Genetically engineered bacterium of high-yield O-succinyl-L-homoserine as well as construction method and application of genetically engineered bacterium

Abstract

The invention belongs to the field of synthetic biology, and particularly relates to a genetically engineered bacterium for producing O-succinyl-L-homoserine at high yield as well as a construction method and application of the genetically engineered bacterium. According to the invention, the large intestine probiotic Nissle 1917 is used as a chassis strain, and compared with other expression hosts, the large intestine probiotic Nissle 1917 can grow and breed more quickly and is safer. The expression of the gene thrB is regulated by using the self-regulation promoter PfliA, so that the synthesis route of threonine is dynamically regulated, the efficient utilization of a carbon source is realized, and the sugar acid conversion rate of OSH synthesis is improved; in addition, by introducing site mutation, feedback inhibition suffered by OSH synthesis key enzyme HST is relieved, the enzyme activity of consumption pathway key enzyme CGS is reduced, and the carbon flow is effectively driven to be redirected to a target product synthesis pathway. According to the microbial cell factory constructed by the invention, the production efficiency of OSH is remarkably improved, the fermentation period is shortened, the production cost is reduced, and the economic feasibility of industrial production is realized.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology, and particularly relates to a genetically engineered bacterium with high yield of O-succinyl-L-homoserine, a construction method thereof, and an application thereof. Background Art

[0002] O-Succinyl-L-homoserine (OSH) is a naturally occurring non-protein amino acid derivative, which is widely involved in microbial metabolic pathways and is a precursor for the synthesis of L-methionine in most bacteria. At the same time, OSH is also an intermediate for the synthesis of the herbicide L-glufosinate and various C4 compounds (such as 1,4-butanediol, succinic acid). Therefore, it shows great application potential in many fields such as agriculture, medicine, and food.

[0003] At present, the production methods of OSH mainly include chemical synthesis method and biosynthesis method. However, the chemical method has problems such as low stereoselectivity, high production cost, harsh reaction conditions, complex subsequent separation and purification process, and environmental pollution, and is not suitable for large-scale production. The biosynthesis method uses cheap and environmentally friendly biomass as raw materials, has mild reaction conditions, is green and environmentally friendly, and has specific stereoselectivity, avoiding the problem of isomer separation. It is superior to the traditional chemical synthesis method in terms of efficiency, sustainability, and process controllability, providing a better solution for the industrial production of OSH. However, there are also some deficiencies in the process of synthesizing OSH by biological methods in the prior art, such as unsatisfactory fermentation yield and long production cycle, resulting in difficulty in industrial production. For example, an engineered Escherichia coli for producing O-succinyl-L-homoserine, a construction method thereof, and an application thereof disclosed in Patent CN 118006519A knock out or weaken the negative regulatory repressor gene (metJ), O-succinyl-L-homoserine lyase gene (metB), and homoserine kinase gene (thrB) in the methionine synthesis pathway of Escherichia coli, and at the same time knock out or weaken at least one of the cystathionine-β-lyase gene (metC), methionine synthase gene (metH), or homocysteine methyltransferase gene (metE), so that the transformed Escherichia coli can produce O-succinyl-L-homoserine. However, the engineered Escherichia coli of this technical solution has a low yield, and the required culture medium contains antibiotics such as chloramphenicol and kanamycin, increasing the cost and not meeting the environmental protection requirements of industrial production. Summary of the Invention

[0004] The present invention aims to overcome the defects in the prior art that the yield of OSH synthesized by biological methods is low, there are many essential amino acids in the fermentation process, and the production cost is high, and provides a genetically engineered bacterium with high yield of O-succinyl-L-homoserine, a construction method thereof, and an application thereof to overcome the above defects.

[0005] To achieve the object of the above invention, the present invention is realized through the following technical solutions: In a first aspect, the present invention discloses a genetically engineered bacterium with high yield of O-succinyl-L-homoserine. The genetically engineered bacterium uses the wild-type Escherichia coli probiotic EcN as the chassis strain, knocks out the genes metI and metJ, dynamically regulates the gene thrB, overexpresses the genes metA and metL, and weakens the gene metB; Introduces the hom gene from Corynebacterium glutamicum to replace the start codon of the gene lysA; Introduces the high-copy plasmid pTrc99A, thereby combinatorially regulating the RBS strength of the aspA gene and the aspC gene, and finally obtaining a genetically engineered bacterium with high yield of O-succinyl-L-homoserine.

[0006] Escherichia coli Nissle 1917 (EcN) is the only beneficial Escherichia coli discovered so far, does not carry any pathogenic factors, and has better performance in terms of growth and tolerance to low iron concentration. In the previous laboratory research, usually directly deleting the key genes encoding by-product amino acids on the genome results in the need to additionally add essential amino acids during the culture fermentation process of the strain, which not only increases the fermentation cost but also greatly increases the operation complexity.

[0007] Therefore, in order to optimize the culture medium components and reduce the operation complexity, the present invention uses the self-regulating promoter P fliA to regulate the expression of the gene thrB, thereby dynamically regulating the threonine synthesis pathway, realizing the efficient utilization of carbon sources, and increasing the sugar-acid conversion rate of OSH synthesis. On this basis, by introducing site mutations to relieve the feedback inhibition of the key enzyme HST for OSH synthesis and reduce the enzyme activity of the key enzyme CGS for the consumption pathway, effectively driving the redirection of carbon flux to the target product synthesis pathway. When using the subsequent recombinant genetically engineered bacterium OSHY3 of the present invention to produce OSH by fermentation method, there is no need to externally add essential amino acids, reducing the fermentation cost, solving the uncertainty of the addition timing of essential amino acids during the fermentation process, and reducing the operation difficulty of fermentation regulation.

[0008] In the present invention, EcN is used as the starting strain. First, the key gene metI of the methionine uptake system is knocked out, the key gene thrB for dynamically regulating the synthesis of threonine is knocked out, the key gene metA for overexpressing OSH synthesis is overexpressed, the key gene metB for weakening the OSH degradation pathway is weakened, and the gene metJ encoding the negative transcriptional regulatory protein is knocked out; then, the gene metL encoding homoserine dehydrogenase is overexpressed, the hom gene from Corynebacterium glutamicum is introduced, and the start codon of the key gene lysA for synthesizing lysine is replaced; at the same time, the high-copy plasmid pTrc99A is introduced, and the RBS strengths of the aspA gene and the aspC gene are combinatorially regulated so that their expression ratio is 1000:4000 (AU), and the carbon flux distribution for synthesizing L-aspartic acid is balanced. In addition, in the present invention, the natural promoter of the gene metL encoding homoserine dehydrogenase is replaced with the strong promoter P trc , and the hom gene from Corynebacterium glutamicum that relieves feedback inhibition is introduced, which has the same function as the thrA gene in Escherichia coli and can promote the synthesis of the important precursor L-homoserine. By replacing the original start codon of the lysA gene with the relatively weak GTG start codon, the protein expression is weakened at the translational level, and the metabolic flux of the lysine pathway is effectively inhibited.

[0009] Furthermore, a copy of the ppc gene is added to the plasmid of the genetically engineered bacterium. By adding a copy of the ppc gene to the plasmid, the supply of oxaloacetic acid is enhanced.

[0010] Furthermore, a copy of the gene pntAB is added to the plasmid of the genetically engineered bacterium. By adding a copy of the gene pntAB encoding pyridine nucleotide transhydrogenase to the plasmid, the intracellular NADPH level is increased, thereby providing sufficient reducing power for the biosynthesis reaction.

[0011] Furthermore, the genetically engineered bacterium dynamically regulates the gene thrB by introducing the self-regulating promoter P fliA . After knocking out the key gene metI of the methionine uptake system in the present invention, the self-regulating promoter P fliA is introduced to dynamically regulate the expression of the gene thrB encoding homoserine kinase, which is the key enzyme for synthesizing the by-product amino acid L-threonine. In the present invention, by using the self-regulating promoters P fliA , P fliC and P flgC or replacing the start codon with a weaker translation efficiency (such as the GTG codon) to replace static knockout, the phenomenon of slow growth of the strain caused by the lack of essential amino acids is avoided.

[0012] Furthermore, the genetically engineered bacterium overexpresses the gene metL through the strong promoter P trc .

[0013] Furthermore, double-site mutations Q64E and T242A were introduced into the genetically engineered bacterium. By introducing the double-site mutations Q64E and T242A, the feedback inhibition of homoserine succinyltransferase (HST), the key enzyme for synthesizing OSH, was relieved, and its overexpression was carried out.

[0014] Furthermore, the genetically engineered bacterium weakened the gene metB by introducing a single-site mutation N148A. By introducing the single-site mutation N148A, cystathionine-γ-synthase (CGS), the key enzyme for catalyzing the degradation of OSH, retained 50% of its original enzyme activity, reducing the consumption of the target product to a certain extent. At the same time, the gene metJ encoding the negative transcriptional regulatory protein was knocked out to relieve its repression on the key enzymes in the L-homoserine synthesis pathway.

[0015] In a second aspect, the present invention also discloses a method for constructing a genetically engineered bacterium with high yield of O-succinyl-L-homoserine, comprising the following steps: S1. Knock out the key gene metI for methionine absorption, and insert the dynamic regulatory promoter P before the gene thrB encoding homoserine kinase, denoted as strain OSHY1; fliA and denote it as strain OSHY1. S2. Regulate the expression of the gene metA encoding homoserine succinyltransferase on the genome of strain OSHY1 with the strong promoter P trc , and introduce double-site mutations Q64E and T242A, denoted as strain OSHY2; S3. Introduce a single-site mutation N148A into cystathionine-γ-synthase (CGS) on the genome of strain OSHY2, and at the same time knock out the gene metJ encoding the negative transcriptional regulatory protein, denoted as strain OSHY3; S4. Regulate the overexpression of the gene metL encoding homoserine dehydrogenase on the genome of strain OSHY3 with the strong promoter P trc , and insert the hom gene that relieves feedback inhibition from Corynebacterium glutamicum at the rph gene locus, denoted as strain OSHY4; S5. Replace the start codon of the gene lysA encoding diaminopimelate decarboxylase from ATG with GTG with relatively weak translation initiation efficiency on the genome of strain OSHY4, denoted as strain OSHY5; S6. Introduce the high-copy plasmid pTrc99A, and combinatorially regulate the RBS strength of the gene aspA encoding aspartase I and the gene aspC encoding aspartate aminotransferase so that their expression ratio is 1000:4000 (AU), denoted as strain OSHY6; S7. Increase a copy of the gene ppc encoding phosphoenolpyruvate carboxylase on the pTrc99A plasmid of strain OSHY6, denoted as strain OSHY7; S8. Add a copy of the pyridine nucleotide transhydrogenase encoding gene pntAB to the pTrc99A plasmid of strain OSHY7, denoted as strain OSHY8, which is the genetically engineered bacterium for high-yield production of O-succinyl-L-homoserine.

[0016] The nucleotide sequence of the metI gene is as shown in SEQ ID NO.1, the nucleotide sequence of the thrB gene is as shown in SEQ ID NO.2, the nucleotide sequence of the P fliA promoter is as shown in SEQ ID NO.3, the nucleotide sequence of the P trc promoter is as shown in SEQ ID NO.4, the nucleotide sequence of the metA gene with double-site mutation introduced is as shown in SEQ ID NO.5, the nucleotide sequence of the metB gene with single-site mutation introduced is as shown in SEQ ID NO.6, the nucleotide sequence of the metJ gene is as shown in SEQ ID NO.7, the nucleotide sequence of the metL gene is as shown in SEQ ID NO.8, the nucleotide sequence of the rph gene is as shown in SEQ ID NO.9, the nucleotide sequence of the exogenous hom gene is as shown in SEQ ID NO.10, the nucleotide sequence of the lysA gene is as shown in SEQ ID NO.11, the nucleotide sequence of the aspA gene is as shown in SEQ ID NO.12, the RBS nucleotide sequence of the aspA gene is as shown in SEQ ID NO.13, the nucleotide sequence of the aspC gene is as shown in SEQ ID NO.14, the RBS nucleotide sequence of the aspC gene is as shown in SEQ ID NO.15, the nucleotide sequence of the ppc gene is as shown in SEQ ID NO.16, and the nucleotide sequence of the pntAB gene is as shown in SEQ ID NO.17.

[0017] In the third aspect, the present invention also discloses the application of a genetically engineered bacterium for high-yield production of O-succinyl-L-homoserine in the microbial fermentation production of OSH.

[0018] In the fourth aspect, the present invention also discloses a method for producing O-succinyl-L-homoserine, comprising the following steps: S1. Streak-culture the genetically engineered bacterium for high-yield production of O-succinyl-L-homoserine according to any one of claims 1-7; S2. Pick a single colony on the plate and inoculate it into an LB liquid medium containing the corresponding resistance for culture to obtain a seed solution; S3. Inoculate the seed solution into a shake-flask fermentation medium, and add Kan resistance and IPTG inducer for fermentation culture to obtain a fermentation broth containing OSH.

[0019] Therefore, the present invention has the following beneficial effects: (1) The present invention uses the colonic probiotic Nissle 1917 as the chassis strain, which grows and reproduces more rapidly and safely compared to other expression hosts; (2) The present invention uses the self-regulating promoter P fliA to regulate the expression of the gene thrB, thereby dynamically regulating the threonine synthesis pathway, achieving efficient utilization of carbon sources, and increasing the sugar-acid conversion rate of OSH synthesis; (3) The present invention introduces site mutations to relieve the feedback inhibition of the key enzyme HST for OSH synthesis and reduce the enzyme activity of the key enzyme CGS in the consumption pathway, effectively driving the redirection of carbon flux to the target product synthesis pathway; (4) The strain OSHY8 obtained in the present invention has a yield of 13.44 g / L in shake flask fermentation. The strain OSHY8 is subjected to fed-batch fermentation in a 5 L fermenter. When the fermentation is carried out for 44 h, the product concentration reaches 80.79 g / L, the sugar-acid conversion rate reaches 0.47 g / g glucose, and the space-time yield reaches 1.84 g / L / h, and the OSH yield is significantly improved; (5) The microbial cell factory constructed in the present invention significantly improves the production efficiency of OSH, shortens the fermentation cycle, reduces the production cost, and has economic feasibility for industrial production. Description of the Drawings

[0020] Figure 1 is a column chart of the biomass OD 600 and yield of strains OSHY1 - OSHY8.

[0021] Figure 2 is a concentration curve chart of the biomass OD 600 and yield of strain OSHY8 in fed-batch fermentation in a 5 L fermenter. Detailed Embodiments

[0022] The present invention will be 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 descriptions are usually only a part of the embodiments of the present invention, rather than all the embodiments. Therefore, all other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention shall fall within the scope of protection of the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all conventional biochemical reagents unless otherwise specified.

[0023] Composition of LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the solvent is deionized water. For solid medium, agar with a final concentration of 20 g / L needs to be added. The chassis bacterium of the present invention is wild-type Escherichia probiotic Nissle 1917 (EPA-, DHA-bacteria).

[0024] The genes involved in gene editing and their corresponding pathways are shown in Table 1: Table 1: Genes involved in strain modification and their corresponding pathways

[0025] The primer sequences used are shown in Table 2: Table 2: Primer sequences

[0026] The determination method of OSH concentration in the following specific embodiments is as follows: Sample treatment: Dilute the sample concentration to between 0.1 - 1.0 g / L with ultrapure water, remove impurities using a 0.22 μm aqueous filter membrane, and then aliquot into the liner tube of an Agilent liquid phase bottle; Standard sample dilution gradient: 0.1 g / L, 0.2 g / L, 0.25 g / L, 0.5 g / L, 1.0 g / L, 2.0 g / L; Derivatization reagent OPA: Take 250 mg of o-phthalaldehyde in a beaker, add 5 mL of absolute ethanol and 100 μL of β-mercaptoethanol in sequence, stir well to completely dissolve o-phthalaldehyde, and then make up the volume to 25 mL with 0.4 mol / L boric acid buffer (pH = 9.5), and store in the dark for later use; Mobile phase A: Weigh a certain amount of sodium acetate solid, prepare a 0.02 mol / L buffer solution with a volume of 1 L, add 180 μL of triethylamine, adjust the pH to 7.20 ± 0.05 with 10% acetic acid solution, then add 3 mL of tetrahydrofuran, mix well, filter through a membrane and ultrasonicate for later use; Mobile phase B: Prepare a 1 L solution by mixing 0.02 mol / L sodium acetate buffer solution, methanol, and acetonitrile in a volume ratio of 1:2:2, adjust the pH to 7.20 ± 0.05 with 10% acetic acid solution, then filter through a membrane and ultrasonicate for later use; Detection method: HPLC model: Thermo Scientific Utimate 3000, the HPLC detection wavelength is 338 nm. The gradient elution program is used to separate OSH. The derivatization reagent OPA and boric acid-borax buffer need to be filtered through a 0.22 μm organic filter membrane to remove impurities before the on-line derivatization reaction can be carried out.

[0027] Example 1: Construction of OSHY1: Knock out the key gene metI of the methionine uptake system on the chassis bacterium, which greatly reduces the uptake efficiency of extracellular methionine by Escherichia coli, thus avoiding the cytotoxicity caused by excessive accumulation of methionine; on this basis, in order to reduce production costs and operation complexity, the self-regulating promoter P fliA dynamically regulates the key gene thrB encoding threonine synthesis, enabling more carbon flux to flow towards the synthesis of the target product.

[0028] Knock out the metI gene in the chassis bacterium genome through the CRISPR / Cas9 system. The specific operations are as follows: (1) Ligation fragment U metI -D metI Using the Escherichia coli EcN genome as a template, the upstream homologous arm and downstream homologous arm of the metI gene are amplified using primers metI-P1 and metI-P2, metI-P3 and metI-P4 respectively. After the PCR products are detected by 1.0% agarose gel electrophoresis, they are treated with DpnI at 37 °C for 1 h. Finally, the purified fragments are recovered and purified using a Clean Up kit. The purified fragments are used to obtain the ligation fragment U metI -D metI by fusion PCR with primers metI-P1 and metI-P4; the PCR reaction conditions are as follows: 95 °C for 5 min; 95 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s, repeat 35 cycles; continue to extend at 72 °C for 10 min. (2) Construction of the pTarget-metI-sg vector Using the pTarget plasmid as a template, and using metI-N20-F and metI-N20-R as primers, amplify the sgRNA capable of expressing the targeted metI gene, construct the pTarget-metI-sg mutant vector, and then linearize the pTarget-metI plasmid with primers pTarget-line-F and pTarget-line-R; the PCR reaction conditions are as follows: 95 °C for 5 min; 95 °C for 30 s, 58 °C for 30 s, 72 °C for 2.5 min, repeat 35 cycles; continue to extend at 72 °C for 10 min. (3) Construction of the pTarget-metI plasmid Connect the linker fragment U in step (1) metI -D metI and the linearized mutant vector pTarget-metI-sg in step (2) for one-step cloning. The reaction program is: 30 min at 37 °C. Transform the cloning product into E. coli DH5α recipient bacteria, coat it on an LB solid plate containing 50 mg / L spectinomycin dihydrochloride (SD) resistance, and culture it at 37 °C for 12 h. After verification using primers pTarget-seq-F and pTarget-seq-R, pick positive clone colonies and transfer them to an LB liquid medium containing 50 mg / L SD resistance, culture at 37 °C for 12 h, and obtain pTarget-metI plasmid using a plasmid extraction kit. (4) Knockout of metI gene Using EcN as the starting strain, knockout the metI gene using the CRISPR / Cas9 gene editing technology. Electrotransform the pTarget-metI plasmid into the EcN strain containing the pCas9 vector and operate according to the following steps: 1) Inoculate the EcN strain transformed with the pCas9 vector into 10 mL of LB liquid medium, add 50 mg / L Kan resistance and 10 mM L-arabinose (L-Ara), and culture overnight at 30 °C and 180 rpm as the seed solution. Pipette 1 mL of the seed solution from the test tube into a 250 mL Erlenmeyer flask containing 50 mL of LB medium, and culture at 30 °C and 180 rpm until the OD 600 value is about 0.6. Transfer the bacterial solution from the Erlenmeyer flask to a pre-chilled 50 mL sterile centrifuge tube, centrifuge at 4 °C and 5000 rpm for 5 min, and discard the supernatant. Add 40 mL of pre-chilled sterile water, gently pipette the bacteria to suspend them, centrifuge at 4 °C and 5000 rpm for 5 min, and discard the supernatant. Repeat the previous step. Add 40 mL of pre-chilled 10% (v:v) glycerol solution, gently pipette the bacteria to suspend them, centrifuge at 4 °C and 5000 rpm for 5 min, and discard the supernatant. Add 1 mL of pre-chilled 10% (v:v) glycerol solution, gently pipette to suspend the bacteria. 2) Place the prepared electrocompetent cells on ice. After they thaw, add 5 μL of plasmid. Transfer the competent cells mixed with the plasmid into a pre-chilled 2 mm electroporation cuvette. Set the operating parameters of the electroporator to 2500 V, 25 μF, and 200 Ω for electroporation. Immediately add 800 mL of LB medium and culture at 30 °C and 180 rpm for 3 h. Spread the electrotransformed bacterial solution onto an LB plate containing 50 mg / L Kan and 50 mg / L SD resistance and culture at 30 °C for 24 h. Pick a single colony as a template and perform PCR with primers metI-YZ-F and metI-YZ-R. The PCR product shows a 1200 bp DNA band in a 1.0% agarose gel, confirming the successful acquisition of a positive clone strain with the metI gene knocked out. 3) Pick the verified correct strain into an LB medium containing 50 mg / L Kan and 5 mM IPTG (isopropyl β-D-thiogalactopyranoside) and culture at 30 °C for 12 h to eliminate the pTarget plasmid. Pick a single colony that has successfully verified the elimination of the pTarget plasmid into a test tube containing 10 mL of LB medium and culture overnight at 42 °C to eliminate the pCas plasmid (the pCas plasmid is temperature-sensitive and is easily lost when cultured above 37 °C); finally, obtain a plasmid-free strain.

[0029] Introduce the self-regulating promoter P into the genome of the previous strain through the CRISPR / Cas9 system fliA to dynamically regulate the thrB gene. The specific operation is as follows: (1) Ligate fragment U thrB -P fliA -D thrB Using the Escherichia coli EcN genome as a template, amplify the upstream homologous arm and downstream homologous arm of the target site with primers thrB-P1 and thrB-P2, thrB-P5 and thrB-P6 respectively, and amplify the gene fragment of promoter P with primers thrB-P3 and thrB-P4. Detect the PCR products by 1.0% agarose gel electrophoresis, then treat with DpnI at 37 °C for 1 h, and finally recover and purify the fragments using a Clean Up kit. Use primers thrB-P1 and thrB-P6 to obtain the ligation fragment U fliA -P thrB -P fliA -D thrB by fusion PCR; the PCR reaction conditions are as follows: 95 °C for 5 min; 95 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s, repeat 35 cycles; continue to extend at 72 °C for 10 min. (2) Construct the pTarget-thrB-sg vector Using the pTarget plasmid as a template and thrB-N20-F and thrB-N20-R as primers, amplify the sgRNA capable of expressing the targeted gene thrB, construct the pTarget-thrB-sg mutant vector, and then linearize the pTarget-thrB plasmid using pTarget-line-F and pTarget-line-R as primers; the PCR reaction conditions are as follows: 95°C for 5 min; 95°C for 30 s, 58°C for 30 s, 72°C for 2.5 min, repeat 35 cycles; continue to extend at 72°C for 10 min. (3) Construct pTarget-P fliA Plasmid Ligate the ligation fragment U thrB -P fliA -D thrB from step (1) and the linearized mutant vector pTarget-thrB-sg from step (2) by one-step cloning. The reaction program is: 37°C for 30 min; transform the cloning product into the E. coli DH5α recipient bacteria, spread on an LB solid plate containing 50 mg / L SD resistance, and culture at 37°C for 12 h. After verification using primers pTarget-seq-F and pTarget-seq-R, pick the positive clone colonies and transfer them to an LB liquid medium containing 50 mg / L SD resistance, culture at 37°C for 12 h, and obtain the pTarget-P fliA plasmid using a plasmid extraction kit. (4) Construct strain OSHY1 Insert the self-regulating promoter P fliA in front of the thrB gene using the CRISPR / Cas9 gene editing technology; electrotransform the pTarget-P fliA plasmid into the above-mentioned strain containing the pCas9 vector, and operate according to the following steps: 1) Inoculate the strain transformed with the pCas9 vector in 10 mL of LB liquid medium, add 50 mg / L of Kan resistance and 10 mM of L-Ara, culture overnight at 30°C and 180 rpm as the seed solution; pipette 1 mL of the seed solution from the test tube into a 250 mL flask containing 50 mL of LB medium, culture at 30°C and 180 rpm until OD 600The value is about 0.6; Transfer the bacterial solution from the shaking flask to a pre-cooled 50 mL sterile centrifuge tube, centrifuge at 4°C and 5000 rpm for 5 min, and discard the supernatant; Add 40 mL of pre-cooled sterile water, gently pipette the bacterial cells to suspend them, centrifuge at 4°C and 5000 rpm for 5 min, and discard the supernatant; Repeat the previous step; Add 40 mL of pre-cooled 10% (v:v) glycerol solution, gently pipette the bacterial cells to suspend them, centrifuge at 4°C and 5000 rpm for 5 min, and discard the supernatant; Add 1 mL of pre-cooled 10% (v:v) glycerol solution, gently pipette to suspend the bacterial cells. 2) Place the prepared electrocompetent cells on ice. After they melt, add 5 μL of plasmid. Add the competent cells mixed with the plasmid to a pre-cooled 2 mm electroporation cuvette. Adjust the working parameters of the electroporator to 2500 V, 25 μF, and 200 Ω for electroporation. Immediately add 800 mL of LB medium and culture at 30°C and 180 rpm for 3 h; Spread the electrotransformed bacterial solution onto an LB plate containing 50 mg / L Kan and 50 mg / L SD resistance, and culture at 30°C for 24 h. Pick a single colony as a template and perform PCR with primer thrB-YZ-F and primer thrB-YZ-R. The PCR product shows a 1300 bp DNA band in a 1.0% agarose gel, confirming the successful insertion of P fliA promoter positive clone strain. 3) Pick the verified correct strain into an LB medium containing 50 mg / L Kan and 5 mM IPTG and culture at 30°C for 12 h to eliminate the pTarget plasmid. Pick a single colony that has successfully verified the elimination of the pTarget plasmid into a test tube containing 10 mL of LB medium and culture overnight at 42°C to eliminate the pCas plasmid; Finally, obtain the plasmid-free strain OSHY1.

[0030] Example 2: Construction of OSHY2: Utilize the strong promoter P trc to overexpress the key gene metA for synthesizing OSH, and introduce double-site mutations Q64E and T242A to relieve the feedback inhibition of homoserine succinyltransferase HST.

[0031] Through the CRISPR / Cas9 system, insert 1 copy of the metA gene controlled by the P trc promoter and introduced with double-site mutations into the in-situ site of the metA gene in the genome of strain OSHY1. The specific operation is as follows: (1) Ligation fragment U metA -P trc -metA-D metA Using the Escherichia coli EcN genome as a template, the upstream homologous arm and downstream homologous arm of the metA gene were amplified using primers metA-P1 and metA-P2, and metA-P5 and metA-P6 respectively. The gene fragment of P trc -metA was amplified using primers metA-P3 and metA-P4. After the PCR products were detected by 1.0% agarose gel electrophoresis, they were treated with DpnI at 37°C for 1 h. Finally, the purified fragments were recovered using a Clean Up kit. The purified fragments were used to obtain the ligation fragment U metA -P trc -metA-D metA by fusion PCR using primers metA-P1 and metA-P6; The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 58°C for 30 s, 72°C for 1 min, repeated for 35 cycles; 72°C for continued extension for 10 min. (2) Construction of the pTarget-metA-sg vector Using the pTarget plasmid as a template and metA-N20-F and metA-N20-R as primers, the sgRNA capable of expressing the targeted gene metA was amplified to construct the pTarget-metA-sg mutant vector. Then, the pTarget-metA plasmid was linearized using primers pTarget-line-F and pTarget-line-R; The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 58°C for 30 s, 72°C for 2.5 min, repeated for 35 cycles; 72°C for continued extension for 10 min. (3) Construction of the pTarget-P trc -metA plasmid The ligation fragment U metA -P trc -metA-D metA from step (1) and the linearized mutant vector pTarget-metA-sg from step (2) were subjected to one-step cloning. The reaction program was: 37°C for 30 min; The cloning products were transformed into E. coli DH5α recipient bacteria and spread on an LB solid plate containing 50 mg / L SD resistance at a final concentration. They were cultured at 37°C for 12 h. After verification using primers pTarget-seq-F and pTarget-seq-R, positive clone colonies were picked and transferred to an LB liquid medium containing 50 mg / L SD resistance at a final concentration. They were cultured at 37°C for 12 h, and the pTarget-P trc -metA plasmid was obtained using a plasmid extraction kit. (4) Construction of the pTarget-P trc -metA(Q64E, T242A) plasmid Using pTarget-P in step (3) trc -metA plasmid as a template, perform Q64E site mutation using primer metA-Q64E-F and primer metA-Q64E-R. After detecting the PCR product by 1.0% agarose gel electrophoresis, treat it with DpnI at 37°C for 1 h, and then recover and purify the product pTarget-P trc -metA(Q64E); Using pTarget-P trc -metA(Q64E) plasmid as a template, perform T242A site mutation using primer metA-T242A-F and primer metA-T242A-R. After detecting the PCR product by 1.0% agarose gel electrophoresis, treat it with DpnI at 37°C for 1 h, and finally recover and purify the product pTarget-P trc -metA(Q64E, T242A); The PCR reaction conditions are as follows: 95°C for 5 min; 95°C for 30 s, 58°C for 30 s, 72°C for 3 min, repeat 35 cycles; continue to extend at 72°C for 10 min; Pipette 5 μL of the purified product pTarget-P trc -metA(Q64E, T242A) was transformed into E.coli DH5α recipient bacteria, spread on an LB solid plate containing 50 mg / L SD resistance at a final concentration, and cultured at 37°C for 12 h. After verification using primers pTarget-seq-F and pTarget-seq-R, pick positive clone colonies and transfer them to an LB liquid medium containing 50 mg / L SD resistance at a final concentration, and culture at 37°C for 12 h. Use a plasmid extraction kit to obtain pTarget-P trc -metA(Q64E, T242A) plasmid. (5) Construct strain OSHY2 Use the CRISPR / Cas9 gene editing technology to insert 1 copy of the metA gene controlled by the P trc promoter and introduced with double point mutations; Electrotransform the pTarget-P trc -metA(Q64E, T242A) plasmid into the OSHY1 strain containing the pCas9 vector, and operate according to the following steps: 1) Inoculate the strain transformed with the pCas9 vector in 10 mL of LB liquid medium, and add 50 mg / L of Kan resistance and 10 mM of L-Ara, culture overnight at 30°C, 180 rpm as the seed solution; Pipette 1 mL of the seed solution from the test tube into a 250 mL flask containing 50 mL of LB medium, culture at 30°C, 180 rpm until OD 600The value is about 0.6; Transfer the bacterial liquid from the shaking flask to a pre-cooled 50 mL sterile centrifuge tube, centrifuge at 4 °C and 5000 rpm for 5 min, and discard the supernatant; Add 40 mL of pre-cooled sterile water, gently pipette the bacterial cells to suspend them, centrifuge at 4 °C and 5000 rpm for 5 min, and discard the supernatant; Repeat the previous step; Add 40 mL of pre-cooled 10% (v:v) glycerol solution, gently pipette the bacterial cells to suspend them, centrifuge at 4 °C and 5000 rpm for 5 min, and discard the supernatant; Add 1 mL of pre-cooled 10% (v:v) glycerol solution, gently pipette to suspend the bacterial cells. 2) Place the prepared electrocompetent cells on ice. After they melt, add 5 μL of plasmid. Add the competent cells mixed with the plasmid to a pre-cooled 2 mm electroporation cuvette. Adjust the working parameters of the electroporator to 2500 V, 25 μF, and 200 Ω for electroporation. Quickly add 800 mL of LB medium and culture at 30 °C and 180 rpm for 3 h; Spread the electrotransformed bacterial liquid onto an LB plate containing 50 mg / L Kan and 50 mg / L SD resistance and culture at 30 °C for 24 h. Pick a single colony as a template and perform PCR with primer metA-YZ-F and primer metA-YZ-R. The PCR product shows a 2300 bp DNA band in a 1.0% agarose gel, confirming the successful acquisition of a positive clone strain with an overexpressed metA double mutant gene copy inserted. 3) Pick the verified correct strain into an LB medium containing 50 mg / L Kan and 5 mM IPTG and culture at 30 °C for 12 h to eliminate the pTarget plasmid. Pick a single colony that has successfully verified the elimination of the pTarget plasmid into a test tube containing 10 mL of LB medium and culture overnight at 42 °C to eliminate the pCas plasmid; Finally, obtain the plasmid-free strain OSHY2.

[0032] Example 3: Construction of OSHY3: Introduce an N148A site mutation into the key gene metB for methionine synthesis, so that it retains 50% of its original enzyme activity, and to a certain extent reduce the consumption of the target product; Further, knockout the key gene metJ for synthesizing the negative transcriptional regulatory protein to relieve its repression on the key enzymes in the product synthesis pathway.

[0033] Introduce an N148A single point mutation into the metB gene of the OSHY2 strain genome through the CRISPR / Cas9 system. The specific operation is as follows: (1) Ligation fragment U metB -D metB (N148A) Using the Escherichia coli EcN genome as a template, the upstream homologous arm and downstream homologous arm containing the N148A site mutation were amplified using primers metB-P1 and metB-P2, and metB-P3 and metB-P4, respectively. After the PCR products were detected by 1.0% agarose gel electrophoresis, they were treated with DpnI at 37°C for 1 h, and finally the purified fragments were recovered using a Clean Up kit. The ligation fragment U was obtained using the same method as the previous steps. metB -D metB (N148A). (2) Construction of the pTarget-metB-sg vector Using the pTarget plasmid as a template and metB-N20-F and metB-N20-R as primers, the sgRNA capable of expressing the targeted gene metB was amplified to construct the pTarget-metB-sg mutant vector. Then, the pTarget-metB plasmid was linearized using pTarget-line-F and pTarget-line-R as primers. (3) Construction of the pTarget-metB(N148A) plasmid The ligation fragment U metB -D metB (N148A) from step (1) and the linearized mutant vector pTarget-metB-sg from step (2) were subjected to one-step cloning, and the pTarget-metB(N148A) plasmid was obtained using the same method as the previous steps. (4) Single-point mutation of the metB gene The N148A site mutation was introduced into the metB gene using the CRISPR / Cas9 gene editing technology; the pTarget-metB(N148A) plasmid was electrotransformed into the OSHY2 strain containing the pCas9 vector. The operation steps were as described above. PCR was performed using primers metB-YZ-F and metB-YZ-R. A 1200 bp DNA band was present in the 1.0% agarose gel of the PCR product, confirming the successful acquisition of the positive clone strain with the N148A mutation introduced into the metB gene. The pTarget plasmid and pCas plasmid were eliminated separately according to the previous steps to obtain a plasmid-free strain.

[0034] The metJ gene was knocked out in the genome of the above strain through the CRISPR / Cas9 system. The specific operation is as follows: (1) Ligation fragment U metJ -D metJ Using the Escherichia coli EcN genome as a template, the upstream homologous arm and downstream homologous arm of the gene metJ were amplified using primers metJ-P1 and metJ-P2, and metJ-P3 and metJ-P4 respectively. Fusion PCR was performed using primers metJ-P1 and metJ-P4, and the ligation fragment U was obtained by the same method as the previous steps. metJ -D metJ 。 (2) Construction of the pTarget-metJ-sg vector Using the pTarget plasmid as a template and metJ-N20-F and metJ-N20-R as primers, the sgRNA capable of expressing the targeted gene metJ was amplified, and the pTarget-metJ-sg mutant vector was constructed. Then, the pTarget-metJ plasmid was linearized using primers pTarget-line-F and pTarget-line-R. (3) Construction of the pTarget-metJ plasmid The ligation fragment U in step (1) metJ -D metJ and the linearized mutant vector pTarget-metJ-sg in step (2) were subjected to one-step cloning, and the pTarget-metJ plasmid was obtained by the same method as the previous steps. (4) Construction of strain OSHY3 The gene metJ was knocked out using the CRISPR / Cas9 gene editing technology; the pTarget-metJ plasmid was electrotransformed into the above-mentioned strain containing the pCas9 vector, and the operation steps were as described above. PCR was performed using primers metJ-YZ-F and metJ-YZ-R, and a DNA band of 1200 bp was present in the 1.0% agarose gel of the PCR product, confirming the successful acquisition of the positive clone strain OSHY3 with the metJ gene knocked out. The pTarget plasmid and pCas plasmid were eliminated respectively according to the previous steps to obtain a plasmid-free strain.

[0035] Example 4: Construction of OSHY4: Introduce the strong promoter P trc Overexpress the key gene metL for synthesizing L-homoserine to increase the accumulation of the precursor L-homoserine; and insert the hom gene from Corynebacterium glutamicum that relieves feedback inhibition at the rph gene locus to promote more metabolic flux into the product synthesis pathway.

[0036] Insert the strong promoter P in front of the metL gene in the genome of strain OSHY3 through the CRISPR / Cas9 system trc , and the specific operation is as follows: (1) Ligation fragment U metL-P trc -D metL Using the Escherichia coli EcN genome as a template, the upstream homologous arm and downstream homologous arm of the target site were amplified using primers metL-P1 and metL-P2, metL-P3 and metL-P4 respectively. Using the downstream homologous arm as a template, a secondary amplification was performed using primers trc-RBS-F and metL-P4 to obtain a downstream homologous arm with the trc promoter sequence. Then, fusion PCR was performed using primers metL-P1 and metL-P4, and the ligation fragment U was obtained using the same method as the previous step. metL -P trc -D metL 。 (2) Construction of the pTarget-metL-sg vector Using the pTarget plasmid as a template and metL-N20-F and metL-N20-R as primers, an sgRNA capable of expressing the target gene metL was amplified, and the pTarget-metL-sg mutant vector was constructed. Then, the pTarget-metL plasmid was linearized using primers pTarget-line-F and pTarget-line-R. (3) Construction of the pTarget-metL plasmid The ligation fragment U in step (1) metL -P trc -D metL and the linearized mutant vector pTarget-metL-sg in step (2) were subjected to one-step cloning, and the pTarget-metL plasmid was obtained using the same method as the previous step. (4) Overexpression of the metL gene Using the CRISPR / Cas9 gene editing technology, a strong promoter P was inserted in front of the metL gene trc ; The pTarget-metL plasmid was electrotransformed into the OSHY3 strain containing the pCas9 vector. The operation steps were as described above. PCR was performed using primers metL-YZ-F and metL-YZ-R. A DNA band of 1300 bp was present in the 1.0% agarose gel of the PCR product, confirming the successful acquisition of a positive clone strain overexpressing the metL gene. The pTarget plasmid and pCas plasmid were eliminated respectively according to the previous steps to obtain a plasmid-free strain.

[0037] The exogenous hom gene was inserted into the rph site of the genome of the above strain through the CRISPR / Cas9 system. The specific operation is as follows: (1) Ligation fragment U rph -hom-Drph Using the Escherichia coli EcN genome as a template, the upstream homologous arm and downstream homologous arm of the target gene rph were amplified using primers rph-P1 and rph-P2, and rph-P5 and rph-P6, respectively; using primers hom-P3 and hom-P4, the plasmid containing the foreign gene was used as a template for amplification to obtain the hom gene fragment, and then fusion PCR was performed using primers rph-P1 and rph-P6, and the ligation fragment U rph -hom-D rph . (2) Construction of the pTarget-rph-sg vector Using the pTarget plasmid as a template and rph-N20-F and rph-N20-R as primers, the sgRNA capable of expressing the targeted gene rph was amplified, and the pTarget-rph-sg mutant vector was constructed. Then, the pTarget-rph plasmid was linearized using pTarget-line-F and pTarget-line-R as primers. (3) Construction of the pTarget-hom(rph) plasmid The ligation fragment U rph -hom-D rph in step (1) and the linearized mutant vector pTarget-rph-sg in step (2) were subjected to one-step cloning, and the pTarget-hom(rph) plasmid was obtained using the same method as in the previous steps. (4) Construction of strain OSHY4 The hom gene was inserted at the rph gene locus using the CRISPR / Cas9 gene editing technology; the pTarget-hom(rph) plasmid was electrotransformed into the above-mentioned strain containing the pCas9 vector, and the operation steps were as described above. PCR was performed using primers rph-YZ-F and rph-YZ-R. The PCR product had a DNA band of 2600 bp in a 1.0% agarose gel, and the positive clone strain OSHY4 with the successfully inserted hom gene was confirmed. The pTarget plasmid and pCas plasmid were eliminated separately according to the previous steps to obtain a plasmid-free strain.

[0038] Example 5: Construction of OSHY5: The start codon of the key gene lysA for synthesizing the essential amino acid lysine was replaced from ATG with the relatively weak GTG to reduce the protein expression level and inhibit the lysine branch pathway.

[0039] Replace the start codon of the lysA gene from ATG to GTG in the genome of strain OSHY4 through the CRISPR / Cas9 system. The specific operations are as follows: (1) Ligation fragment U lysA -(GTG)-D lysA Using the Escherichia coli EcN genome as a template, amplify the upstream homologous arm and downstream homologous arm with the GTG start codon using primers lysA-P1 and lysA-P2, and lysA-P3 and lysA-P4 respectively. Then perform fusion PCR using primers lysA-P1 and lysA-P4, and obtain ligation fragment U using the same method as the previous steps. lysA -(GTG)-D lysA . (2) Construct the pTarget-lysA-sg vector Using the pTarget plasmid as a template and lysA-N20-F and lysA-N20-R as primers, amplify the sgRNA that can express the targeted gene lysA, construct the pTarget-lysA-sg mutant vector, and then linearize the pTarget-lysA plasmid using primers pTarget-line-F and pTarget-line-R. (3) Construct the pTarget-(GTG)lysA plasmid Ligate the ligation fragment U in step (1) lysA -(GTG)-D lysA and the linearized mutant vector pTarget-lysA-sg in step (2) by one-step cloning, and obtain the pTarget-(GTG)lysA plasmid using the same method as the previous steps. (4) Construct strain OSHY5 Use the CRISPR / Cas9 gene editing technology to replace the start codon of the lysA gene from ATG to GTG; electrotransform the pTarget-(GTG)lysA plasmid into the OSHY4 strain containing the pCas9 vector. The operation steps are as described above. Perform PCR using primer lysA-YZ-F and primer lysA-YZ-R. The PCR product has a 1200 bp DNA band in a 1.0% agarose gel, and confirm the successful acquisition of the positive clone strain OSHY5 with the start codon of the lysA gene replaced. Eliminate the pTarget plasmid and pCas plasmid respectively according to the previous steps to obtain a plasmid-free strain.

[0040] Example 6: Construction of OSHY6: The high-copy plasmid pTrc99A was introduced, and the RBS expression intensities of the aspartase I-encoding gene aspA and the aspartate aminotransferase-encoding gene aspC were combinatorially regulated to promote the biosynthesis of L-aspartic acid.

[0041] The pTrc99A plasmid was introduced into the strain OSHY5 by transformation, so that the RBS intensity ratio of the aspA and aspC genes was 1000:4000 (AU). The specific operations are as follows: (1) Construct the pTrc99A-RBS(4000)-aspC plasmid Using the Escherichia coli EcN genome as a template, the target gene aspC was amplified using the primers aspC-F and aspC-R. The pTrc99A plasmid was linearized using the primers pTrc99A-line-F and pTrc99A-line-R. The aspC gene fragment was subjected to one-step cloning with the linearized vector pTrc99A, and the pTrc99A-aspC plasmid was obtained by the same method as the previous steps. Using the extracted pTrc99A-aspC plasmid as a template, the original RBS on the plasmid was replaced with an RBS sequence with an intensity of 4000 AU using the primers aspC-4000RBS-F and aspC-4000RBS-R. The mutated plasmid was purified by the same method as the previous steps and transformed into the E. coli DH5α recipient bacterium, and finally the pTrc99A-RBS(4000)-aspC plasmid was obtained. (2) Construct the pTrc99A-RBS(1000)-aspA plasmid Using the Escherichia coli EcN genome as a template, the target gene aspA was amplified using the primers aspA-F and aspA-R. The pTrc99A plasmid was linearized using the primers pTrc99A-line-F and pTrc99A-line-R. The aspA gene fragment was subjected to one-step cloning with the linearized vector pTrc99A, and the pTrc99A-aspA plasmid was obtained by the same method as the previous steps. Using the extracted pTrc99A-aspA plasmid as a template, the original RBS on the plasmid was replaced with an RBS sequence with an intensity of 1000 AU using the primers aspA-1000RBS-F and aspA-1000RBS-R. The mutated plasmid was purified by the same method as the previous steps and transformed into the E. coli DH5α recipient bacterium, and finally the pTrc99A-RBS(1000)-aspA plasmid was obtained. (3) Construct the pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA plasmid Using the pTrc99A-RBS(4000)-aspC plasmid obtained in step (1) as a template, linearize it using primer pTrc99A-aspC-XF and primer pTrc99A-aspC-XR; using the pTrc99A-RBS(1000)-aspA plasmid obtained in step (2) as a template, amplify the aspA gene sequence with RBS(1000AU) using primer aspA-1000-P1 and primer aspA-1000-P2; perform one-step cloning of the obtained aspA gene fragment with the linearized vector pTrc99A-RBS(4000)-aspC, and obtain the pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA plasmid by the same method as the previous steps. (4) Construct strain OSHY6 Electrotransform the pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA plasmid into the plasmid-free strain OSHY5, and operate according to the following steps: 1) Inoculate the plasmid-free strain OSHY5 in 10 mL of LB liquid medium and culture overnight at 37 °C and 180 rpm as a seed solution; pipette 1 mL of the seed solution from the test tube into a 250 mL Erlenmeyer flask containing 50 mL of LB medium and culture at 37 °C and 180 rpm until the OD 600 value is about 0.6; transfer the bacterial solution from the Erlenmeyer flask to a pre-cooled 50 mL sterile centrifuge tube, centrifuge at 4 °C and 5000 rpm for 5 min, discard the supernatant; add 40 mL of pre-cooled sterile water in advance, gently pipette the bacterial cells to suspend them, centrifuge at 4 °C and 5000 rpm for 5 min, discard the supernatant; repeat the previous step; add 40 mL of pre-cooled 10% (v:v) glycerol solution in advance, gently pipette the bacterial cells to suspend them, centrifuge at 4 °C and 5000 rpm for 5 min, discard the supernatant; add 1 mL of pre-cooled 10% (v:v) glycerol solution in advance, gently pipette to suspend the bacterial cells. 2) Place the prepared electrocompetent cells on ice. After they thaw, add 5 μL of plasmid. Add the competent cells mixed with the plasmid to a pre-chilled 2 mm electroporation cuvette. Adjust the working parameters of the electroporator to 2500 V, 25 μF, and 200 Ω for electroporation. Quickly add 800 mL of LB medium and culture at 37 °C and 180 rpm for 3 h. Spread the electrotransformed bacterial solution onto an LB plate containing 50 mg / L Kan resistance and culture at 37 °C for 12 h. Pick a single colony as a template and perform PCR with primers pTrc99A-seq-F and pTrc99A-seq-R. The PCR product shows a 3000 bp DNA band in a 1.0% agarose gel, confirming the successful acquisition of the positive clone strain OSHY6 that has been successfully introduced with the pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA plasmid.

[0042] Example 7: Construction of OSHY7: Add a copy of the phosphoenolpyruvate carboxylase encoding gene ppc to the high-copy plasmid pTrc99A to further enhance the bioavailability of oxaloacetate.

[0043] Add a copy of the ppc gene to the previously obtained pTrc99A plasmid by recombinant methods. The specific operations are as follows: (1) Construct the pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA-ppc plasmid Using the Escherichia coli EcN genome as a template, amplify the target gene ppc using primers ppc-F and ppc-R. Using the pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA plasmid as a template, linearize the plasmid using primers pTrc99A-ppc-XF and pTrc99A-ppc-XR. Perform one-step cloning of the ppc gene fragment with the linearized vector pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA, and obtain the pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA-ppc plasmid using the same method as the previous steps. (2) Construct the strain OSHY7 The plasmid pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA-ppc was electrotransformed into the plasmid-free strain OSHY5. The operation steps were the same as those for constructing the strain OSHY6. PCR was performed using the primers pTrc99A-seq-F and pTrc99A-seq-R. A DNA band of 5600 bp was present in the 1.0% agarose gel of the PCR product, confirming the successful acquisition of the positive clone strain OSHY7 into which the plasmid pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA-ppc was introduced.

[0044] Example 8: Construction of OSHY8: Add a copy of the pyridine nucleotide transhydrogenase-encoding gene pntAB to the high-copy plasmid pTrc99A to increase the intracellular NADPH level and provide sufficient reducing power for anabolic reactions in the organism.

[0045] Add a copy of the pntAB gene to the above-obtained pTrc99A plasmid by recombinant methods. The specific operations are as follows: (1) Construction of the plasmid pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA-ppc-pntAB Using the Escherichia coli EcN genome as a template, the target gene pntAB was amplified using the primers pntAB-F and pntAB-R; using the plasmid pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA-ppc as a template, the plasmid was linearized using the primers pTrc99A-pntAB-XF and pTrc99A-pntAB-XR; the pntAB gene fragment was subjected to one-step cloning with the linearized vector pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA-ppc, and the plasmid pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA-ppc-pntAB was obtained by the same method as the previous steps. (2) Construction of the strain OSHY8 The plasmid pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA-ppc-pntAB was electrotransformed into the plasmid-free strain OSHY5. The operating steps were the same as those for constructing the strain OSHY6. PCR was performed using the primers pTrc99A-seq-F and pTrc99A-seq-R. The PCR product showed an 8600 bp DNA band in a 1.0% agarose gel, confirming the successful acquisition of the positive clone strain OSHY8 into which the plasmid pTrc99A-RBS(4000)-aspC-RBS(1000)-aspA-ppc-pntAB was introduced.

[0046] Flask fermentation experiment of the example: Fermentation experiments were carried out on the chassis bacterium EcN and the strains constructed in Examples 1-8 above (OSHY1, OSHY2, OSHY3, OSHY4, OSHY5, OSHY6, OSHY7, OSHY8) in flasks to compare the ability of each genotype strain to produce OSH.

[0047] First, pick a single colony into 10 mL of LB liquid medium and culture it in a constant temperature shaking incubator at 37 °C and 200 rpm for 14 h as the seed liquid for flask fermentation. Pipette 1 mL of the seed liquid and inoculate it into a 500 mL flask containing 30 mL of fermentation medium, and then add 0.35 g of sterile CaCO3. If the strain contains the pTrc99A plasmid, 0.1% Kan resistance should be added when culturing the seed liquid, and 0.1% Kan resistance and 0.02% IPTG inducer should be added during fermentation. Culture in a constant temperature shaker at 30 °C and 180 rpm for 48 h, and set three parallel experiments for each genotype strain. After the fermentation process is completed, take 1 mL of the fermentation broth from the flask into a 2 mL centrifuge tube and centrifuge at 12000 rpm for 2 min at room temperature. Transfer the supernatant to a new 2 mL centrifuge tube, then add 1 mL of 2 M hydrochloric acid to react with the CaCO3 in the remaining bacteria for 5 min, and then centrifuge at 12000 rpm for 2 min at room temperature and discard the supernatant. Resuspend the precipitate with 1 mL of ultrapure water again, centrifuge and discard the supernatant. Finally, dilute it with ultrapure water by an appropriate multiple and measure its OD 600 value. To ensure the accuracy of the measurement, each sample was measured three times to reduce the error caused by the operation. The biomass OD 600 and the content of OSH of the strains OSHY1-OSHY8 are as Figure 1As shown. Compared with the wild strain EcN, the recombinant Escherichia coli OSHY8 has a significantly improved ability to produce OSH, with a yield of 13.44 g / L in shake flask fermentation. Composition of LB medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, and the solvent is deionized water. Solid medium requires the addition of agar at a final concentration of 20 g / L. Composition of shake flask fermentation medium: glucose 30 g / L, (NH4)2SO4 16 g / L, yeast extract 2.5 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, FeSO4·4H2O 0.01 g / L, MnSO4 0.005 g / L, ZnSO4·7H2O 0.0025 g / L, CaCO3 0.35 g / bottle, and the solvent is deionized water, with a pH value of 6.8.

[0048] Fermentation verification of the non-auxotrophic strain OSHY8 in a 5 L fermenter: Fed-batch fermentation was carried out in a 5 L fermenter. Figure 2 The fermentation process is shown, including the residual sugar, biomass (OD 600 ) and the change of OSH yield over time. The initial 25 g / L of glucose in the OSHY8 fermentation broth was basically consumed at 6 - 7 h. When the glucose was consumed, the pH value of the fermentation broth increased. When the pH was greater than 6.81, automatic feeding was started. With the addition of the feeding medium, the pH value gradually decreased and was maintained at 6.80, keeping the glucose concentration in the medium at a relatively low level (<1 g / L) to avoid the inhibitory effect of high-concentration glucose on the growth of strain cells. The OSH yield was the highest at 44 h of fermentation, reaching 80.79 g / L, with a sugar-acid conversion rate of 0.47 g / g glucose and a space-time yield of 1.84 g / L / h, which is the highest reported OSH yield to date. In summary, the fermentation process regulation of the recombinant Escherichia coli OSHY8 is relatively simple, the fermentation cost is reduced, the fermentation cycle is greatly shortened, laying a certain foundation for the industrial production of OSH.

[0049] Finally, it should be noted that the above experiments and examples are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A genetically engineered bacterium with high yield of O-succinyl-L-homoserine, characterized in that: The genetically engineered bacterium uses the wild-type Escherichia coli probiotic EcN as the chassis strain, knocks out the genes metI and metJ, dynamically regulates the gene thrB, overexpresses the genes metA and metL, and weakens the gene metB; Introduce the hom gene from Corynebacterium glutamicum to replace the start codon of the gene lysA; Introduce the high-copy plasmid pTrc99A, thereby combinatorially regulating the RBS strengths of the aspA gene and the aspC gene, and finally obtaining a genetically engineered bacterium with high production of O-succinyl-L-homoserine.

2. The genetically engineered bacterium for highly producing O-succinyl-L-homoserine according to claim 1, characterized in that: The genetically engineered bacterium adds a copy of the ppc gene to the plasmid.

3. The genetically engineered bacterium for highly producing O-succinyl-L-homoserine according to claim 2, characterized in that: The genetically engineered bacterium increases a copy of the gene pntAB on the plasmid.

4. The genetically engineered bacterium for highly producing O-succinyl-L-homoserine according to claim 1, wherein: The genetically engineered bacterium dynamically regulates the gene thrB by introducing the self-regulating promoter P fliA ​ 5. The genetically engineered bacterium for highly producing O-succinyl-L-homoserine according to claim 1, characterized in that: The genetically engineered bacterium overexpresses the gene metL through the strong promoter P trc ​ 6. The genetically engineered bacterium for highly producing O-succinyl-L-homoserine according to claim 1, characterized in that: The genetically engineered bacterium also introduces the Q64E, T242A double-site mutation.

7. The genetically engineered bacterium for highly producing O-succinyl-L-homoserine according to claim 1, characterized in that: The genetically engineered bacterium weakens the gene metB by introducing the N148A single-site mutation.

8. A method for constructing a genetically engineered bacterium with high yield of O-succinyl-L-homoserine, characterized in that, It includes the following steps: S1. Knock out the key gene metI for methionine absorption, and insert the dynamic regulation promoter P before the gene thrB encoding homoserine kinase fliA , denoted as strain OSHY1; S2. On the genome of strain OSHY1, use a strong promoter P trc to regulate the expression of the homoserine succinyltransferase-encoding gene metA, and introduce double-site mutations Q64E and T242A, denoted as strain OSHY2; S3. Introduce the N148A single-site mutation into the cystathionine-γ-synthase CGS on the genome of the strain OSHY2, and at the same time knock out the negative transcriptional regulatory protein-encoding gene metJ, denoted as the strain OSHY3; S4. On the genome of strain OSHY3, use the strong promoter P trc to regulate the overexpression of the homoserine dehydrogenase-encoding gene metL, and insert the hom gene from Corynebacterium glutamicum that relieves feedback inhibition at the rph gene locus, denoted as strain OSHY4; S5. Replace the start codon of the diaminopimelate decarboxylase-encoding gene lysA on the genome of the strain OSHY4 from ATG with GTG with relatively weak translation initiation efficiency, denoted as the strain OSHY5; S6. Introduce the high-copy plasmid pTrc99A, combinatorially regulate the RBS strengths of the aspartase I-encoding gene aspA and the aspartate aminotransferase-encoding gene aspC so that their expression ratio is 1000:4000 (AU), denoted as the strain OSHY6; S7. Add a copy of the phosphoenolpyruvate carboxylase-encoding gene ppc to the pTrc99A plasmid of the strain OSHY6, denoted as the strain OSHY7; S8. Add a copy of the pyridine nucleotide transhydrogenase-encoding gene pntAB to the pTrc99A plasmid of the strain OSHY7, denoted as the strain OSHY8, which is the described genetically engineered bacterium with high production of O-succinyl-L-homoserine.

9. Application of a genetically engineered bacterium with high production of O-succinyl-L-homoserine in the microbial fermentation production of OSH.

10. A method for producing O-succinyl-L-homoserine, characterized in that, It includes the following steps: S1. Streak-culture the genetically engineered bacterium with high production of O-succinyl-L-homoserine according to any one of claims 1-7; S2. Pick a single colony on the plate and inoculate it into an LB liquid medium containing the corresponding resistance for culture to obtain a seed solution; S3. Inoculate the seed solution into a shake flask for fermentation culture, and add Kan resistance and IPTG inducer for fermentation culture to obtain a fermentation broth containing OSH.

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