A genetically engineered bacterium with high yield of o-succinyl-l-homoserine and a construction method and application thereof

Abstract

The application provides a genetically engineered bacterium with high O-succinyl-L-homoserine yield and a construction method and application thereof. First, the CRP mutant capable of removing glucose repression is introduced to promote the utilization of glucose and biosynthesis. The E. coli regulated by the beneficial CRP mutant can be used as a favorable chassis for amino acid biosynthesis. Second, the exogenous glycerol-3-phosphate dehydrogenase and its mutant are introduced to provide more NADPH supply for the metabolic pathway, realize the balance of intracellular reducing power, reduce the pressure on cell growth caused by the consumption of NADPH in the homoserine synthesis process, and further improve the yield of OSH. Finally, an OSH production strain suitable for industrial production is obtained, which has important significance for expanding the industrial application of E. coli.

Description

Technical Field

[0001] This invention belongs to the field of microbial metabolic engineering, specifically relating to a genetically engineered bacterium that produces high levels of O-succinyl-L-homoserine and its construction method, as well as the application of this genetically engineered bacterium in the microbial fermentation preparation of O-succinyl-L-homoserine. Background Technology

[0002] With the development of the amino acid industry and the continuous expansion of its application fields, the amino acid market has gradually grown. Statistics show that the global amino acid market size has exceeded US$20 billion and is expected to continue its rapid growth in the coming years. China is one of the world's major producers and consumers of amino acids, and the amino acid industry has become one of China's strategic emerging industries. O-succinyl-L-homoserine (OSH) is an important C4 platform compound; using it as a substrate, other high-value chemicals can be produced. Therefore, building efficient cell factories for OSH production has broad industrial application prospects.

[0003] Acylhomoserine, first discovered by Rowbury in 1964, is a precursor for the microbial synthesis of L-methionine. In microorganisms, the substrate homoserine undergoes acylation catalyzed to yield acylhomoserine, which is then used to synthesize methionine via a specific pathway. The key enzyme in this acylation process is homoserine O-acyltransferase, which exhibits strong substrate specificity. Based on different acyl donors, it can be classified into homoserine succinyltransferase and homoserine acetyltransferase. Most fungi and a few bacteria, such as Corynebacterium glutamicum, use homoserine acetyltransferase for catalysis; most bacteria, such as Escherichia coli, use homoserine succinyltransferase. While these two enzymes do not share sequence similarity, they both follow a ping-pong reaction enzymatic chemical mechanism when catalyzing similar acylation reactions. When succinyl-Coenzyme A acts as an acyl donor, it transfers the succinyl group to a nucleophilic residue, which is then transferred to homoserine to form O-succinyl-L-homoserine.

[0004] Because the synthesis pathway of OSH is strictly regulated, traditional mutagenesis has not yielded ideal results. With the development of synthetic biology, it has become possible to modify microorganisms using metabolic engineering techniques to maximize the accumulation of target products and minimize the content of metabolites from unrelated pathways. In the metabolic synthesis pathway of OSH in *E. coli*, glucose first enters the EMP pathway and is converted to pyruvate. Pyruvate is then converted to acetyl-CoA by the pyruvate decarboxylase system, and then to oxaloacetate through the TCA cycle. Oxaloacetate is a common precursor of the aspartate family of amino acids. Through a series of catalytic transformations, it flows to the homoserine pathway. Homoserine combines with succinyl-CoA under the catalysis of homoserine-O-succinyl transferase (encoded by the metA gene) to generate OSH. To increase OSH biosynthesis, several modification strategies are commonly used, such as increasing precursor supply, blocking the destination pathway, and weakening the pathway. One key challenge is to develop an effective strategy to maximize the synthesis of the target product without sacrificing cell growth, because simply knocking out or overexpressing essential genes can easily damage the intracellular metabolic network, leading to metabolic disorders and the accumulation of intermediates and byproducts.

[0005] Therefore, a new approach is needed for more effective metabolic engineering optimization to promote OSH synthesis and maximize substrate conversion. Summary of the Invention

[0006] To address the problem of low OSH production in existing Escherichia coli metabolic synthesis technologies, this invention provides a genetically engineered bacterium with excellent biosynthetic capabilities, its construction method, and its applications. To obtain an E. coli strain with satisfactory OSH biosynthetic capacity, this invention first introduces a CRP mutant that can relieve glucose repression (this type of CRP mutant activates glycolysis and the TCA cycle during glucose-based fermentation), thereby promoting glucose utilization and biosynthesis. E. coli regulated by this beneficial CRP mutant can serve as a favorable substrate for amino acid biosynthesis. Secondly, exogenous glyceraldehyde-3-phosphate dehydrogenase and its mutant are introduced to provide more NADPH for the metabolic pathway, achieving a balance in intracellular reducing power and reducing the stress on cell growth caused by NADPH consumption during homoserine synthesis, further increasing OSH production. Finally, an OSH-producing strain suitable for industrial production is obtained, which is of great significance for expanding the industrial application of E. coli.

[0007] The technical solution adopted in this invention is:

[0008] A method for constructing a genetically engineered bacterium that produces high levels of O-succinyl-L-homoserine, the method comprising: using CRISPR-CAS9 technology to introduce the coding gene of a CRP mutant that relieves glucose repression into the genome of a sclerotium.

[0009] Furthermore, the method also includes: using CRISPR-CAS9 technology to introduce the coding gene of a heterologous GAPDH mutant into the genome of *Bacillus thuringiensis*; the GAPDH mutant is used to increase the supply of NADPH.

[0010] The present invention constructed the genetically engineered bacterium that produces high levels of O-succinyl-L-homoserine according to the following approach:

[0011] (1) First, the cAMP receptor protein (CRP) is a known major global regulator of carbon source catabolism. As a global regulatory master protein that senses cellular energy status through cAMP levels, CRP regulates more than 400 genes, many of which are essential for the breakdown of various carbon sources other than glucose. The CRP-cAMP complex can activate multiple operators encoding enzymes, transporters, and enzymes that initiate carbon metabolism in central metabolic pathways such as the tricarboxylic acid (TCA) cycle and glycolysis. The protein structure of CRP has been comprehensively studied; it contains an N-terminal domain that binds to cAMP and a C-terminal domain that binds to DNA. It has been reported to effectively improve the strain's tolerance to various stresses, such as alcohol, acid, temperature, organic solvents, and oxidative stress. To obtain *E. coli* cells with good biosynthetic capacity, this invention selects to introduce CRP mutants that relieve "glucose repression." These CRP mutants are expected to activate glycolysis and the TCA cycle during glucose-based fermentation, thereby promoting glucose utilization and biosynthesis. Simultaneously, strains containing the CRP mutant with poor cell growth will be eliminated throughout the iterative screening process. E. coli cells regulated by this beneficial CRP mutant can serve as a favorable chassis for natural product biosynthesis. Therefore, this invention utilizes CRISPR-CAS9 technology to introduce the CRP mutant at the genome level. E73G / R83L / G142H CRP R83F / G142P This enables the construction of a highly efficient chassis microbial community.

[0012] (2) Secondly, cofactor recovery is considered a crucial process in biochemical production. NADPH cofactors are used as reducing agents in the biosynthesis of amino acids and other valuable chemicals. To address the prevalent cofactor imbalance, a sufficient supply of NADPH cofactors is needed. Therefore, improving the efficiency of E. coli in producing target chemicals by increasing the NADPH / NADP+ ratio is essential. E. coli, as an OSH-producing strain, produces NADPH mainly through two pathways: the pentose phosphate pathway and the citric acid cycle. However, the ability of these two pathways to produce NADPH within the bacteria is limited. To obtain a greater supply of NADPH, it is necessary to introduce exogenous, highly efficient NADPH synthases. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the central carbon metabolism pathway, catalyzing the conversion of NADP+ and glyceraldehyde-3-phosphate (G3P) into NADPH and 3-phosphate-D-glyceryl phosphate. It is a major target for NADPH supply and addressing the cofactor imbalance problem. This invention utilizes molecular modification technology to mutate glyceraldehyde-3-phosphate dehydrogenase derived from Clostridium acetobutylicum and Corynebacterium glutamicum to obtain a beneficial mutant (GAPDH). R233A GAPDH F100V GAPDH F100V / P192S The mutant was then heterologously expressed in the previously constructed E. coli OSH-producing strain to increase the supply of NADPH during OSH synthesis, thereby enhancing the fermentation production capacity of OSH.

[0013] The genes involved in the modification of this invention and their corresponding pathways are shown in Table 1.

[0014] Table 1. Genes involved in strain modification and their corresponding pathways

[0015]

[0016] Preferably, the CRP mutant is selected from CRP. E73G / R83L / G142H CRP R83F / G142P Any one of them.

[0017] Preferably, the GAPDH mutant is selected from GAPDH derived from Clostridium acetobutylicum. ca R233A GAPDH derived from Corynebacterium glutamicum cg F100V GAPDH derived from Corynebacterium glutamicumcg F100V / P192S Any one of them.

[0018] Preferably, the substrate bacteria is *E. coli* W3110ΔmetIΔmetJΔthrBΔmetBΔldhAΔadhEΔpflB Ptrc-metLΔarcAΔiclR / pTrc99a-metA-yjeH. The method for constructing the substrate bacteria includes: using *E. coli* W3110ΔmetIΔmetJΔthrBΔmetB as the starting strain (patent CN 109055290 B), firstly, knocking out the ldhA gene encoding lactate dehydrogenase, the adhE gene encoding glycolate dehydrogenase, and the pflB gene encoding pyruvate-formate lyase in its genome to block the synthesis pathways of formic acid, lactate, and ethanol, reducing the accumulation of organic acid metabolic byproducts such as formic acid, lactate, and ethanol, concentrating carbon flux on the conversion of pyruvate to oxaloacetate, and further improving the sugar-acid conversion rate of the engineered strain. Secondly, to further increase the supply of the precursor L-homoserine, the expression of the metL gene encoding homoserine dehydrogenase was enhanced. To increase the supply of the precursor succinyl-CoA, the gene encoding the global regulatory factor arcA and the gene encoding the DNA-binding transcriptional repressor iclR were knocked out, accelerating the TCA cycle and increasing the supply of succinyl-CoA. Finally, to overcome the problem of insufficient expression of the key genes metA (homoserine transsuccinylase) and yjeH (L-methionine / branched-chain amino acid transporter), the overexpression plasmid pTrc99a-metA-yjeH was introduced into the chassis strain via tandem plasmid expression to achieve overexpression of these two genes. The detailed construction method of the chassis strain is described in Chinese Patent 202311767745.6.

[0019] Preferably, the CRP mutant CRP E73G / R83L / G142H The nucleotide sequence is shown in SEQ ID NO.2.

[0020] Preferably, the CRP mutant CRP R83F / G142P The nucleotide sequence is shown in SEQ ID NO.3.

[0021] Preferably, the GAPDH mutant GAPDH ca R233AThe nucleotide sequence is shown in SEQ ID NO. 6. The preferred amino acid sequence of the glyceraldehyde-3-phosphate dehydrogenase derived from *Clostridium acetobutylicum* is shown in SEQ ID NO. 4. The glyceraldehyde-3-phosphate dehydrogenase mutant GAPDH is described above. ca R233A The nucleotide sequence of the mutant is preferably shown in SEQ ID NO. 6, where the arginine at position 233 of the amino acid sequence shown in SEQ ID NO. 4 is mutated to alanine.

[0022] Preferably, the GAPDH mutant GAPDH cg F100V The nucleotide sequence is shown in SEQ ID NO.7. The preferred amino acid sequence of the glyceraldehyde-3-phosphate dehydrogenase derived from *Corynebacterium glutamicum* is shown in SEQ ID NO.5. The glyceraldehyde-3-phosphate dehydrogenase mutant GAPDH... cg F100V The 100th phenylalanine in the amino acid sequence shown in SEQ ID NO.5 is mutated to valine, and the nucleotide sequence of the mutant is shown in SEQ ID NO.7.

[0023] Preferably, the GAPDH mutant GAPDH cg F100V / P192S The nucleotide sequence is shown in SEQ ID NO. 8. Similarly, the glyceraldehyde-3-phosphate dehydrogenase mutant GAPDH... cg F100V / P192S The amino acid sequence shown in SEQ ID NO.7 is modified by mutating proline at position 192 to serine, and the nucleotide sequence of the mutant is shown in SEQ ID NO.8.

[0024] The present invention also provides a genetically engineered bacterium that produces high levels of O-succinyl-L-homoserine by constructing the bacterium using the method described above.

[0025] Preferably, the genetically engineered bacterium that produces high levels of O-succinyl-L-homoserine is:

[0026] E.coli W3110ΔmetIΔmetJΔthrBΔmetBΔldhAΔadhEΔpflB Ptrc-metLΔarcAΔiclR crp R83F / G142P / pTrc99a-metA-yjeH. This genetically engineered bacterium, which produces high levels of O-succinyl-L-homoserine, was created by introducing a CRP mutant, CRP, into the genome of *E. coli* W3110ΔmetIΔmetJΔthrBΔmetBΔldhAΔadhEΔpflBPtrc-metLΔarcAΔiclR using CRISPR-CAS9 technology. R83F / G142P After the encoding gene was obtained, the OSH yield was constructed by introducing the overexpression plasmid pTrc99a-metA-yjeH. After fed-batch fermentation in a 5L fermenter, the OSH yield reached 101.9 g / L.

[0027] Preferably, the genetically engineered bacterium that produces high levels of O-succinyl-L-homoserine is:

[0028] E.coli W3110ΔmetIΔmetJΔthrBΔmetBΔldhAΔadhEΔpflB Ptrc-metLΔarcAΔiclR crp R83F / G142P gapdh cg F100V / P192S / pTrc99a-metA-yjeH. This genetically engineered bacterium, which produces high levels of O-succinyl-L-homoserine, was created by introducing a CRP mutant, CRP, into the genome of *E. coli* W3110ΔmetIΔmetJΔthrBΔmetBΔldhAΔadhEΔpflB Ptrc-metLΔarcAΔiclR using CRISPR-CAS9 technology. R83F / G142P The encoding gene and the glyceraldehyde-3-phosphate dehydrogenase mutant GAPDH derived from Corynebacterium glutamicum cg F100V / P192S Subsequently, the OSH yield was obtained by introducing the overexpression plasmid pTrc99a-metA-yjeH, and after fed-batch fermentation in a 5L fermenter, the yield reached 107.1 g / L.

[0029] This invention also provides the application of the genetically engineered bacteria that produce high levels of O-succinyl-L-homoserine in the production of O-succinyl-L-homoserine. The preferred method of application includes: inoculating the genetically engineered bacteria that produce high levels of O-succinyl-L-homoserine into a fermentation medium, fermenting at 28–37°C and 100–500 rpm for 70–100 h, and then isolating O-succinyl-L-homoserine after fermentation.

[0030] Specifically, the genetically engineered bacteria that produce high levels of O-succinyl-L-homoserine produce O-succinyl-L-homoserine through shake-flask fermentation or 5L fermenter fermentation.

[0031] The shake-flask fermentation includes: inoculating the genetically engineered bacteria into a fermentation medium containing 50 mg / L kanamycin, culturing at 30°C and 180 rpm for 48 h until the end of fermentation, obtaining a fermentation broth containing O-succinyl-L-homoserine, separating and purifying the fermentation broth to obtain O-succinyl-L-homoserine.

[0032] The 5L fermenter fermentation process includes: before fermentation, inoculating the genetically engineered bacteria into LB medium and incubating overnight at 37°C and 180 rpm to prepare a seed culture; then inoculating the seed culture into the fermentation medium at a volume concentration of 5%. The genetically engineered bacteria are then inoculated into the fermentation medium and fermented at 28–37°C and 100–500 rpm for 70–100 hours. After fermentation, O-succinyl-L-homoserine is isolated. The fermentation is carried out in a 5L fermenter. To further improve fermentation efficiency, this invention preferably employs fed-batch fermentation to culture the genetically engineered bacteria. Here, fed-batch fermentation generally refers to automatically starting feeding when the initial sugar in the fermenter is consumed and the pH of the fermentation system is higher than 6.80, adding feeding medium at a rate of 0–50 mL / h until the pH is lower than 6.80, at which point feeding stops, maintaining the residual sugar in the fermenter at a low level, with a sugar concentration of 0–5 g / L.

[0033] The beneficial effects of this invention are:

[0034] To obtain *E. coli* cells with good biosynthetic capabilities, this invention first screened for CRP mutants that relieved glucose repression. These CRP mutants activate glycolysis and the TCA cycle during glucose-based fermentation, thereby promoting glucose utilization and biosynthesis, which is essential for maximizing the generation of cellular metabolic networks and optimizing pathway expression. Secondly, to increase the intracellular NADPH pool, molecular engineering techniques were used to mutate glyceraldehyde-3-phosphate dehydrogenase from *Clostridium acetobutylicum* and *Corynebacterium glutamicum* to obtain a beneficial mutant (GAPDH). ca R233A GAPDH cg F100V GAPDH cg F100V / P192S The mutant was then heterologously expressed in the previously constructed E. coli OSH-producing strain to increase the supply of NADPH during OSH synthesis, thereby enhancing the fermentation production capacity of OSH.

[0035] Compared with existing technologies, this invention has certain economic value in industrial production because the genetically engineered bacteria can synthesize the target product to the maximum extent without sacrificing cell growth. This is because simply knocking out or overexpressing essential genes easily disrupts the intracellular metabolic network, leading to metabolic disorders and the accumulation of intermediates and byproducts. Besides the metabolic network, the recombinant expression level and cell growth also need optimization. In *E. coli* cells, glucose is the main carbon source in high-density fermentation. Carbon metabolite repression, including glucose repression, is regulated by the cAMP-CRP complex. In the presence of readily metabolizable carbon sources (glucose), the intracellular cAMP concentration is low, thus the expression of genes involved in the catabolism of other carbon sources is not activated. Some enzymes encoding central metabolic pathways (e.g., the tricarboxylic acid cycle, glycolysis), transport proteins, and operons of enzymes initiating carbon metabolism are activated by the CRP-cAMP complex. Furthermore, this invention increases the NADPH / NADP ratio in *E. coli*. + To improve OSH production efficiency, a beneficial mutant (GAPDH) was obtained by mutating glyceraldehyde-3-phosphate dehydrogenase derived from Clostridium acetobutylicum and Corynebacterium glutamicum using molecular modification technology. ca R233A GAPDH cg F100V GAPDH cg F100V / P192S The mutant was then heterologously expressed in the previously constructed E. coli OSH-producing strain to increase the supply of NADPH during OSH synthesis, thereby enhancing the fermentation production capacity of OSH.

[0036] The genetically engineered strains obtained through the above-mentioned systemic metabolic engineering modification strategy can effectively accumulate OSH, with an OSH yield of 16.19 g / L in shake flasks and 107.1 g / L in fed-batch fermentation in a 5L fermenter, laying the foundation for the subsequent construction of high-yield OSH engineered strains. Attached Figure Description

[0037] Figure 1 The bar chart shows the biomass OD600 and OSH concentration of strains OSH-0A, OSH-1A, OSH-2, OSH-3A, OSH-4A and OSH-5A in Example 6.

[0038] Figure 2 The graph shows the biomass OD600, residual sugar, and OSH concentration curves of strain OSH-2A in Example 7 during fed-batch fermentation in a 5L fermenter.

[0039] Figure 3The graph shows the biomass OD600, residual sugar, and OSH concentration curves of strain OSH-5A in Example 7 during fed-batch fermentation in a 5L fermenter. Detailed Implementation

[0040] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0041] In the examples, the final concentration of kanamycin (Kan) in both liquid and solid culture media was 50 mg / L, and the final concentration of spectinomycin was 50 mg / L.

[0042] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in deionized water, pH at natural.

[0043] Shake-flask fermentation medium: glucose 25 g / L, ammonium sulfate 16 g / L, yeast extract 2.5 g / L, KH₂PO₄ 1 g / L, MgSO₄ 0.5 g / L, CaCO₃ 15 g / L, salt solution 1 mL / L, with CaCO₃ separately aliquoted and sterilized. Add CaCO₃ at inoculation.

[0044] Culture medium for 5L fermenter: glucose 25g / L, ammonium sulfate 16g / L, yeast extract 2.5g / L, KH2PO4 1g / L, MgSO4 0.5g / L, trace metal salt solution 1mL / L.

[0045] Feeding medium: glucose 500 g / L, (NH4)2SO4 16 g / L, KH2PO4 12.5 g / L, pH adjusted to 6.8 with 50% ammonia.

[0046] This invention involves genetic engineering modification of the starting strain E. coli W3110ΔmetIΔmetJΔthrBΔmetBΔldhAΔadhEΔpflB Ptrc-metLΔarcAΔiclR (denoted as OSH0). The genes involved and their corresponding pathways are shown in Table 1. A mutant CRP was constructed. mu2 E73G / R83L / G142H CRP mu9 R83F / G142P Primers and construction of the GAPDH mutantca R233A GAPDH cg F100V GAPDH cg F100V / P192S The primers are shown in Table 2. After introducing a specific mutant coding gene into the genome, the overexpression plasmid pTrc99a-metA-yjeH was then introduced.

[0047] Example 1: Construction of a genetically engineered bacterium OSH-1 that produces high levels of O-succinyl-L-homoserine

[0048] Starting with OSH-0 strain, the crp gene was mutated at different sites using CRISPR / Cas9 gene editing technology to generate mutant CRP. mu2 E73G / R83L / G142H To increase glucose utilization, the specific steps are as follows:

[0049] (1) Constructing the pT-CRP plasmid:

[0050] Using plasmid pTarget as a template and pT-CRP-F / pT-CRP-R as primers, plasmid pTarget-CRP was amplified. The PCR product was digested with Dpn I at 37°C for 1 hour, purified using a Clean Up kit, and then transformed into E. coli DH5α. The transformed product was plated onto LB agar plates containing 50 mg / L spectinomycin and incubated upside down at 37°C for 20 hours. Colony PCR was used for preliminary verification, followed by sequencing to confirm the correctness of the pT-CRP plasmid, thus obtaining plasmid pT-CRP. Using pT-line-F / pT-line-R as primers in Table 2 and plasmid pT-CRP as a template, the linearized fragment of the plasmid was amplified by PCR. The PCR product was digested with Dpn I and incubated at 37°C for 3 hours. The DNA fragment was recovered using a Clean Up kit to obtain the linearized plasmid pT-CRP.

[0051] (2) Constructing a pTD-CRP plasmid containing Donor:

[0052] Using the E. coli W3110 genome as a template, and CRP-F1 / CRP-R1 and CRP-F2 / CRP-R2 as primers, PCR amplification yielded upstream and downstream homologous arms CRP-1 and CRP-2. Using CRP-1 and CRP-2 as templates, and CRP-F1 and CRP-R2 as primers, fusion PCR amplification yielded CRP-Donor. Using a one-step cloning technique, the linearized plasmid pT-CRP-Line and CRP-Donor were ligated to form a complete plasmid, which was then transformed into DH5α. After colony PCR verification using primers pT-VF / pT-VR, correct positive clones were picked, cultured in LB tubes, and plasmids were extracted to obtain pTD-CRP.

[0053] (3) Constructing pTD-CRP mu2 E73G Mutant plasmid:

[0054] Using pTD-CRP as a template, and CRP R233A -F / CRP R233A -R is a primer; PCR amplification yields pTD-CRP. mu2 E73G The PCR product was digested with Dpn I at 37°C for 1 hour, purified using a Clean Up kit, and then transformed into E. coli DH5α. The product was plated onto LB agar plates containing 50 mg / L spectinomycin and incubated upside down at 37°C for 20 hours. Preliminary colony PCR verification was performed, followed by sequencing verification of pTD-CRP. mu2 E73G The correctness of the plasmid, obtaining plasmid pTD-CRP mu2 E73G

[0055] (4) Constructing pTD-CRP mu2 E73G / R83L Mutant plasmid:

[0056] pTD-CRP mu2 E73G Using CRP as a template R83L -F / CRP R83L -R is a primer; PCR amplification yields pTD-CRP. mu2 E73G / R83L The PCR product was digested with Dpn I at 37°C for 1 hour, purified using a Clean Up kit, and then transformed into E. coli DH5α. The product was plated onto LB agar plates containing 50 mg / L spectinomycin and incubated upside down at 37°C for 20 hours. Preliminary colony PCR verification was performed, followed by sequencing verification of pTD-CRP. mu2 E73G / R83LThe correctness of the plasmid, obtaining plasmid pTD-CRP mu2 E73G / R83L ;

[0057] (5) Constructing pTD-CRP mu2 E73G / R83L / G142H Mutant plasmid:

[0058] pTD-CRP mu2 E73G / R83L Using CRP as a template G142H -F / CRP G142H -R is a primer; PCR amplification yields pTD-CRP. mu2 E73G / R83L / G142H The PCR product was digested with Dpn I at 37°C for 1 hour, purified using a Clean Up kit, and then transformed into E. coli DH5α. The product was plated onto LB agar plates containing 50 mg / L spectinomycin and incubated upside down at 37°C for 20 hours. Preliminary colony PCR verification was performed, followed by sequencing verification of pTD-CRP. mu2 E73G / R83L / G142H The correctness of the plasmid, obtaining plasmid pTD-CRP mu2 E73G / R83L / G142H .

[0059] (6) Transform plasmid pCas9 into competent OSH-0 cells, spread them onto LB agar plates containing 50 mg / L kanamycin, and incubate overnight at 30°C. Pick single colonies and transfer them to LB tubes containing 50 mg / L kanamycin, and incubate overnight at 30°C. Then, inoculate 1% (v / v) into 50 mL of LB medium, add kanamycin to a final concentration of 50 mg / L and 10 mM L-arabinose, and incubate at 30°C and 180 rpm until OD600 = 0.4-0.6. Centrifuge at 4°C and 4000 rpm. Wash twice with pre-cooled ultrapure water at 4°C, then wash once with 10% cold glycerol, and finally resuspend in 10% glycerol, aliquot and store to obtain electroporated competent cells for later use.

[0060] (7) Take 5 μL of the pTD-CRP prepared by the method in step (5). mu2 E73G / R83L / G142HThe plasmid was mixed with 100 μL of electroporation competent cells from step (6) and transferred into a 2 mm electroporation cuvette. The mixture was incubated on ice for 1 min and then electroporated using a MicroPluser™ (BIO-RAD) at 2500 V. Immediately after electroporation, 700 μL of pre-cooled LB medium (4°C) was added, mixed thoroughly, and immediately transferred to a new sterile 1.5 mL EP tube. The tube was incubated at 30°C and 150 rpm for 3 h with shaking. The culture was then spread onto LB solid medium containing 50 mg / L kanamycin and 50 mg / L spectinomycin and incubated upside down at 30°C for 24 h. Colony PCR was performed using YZ-CRP-F / YZ-CRP-R primers to verify the colony PCR, and sequencing was used to verify the correctness of the strain construction. The strain OSH-1 (OSH-0, CRP) was successfully constructed. mu2 E73G / R83L / G142H ).

[0061] (8) Elimination of pTarget and pCas9 plasmids: A single positive colony from step (7) is picked and inoculated into a test tube containing 2 mM IPTG and 50 mg / L kanamycin in LB liquid medium. The culture is incubated overnight at 30°C. The bacterial culture is then streaked onto LB solid medium containing 50 mg / L kanamycin and incubated upside down at 30°C for 20 hours until a single colony appears. A single colony is then picked and incubated onto LB solid medium containing 50 mg / L spectinomycin and incubated upside down at 30°C for 20 hours. If no single colony appears, the pTarget plasmid has been eliminated from this strain. The strain with the pTarget plasmid eliminated is then inoculated into an antibiotic-free LB liquid medium test tube and incubated at 42°C for 10 hours. The bacterial culture is then streaked onto LB solid medium containing 50 mg / L kanamycin and incubated upside down at 37°C for 10 hours. If no single colony appears, the pCas9 plasmid has been successfully eliminated, resulting in the plasmid-free strain OSH-1 (OSH-0, CRP). mu2 E73G / R83L / G142H ).

[0062] Example 2: Construction of a genetically engineered bacterium OSH-2 that produces high levels of O-succinyl-L-homoserine

[0063] Starting with OSH-0 strain, the crp gene was mutated at different sites using CRISPR / Cas9 gene editing technology to generate mutant CRP. mu9 R83F / G142P To increase glucose utilization, the specific steps are as follows:

[0064] (1) Constructing pTD-CRP mu9 R83F Mutant plasmid:

[0065] Using pTD-CRP as a template, and CRP R83F -F / CRP R83F-R is a primer; PCR amplification yields pTD-CRP. mu9 R83F The PCR product was digested with Dpn I at 37°C for 1 hour, purified using a Clean Up kit, and then transformed into E. coli DH5α. The product was plated onto LB agar plates containing 50 mg / L spectinomycin and incubated upside down at 37°C for 20 hours. Preliminary colony PCR verification was performed, followed by sequencing verification of pTD-CRP. mu9 R83F The correctness of the plasmid, obtaining plasmid pTD-CRP mu9 R83F .

[0066] (2) Constructing pTD-CRP mu9 R83F / G142P Mutant plasmid:

[0067] pTD-CRP mu9 R83F Using CRP as a template G142P -F / CRP G142P -R is a primer; PCR amplification yields pTD-CRP. mu9 R83F / G142P The PCR product was digested with Dpn I at 37°C for 1 hour, purified using a Clean Up kit, and then transformed into E. coli DH5α. The product was plated onto LB agar plates containing 50 mg / L spectinomycin and incubated upside down at 37°C for 20 hours. Preliminary colony PCR verification was performed, followed by sequencing verification of pTD-CRP. mu9 R83F / G142P The correctness of the plasmid, obtaining plasmid pTD-CRP mu9 R83F / G142P ;

[0068] (3) Take 5 μL of the pTD-CRP prepared by the method in step (2). mu9 R83F / G142P The plasmid was mixed with 100 μL of electroporation competent cells from step (6) and transferred into a 2 mm electroporation cuvette. The mixture was incubated on ice for 1 min and then electroporated using a MicroPluser™ (BIO-RAD) at 2500 V. Immediately after electroporation, 700 μL of pre-cooled LB medium (4°C) was added, mixed thoroughly, and immediately transferred to a new sterile 1.5 mL EP tube. The tube was incubated at 30°C and 150 rpm for 3 h with shaking. The culture was then spread onto LB solid medium containing 50 mg / L kanamycin and 50 mg / L spectinomycin and incubated upside down at 30°C for 24 h. Colony PCR was performed using YZ-CRP-F / YZ-CRP-R primers to verify the colony PCR, and sequencing was used to verify the correctness of the strain construction. The strain OSH-2 (OSH-0, CRP) was successfully constructed. mu9R83F / G142P ).

[0069] (4) Elimination of pTarget and pCas9 plasmids: A single positive colony from step (3) was picked and inoculated into a test tube containing 2 mM IPTG and 50 mg / L kanamycin in LB liquid medium. The culture was incubated overnight at 30°C. The bacterial culture was then streaked onto LB solid medium containing 50 mg / L kanamycin and incubated upside down at 30°C for 20 hours until a single colony appeared. A single colony was then picked and incubated onto LB solid medium containing 50 mg / L spectinomycin and incubated upside down at 30°C for 20 hours. If no single colony appeared, it indicated that the pTarget plasmid had been eliminated from the strain. The strain with the pTarget plasmid eliminated was then inoculated into an antibiotic-free LB liquid medium test tube and incubated at 42°C for 10 hours. The bacterial culture was then streaked onto LB solid medium containing 50 mg / L kanamycin and incubated upside down at 37°C for 10 hours. If no single colony appeared, it indicated that the pCas9 plasmid had been successfully eliminated, resulting in the plasmid-free strain OSH-2 (OSH-0, CRP). mu9 R83F / G142P ).

[0070] Example 3: Construction of a genetically engineered bacterium OSH-3 that produces high levels of O-succinyl-L-homoserine

[0071] Using strain OSH-2 as the starting strain, the GAPDH gene was mutated at different sites using CRISPR / Cas9 gene editing technology to generate mutant GAPDH. R233A To increase the utilization rate of NADPH, the specific steps are as follows:

[0072] (1) Construction of pT-GAPDH plasmid:

[0073] Using plasmid pTarget as a template and pT-GAPDH-F / pT-GAPDH-R as primers, plasmid pT-GAPDH was amplified. The PCR product was digested with Dpn I at 37°C for 1 hour, purified using a Clean Up kit, and then transformed into E. coli DH5α. The transformed product was plated on LB agar plates containing 50 mg / L spectinomycin and incubated upside down at 37°C for 20 hours. Colony PCR was used for preliminary verification, followed by sequencing to confirm the correctness of the pT-GAPDH plasmid. Using pT-line-F / pT-line-R as primers in Table 2 and pT-GAPDH as a template, the linearized fragment of the plasmid was amplified by PCR. The PCR product was digested with Dpn I and incubated at 37°C for 3 hours. The DNA fragment was recovered using a Clean Up kit to obtain the linearized plasmid pT-GAPDH.

[0074] (2) Constructing the pTD-GAPDH plasmid containing Donor:

[0075] Using the E. coli W3110 genome as a template, and GAPDH-F1 / GAPDH-R1 and GAPDH-F2 / GAPDH-R2 as primers, PCR amplification yielded upstream and downstream homologous arms GAPDH-1 and GAPDH-2. Using GAPDH-1 and GAPDH-2 as templates, and GAPDH-F1 and GAPDH-R2 as primers, fusion PCR amplification yielded GAPDH-Donor. Using a one-step cloning technique, the linearized plasmid pTarget-GAPDH-Line and GAPDH-Donor were ligated to form a complete plasmid, which was then transformed into DH5α. After verification by colony PCR, correct positive clones were picked, cultured in LB tubes, and the plasmid was extracted to obtain pTD-GAPDH.

[0076] (3) Constructing pTD-GAPDH containing Clostridium acetobutylicum ca plasmid:

[0077] Clostridium acetobutylicum GAPDH synthesized through gene synthesis ca Using GAPDH as a template ca -F / GAPDH ca -R is a primer, and PCR amplification yields GAPDH. ca Using a one-step cloning technique, the linearized plasmids pTarget-GAPDH-Line and GAPDH were cloned. ca After ligation to form a complete plasmid, it was transformed into DH5α; after verification by colony PCR, the correct positive clones were picked and cultured in LB tubes, and the plasmid was extracted to obtain pTD-GAPDH. ca ;

[0078] (4) Constructing pTD-GAPDH ca R233A Mutant plasmid:

[0079] pTD-GAPDH ca Using GAPDH as a template ca R233A -F / GAPDH ca R233A -R is a primer, and PCR amplification yields pTD-GAPDH. R233AThe PCR product was digested with Dpn I at 37°C for 1 hour, purified using a Clean Up kit, and then transformed into E. coli DH5α. The product was plated onto LB agar plates containing 50 mg / L spectinomycin and incubated upside down at 37°C for 20 hours. Preliminary colony PCR verification was performed, followed by sequencing verification of pTD-GAPDH. ca R233A The correctness of the plasmid was verified, and the plasmid pTD-GAPDH was obtained. ca R233A ;

[0080] (5) The pCas9 plasmid was introduced into the competent cells of strain OSH-2 constructed according to the method for preparing competent cells in Example 1 (6).

[0081] (6) Take 5 μL of the pTD-GAPDH constructed in step (4). R233A The plasmid was mixed with 100 μL of electroporation competent cells from step (5) and transferred into a 2 mm electroporation cuvette. The mixture was incubated on ice for 1 min and then electroporated using a MicroPluser™ (BIO-RAD) at 2500 V. Immediately after electroporation, 700 μL of pre-cooled LB medium at 4°C was added, mixed thoroughly, and immediately transferred to a new sterile 1.5 mL EP tube. The tube was incubated at 30°C with shaking at 150 rpm for 3 h. The culture was then spread onto LB solid medium containing 50 mg / L kanamycin and 50 mg / L spectinomycin and incubated upside down at 30°C for 24 h. Colony PCR was performed using YZ-GAPDH-F / YZ-GAPDH-R primers to verify the colony PCR, and sequencing was used to verify the correctness of the strain construction. The strain OSH-3 (OSH-2, GAPDH) was successfully constructed. ca R233A ).

[0082] (6) pTarget and pCas9 plasmid elimination: As shown in Example 1(8), the final plasmid-free strain OSH-3 (OSH-2, GAPDH) was obtained. ca R233A ).

[0083] Example 4: Construction of a genetically engineered bacterium OSH-4 that produces high levels of O-succinyl-L-homoserine

[0084] Using strain OSH-2 as the starting strain, the GAPDH gene was mutated at different sites using CRISPR / Cas9 gene editing technology to generate mutant GAPDH. cg F100V To increase the utilization rate of NADPH, the specific steps are as follows:

[0085] (1) Constructing pTD-GAPDH containing Corynebacterium glutamicum cg plasmid:

[0086] Corynebacterium glutamicum GAPDH synthesized through gene synthesis cg Using GAPDH as a template cg -F / GAPDH cg -R is a primer, and PCR amplification yields GAPDH. cg Using a one-step cloning technique, the linearized plasmids pTarget-GAPDH-Line and GAPDH were cloned. cg After ligation to form a complete plasmid, it was transformed into DH5α; after verification by colony PCR, the correct positive clones were picked and cultured in LB tubes, and the plasmid was extracted to obtain pTD-GAPDH. cg ;

[0087] (2) Constructing pTD-GAPDH cg F100V Mutant plasmid:

[0088] pTD-GAPDH cg Using GAPDH as a template cg F100V -F / GAPDH cg F100V -R is a primer, and PCR amplification yields pTD-GAPDH. cg F100V The PCR product was digested with Dpn I at 37°C for 1 hour, purified using a Clean Up kit, and then transformed into E. coli DH5α. The product was plated onto LB agar plates containing 50 mg / L spectinomycin and incubated upside down at 37°C for 20 hours. Preliminary colony PCR verification was performed, followed by sequencing verification of pTD-GAPDH. cg F100V The correctness of the plasmid was verified, and the plasmid pTD-GAPDH was obtained. cg F100V ;

[0089] (3) The pCas9 plasmid was introduced into the competent cells of strain OSH-2 constructed according to the method for preparing competent cells in Example 1 (6).

[0090] (4) Take 5 μL of the pTD-GAPDH constructed by the method in step (2). cg F100VThe plasmid was mixed with 100 μL of electroporation competent cells from step (3) and transferred into a 2 mm electroporation cuvette. After incubation on ice for 1 min, the cells were electroporated using a MicroPluser™ (BIO-RAD) at a voltage of 2500 V. Immediately after electroporation, 700 μL of pre-cooled LB medium at 4 °C was added and mixed thoroughly. The mixture was then immediately transferred to a new sterile 1.5 mL EP tube and cultured at 30 °C with shaking at 150 rpm for 3 h. The cultured cells were then plated onto LB solid medium containing 50 mg / L kanamycin and 50 mg / L spectinomycin and incubated upside down at 30 °C for 24 h. Colony PCR was performed using YZ-GAPDH-F / YZ-GAPDH-R primers to verify the colony PCR and sequencing was used to verify the correctness of the strain construction. The strain OSH-4 (OSH-2, GAPDH) was successfully constructed. cg F100V ).

[0091] (5) pTarget and pCas9 plasmid elimination: As shown in Example 1(8), the final plasmid-free strain OSH-4 (OSH-2, GAPDH) was obtained. cg F100V ).

[0092] Example 5: Construction of a genetically engineered bacterium OSH-5 that produces high levels of O-succinyl-L-homoserine

[0093] Using strain OSH-4 as the starting strain, the GAPDH gene was mutated at different sites using CRISPR / Cas9 gene editing technology to generate mutant GAPDH. cg F100V / P192S To increase the utilization rate of NADPH, the specific steps are as follows:

[0094] (1) Constructing pTD-GAPDH cg F100V / P192S Mutant plasmid:

[0095] pTD-GAPDH cg F100V Using GAPDH as a template cg F100V / P192S -F / GAPDH cg F100V / P192S -R is a primer, and PCR amplification yields pTD-GAPDH. cg F100V / P192S The PCR product was digested with Dpn I at 37°C for 1 hour, purified using a Clean Up kit, and then transformed into E. coli DH5α. The product was plated onto LB agar plates containing 50 mg / L spectinomycin and incubated upside down at 37°C for 20 hours. Preliminary colony PCR verification was performed, followed by sequencing verification of pTD-GAPDH. cgF100V / P192S The correctness of the plasmid was verified, and the plasmid pTD-GAPDH was obtained. cg F100V / P192S ;

[0096] (2) The pCas9 plasmid was introduced into the competent cells of strain OSH-2 constructed according to the method for preparing competent cells in Example 1 (6).

[0097] (3) Take 5 μL of the pTD-GAPDH constructed in step (1). cg F100V / P192S The plasmid was mixed with 100 μL of electroporation competent cells from step (2) and transferred into a 2 mm electroporation cuvette. The mixture was incubated on ice for 1 min and then electroporated using a MicroPluser™ (BIO-RAD) at 2500 V. Immediately after electroporation, 700 μL of pre-cooled LB medium (4°C) was added, mixed thoroughly, and immediately transferred to a new sterile 1.5 mL EP tube. The tube was incubated at 30°C and 150 rpm for 3 h with shaking. The culture was then spread onto LB solid medium containing 50 mg / L kanamycin and 50 mg / L spectinomycin and incubated upside down at 30°C for 24 h. Colony PCR was performed using YZ-GAPDH-F / YZ-GAPDH-R primers to verify the colony PCR, and sequencing was used to verify the correctness of the strain construction. The strain OSH-5 (OSH-2, GAPDH) was successfully constructed. cg F100V / P192S ).

[0098] Example 6: Shake-flask fermentation of OSH engineered strain

[0099] (1) The plasmid pTrc99a-metA-yjeH was transformed into competent cells of strains OSH0 to OSH5 to construct strains OSH-0 / pTrc99a-metA-yjeH, OSH-1 / pTrc99a-metA-yjeH, OSH-2 / pTrc99a-metA-yjeH, OSH-3 / pTrc99a-metA-yjeH, OSH-4 / pTrc99a-metA-yjeH, and OSH-5 / pTrc99a-metA-yjeH, which were denoted as strains OSH-0A, OSH-1A, OSH-2A, OSH-3A, OSH-4A, and OSH-5A.

[0100] (2) The constructed strains OSH-0A, OSH-1A, OSH-2A, OSH-3A, OSH-4A, and OSH-5A were verified in fermentation medium. Two single colonies were picked and cultured overnight at 37°C and 180 rpm in LB broth to prepare seed culture. 1 mL of seed culture was inoculated into a 500 mL shake flask containing 20 mL of fermentation medium and shaken at 30°C and 180 rpm until OD was reached.600 =0.5, add 0.2mM IPTG to a final concentration, and incubate at 30℃ with shaking at 180rpm for 48h. After fermentation, take 1mL of fermentation broth, centrifuge at 12000rpm for 3min, discard all supernatant, add 1mL of distilled water to resuspend the bacterial cells and calcium carbonate, centrifuge at 12000rpm for 3min and discard supernatant, add another 1mL of distilled water to resuspend the bacterial cells and calcium carbonate, centrifuge at 12000rpm for 3min and discard supernatant. Finally, add 800μL of distilled water to resuspend the bacterial cells and calcium carbonate, then add 200μL of 20% acetic acid aqueous solution, and let stand at room temperature for 5min to dissolve the calcium carbonate. Take 100μL of the bacterial solution with dissolved calcium carbonate and add it to 1900μL of distilled water, dilute 20 times, and finally measure the biomass OD using a spectrophotometer. 600 OSH was detected using a Hitachi L8080 amino acid analyzer; specific methods can be found in the L8080 operation manual.

[0101] Biomass OD of strains OSH-0A, OSH-1A, OSH-2A, OSH-3A, OSH-4A, and OSH-5A 600 And the content of OSH, such as Figure 1 As shown. Biomass OD of strain OSH-2A 600 The OD value was 5.58, and the OSH content was 15.27 g / L. The biomass OD of strain OSH-5A was... 600 The value was 5.56, and the OSH content was 16.19 g / L. (From...) Figure 1 As can be seen, compared with strain OSH-0A, strain OSH-5A increased the shake-flask yield of OSH from 13.33 g / L to 16.19 g / L, representing a 21.5% increase in OSH yield. This demonstrates that by utilizing the CRP mutant that relieves glucose repression, glucose utilization and biosynthesis can be promoted. E. coli cells regulated by this beneficial CRP mutant can serve as a favorable substrate for the biosynthesis of natural products. Secondly, the introduction of exogenous glyceraldehyde-3-phosphate dehydrogenase and its mutant provides more NADPH supply to the metabolic pathway, effectively enhancing OSH accumulation.

[0102] Example 7: Fed-feed fermentation of strains OSH-2A and OSH-5A in a 5 L fermenter

[0103] (1) Strawberries OSH-2A and OSH-5A were streaked onto LB agar plates containing 50 mg / L kanamycin resistance and incubated overnight at 37°C. Single colonies were picked and transferred to LB tubes containing 50 mg / L kanamycin resistance and incubated overnight at 37°C and 150 rpm to prepare seed culture. The OSH-2A and OSH-5A seed cultures were inoculated into 100 mL of LB medium at a volume concentration of 5% and incubated overnight at 37°C and 150 rpm to prepare secondary seed cultures. The OSH-2A and OSH-5A secondary seed cultures were inoculated into 5-L fermenters containing 2 L of fermentation medium at a volume concentration of 15% and IPTG was added to a final concentration of 0.2 mM. Fermentation was carried out at 30℃, 500 rpm, and an aeration rate of 0.5 V / V·min. When the pH value was higher than 6.80 (the initial sugar in the fermenter was consumed), the automatic feeding was turned on, and the feeding medium was added at a rate of 25 mL / h until the pH value was lower than 6.80. The feeding was then stopped. The residual sugar in the fermenter was kept at a low level, and the sugar concentration was maintained at 0-5 g / L.

[0104] (2) After genetic engineering, the genetically engineered bacteria OSH-2A and OSH-5A, which produce high levels of O-succinyl-L-homoserine, achieved OSH yields of 101.9 g / L and 107.1 g / L, respectively, through fed-batch fermentation in a 5L fermenter. Figure 2 , Figure 3 Therefore, the OSH-producing genetically engineered bacteria constructed in this invention can effectively accumulate OSH in the fermentation broth during fermentation, laying the foundation for constructing genetically engineered strains that produce high levels of OSH.

[0105] Table 2. Primer sequences required for genome editing

[0106]

[0107] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A method for constructing a genetically engineered bacterium that produces high levels of O-succinyl-L-homoserine, characterized in that, The method includes: using CRISPR-CAS9 technology to introduce the coding gene of a CRP mutant that relieves glucose repression into the genome of Chameleon spp. The CRP mutant is a CRP with a nucleotide sequence as shown in SEQ ID NO.

3. R83F / G142P ; The chassis bacteria are E. coli W3110 ΔmetI ΔmetJ ΔthrB ΔmetB ΔldhA ΔadhE ΔpflB Ptrc-metL ΔarcA ΔiclR / pTrc99a-metA-yjeH .

2. The method as described in claim 1, characterized in that, The method further includes: using CRISPR-CAS9 technology to introduce the coding gene of a heterologous GAPDH mutant into the genome of *Bacillus subtilis*; the GAPDH mutant is used to increase the supply of NADPH; The GAPDH mutant is Corynebacterium glutamicum The nucleotide sequence of the source is GAPDH as shown in SEQ ID NO.

7. cg F100V ,or Corynebacterium glutamicum The nucleotide sequence of the source is GAPDH as shown in SEQ ID NO.

8. cg F100V / P192S .

3. The genetically engineered bacterium that produces high levels of O-succinyl-L-homoserine, constructed by the method described in any one of claims 1 or 2.

4. The genetically engineered bacterium producing high levels of O-succinyl-L-homoserine as described in claim 3, characterized in that, The genetically engineered bacterium that produces high levels of O-succinyl-L-homoserine is: E. coli W3110 ΔmetI ΔmetJ ΔthrB ΔmetB ΔldhA ΔadhE ΔpflB Ptrc-metL ΔarcA ΔiclR crp R83F / G142P / pTrc99a -metA-yjeH ;or E. coli W3110 ΔmetI ΔmetJ ΔthrB ΔmetB ΔldhA ΔadhE ΔpflB Ptrc-metL ΔarcA ΔiclR crp R83F / G142P gapdh cg F100V / P192S / pTrc99a -metA-yjeH 。 5. The application of the genetically engineered bacteria with high O-succinyl-L-homoserine production as described in claim 4 in the production of O-succinyl-L-homoserine.

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