above methods. The present invention further provides a use of Corynebacterium glutamicum producing D-pantothenic acid in the fermentation production of D-pantothenic acid.

[0016] In a third aspect, the present invention further provides a use of a genetically engineered bacterium for efficiently producing O-acetyl-L-homoserine in the fermentation of producing O-acetyl-L-homoserine.

[0017] Preferably, the application is to inoculate the genetically engineered bacteria into a fermentation medium, ferment and culture at 30-33° C. and 180-220 rpm, separate and purify the culture solution to obtain O-acetyl-L-homoserine.

[0018] The final concentration of the fermentation medium of the present invention is as follows: 30 g / L of glucose, 2 g / L of potassium dihydrogen phosphate, 20 g / L of ammonium sulfate, 4 g / L of yeast powder, 20 g / L of calcium carbonate, 0.20 g / L of L-threonine, 0.0001 g / L of vitamin B1, 0.0001 g / L of vitamin B12, 0.2 g / L of L-isoleucine, 2 g / L of MgSO4, 0.005 g / L of FeSO4, 0.005 g / L of MnSO4 and 0.005 g / L of ZnSO4, the solvent is deionized water, and the pH value is 6.8.

[0019] Before the fermentation culture, slant activation and seed culture are performed, and the seed liquid is inoculated into the fermentation medium. The slant activation method is: inoculate the recombinant Escherichia coli on the LB plate, culture it overnight at 37°C to obtain the slant bacteria; the seed culture method is: pick a single colony of the slant bacteria and inoculate it into the LB liquid culture medium, culture it overnight at 37°C and 200rpm to obtain the seed liquid. The seed liquid is inoculated into the fermentation medium at an inoculation amount of 2% by volume.

[0020] The present invention has the following beneficial effects: (1) Compared with the wild-type and chassis strains, the modified recombinant Escherichia coli provided by the present invention can better utilize carbon sources such as glucose to produce O-acetyl-L-homoserine, and has higher yield, productivity and sugar-acid conversion rate. (2) The production of O-acetyl-L-homoserine increased from 12.66 g / L at the bottom to 17.23 g / L, an increase of 36.1%. (3) Therefore, the genetically engineered bacteria provided by the present invention have important industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a fermentation result diagram of shake flask fermentation of all strains in the present invention. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with specific embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0023] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0024] Unless otherwise specified, the test materials used in the following examples are all conventional biochemical reagents.

[0025] LB liquid culture medium composition: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, solvent is deionized water, pH value is natural.

[0026] LB plates were prepared by adding agar to LB liquid medium at a final concentration of 2 g / L. Example 1 Construction of genetically engineered bacteria for efficient production of O-acetyl-L-homoserine (1) Replacement of methionine synthesis pathway

[0027] The methionine transsulfurization pathway requires additional consumption of succinyl-CoA. When replaced with the direct sulfurization pathway, the generation of extra byproducts can be avoided and the foundation can be laid for weakening the TCA cycle without affecting growth. cm The purpose is to reduce product degradation. Therefore, the homoserine O-succinyltransferase encoding gene metA, the γ-cystathionine synthase encoding gene metB and the cysteine ​​lyase encoding gene metC were knocked out in the genome, and the O-acetyl-L-homoserine thiolase encoding gene metY from Cyclobacter marineus was integrated in the place of metA using the PfliC promoter. cm . It is realized by using CRISPR-Cas9 system, see Jiang Y, Chen B, Duan C, et al. Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System [J]. Applied & Environmental Microbiology, 2015, 81 (7): 2506.

[0028] The pTargetF vector (plasmid from Addgene) was diluted and used as a template. Primers 1 and 2 were used to construct the pTarget-ΔmetA mutant vector that can express the sgRNA targeting the target gene metA by PCR. The PCR reaction conditions are as follows: 95℃5min; 95℃30s, 55℃30s, 72℃1min, 30 cycles; 72℃5min. The PCR product was transformed into E.coli DH5α competent medium and screened on a plate with spectinomycin hydrochloride (SD) resistance to obtain the pTarget-ΔmetA vector with correct sequencing.

[0029] Then, primers 3 and 4 were used to amplify the upstream homologous sequence of metA. The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 5 min. In the same way, primers 5 and 6 were used to amplify the downstream homologous sequence of metA. The PCR product was purified using a purification kit.

[0030] Then, according to the fliC gene of Escherichia coli published on NCBI, the promoter PfliC was amplified using primers 7 and 8. The PCR reaction conditions were as follows: 95℃5min; 95℃30s, 55℃30s, 72℃1min, 30 cycles; 72℃5min. cm Gene, metY was amplified using primers 9 and 10 cm The PCR product was purified using a purification kit.

[0031] The purified metA upstream and downstream homologous fragments, promoters PfliC and metY cm Gene fragment, using primer 3 and primer 6 for fusion PCR to obtain the knock-in frame, the PCR reaction conditions are as follows: 95℃5min; 95℃30s, 55℃30s, 72℃1min, 30 cycles; 72℃5min.

[0032] The process of preparing competent cells by chemical transformation is as follows: pick a single colony of Escherichia coli W3110 and inoculate it into a test tube containing 5 mL of LB medium, and culture it overnight at 37°C and 200 rpm in a constant temperature shaker. Pipette 1 mL of the overnight culture solution and transfer it to a 250 mL conical flask containing 40 mL of LB medium, and culture it at 37°C and 200 rpm in a constant temperature shaker for 1-2 hours until the OD value of the culture solution reaches 0. 600 The pH value is between 0.4 and 0.6. Place the cultured bacterial solution on ice to cool for about 10 minutes, pour into a 50mL centrifuge tube, centrifuge at 4°C, 5500rpm for 5 minutes, remove the supernatant and retain the bacteria in a clean bench, add about 40mL of pre-sterilized and pre-cooled 0.1M CaCl2 solution, resuspend in an ice-water bath, and ice-bath for 30 minutes. After centrifugation at 6000rpm for 5 minutes, add 1mL of pre-cooled 0.1M CaCl2 and 15% glycerol, resuspend in an ice-water bath, and dispense into 1.5mL sterile centrifuge tubes (100μL per tube), keep one tube for use, and store the rest at -80°C.

[0033] The process of chemical transformation of pCas plasmid is as follows: take a competent cell and place it on ice, add 1 μL of plasmid to the competent cell in a clean bench, mix well and then place it in an ice bath for 30 minutes. Then place the EP tube containing competent cells in a 42°C constant temperature water bath for 90 seconds, and quickly insert it into ice to cool for 5 minutes. Add 700 μL of pre-cooled LB culture medium to the EP tube in the clean bench, and culture it in a constant temperature shaker at 37°C and 200rpm for 1-2 hours. Pipette 200 μL of bacterial solution and spread it on the LB solid plate of the corresponding resistance, invert it in a 37°C constant temperature incubator and culture it overnight to obtain the Escherichia coli W3110 strain containing the pCas vector.

[0034] The electroporation competent preparation process is as follows: pick a single colony from the Escherichia coli W3110 pCas streak plate, inoculate it into a 5mL LLB tube (kan resistance), add 100μL L-Ara mother solution, and culture it at 30℃, 180rpm overnight as seed solution. Inoculate 500μL of the seed solution into a 50mL shake flask LLB (kan resistance), add 500μL L-Ara mother solution, and culture it at 30℃, 180rpm for 3-4h until the OD reaches 0. 600 =0.6. Precool the shake flask and centrifuge tube in an ice bath for 10 minutes, pour the bacterial solution into the centrifuge tube in the clean bench, and centrifuge at 4°C, 6000rpm for 5 minutes. Discard the supernatant in the clean bench and add 40mL of precooled sterile ultrapure water. Gently resuspend the cells in an ice water bath, centrifuge at 4°C, 6000rpm for 6 minutes, and repeat once. Discard the supernatant in the clean bench (pay attention to the loss of bacteria), add 20mL of precooled 10% glycerol to a centrifuge tube, and resuspend in an ice water bath. Centrifuge at 4°C, 6000rpm for 5 minutes. Discard the supernatant in the clean bench (pay attention to the loss of bacteria), add 1mL of precooled 10% glycerol, resuspend in an ice water bath, and dispense 100μL into sterile 2mL EP tubes in each tube, keep one tube for use, and store the rest at -80°C.

[0035] The electroporation process is as follows: Take an electroporation competent cell and place it in an ice bath. Add 2μL of pTarget-ΔmetA vector and 2μL of knock-in frame PCR purification product in the clean bench, mix gently with a medium gun, and place on ice for 1 minute. Use a medium gun to transfer to a pre-cooled 2mm electroporation cup and place on ice for 1 minute. Use a paper towel to wipe off the water mist on the outside of the electroporation cup, place it in the electroporator, and use ECO 2 for electroporation. In the clean bench, add 1mL of pre-cooled LLB culture medium to the groove of the electroporation cup, tilt the electroporation cup, absorb all the bacterial solution from the mouth of the electroporation cup, and transfer it to a 2mL sterile ep tube. Resuscitate at 30℃, 180rpm for 3h. Take 400μL and spread it on LLB solid culture medium (SD+kan resistance) and culture overnight at 30℃.

[0036] A single colony was selected as a template for colony PCR verification to confirm the knockout of metA and metY cm The strain confirmed by this was cultured in LB medium containing 50 mg / L kanamycin and 5 mM IPTG at 30°C overnight to remove the pTarget-ΔmetA vector. The strain from which the pTarget-ΔmetA vector had been removed was then cultured in LB medium at 37°C overnight to remove the pCas vector. In this way, the strain Escherichia coli W3110ΔmetA::PfliC-metY was obtained. cm .

[0037] Next, the pTargetF vector (plasmid from Addgene) was diluted and used as a template. Primers 11 and 12 were used to construct the pTarget-ΔmetB mutant vector that can express the sgRNA targeting the target gene metB by PCR. The PCR reaction conditions are as follows: 95℃5min; 95℃30s, 55℃30s, 72℃1min, 30 cycles; 72℃5min. The PCR product was transformed into E.coli DH5α competent medium and screened on a plate with spectinomycin hydrochloride (SD) resistance to obtain the pTarget-ΔmetB vector with correct sequencing.

[0038] Then, primers 13 and 14 were used to amplify the upstream homologous sequence of metB. The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 5 min. In the same way, primers 15 and 16 were used to amplify the downstream homologous sequence of metB. The PCR product was purified using a purification kit.

[0039] The purified upstream and downstream homologous fragments of metB were subjected to fusion PCR using primers 13 and 16 to obtain the knockout frame. The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 5 min.

[0040] The process of preparing competent cells by chemical transformation is as follows: Pick Escherichia coli W3110ΔmetA::PfliC-metY cm A single colony was inoculated into a test tube containing 5 mL of LB medium and cultured overnight at 37°C, 200 rpm in a constant temperature shaker. 1 mL of the overnight culture was transferred to a 250 mL conical flask containing 40 mL of LB medium and cultured at 37°C, 200 rpm in a constant temperature shaker for 1-2 hours until the OD value of the culture reached 0. 600The pH value is between 0.4 and 0.6. Place the cultured bacterial solution on ice to cool for about 10 minutes, pour into a 50mL centrifuge tube, centrifuge at 4°C, 5500rpm for 5 minutes, remove the supernatant and retain the bacteria in a clean bench, add about 40mL of pre-sterilized and pre-cooled 0.1M CaCl2 solution, resuspend in an ice-water bath, and ice-bath for 30 minutes. After centrifugation at 6000rpm for 5 minutes, add 1mL of pre-cooled 0.1M CaCl2 and 15% glycerol, resuspend in an ice-water bath, and dispense into 1.5mL sterile centrifuge tubes (100μL per tube), keep one tube for use, and store the rest at -80°C.

[0041] The process of chemical transformation of pCas plasmid is as follows: take a competent cell and place it on ice, add 1 μL of plasmid to the competent cell in a clean bench, mix well and then place it in an ice bath for 30 minutes. Then place the EP tube containing the competent cells in a 42°C constant temperature water bath for heat shock for 90 seconds, and quickly insert it into ice to cool for 5 minutes. Add 700 μL of pre-cooled LB culture medium to the EP tube in the clean bench, and culture it in a constant temperature shaker at 37°C and 200rpm for 1-2 hours. Pipette 200 μL of bacterial solution and spread it on the LB solid plate with the corresponding resistance, invert it in a 37°C constant temperature incubator and culture it overnight to obtain Escherichia coli W3110ΔmetA::PfliC-metY containing the pCas vector. cm strains.

[0042] The electroporation competent cell preparation process is as follows: Escherichia coli W3110ΔmetA::PfliC-metY cm A single colony was picked from the pCas streaked plate and inoculated into a 5 mL LLB tube (kan resistance), and 100 μL of L-Ara stock solution was added, and cultured overnight at 30°C, 180 rpm as the seed solution. 500 μL of the seed solution was inoculated into a 50 mL shake flask LLB (kan resistance), and 500 μL of L-Ara stock solution was added, and cultured at 30°C, 180 rpm for 3-4 h until the OD 600 =0.6. Precool the shake flask and centrifuge tube in an ice bath for 10 minutes, pour the bacterial solution into the centrifuge tube in the clean bench, and centrifuge at 4°C, 6000rpm for 5 minutes. Discard the supernatant in the clean bench and add 40mL of precooled sterile ultrapure water. Gently resuspend the cells in an ice water bath, centrifuge at 4°C, 6000rpm for 6 minutes, and repeat once. Discard the supernatant in the clean bench (pay attention to the loss of bacteria), add 20mL of precooled 10% glycerol to a centrifuge tube, and resuspend in an ice water bath. Centrifuge at 4°C, 6000rpm for 5 minutes. Discard the supernatant in the clean bench (pay attention to the loss of bacteria), add 1mL of precooled 10% glycerol, resuspend in an ice water bath, and dispense 100μL into sterile 2mL EP tubes in each tube, keep one tube for use, and store the rest at -80°C.

[0043] The electroporation process is as follows: Take an electroporation competent cell and place it in an ice bath. Add 2μL of pTarget-ΔmetB vector and 2μL of knockout box PCR purification product in the clean bench, mix gently with a medium gun, and place on ice for 1 minute. Use a medium gun to transfer to a pre-cooled 2mm electroporation cup and place on ice for 1 minute. Use a paper towel to wipe off the water mist on the outside of the electroporation cup, place it in the electroporator, and use ECO 2 for electroporation. In the clean bench, add 1mL of pre-cooled LLB culture medium to the groove of the electroporation cup, tilt the electroporation cup, absorb all the bacterial solution from the mouth of the electroporation cup, and transfer it to a 2mL sterile ep tube. Resuscitate at 30℃, 180rpm for 3h. Take 400μL and spread it on LLB solid culture medium (SD+kan resistance) and culture it at 30℃ overnight.

[0044] A single colony was picked as a template for colony PCR verification to confirm the knockout of metB. The strain that passed this confirmation was cultured overnight at 30°C in LB medium containing 50 mg / L kanamycin and 5 mM IPTG to remove the pTarget-ΔmetB vector. The strain from which the pTarget-ΔmetB vector had been removed was then cultured overnight in LB medium at 37°C to remove the pCas vector. In this way, the strain Escherichia coli W3110ΔmetA::PfliC-metY was obtained. cm ΔmetB.

[0045] Finally, the pTargetF vector (plasmid from Addgene) was diluted and used as a template. Primers 17 and 18 were used to construct the pTarget-ΔmetC mutant vector that can express the sgRNA targeting the target gene metC by PCR. The PCR reaction conditions are as follows: 95℃5min; 95℃30s, 55℃30s, 72℃1min, 30 cycles; 72℃5min. The PCR product was transformed into E.coli DH5α competent medium and screened on a plate with spectinomycin hydrochloride (SD) resistance to obtain the pTarget-ΔmetC vector with correct sequencing.

[0046] Then, primers 19 and 20 were used to amplify the upstream homologous sequence of metC. The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 5 min. In the same way, primers 21 and 22 were used to amplify the downstream homologous sequence of metC. The PCR product was purified using a purification kit.

[0047] The purified upstream and downstream homologous fragments of metC were subjected to fusion PCR using primers 19 and 22 to obtain the knockout frame. The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 5 min.

[0048] The process of preparing competent cells by chemical transformation is as follows: Pick Escherichia coli W3110ΔmetA::PfliC-metY cm A single colony of ΔmetB was inoculated into a test tube containing 5 mL of LB medium and cultured overnight at 37°C, 200 rpm in a constant temperature shaker. 1 mL of the overnight culture was transferred to a 250 mL conical flask containing 40 mL of LB medium and cultured at 37°C, 200 rpm in a constant temperature shaker for 1-2 hours until the OD value of the culture reached 0. 600 The pH value is between 0.4 and 0.6. Place the cultured bacterial solution on ice to cool for about 10 minutes, pour into a 50mL centrifuge tube, centrifuge at 4°C, 5500rpm for 5 minutes, remove the supernatant and retain the bacteria in a clean bench, add about 40mL of pre-sterilized and pre-cooled 0.1M CaCl2 solution, resuspend in an ice-water bath, and ice-bath for 30 minutes. After centrifugation at 6000rpm for 5 minutes, add 1mL of pre-cooled 0.1M CaCl2 and 15% glycerol, resuspend in an ice-water bath, and dispense into 1.5mL sterile centrifuge tubes (100μL per tube), keep one tube for use, and store the rest at -80°C.

[0049] The process of chemical transformation of pCas plasmid is as follows: take a competent cell and place it on ice, add 1 μL of plasmid to the competent cell in a clean bench, mix well and then place it in an ice bath for 30 minutes. Then place the EP tube containing the competent cells in a 42°C constant temperature water bath for heat shock for 90 seconds, and quickly insert it into ice to cool for 5 minutes. Add 700 μL of pre-cooled LB culture medium to the EP tube in the clean bench, and culture it in a constant temperature shaker at 37°C and 200rpm for 1-2 hours. Pipette 200 μL of bacterial solution and spread it on the LB solid plate with the corresponding resistance, invert it in a 37°C constant temperature incubator and culture it overnight to obtain Escherichia coli W3110ΔmetA::PfliC-metY containing the pCas vector. cm ΔmetB strain.

[0050] The electroporation competent cell preparation process is as follows: Escherichia coli W3110ΔmetA::PfliC-metY cmA single colony was picked from the ΔmetB pCas streaked plate, inoculated into a 5 mL LLB tube (kan resistance), and 100 μL of L-Ara stock solution was added, and cultured overnight at 30°C, 180 rpm as the seed solution. 500 μL of the seed solution was inoculated into a 50 mL shake flask LLB (kan resistance), and 500 μL of L-Ara stock solution was added, and cultured at 30°C, 180 rpm for 3-4 h until the OD 600 =0.6. Precool the shake flask and centrifuge tube in an ice bath for 10 minutes, pour the bacterial solution into the centrifuge tube in the clean bench, and centrifuge at 4°C, 6000rpm for 5 minutes. Discard the supernatant in the clean bench and add 40mL of precooled sterile ultrapure water. Gently resuspend the cells in an ice water bath, centrifuge at 4°C, 6000rpm for 6 minutes, and repeat once. Discard the supernatant in the clean bench (pay attention to the loss of bacteria), add 20mL of precooled 10% glycerol to a centrifuge tube, and resuspend in an ice water bath. Centrifuge at 4°C, 6000rpm for 5 minutes. Discard the supernatant in the clean bench (pay attention to the loss of bacteria), add 1mL of precooled 10% glycerol, resuspend in an ice water bath, and dispense 100μL into sterile 2mL EP tubes in each tube, keep one tube for later use, and store the rest at -80°C

[0051] The electroporation process is as follows: Take an electroporation competent cell and place it in an ice bath. Add 2μL of pTarget-ΔmetC vector and 2μL of knockout box PCR purification product in the clean bench, mix gently with a medium gun, and place on ice for 1 minute. Use a medium gun to transfer to a pre-cooled 2mm electroporation cup and place on ice for 1 minute. Use a paper towel to wipe off the water mist on the outside of the electroporation cup, place it in the electroporator, and use ECO 2 for electroporation. In the clean bench, add 1mL of pre-cooled LLB culture medium to the groove of the electroporation cup, tilt the electroporation cup, absorb all the bacterial solution from the mouth of the electroporation cup, and transfer it to a 2mL sterile ep tube. Resuscitate at 30℃, 180rpm for 3h. Take 400μL and spread it on LLB solid culture medium (SD+kan resistance) and culture it at 30℃ overnight.

[0052] A single colony was picked as a template for colony PCR verification to confirm the knockout of metC. The strain that passed this confirmation was cultured overnight at 30°C in LB medium containing 50 mg / L kanamycin and 5 mM IPTG to remove the pTarget-ΔmetC vector. The strain from which the pTarget-ΔmetC vector had been removed was then cultured overnight in LB medium at 37°C to remove the pCas vector. In this way, the strain Escherichia coli W3110ΔmetA::PfliC-metY was obtained. cm ΔmetBΔmetC and named it OAHS1. (2) Introduction of carbon fixation pathways

[0053] The rbcLS gene encoding ribulose bisphosphate carboxylase from Synechococcus elongatus se and the phosphoribosyl kinase encoding gene prk se It can fix one molecule of carbon dioxide and generate triphosphoglycerate, thereby increasing the cell's carbon fixation capacity and enhancing the flux of glycolysis. se and prk se Gene, see JiangY, ChenB, Dua n C, et al.Multigene Editing in the Escherichia coli Genome via theCRISPR-Cas9System[J]. Applied&Environmental Microbiology, 2015, 81(7):2506.

[0054] The pTargetF vector (plasmid from Addgene) was diluted and used as a template. Primers 23 and 24 were used to construct the pTarget-ΔefeU mutant vector that can express the sgRNA targeting the target gene efeU by PCR. The PCR reaction conditions are as follows: 95℃5min; 95℃30s, 55℃30s, 72℃1min, 30 cycles; 72℃5min. The PCR product was transformed into E.coli DH5α competent cells and screened on a plate with spectinomycin hydrochloride (SD) resistance to obtain the pTarget-ΔefeU vector with correct sequencing.

[0055] Subsequently, primers 25 and 26 were used to amplify the upstream homologous sequence of efeU. The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 5 min. In the same way, primers 27 and 28 were used to amplify the downstream homologous sequence of efeU. The PCR product was purified using a purification kit.

[0056] Then, rbcLS was performed according to the Synechococcus elongatus published in NCBI. se and prk se The gene was synthesized and codon optimized, and primers 29 and 30 were used as templates to amplify rbcLS se The strong promoter Ptrc was introduced and primers 31 and 32 were used to amplify prk se Gene. PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 5 min. PCR products were purified using a purification kit.

[0057] The purified efeU upstream and downstream homologous fragments, rbcLS se and prk se The knock-in frame was obtained by fusion PCR using primers 25 and 28. The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 5 min.

[0058] The process of preparing competent cells by chemical transformation is as follows: pick a single colony of OAHS1 and inoculate it into a test tube containing 5 mL of LB medium, and culture it overnight at 37°C and 200 rpm in a constant temperature shaker. Pipette 1 mL of the overnight culture solution and transfer it to a 250 mL conical flask containing 40 mL of LB medium, and culture it at 37°C and 200 rpm in a constant temperature shaker for 1-2 hours until the OD value of the bacterial solution reaches 0. 600 The pH value is between 0.4 and 0.6. Place the cultured bacterial solution on ice to cool for about 10 minutes, pour into a 50mL centrifuge tube, centrifuge at 4°C, 5500rpm for 5 minutes, remove the supernatant and retain the bacteria in a clean bench, add about 40mL of pre-sterilized and pre-cooled 0.1M CaCl2 solution, resuspend in an ice-water bath, and ice-bath for 30 minutes. After centrifugation at 6000rpm for 5 minutes, add 1mL of pre-cooled 0.1M CaCl2 and 15% glycerol, resuspend in an ice-water bath, and dispense into 1.5mL sterile centrifuge tubes (100μL per tube), keep one tube for use, and store the rest at -80°C.

[0059] The process of chemical transformation of pCas plasmid is as follows: take a competent cell and place it on ice, add 1 μL of plasmid to the competent cell in a clean bench, mix well and then place it in an ice bath for 30 minutes. Then place the EP tube containing competent cells in a 42°C constant temperature water bath for 90 seconds, and quickly insert it into ice to cool for 5 minutes. Add 700 μL of pre-cooled LB culture medium to the EP tube in the clean bench, and culture it in a constant temperature shaker at 37°C and 200rpm for 1-2 hours. Pipette 200 μL of bacterial solution and spread it on the LB solid plate of the corresponding resistance, invert it in a 37°C constant temperature incubator and culture it overnight to obtain the OAHS1 strain containing the pCas vector.

[0060] The electroporation competent preparation process is as follows: pick a single colony from the OAHS1 pCas streaked plate, inoculate it into a 5mL LLB test tube (kan resistance), add 100μL L-Ara mother solution, and culture it at 30℃, 180rpm overnight as seed solution. Inoculate 500μL of the seed solution into a 50mL shake flask LLB (kan resistance), add 500μL L-Ara mother solution, and culture it at 30℃, 180rpm for 3-4h until the OD reaches 0. 600=0.6. Precool the shake flask and centrifuge tube in an ice bath for 10 minutes, pour the bacterial solution into the centrifuge tube in the clean bench, and centrifuge at 4°C, 6000rpm for 5 minutes. Discard the supernatant in the clean bench and add 40mL of precooled sterile ultrapure water. Gently resuspend the cells in an ice water bath, centrifuge at 4°C, 6000rpm for 6 minutes, and repeat once. Discard the supernatant in the clean bench (pay attention to the loss of bacteria), add 20mL of precooled 10% glycerol to a centrifuge tube, and resuspend in an ice water bath. Centrifuge at 4°C, 6000rpm for 5 minutes. Discard the supernatant in the clean bench (pay attention to the loss of bacteria), add 1mL of precooled 10% glycerol, resuspend in an ice water bath, and dispense 100μL into sterile 2mL EP tubes in each tube, keep one tube for use, and store the rest at -80°C.

[0061] The electroporation process is as follows: Take an electroporation competent cell and place it in an ice bath. Add 2μL of pTarget-ΔefeU vector and 2μL of knock-in frame PCR purification product in the clean bench, mix gently with a medium gun, and place on ice for 1 minute. Use a medium gun to transfer to a pre-cooled 2mm electroporation cup and place on ice for 1 minute. Use a paper towel to wipe off the water mist on the outside of the electroporation cup, place it in the electroporator, and use ECO 2 for electroporation. In the clean bench, add 1mL of pre-cooled LLB culture medium to the groove of the electroporation cup, tilt the electroporation cup, absorb all the bacterial solution from the mouth of the electroporation cup, and transfer it to a 2mL sterile ep tube. Resuscitate at 30℃, 180rpm for 3h. Take 400μL and spread it on LLB solid culture medium (SD+kan resistance) and culture it at 30℃ overnight.

[0062] Pick a single colony as a template and perform colony PCR verification to confirm rbcLS se and prk se Knock-in of the gene. The strain confirmed by this was cultured in LB medium containing 50 mg / L of kanamycin and 5 mM IPTG at 30°C overnight to remove the pTarget-ΔefeU vector. The strain from which the pTarget-ΔefeU vector had been removed was then cultured in LB medium at 37°C overnight to remove the pCas vector. This resulted in strain OAHS2. (3) Weakness of the TCA pathway

[0063] Weakening the TCA cycle can not only reduce the extra consumption of acetyl-CoA, provide more acetyl-CoA for the synthesis of O-acetyl-L-homoserine, but also strengthen the L-homoserine synthesis pathway. The key to entering the TCA cycle is citrate synthase, whose encoding gene is gltA. Direct knockout will directly affect growth, so using the dynamic regulation promoter PfliC is a feasible method. The CRISPR-Cas9 system is used to replace the gltA promoter with PfliC, see Jiang Y, Chen B, Duan C, et al. Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System [J]. Applied & Environmental Microbiology, 2015, 81 (7): 2506.

[0064] The pTargetF vector (plasmid from Addgene) was diluted and used as a template. Primers 33 and 34 were used to construct the pTarget-ΔgltA mutant vector that can express sgRNA targeting the non-coding region of the target gene gltA by PCR. The PCR reaction conditions are as follows: 95℃5min; 95℃30s, 55℃30s, 72℃1min, 30 cycles; 72℃5min. The PCR product was transformed into E.coli DH5α competent cells and screened on a plate with spectinomycin hydrochloride (SD) resistance to obtain the pTarget-ΔgltA vector with correct sequencing.

[0065] Subsequently, primers 35 and 36 were used to amplify the upstream homologous sequence of gltA. The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 5 min. In the same way, primers 37 and 38 were used to amplify the downstream homologous sequence of gltA. The PCR product was purified using a purification kit.

[0066] According to the fliC gene of Escherichia coli published on NCBI, the promoter PfliC was amplified using primers 39 and 40. The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 5 min. The PCR product was purified using a purification kit.

[0067] According to the fliC gene of Escherichia coli published on NCBI, the promoter PfliC was amplified using primers 39 and 40. The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 5 min. The PCR product was purified using a purification kit.

[0068] The process of preparing competent cells by chemical transformation is as follows: pick a single colony of OAHS2 and inoculate it into a test tube containing 5 mL of LB medium, and culture it overnight at 37°C and 200 rpm in a constant temperature shaker. Pipette 1 mL of the overnight culture solution and transfer it to a 250 mL conical flask containing 40 mL of LB medium, and culture it at 37°C and 200 rpm in a constant temperature shaker for 1-2 hours until the OD value of the bacterial solution reaches 0. 600 The pH value is between 0.4 and 0.6. Place the cultured bacterial solution on ice to cool for about 10 minutes, pour into a 50mL centrifuge tube, centrifuge at 4°C, 5500rpm for 5 minutes, remove the supernatant and retain the bacteria in a clean bench, add about 40mL of pre-sterilized and pre-cooled 0.1M CaCl2 solution, resuspend in an ice-water bath, and ice-bath for 30 minutes. After centrifugation at 6000rpm for 5 minutes, add 1mL of pre-cooled 0.1M CaCl2 and 15% glycerol, resuspend in an ice-water bath, and dispense into 1.5mL sterile centrifuge tubes (100μL per tube), keep one tube for use, and store the rest at -80°C.

[0069] The process of chemical transformation of pCas plasmid is as follows: take a competent cell and place it on ice, add 1 μL of plasmid to the competent cell in a clean bench, mix well and then place it in an ice bath for 30 minutes. Then place the EP tube containing competent cells in a 42°C constant temperature water bath for 90 seconds, and quickly insert it into ice to cool for 5 minutes. Add 700 μL of pre-cooled LB culture medium to the EP tube in the clean bench, and culture it in a constant temperature shaker at 37°C and 200rpm for 1-2 hours. Pipette 200 μL of bacterial solution and spread it on the LB solid plate of the corresponding resistance, invert it in a 37°C constant temperature incubator and culture it overnight to obtain the OAHS2 strain containing the pCas vector.

[0070] The electroporation competent preparation process is as follows: pick a single colony from the OAHS2 pCas streak plate, inoculate it into a 5mL LLB tube (kan resistance), add 100μL L-Ara mother solution, and culture it at 30℃, 180rpm overnight as seed solution. Inoculate 500μL of the seed solution into a 50mL shake flask LLB (kan resistance), add 500μL L-Ara mother solution, and culture it at 30℃, 180rpm for 3-4h until the OD reaches 0. 600=0.6. Precool the shake flask and centrifuge tube in an ice bath for 10 minutes, pour the bacterial solution into the centrifuge tube in the clean bench, and centrifuge at 4°C, 6000rpm for 5 minutes. Discard the supernatant in the clean bench and add 40mL of precooled sterile ultrapure water. Gently resuspend the cells in an ice water bath, centrifuge at 4°C, 6000rpm for 6 minutes, and repeat once. Discard the supernatant in the clean bench (pay attention to the loss of bacteria), add 20mL of precooled 10% glycerol to a centrifuge tube, and resuspend in an ice water bath. Centrifuge at 4°C, 6000rpm for 5 minutes. Discard the supernatant in the clean bench (pay attention to the loss of bacteria), add 1mL of precooled 10% glycerol, resuspend in an ice water bath, and dispense 100μL into sterile 2mL EP tubes in each tube, keep one tube for use, and store the rest at -80°C.

[0071] The electroporation process is as follows: Take an electroporation competent cell and place it in an ice bath. Add 2μL of pTarget-ΔgltA vector and 2μL of knock-in frame PCR purification product in the clean bench, mix gently with a medium gun, and place on ice for 1 minute. Use a medium gun to transfer to a pre-cooled 2mm electroporation cup and place on ice for 1 minute. Use a paper towel to wipe off the water mist on the outside of the electroporation cup, place it in the electroporator, and use ECO 2 for electroporation. In the clean bench, add 1mL of pre-cooled LLB culture medium to the groove of the electroporation cup, tilt the electroporation cup, absorb all the bacterial solution from the mouth of the electroporation cup, and transfer it to a 2mL sterile ep tube. Resuscitate at 30℃, 180rpm for 3h. Take 400μL and spread it on LLB solid culture medium (SD+kan resistance) and culture it at 30℃ overnight.

[0072] A single colony was picked as a template, and colony PCR verification was performed and sent for testing to confirm the replacement of the PfliC promoter. The strain that passed this confirmation was cultured overnight at 30°C in LB medium containing 50 mg / L of kanamycin and 5 mM IPTG to remove the pTarget-ΔgltA vector. The strain from which the pTarget-ΔgltA vector had been removed was then cultured overnight in LB medium at 37°C to remove the pCas vector. This resulted in strain OAHS3. Table 1 Primer list Example 2 Shake flask fermentation experiment

[0073] The above-mentioned bacteria were tested in a fermentation experiment in a shake flask to compare the ability of each genotype strain to produce O-acetyl-L-homoserine. The shake flask fermentation experiment was carried out according to the following scheme: each strain was streaked on an LB plate, cultured in a 37°C incubator overnight, a single colony was picked and inoculated into 5 ml of LB medium, and cultured in a 37°C incubator overnight at a speed of 200 rpm to obtain a seed solution.

[0074] Add 50 ml of fermentation medium to a 500 ml shake flask, and inoculate 1 ml of seed solution of each strain into the fermentation medium. The final concentration of the fermentation medium is as follows: glucose 30 g / L, potassium dihydrogen phosphate 2 g / L, ammonium sulfate 20 g / L, yeast powder 4 g / L, calcium carbonate 20 g / L, L-threonine 0.20 g / L, vitamin B1 0.0001 g / L, vitamin B12 0.0001 g / L, L-isoleucine 0.2 g / L, MgSO4 2 g / L, FeSO4 0.005 g / L, MnSO4 0.005 g / L, ZnSO4 0.005 g / L, the solvent is deionized water, and the pH value is 6.8. Cultivate at 200 rpm in an incubator at 30°C for 48 hours, take a certain amount of fermentation liquid from the shake flask, dilute it 100 times with ultrapure water, filter it through a membrane, and use an amino acid analyzer to detect the content of O-acetyl-L-homoserine in the fermentation liquid. Finally, compare the amount of O-acetyl-L-homoserine obtained by strains of different genotypes. The results of the shake flask fermentation experiment of the metabolically modified strains are shown in Figure 2. Figure 1 shown.

[0075] according to Figure 1 It can be seen that the metabolically modified OAHS3 strain has the ability to produce and accumulate O-acetyl-L-homoserine extracellularly, and can better utilize carbon sources such as glucose to produce O-acetyl-L-homoserine compared to the wild-type and chassis strains. The strain with the best performance after modification increased the level of O-acetyl-L-homoserine fermentation from 12.66g / L to 17.23g / L compared to the starting strain, an increase of 36.1%. The levels of O-acetyl-L-homoserine production of other modified strains have been improved to varying degrees compared to the original strains.

Claims

1. A method for constructing a genetically engineered bacterium for efficiently producing O-acetyl-L-homoserine, characterized in that: The specific steps include: (1) Using the strain Escherichia coli W3110 as the chassis, the metA gene encoding homoserine O-succinyltransferase was knocked out; (2) Knockout of metB, the gene encoding γ-cystathionine synthase; (3) Knockout of metC, the gene encoding cysteine ​​cleavage enzyme; (4) Weakly expressed metYcm, the gene encoding O-acetyl-L-homoserine thiolase from Cyclobacter maritima; (5) Overexpression of the ribulose bisphosphate carboxylase encoding gene rbcLSse from Synechococcus elongatus; (6) Overexpression of the phospholipid kinase encoding gene prkse from Synechococcus elongatus; (7) Weakening the expression of the citrate synthase encoding gene gltA to obtain the genetically engineered bacteria that efficiently produces O-acetyl-L-homoserine.

2. The method for constructing a genetically engineered bacterium for efficiently producing O-acetyl-L-homoserine according to claim 1, characterized in that: In step 5, rbcLS se 、Step 6 From prk se A gene has one more copy added to the genome to enhance its expression; The rbcLS se 、prk se The gene is expressed from a strong promoter.

3. The method for constructing a genetically engineered bacterium for efficiently producing O-acetyl-L-homoserine according to claim 2, characterized in that: The strong promoter in step 5 is Ptrc, and its nucleotide sequence is SEQ ID NO.

1.

4. The method for constructing a genetically engineered bacterium for efficiently producing O-acetyl-L-homoserine according to claim 1, characterized in that: metY in step 4 cm The weakened expression of is achieved by using a dynamic regulation promoter, and the weakened expression of gltA in step 7 is achieved by replacing the original promoter with a dynamic regulation promoter.

5. The method for constructing a genetically engineered bacterium for efficiently producing O-acetyl-L-homoserine according to claim 4, characterized in that: The dynamic regulation promoter is PfliC, and its nucleotide sequence is SEQ ID NO.

2.

6. The method for constructing a genetically engineered bacterium for efficiently producing O-acetyl-L-homoserine according to claim 1, characterized in that: The metY cm Integrated into the metA gene locus, the rbcLS se and prk se Integrated into the efeU gene locus.

7. The method for constructing a genetically engineered bacterium for efficiently producing O-acetyl-L-homoserine according to claim 6, characterized in that: The metY cm From Cyclobacter maritima, the rbcLS se and prk se From Synechococcus elongatus.

8. A genetically engineered bacterium for efficiently producing O-acetyl-L-homoserine, characterized in that: It is prepared by the method described in any one of claims 1 to 7.

9. Use of the genetically engineered bacteria for efficiently producing O-acetyl-L-homoserine according to claim 8 in fermentation for producing O-acetyl-L-homoserine.

10. The use according to claim 9, characterized in that The application is to inoculate the genetically engineered bacteria into a fermentation medium, ferment and culture at 30-33°C and 180-220rpm, separate and purify the culture solution to obtain O-acetyl-L-homoserine.

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