Recombinant escherichia coli with high yield of o-succinyl-l-homoserine and construction method and application thereof

Abstract

The application discloses a kind of high-yield O-succinyl-L-homoserine recombinant escherichia coli and its construction method and application, belong to genetic engineering and fermentation engineering technical field.The recombinant escherichia coli is expressed by expressing anti-feedback inhibition homoserine O-succinyltransferase metA fbr , aspartate ammonia-lyase aspA, aspartate transaminase aspC, 2 ketoglutarate decarboxylase sucA, dihydrothioctic acid succinyltransferase sucB and efflux protein yjeH, knock out metJ, improve intracellular NADPH availability, to enhance OSH synthesis pathway metabolic flow, precursor succinyl coenzyme A supply and product efflux.Using 5 L bioreactor for fed-batch fermentation, after fermentation 60 h, OSH production is as high as 137.4 g / L, and sugar acid conversion rate reaches 50.5%.The application provides engineering strain and technical realization path for the efficient fermentation of OSH.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a recombinant Escherichia coli that produces high levels of O-succinyl-L-homoserine, its construction method, and its applications. Background Technology

[0002] O-succinyl-L-homoserine (OSH) is an important intermediate in the microbial L-methionine synthesis pathway, playing a crucial metabolic role in microorganisms such as *Escherichia coli*. OSH can not only be used for L-methionine synthesis but can also be further converted to synthesize various C4 compounds such as succinic acid, homoserine lactone, γ-butyrolactone, and 1,4-butanediol, demonstrating high application value and wide applicability in pharmaceuticals, animal feed, agriculture, and food. Furthermore, OSH can react with methanethiol under the catalysis of O-succinyl-L-homoserine thiotransferase to produce L-methionine and succinic acid; therefore, OSH is also an important precursor in the microbial manufacturing of L-methionine. The global market demand for L-methionine continues to grow. As an essential amino acid, its applications in food, animal feed, cosmetics, and pharmaceuticals are increasingly widespread, with a global market size reaching approximately US$5 billion. Against this backdrop, the development of green and efficient OSH biomanufacturing technology has become a focus of attention for both academia and industry.

[0003] With the development of green manufacturing concepts, the production of OSH using biological methods has received increasing attention. In recent years, studies have constructed OSH-producing strains by knocking out negative transcriptional regulators, blocking OSH consumption pathways, and inhibiting competitive bypass pathways. Furthermore, by enhancing the precursor synthesis pathways of aspartic acid and homoserine, modifying the pentose phosphate pathway and glycolysis pathway to increase NADPH supply, and combining adaptive evolution and fermentation regulation strategies, a high level of OSH fermentation production has been achieved, with an OSH yield of 121.7 g / L in a 50 L fermenter (J Agric Food Chem. 2025, 73(3): 2068-2076). Other studies have improved OSH fermentation yield by systematically optimizing precursor accumulation via the L-aspartic acid and L-homoserine pathways, achieving OSH yields of 131.99 g / L (1.18 g / L / h) and 131.06 g / L in 5 L and 50 L fermenters, respectively (Biochem Eng J. 2025, 223: 109864). Furthermore, studies have utilized a layered modification strategy to construct a non-auxotrophic OSH-producing strain in probiotic *E. coli* Nissle 1917, obtaining an OSH yield of 80.79 g / L and a production rate of 1.84 g / L / h in a 5 L bioreactor. Biochem Eng J. 2025, 222: 109852).

[0004] Nevertheless, existing OSH biosynthesis technologies still suffer from problems such as insufficient fermentation yield and sugar-acid conversion rate, inadequate metabolic flux distribution, carbon flux loss due to byproduct accumulation, and insufficient precursor supply, NADPH balance, and product efflux capacity. Therefore, developing a recombinant Escherichia coli and its fermentation production method that can further improve OSH yield and sugar-acid conversion rate remains a pressing technical problem to be solved in this field. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention enhances the metabolic flux of the O-succinyl-L-homoserine (OSH) synthesis pathway in *E. coli* through metabolic pathway engineering, transport engineering, cofactor engineering, and fermentation process optimization. This improves precursor supply, product efflux capacity, and intracellular reducing power, thereby increasing OSH fermentation yield and sugar-acid conversion rate. Specifically, this invention utilizes the pTrc99a plasmid to express the feedback-inhibition-resistant homoserine O-succinyltransferase encoding gene. metA fbr Knockout of the gene encoding the repressor protein metJ Strengthen the gene encoding aspartate aminopyrate lyase aspA and aspartate transaminase encoding gene aspC The expression of [the gene] was enhanced to increase the carbon flux for OSH synthesis; the expression of the 2-ketoglutarate decarboxylase encoding gene was strengthened. sucA and the gene encoding dihydrolipoic acid succinyltransferase sucB The expression of these genes is increased to enhance the supply of the precursor succinyl-CoA and to increase the expression of genes encoding efflux proteins. yjeH The expression of [the gene] promotes the efflux of OSH; simultaneously, it enhances the expression of the pyridine nucleotide transhydrogenase-encoding gene. pntAB The expression of [a specific ingredient] was enhanced to improve the availability of intracellular reducing power NADPH and maintain cofactor balance. Further optimization of fed-batch fermentation conditions in a 5 L bioreactor resulted in an engineered strain with a clear genetic background capable of stable and efficient OSH production. After 60 h of fermentation in a 5 L fermenter, the OSH yield reached 137.4 g / L, with a sugar-acid conversion rate of 50.5%.

[0006] This invention provides a recombinant *E. coli* strain that increases the production of O-succinyl-L-homoserine, wherein the recombinant *E. coli* strain is an L-homoserine-producing *E. coli* strain with the repressor protein coding gene knocked out. metJ It also expressed homoserine O-succinyltransferase, aspartate aminotransferase, aspartate aminotransferase, 2-ketoglutarate decarboxylase, dihydrolipoamide succinyltransferase, OSH transporter, and pyridine nucleotide transhydrogenase derived from Escherichia coli.

[0007] In one embodiment of the present invention, the amino acid sequence of the homoserine O-succinyl transferase is shown in SEQ ID NO. 8, the amino acid sequence of the aspartate amino acid lyase is shown in SEQ ID NO. 9, the amino acid sequence of the aspartate transaminase is shown in SEQ ID NO. 10, the amino acid sequence of the 2-ketoglutarate decarboxylase is shown in SEQ ID NO. 11, the amino acid sequence of the dihydrolipoamide succinyl transferase is shown in SEQ ID NO. 12, the amino acid sequence of the OSH transporter is shown in SEQ ID NO. 13, and the amino acid sequence of the pyridine nucleotide transhydrogenase is shown in SEQ ID NO. 14-15.

[0008] In one embodiment of the present invention, the gene encoding the homoserine O-succinyltransferase is... metA fbr The nucleotide sequence is shown in SEQ ID NO.2, and the gene encoding the aspartate aminopyase is... aspA The nucleotide sequence is shown in SEQ ID NO.3, and the gene encoding the aspartate transaminase is... aspC The nucleotide sequence is shown in SEQ ID NO.4, which encodes the genes for 2-ketoglutarate decarboxylase and dihydrolipoic acid succinyltransferase. sucAB The nucleotide sequence is shown in SEQ ID NO.5, and the gene encoding the OSH transporter protein is... yjeH The nucleotide sequence is shown in SEQ ID NO.6, and the gene encoding the pyridine nucleotide transhydrogenase is... pntAB The nucleotide sequence is shown in SEQ ID NO.7.

[0009] In one embodiment of the present invention, the aspartate aminotransferase, aspartate transaminase, 2-ketoglutarate decarboxylase, dihydrolipoamide succinyltransferase, OSH transporter, and pyridine nucleotide transhydrogenase all employ promoters. P trc Regulation is used to express this.

[0010] In one embodiment of the present invention, the aspA Integration ycdN Gene loci, the aspC Integration yeeP Gene loci, the sucAB Integration ygaY Gene loci, the yjeH Integration metI Gene loci, the pntAB Integrate separately into ilvG , yghX and / or yjiP Gene loci.

[0011] In one embodiment of the present invention, the promoter P trc The nucleotide sequence is shown in SEQ ID NO.1.

[0012] In one embodiment of the present invention metJ Gene, ycdN Gene, yeeP Gene, ygaY Gene, metI Gene, ilvG Gene, yghX Gene, yjiP The gene IDs on NCBI are: 948435, 948956, 946524, 2847696, 944894, 2847699, 2847694, and 38094982.

[0013] In one embodiment of the present invention, the recombinant Escherichia coli is constructed using Escherichia coli H0 as the starting strain, and the feedback-inhibition-resistant homoserine O-succinyltransferase encoding gene is expressed via the pTrc99a plasmid. metA fbr Knockout of the gene encoding the repressor protein metJ and overexpress the gene encoding aspartate aminopyase. aspA Aspartate transaminase encoding gene aspC 2-Ketoglutarate decarboxylase encoding gene sucA The gene encoding dihydrolipoamide succinyltransferase sucB OSH transporter protein encoding gene yjeH and the gene encoding pyridine nucleotide transhydrogenase pntAB ; The homoserine O-succinyltransferase encoding gene is described above. metA fbr The mutation sites are R27C, I296S, and P298L, and plasmid pTrc99a- was constructed based on these sites. metA fbr The promoter P trc Controlled aspA Integration ycdN Gene locus, the promoter P trc Controlled aspC Integration yeeP Gene locus, the promoter P trc Controlled sucAB Integration ygaY Gene locus, the promoter Ptrc Controlled yjeH Integration metI Gene locus, the promoter P trc Controlled pntAB Integrate separately into ilvG , yghX and yjiP Gene loci.

[0014] In one embodiment of the present invention, the gene encoding the homoserine O-succinyltransferase is... metA fbr The nucleotide sequence is shown in SEQ ID NO.2, and the gene encoding the aspartate aminopyase is... aspA The nucleotide sequence is shown in SEQ ID NO.3, and the gene encoding the aspartate transaminase is... aspC The nucleotide sequence is shown in SEQ ID NO.4, which encodes the genes for 2-ketoglutarate decarboxylase and dihydrolipoic acid succinyltransferase. sucAB The nucleotide sequence is shown in SEQ ID NO.5, and the gene encoding the OSH transporter protein is... yjeH The nucleotide sequence is shown in SEQ ID NO.6, and the gene encoding the pyridine nucleotide transhydrogenase is... pntAB The nucleotide sequence is shown in SEQ ID NO.7.

[0015] In one embodiment of the present invention, the starting strain is Escherichia coli H0.

[0016] This invention also provides a method for increasing the yield of O-succinyl-L-homoserine from Escherichia coli, the method comprising: Starting with L-homoserine-producing *Escherichia coli* strains, the gene encoding the repressor protein in the genome was knocked out. metJ It also expressed homoserine O-succinyltransferase, aspartate aminotransferase, aspartate aminotransferase, 2-ketoglutarate decarboxylase, dihydrolipoamide succinyltransferase, OSH transporter, and pyridine nucleotide transhydrogenase derived from Escherichia coli.

[0017] In one embodiment of the present invention, the amino acid sequence of the homoserine O-succinyl transferase is shown in SEQ ID NO. 8, the amino acid sequence of the aspartate amino acid lyase is shown in SEQ ID NO. 9, the amino acid sequence of the aspartate transaminase is shown in SEQ ID NO. 10, the amino acid sequence of the 2-ketoglutarate decarboxylase is shown in SEQ ID NO. 11, the amino acid sequence of the dihydrolipoamide succinyl transferase is shown in SEQ ID NO. 12, the amino acid sequence of the OSH transporter is shown in SEQ ID NO. 13, and the amino acid sequence of the pyridine nucleotide transhydrogenase is shown in SEQ ID NO. 14-15.

[0018] In one embodiment of the present invention, the gene encoding the homoserine O-succinyltransferase is... metA fbr The nucleotide sequence is shown in SEQ ID NO.2, and the gene encoding the aspartate aminopyase is... aspA The nucleotide sequence is shown in SEQ ID NO.3, and the gene encoding the aspartate transaminase is... aspC The nucleotide sequence is shown in SEQ ID NO.4, which encodes the genes for 2-ketoglutarate decarboxylase and dihydrolipoic acid succinyltransferase. sucAB The nucleotide sequence is shown in SEQ ID NO.5, and the gene encoding the OSH transporter protein is... yjeH The nucleotide sequence is shown in SEQ ID NO.6, and the gene encoding the pyridine nucleotide transhydrogenase is... pntAB The nucleotide sequence is shown in SEQ ID NO.7.

[0019] In one embodiment of the present invention, the aspartate aminotransferase, aspartate transaminase, 2-ketoglutarate decarboxylase, dihydrolipoamide succinyltransferase, OSH transporter, and pyridine nucleotide transhydrogenase all employ promoters. P trc Regulation is used to express this.

[0020] In one embodiment of the present invention, the aspA Integration ycdN Gene loci, the aspC Integration yeeP Gene loci, the sucAB Integration ygaY Gene loci, the yjeH Integration metI Gene loci, the pntAB Integrate separately into ilvG , yghX and / or yjiP Gene loci.

[0021] In one embodiment of the present invention, the promoter P trc The nucleotide sequence is shown in SEQ ID NO.1.

[0022] In one embodiment of the present invention metJ Gene, ycdN Gene, yeeP Gene, ygaY Gene, metI Gene, ilvG Gene, yghX Gene, yjiP The gene IDs on NCBI are: 948435, 948956, 946524, 2847696, 944894, 2847699, 2847694, and 38094982.

[0023] The present invention also provides a method for constructing the above-mentioned recombinant Escherichia coli, the method comprising the following steps, wherein the steps may be performed in any order: (1) Knockout of E. coli H0 metJ Gene; (2) P trc - aspA Integration ycdN Gene loci; (3) P trc - aspC Integration yeeP Gene loci; (4) P trc - sucAB Integration ygaY Gene loci; (5) P trc - yjeH Integration metI Gene loci; (6) P trc - pntAB Integrate separately into ilvG , yghX and yjiP Gene loci.

[0024] This invention uses Escherichia coli as the starting strain and systematically modifies and reconstructs the OSH biosynthesis pathway and related metabolic pathways to obtain a genetically engineered strain with a clear genetic background that can stably and efficiently produce OSH.

[0025] The genetically engineered strain obtained in this invention enhances OSH synthesis throughput and precursor supply, optimizes central metabolic carbon allocation, promotes product efflux, and improves intracellular reducing power NADPH availability, thereby improving the fermentation production level of OSH.

[0026] The present invention also provides the application of the above-mentioned recombinant Escherichia coli in the preparation of OSH.

[0027] In one embodiment of the present invention, glucose is used as a substrate and the above-mentioned recombinant Escherichia coli is fermented to prepare OSH.

[0028] In one embodiment of the present invention, during shake-flask fermentation, the activated strain is cultured at 35-37°C and 180-220 r / min to obtain a seed culture, which is then inoculated into the fermentation medium at an inoculum rate of 10-20%. The fermentation temperature is 35-37°C, the rotation speed is 180-220 r / min, and the pH is 6.5-6.7. When the glucose in the culture medium is depleted, 600 g / L glucose solution is added. The induction culture temperature was 30-32℃, the rotation speed was 180-220 r / min, and the pH was 6.5-6.7. When the glucose in the culture medium was depleted, 600 g / L glucose solution was added to maintain fermentation. The fermentation time was 24-48 h. The seed culture medium consisted of: 30 g / L glucose, 10 g / L yeast extract, 4 g / L (NH4)2SO4, 3 g / L KH2PO4, 2 g / L MgSO4·7H2O, 2 g / L sodium citrate, 30 mg / L FeSO4·7H2O, and 1 mg / L L sodium citrate. H and 1 mg / LV B1 ; The fermentation medium comprises the following components: 10-20 g / L glucose, 5-10 g / L yeast extract, 1-5 g / L (NH4)2SO4, 1-3 g / L KH2PO4, 1-2 g / L MgSO4·7H2O, 1-2 g / L citric acid or citrate, 20-30 mg / L FeSO4·7H2O, and 0.5-1.0 mg / L L... H and 0.5~1.0 mg / LV B1 Preferably, the fermentation medium consists of: 20 g / L glucose, 5 g / L yeast extract, 5 g / L (NH4)2SO4, 3 g / L KH2PO4, 2 g / L MgSO4·7H2O, 2 g / L sodium citrate, 30 mg / L FeSO4·7H2O, and 1 mg / L sodium citrate. H and 1 mg / LV B1 .

[0029] In one embodiment of the present invention, during fermentation in a fermenter, activated recombinant *E. coli* is cultured in a seed culture medium to obtain a seed solution, which is then inoculated into the fermentation culture medium at an inoculation rate of 10-20% for fermentation. During the seed culture stage, the culture temperature is 35-37℃, the pH is 6.5-6.7, and the dissolved oxygen is controlled at 25-35% by adjusting the stirring speed and aeration rate. During the fermentation stage, the culture temperature is 35-37℃, the pH is 6.5-6.7, the dissolved oxygen is controlled at 25-35%, and the glucose residual concentration in the fermentation broth is controlled at 1-10 g / L using a fed-batch feeding method. When the OD600 reaches 20, IPTG is added for induction, with a final IPTG concentration of 0.5 mM. After induction, the culture temperature is 30-32℃, the pH is 6.5-6.7, the dissolved oxygen is controlled at 25-35%, and the glucose residual concentration in the fermentation broth is controlled at 1-10 g / L using a fed-batch feeding method. The fermentation time is preferably 48-72 h. The seed culture medium consisted of: 30 g / L glucose, 10 g / L yeast extract, 4 g / L (NH4)2SO4, 3 g / L KH2PO4, 2 g / L MgSO4·7H2O, 2 g / L sodium citrate, 30 mg / L FeSO4·7H2O, and 1 mg / L sodium citrate. H and 1 mg / LV B1 ; The fermentation medium comprises the following components: 10-20 g / L glucose, 5-10 g / L yeast extract, 1-5 g / L (NH4)2SO4, 1-3 g / L KH2PO4, 1-2 g / L MgSO4·7H2O, 1-2 g / L citric acid or citrate, 20-30 mg / L FeSO4·7H2O, and 0.5-1.0 mg / L L... H and 0.5~1.0 mg / LV B1 Preferably, the fermentation medium comprises: 20 g / L glucose, 5 g / L yeast extract, 5 g / L (NH4)2SO4, 3 g / L KH2PO4, 2 g / L MgSO4·7H2O, 2 g / L sodium citrate, 30 mg / L FeSO4·7H2O, 1 mg / L L... H and 1 mg / LV B1 ; The fermentation process can be carried out using a fed-batch culture method. The composition of the culture medium is: 5-10 g / L yeast extract, 1-5 g / L (NH4)2SO4, 1-3 g / L KH2PO4, 1-2 g / L MgSO4·7H2O, 1-2 g / L citric acid or citrate, 20-30 mg / L FeSO4·7H2O, and 0.5-1.0 mg / L LV.H and 0.5~1.0 mg / LV B1 The feeding time of the fed-batch medium is 14-20 h after the start of fermentation.

[0030] The present invention also provides the application of the above-mentioned recombinant Escherichia coli in the preparation of O-succinyl-L-homoserine or products containing O-succinyl-L-homoserine.

[0031] Beneficial effects By adopting the above technical solution, the present invention has at least the following beneficial effects: This invention provides a recombinant *Escherichia coli* strain that enhances O-succinyl-L-homoserine (OSH) production, its construction method, and its applications. The recombinant *E. coli* strain expresses a feedback-inhibition-resistant homoserine O-succinyltransferase encoding gene. metA fbr Knockout of the gene encoding the repressor protein metJ and strengthen aspA , aspC , sucA , sucB , yjeH and pntAB The expression of [specific enzyme name] was enhanced, thereby increasing the carbon flux of the OSH synthesis pathway, improving the supply of succinyl-CoA, promoting OSH efflux, and increasing intracellular NADPH availability to maintain cofactor balance. Further optimization of fed-batch fermentation conditions in a 5 L bioreactor resulted in a genetically engineered strain with a clear genetic background capable of stable and efficient OSH production. After 60 h of fermentation in a 5 L fermenter, the OSH yield reached 137.4 g / L, and the sugar-acid conversion rate reached 50.5%.

[0032] The above description is only an overview of the technical solution of the present invention. In order to make the technical means, technical features, objectives and beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0033] To make the objectives, technical solutions, and beneficial concepts of this invention clearer, the invention will be further described below with reference to the accompanying drawings.

[0034] Figure 1 This is a diagram showing the results of shake-flask fermentation of the OSH genetically engineered strain. Figure 2 This is a graph showing the fermentation process of strain W8 in a 5 L fermenter using a fed-batch method. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0036] To further illustrate the technical means and effects adopted by the present invention to achieve its intended purpose, the specific embodiments, structures, features, and effects of the present invention will be described in detail below with reference to the accompanying drawings and preferred embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0037] The starting strains involved in the following examples Escherichia coli The genotype of H0 is: E. coli W3110, Δ lacI, ycgH::P trc -thrA fbr Δ thrB P thrABC ::P fliC ydeU::P trc -thrA fbr yjhE::P trc - thrA fbr P ppc ::P trc ,in, lacI, ycgH, thrB, ydeU, yjhE, The Gene IDs on NCBI (genome NC_000913.3) are 945007, 2847703, 947498, 7751623, and 948810, respectively; Escherichia coli H 0 is described in the paper titled "Development of a nonauxotrophic L-homoserine hyperproducer in Escherichiacoli by systems metabolic engineering," and is named H06 in the paper.

[0038] The matters involved thrA fbr The amino acid sequence is as follows: MRVLKFGGTSVANAERFLRVADILESNARQGQVATVLSAPAKITNHLVAMIEKTISGQDALPNISDAERIFAELLTGLAAAQPGFPLAQLKTFVDQEFAQIKHVLHGISLLGQCPDSINAALICRGEKMSIAIMAGVLEARGHNVTVIDPVEKLLAVGHYLESTVDIAESTRRIAASRIPADHMVLMAGFTAGNEKGELVVLGRNGSDYSAAVLAACLRADCCEIWTDVDGVYTCDPRQVPDARLLKSMSYQEAMELSYFGAKVLHPRTITPIAQFQIPCLIKNTGNPQAPGTLIGASRDEDELPVKGISNLNNMAMFSVSGPGMKGMVGMAARVFAAMSRARIFVVLITQSSSEYSISFCVPQSDCVRAERAMQEEFYLELKEGLLEPLAVTERLAIISVVGDGMRTLRGISAKFFAALARANINIVAIAQGSSERSISVVVNNDDATTGVRVTHQMLFNTDQVIEVFVIGVGGVGGALLEQLKRQQSWLKNKHIDLRVCGVANSKALLTNVHGLNLENWQEELAQAKEPFNLGRLIRLVKEYHLLNPVIVDCTSSQAVADQYADFLREGFHVVTPNKKANTSSMDYYHQLRYAAEKSRRKFLYDTNVGAGLPVIENLQNLLNAGDELMKFSGILSGSLSYIFGKLDEGMSFSEATTLAREMGYTEPDPRDDLSGMDVARKLLILARETGRELELADIEIEPVLPAEFNAEGDVAAFMANLSQLDDLFAARVAKARDEGKVLRYVGNIDEDGVCRVKIAEVDGNDPLFKVKNGENALAFYSHYYQPLPLVLRGYGAGNDVTAAGVFADLLRTLSWKLGV* The promoter P involved flic The nucleotide sequence is as follows: aaaaaatggctgtttttgaaaaaaattctaaaggttgttttacgacagacgataacagggttgacggcgattgagccgacgggtggaaacccaatacgtaatcaacgacttgcaatataggataacgaatc The promoter P involved thrABC The nucleotide sequence is as follows: cgcgtacaggaaacacagaaaaaagcccgcacctgacagtgcgggctttttttttcgaccaaaggtaacgaggtaacaacc The detection methods involved in the following embodiments are as follows: OSH production measurement: (1) Sample preparation: The fermentation broth was centrifuged at 12000 rpm for 10 min and the supernatant was collected. The supernatant was diluted with deionized water and then filtered through an aqueous filter membrane with a pore size of 0.22 μm.

[0039] (2) Sample detection: The amino acids in the fermentation broth were determined using an Agilent 1260 high-performance liquid chromatography (HPLC) system via pre-column derivatization with o-phthalaldehyde (OPA). The column model was Poroshell HPH-C18. The mobile phase preparation method was as follows: Phase A: 8 g of anhydrous sodium acetate was dissolved in 1 L of ddH2O, 225 μl of triethylamine was added, the pH was adjusted to 7.2 ± 0.5, and finally 5 ml of tetrahydrofuran was added. Phase B: 86 g of anhydrous sodium acetate was dissolved in 200 mL of ddH2O, the pH was adjusted to 7.2 ± 0.5, and finally 400 ml of methanol and 400 ml of acetonitrile were added.

[0040] The program settings are shown in Table 1 below: Table 1: Program Parameters

[0041] The following examples illustrate the calculation method for sugar-acid conversion rate:

[0042] The primers used in the following examples are shown in Table 2 below: Table 2: Primers

[0043] Example 1: Construction of genetically engineered strains W1-W8 1) Plasmid pTrc99a-metA fbr Construction by E . coli Using the W3110 genome as a template, according to metA The gene sequence was determined, primers metA-F and metA-R were designed, and the gene fragment was obtained by PCR amplification. metA The gene fragment was ligated into plasmid pTrc99a to construct plasmid pTrc99a- metA To construct plasmid pTrc99a- metA Using the template, site-directed mutagenesis was performed using the mutation primers R27C-F and R27C-R to obtain the plasmid pTrc99a- metA R27C Then, using plasmid pTrc99a- metA R27C The template was used for site-directed mutagenesis with the mutation primers I296S-F and P298L-R to obtain the plasmid pTrc99a- metA fbr (pTrc99a- metA R27C / I296S / P298L ). pTrc99a- metA fbr Transformed into strain E . coli From W3110 H0, the engineered strain W0 ( E . coli W3110 H0 / pTrc99a- metA fbr ).

[0044] Homoserine O-succinyltransferase encoding gene metA fbrNucleotide sequence (SEQ ID NO.2): atgccgattcgtgtgccggacgagctacccgccgtcaatttcttgcgtgaagaaaacgtctttgtgatgacaacttcttgtgcgtctggtcaggaaattcgtccacttaaggttctgatccttaacctgatgccgaagaagattgaaactgaaaatcagtttctgcgcctgctttcaaactcacctttgcaggtcgatattcagctgttgcgcatcgattcccgtgaatcgcgcaacacgcccgcagagcatctgaacaacttctactgtaactttgaagatattcaggatcagaactttgacggtttgattgtaactggtgcgccgctgggcctggtggagtttaatgatgtcgcttactggccgcagatcaaacaggtgctggagtggtcgaaagatcacgtcacctcgacgctgtttgtctgctgggcggtacaggccgcgctcaatatcctctacggcattcctaagcaaactcgcaccgaaaaactctctggcgtttacgagcatcatattctccatcctcatgcgcttctgacgcgtggctttgatgattcattcctggcaccgcattcgcgctatgctgactttccggcagcgttgattcgtgattacaccgatctggaaattctggcagagacggaagaaggggatgcatatctgtttgccagtaaagataagcgcattgcctttgtgacgggccatcccgaatatgatgcgcaaacgctggcgcaggaatttttccgcgatgtggaagccggactagacccggatgtaccgtataactatttcccgcacaatgatccgcaaaatacaccgcgagcgagctggcgtagtcacggtaatttactgtttaccaactggctcaactattacgtctaccagagcacgttatacgatctacggcacatgaatccaacgctggattaa The amino acid sequence of homoserine O-succinyltransferase is as follows (SEQ ID NO.8): MPIRVPDELPAVNFLREENVFVMTTTSRASGQEIRPLKVLILNLMPKKIETENQFLRLLSNSPLQVDIQLLRIDSRESRNTPAEHLNNFYCNFEDIQDQNFDGLIVTGAPLGLVEFNDVAYWPQIKQVLEWSKDHVTSTLFVCWAVQAALNILYG IPKQTRTEKLSGVYEHHILHPHALLTRGFDDSFLAPHSRYADFPAALIRDYTDLEILAETEEGDAYLFASKDKRIAFVTGHPEYDAQTLAQEFFRDVEAGLDPDVPYNYFPHNDPQNTPRASWRSHGNLLFTNWLNYYVYQSTLYDLRHMNPTLD 2) Knockout E . coli W3110 H0 metJ Gene by E . coli Using the W3110 genome as a template, based on its metJ Primers metJ-1 and metJ-2 for the upstream homologous arm and metJ-3 and metJ-4 for the downstream homologous arm of the gene (Gene ID 948435) were designed, and the gene was amplified by PCR. metJ The upstream and downstream homologous arm fragments of the gene were fused using overlap PCR to obtain... metJ Gene knockout fragment. The DNA fragment formed after annealing primers N20-metJ-F and N20-metJ-R was ligated to plasmid pGRB to construct plasmid pGRB-. metJ Plasmid pGRB- metJ and stated metJ The knockout fragments of the gene were co-electroporated into a gene containing pREDCas9. E . coli Positive transformants were obtained by screening W3110 H0 electrocompetent cells. After plasmid elimination, strain H1 was obtained. E . coli W3110 H0 Δ metJ The plasmid pTrc99a- obtained in step 1 was used... metA fbr Transformed into strain H1, resulting in engineered strain W1 ( E . coli W3110 H0 Δ metJ / pTrc99a- metA fbr ).

[0045] 3) P trc- aspA Integration ycdN gene locus by E . coli Using the W3110 genome as a template, according to ycdN Primers ycdN-1 and ycdN-2 for the upstream homologous arm and ycdN-3 and ycdN-4 for the downstream homologous arm were designed based on the gene sequence (Gene ID: 948956), and obtained by PCR amplification. ycdN Upstream and downstream homologous arms of the gene. Additionally... E . coli Using the W3110 genome as a template, according to aspA Primers aspA-F and aspA-R were designed based on the gene sequence, among which... P trc Promoter sequences were designed in primers ycdN-2 and aspA-F. The corresponding fragments were obtained by PCR amplification, and fusion PCR was performed using each fragment as a template to obtain... P trc - aspA Integrating the fragment. The DNA fragment formed after annealing primers N20-ycdN-F and N20-ycdN-R was ligated to plasmid pGRB to construct plasmid pGRB- ycdN Plasmid pGRB- ycdN and P trc - aspA The integrated fragments were co-electroporated into strain H1 containing pREDCas9 ( E . coli W3110 H0 Δ metJ Positive transformants were obtained by electroporation of competent cells, and after plasmid elimination, strain H2 was obtained. E . coli W3110 H0Δ metJ Δ ycdN ::P trc - aspA The plasmid pTrc99a- obtained in step 1 was used... metA fbr Transformed into strain H2 to obtain engineered strain W2 ( E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA / pTrc99a- metA fbr ).

[0046] promoter P trc The nucleotide sequence is shown below (SEQ ID NO.1): ttgacaattaatcatccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagacc The aspartate aminopyrase encoding gene aspA The nucleotide sequence is shown below (SEQ ID NO.3): The amino acid sequence of aspartate amino acid lyase is as follows (SEQ ID NO.9): MSNNIRIEEDLLGTREVPADAYYGVHTLRAIENFYISNNKISDIPEFVRGMVMVKKAAAMANKELQTIPKSVANAIIAACDEVLNNGKCMDQFPVDVYQGGAGTSVNMNTNEVLANIGL ELMGHQKGEYQYLNPNDHVNKCQSTNDAYPTGFRIAVYSSLIKLVDAINQLREGFERKAVEFQDILKMGRTQLQDAVPMTLGQEFRAFSILLKEEVKNIQRTAELLLEVNLGATAIGTGL NTPKEYSPLAVKKLAEVTGFPCVPAEDLIEATSDCGAYVMVHGALKRLAVKMSKICNDLRLLSSGPRAGLNEINLPELQAGSSIMPAKVNPVVPEVVNQVCFKVIGNDTTVTMAAEAGQL QLNVMEPVIGQAMFESVHILTNACYNLLEKCINGITANKEVCEGYVYNSIGIVTYLNPFIGHHNGDIVGKICAETGKSVREVVLERGLLTEAELDDIFSVQNLMHPAYKAKRYTDESEQ* 4) P trc- aspC Integration yeeP gene locus by E . coli Using the W3110 genome as a template, according to yeeP Design upstream homologous arm primers based on gene (Gene ID: 946524) sequence. yeeP -1h and yeeP -2 and downstream homologous arm primers yeeP -3 and yeeP -4, obtained through PCR amplification yeeP Upstream and downstream homologous arms of the gene. Additionally... E . coli Using the W3110 genome as a template, according to aspC Gene sequence design primers aspC -F and aspC -R, where P trc Promoter sequence design in primers yeeP -2 and aspC-F in. The corresponding fragments are obtained through PCR amplification, and fusion PCR is performed using each fragment as a template to obtain... P trc - aspC Integrate the fragment. Introduce primer N20- yeeP -F and N20- yeeP The DNA fragment formed after -R annealing was ligated to plasmid pGRB to construct plasmid pGRB- yeeP Plasmid pGRB- yeeP and stated P trc - aspC The integrated fragments are electrochemically converted into H2 containing pREDCas9 ( E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Positive transformants were obtained by screening electrocompetent cells of ) and, after plasmid elimination, strain H3 was obtained. E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Δ yeeP :: P trc - aspC The plasmid pTrc99a- obtained in step 1 was used... metA fbr Transformed into strain H3, resulting in engineered strain W3 ( E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Δ yeeP :: P trc - aspC / pTrc99a- metA fbr ).

[0047] The aspartate transaminase encoding gene aspC The nucleotide sequence is shown below (SEQ ID NO.4): The amino acid sequence of aspartate transaminase is as follows (SEQ ID NO.10): MFENITAAPADPILGLADLFRADERPGKINLGIGVYKDETGKTPVLTSVKKAEQYLLENETTKNYLGIDGIPEFGRCTQELLFGKGSALINDKRARTAQTPGGTGALRVAADFLAKNTSVKRVWVSNPSWPNHKSVFNSAGLEVREYAYYDAENHTLDFDALINSLNEAQAGDVVLFHGCCHNPTGIDPTLEQWQTLA QLSVEKGWLPLFDFAYQGFARGLEEDAEGLRAFAAMHKELIVASSYSKNFGLYNERVGACTLVAADSETVDRAFSQMKAAIRANYSNPPAHGASVVATILSNDALRAIWEQELTDMRQRIQRMRQLFVNTLQEKGANRDFSFIIKQNGMFSFSGLTKEQVLRLREEFGVYAVASGRVNVAGMTPDNMAPLCEAIVAVL* 5) P trc- sucAB Integration ygaY gene locus by E . coli Using the W3110 genome as a template, according to ygaY Design upstream homologous arm primers based on gene (Gene ID: 2847696) sequence. ygaY -1 and ygaY -2 and downstream homologous arm primers ygaY -3 and ygaY -4 was obtained through PCR amplification. ygaY Upstream and downstream homologous arms of the gene. Additionally... E . coli Using the W3110 genome as a template, according to sucAB Gene sequence design primers sucAB -F and sucAB -R, where P trc Promoter sequence design in primers ygaY -2 and sucAB In -F, the corresponding fragments are obtained through PCR amplification, and fusion PCR is performed using each fragment as a template to obtain... P trc - sucAB Integrate the fragment. Introduce primer N20- ygaY -F and N20- ygaY The DNA fragment formed after -R annealing was ligated to plasmid pGRB to construct plasmid pGRB- ygaY Plasmid pGRB- ygaY and stated P trc - sucAB The integrated fragments are electrochemically converted to H3 containing pREDCas9 ( E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Δ yeeP :: P trc - aspC Positive transformants were obtained by screening electrocompetent cells of ) and, after plasmid elimination, strain H4 was obtained. E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Δ yeeP :: P trc - aspC Δ ygaY :: P trc - sucAB The plasmid pTrc99a- obtained in step 1 was used... metA fbr Transformed into strain H4 to obtain engineered strain W4 ( E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Δ yeeP :: P trc - aspC Δ ygaY :: P trc - sucAB / pTrc99a- metA fbr ).

[0048] The genes encoding 2-ketoglutarate decarboxylase and dihydrolipoamide succinyltransferase sucAB The nucleotide sequence is shown below (SEQ ID NO.5): The amino acid sequence of 2-ketoglutaric acid decarboxylase is as follows (SEQ ID NO.11): MQNSALKAWLDSSYLSGANQSWIEQLYEDFLTDPDSVDANWRSTFQQLPGTGVKPDQFHSQTREYFRRLAKDASRYSSTISDPDTNVKQVKVLQLINAYRFRGHQHANLDPLGLWQQDKVADLDPSFHDLTEADFQETFNVGSFASGKETMKLGELLEALKQTYCGPIGAEYMHITSTEEKRWIQQRIESGRATFNSEEKKRFLSELTAAEGLERYLGAKFPGAKRFSLEGGDALIPMLKEMIRHAGNSGTREVVLGMAHRGRLNVLVNVLGKKPQDLFDEFAGKHKEHLGTGDVKYHMGFSSDFQTDGGLVHLALAFNPSHLEIVSPVVIGSVRARLDRLDEPSSNKVLPITIHGDAAVTGQGVVQETLNMSKARGYEVGGTVRIVINNQVGFTTSNPLDARSTPYCTDIGKMVQAPIFHVNADDPEAVAFVTRLALDFRNTFKRDVFIDLVCYRRHGHNEADEPSATQPLMYQKIKKHPTPRKIYADKLEQEKVATLEDATEMVNLYRDALDAGDCVVAEWRPMNMHSFTWSPYLNHEWDEEYPNKVEMKRLQELAKRISTVPEAVEMQSRVAKIYGDRQAMAAGEKLFDWGGAENLAYATLVDEGIPVRLSGEDSGRGTFFHRHAVIHNQSNGSTYTPLQHIHNGQGAFRVWDSVLSEEAVLAFEYGYATAEPRTLTIWEAQFGDFANGAQVVIDQFISSGEQKWGRMCGLVMLLPHGYEGQGPEHSSARLERYLQLCAEQNMQVCVPSTPAQVYHMLRRQALRGMRRPLVVMSPKSLLRHPLAVSSLEELANGTFLPAIGEIDELDPKGVKRVVMCSGKVYYDLLEQRRKNNQHDVAIVRIEQLYPFPHKAMQEVLQQFAHVKDFVWCQEEPLNQGAWYCSQHHFREVIPFGASLRYAGRPASASPAVGYMSVHQKQQQDLVNDALNVE* The amino acid sequence of dihydrolipoamide succinyltransferase is as follows (SEQ ID NO.12): MSSVDILVPDLPESVADATVATWHKKPGDAVVRDEVLVEIETDKVVLEVPASADGILDAVLEDEGTTVTSRQILGRLREGNSAGKETSAKSEEKASTPAQRQQASLEEQNNDALSPAIRRLLAEHNLDASAIKGTGVGGRLTREDVEKHLAKAPAKESAPAAAAPAAQPALAARSEKRVPMTRLRKRVAERLLEAKNSTAMLT TFNEVNMKPIMDLRKQYGEAFEKRHGIRLGFMSFYVKAVVEALKRYPEVNASIDGDDVVYHNYFDVSMAVSTPRGLVTPVLRDVDTLGMADIEKKIKELAVKGRDGKLTVEDLTGGNFTITNGGVFGSLMSTPIINPPQSAILGMHAIKDRPMAVNGQVEILPMMYLALSYDHRLIDGRESVGFLVTIKELLEDPTRLLLDV* 6) P trc - yjeH Integration metI gene locus by E . coli Using the W3110 genome as a template, according to metI Design upstream homologous arm primers based on gene (Gene ID: 944894) sequence. metI -1 and metI -2 and downstream homologous arm primers metI -3 and metI -4, obtained through PCR amplification metI Upstream and downstream homologous arms of the gene. Additionally... E . coli Using the W3110 genome as a template, according to yjeH Gene sequence design primers yjeH -F and yjeH -R, where, according to P trc Promoter sequence design in primers metI -2 and yjeH In -F, after obtaining the corresponding fragments through PCR amplification, fusion PCR is performed using each fragment as a template to obtain P. trc - yjeH Integrate the fragment. Introduce primer N20- metI-F and N20- metI The DNA fragment formed after -R annealing was ligated to plasmid pGRB to construct plasmid pGRB- metI Plasmid pGRB- metI and stated P trc - yjeH The integrated fragments are electrochemically converted to H4 containing pREDCas9 ( E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Δ yeeP :: P trc - aspC Δ ygaY :: P trc - sucAB Positive transformants were obtained by screening electrocompetent cells of ) and, after plasmid elimination, strain H5 was obtained. E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Δ yeeP :: P trc - aspC Δ ygaY :: P trc - sucAB Δ metI :: P trc - yjeH The plasmid pTrc99a- obtained in step 1 was used... metA fbr Transformed into strain H5 to obtain engineered strain W5 ( E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Δ yeeP :: P trc - aspC Δ ygaY :: P trc - sucAB Δ metI :: P trc - yjeH / pTrc99a- metA fbr ).

[0049] The OSH transporter protein encoding gene yjeH The nucleotide sequence is shown below (SEQ ID NO. 6): The amino acid sequence of the OSH transporter protein is as follows (SEQ ID NO.13): MSGLKQELGLAQGIGLLSTSLLGTGVFAVPALAALVAGNNSLWAWPVLIILVFPIAIVFAILGRHYPSAGGVAHFVGMAFGSRLERVTGWLFLSVIPVGLPAALQIAAGFGQAMFGWHSWQLLLAELGTLALVWYIGTRGASSSANLQTVIAGLIVALIVAIWWAGDIKPANIPFPAPGNIELTGLFAALSVMFWCFVGLEAFAHLASE FKNPERDFPRALMIGLLLAGLVYWGCTVVVLHFDAYGEKMAAAASLPKIVVQLFGVGALWIACVIGYLACFASLNIYIQSFARLVWSQAQHNPDHYLARLSSRHI PNNALNAVLGCCVVSTLVIHALEINLDALIIYANGIFIMIYLLCMLAGCKLLQGRYRLLAVVGGLLCVLLLAMVGWKSLYALIMLAGLWLLLPKRKTPENGITT* 7) P trc - pntAB Integrate separately into ilvG gene locus by E . coli Using the W3110 genome as a template, according to ilvG Design upstream homologous arm primers based on gene (Gene ID: 2847699) sequence. ilvG -1 and ilvG -2 and downstream homologous arm primers ilvG -3 and ilvG -4, obtained through PCR amplification ilvG Upstream and downstream homologous arms of the gene. Additionally... E . coli Using the W3110 genome as a template, according to pntAB Gene sequence design primers pntAB -F and pntAB -R, where P trc Promoter sequence design in primers ilvG -2 and pntAB -F in. The corresponding fragments are obtained through PCR amplification, and fusion PCR is performed using each fragment as a template to obtain... P trc - pntABIntegrate the fragment. Introduce primer N20- ilvG -F and N20- ilvG The DNA fragment formed after -R annealing was ligated to plasmid pGRB to construct plasmid pGRB- ilvG Plasmid pGRB- ilvG and stated P trc - pntAB The integrated fragments were co-electroplated into H5 electrocompetent cells containing pREDCas9, and positive transformants were obtained through screening. After plasmid elimination, strain H6 was obtained. E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Δ yeeP :: P trc - aspC Δ ygaY :: P trc - sucAB Δ metI :: P trc - yjeH Δ ilvG :: P trc - pntAB The plasmid pTrc99a- obtained in step 1 was used... metA fbr Transformation of strain H6 yielded engineered strain W6 ( E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Δ yeeP :: P trc - aspC Δ ygaY :: P trc - sucAB Δ metI :: P trc - yjeH Δ ilvG :: P trc - pntAB / pTrc99a- metA fbr ).

[0050] The pyridine nucleotide transhydrogenase encoding gene pntABThe nucleotide sequence is shown below (SEQ ID NO. 7): The amino acid sequence of pntA is as follows (SEQ ID NO.14): MRIGIPRERLTNETRVAATPKTVEQLLKLGFTVAVESGAGQLASFDDKAFVQAGAEIVEGNSVWQSEIILKVNAPLDDEIALLNPGTTLVSFIWPAQNPELMQKLAERNVTVMAMDSVPRISRAQSLDALSSMANIAGYRAIVEAAHEFGRFFTGQITAAGKVPPAKVMVIGAGVAGLAAIGAANSLGAIVRAFDTRPEVKEQVQSMGAEFLELDFKEEAGSGDGYAKVMSDAFIKAEMELFAAQAKEVDIIVTTALIPGKPAPKLITREMVDSMKAGSVIVDLAAQNGGNCEYTVPGEIFTTENGVKVIGYTDLPGRLPTQSSQLYGTNLVNLLKLLCKEKDGNITVDFDDVVIRGVTVIRAGEITWPAPPIQVSAQPQAAQKAAPEVKTEEKCTCSPWRKYALMALAIILFGWMASVAPKEFLGHFTVFALACVVGYYVVWNVSHALHTPLMSVTNAISGIIVVGALLQIGQGGWVSFLSFIAVLIASINIFGGFTVTQRMLKMFRKN* The amino acid sequence of pntB is as follows (SEQ ID NO.15): MSGGLVTAAYIVAAILFIFSLAGLSKHETSRQGNNFGIAGMAIALIATIFGPDTGNVGWILLAMVIGGAIGIRLAKKVEMTEMPELVAILHSFVGLAAVLVGFNSYLHHDAGMAP ILVNIHLTEVFLGIFIGAVTFTGSVVAFGKLCGKISSKPLMLPNRHKMNLAALVVSFLLLIVFVRTDSVGLQVLALLIMTAIALVFGWHLVASIGGADMPVVVSMLNSYSGWAAAA AGFMLSNDLLIVTGALVGSSGAILSYIMCKAMNRSFISVIAGGFGTDGSSTGDDQEVGEHREITAEETAELLKNSHSVIITPGYGMAVAQAQYPVAEITEKLRARGINVRFGIHPV AGRLPGHMNVLLAEAKVPYDIVLEMDEINDDFADTDTVLVIGANDTVNPAAQDDPKSPIAGMPVLEVWKAQNVIVFKRSMNTGYAGVQNPLFFKENTHMLFGDAKASVDAILKAL* 8) P trc - pntAB Integrate separately into yghX gene locus by E . coli Using the W3110 genome as a template, according to yghX Design upstream homologous arm primers based on gene (Gene ID: 2847694) sequence. yghX -1 and yghX -2 and downstream homologous arm primers yghX -3 and yghX -4, obtained through PCR amplification yghX Upstream and downstream homologous arms of the gene. Additionally... E . coli Using the W3110 genome as a template, according to pntAB Gene sequence design primers pntAB -F and pntAB -R, where P trc Promoter sequence design in primers yghX -2 and pntAB In -F, the corresponding fragments are obtained through PCR amplification, and then fusion PCR is performed using each fragment as a template to obtain... P trc - pntAB Integrate the fragment. Introduce primer N20- yghX -F and N20- yghX The DNA fragment formed after -R annealing was ligated to plasmid pGRB to construct plasmid pGRB- yghX Plasmid pGRB- yghX and P trc - pntAB The integrated fragments were co-electroplated into H6 electrocompetent cells containing pREDCas9, and positive transformants were obtained through screening. After plasmid elimination, strain H7 was obtained. E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Δ yeeP :: P trc - aspC Δ ygaY :: P trc - sucAB Δ metI :: P trc - yjeH Δ ilvG :: P trc - pntAB Δ yghX :: P trc - pntAB The plasmid pTrc99a- obtained in step 1 was used... metA fbr Transformed into strain H7 to obtain engineered strain W7 ( E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Δ yeeP :: P trc - aspC Δ ygaY :: P trc - sucAB Δ metI :: P trc - yjeH Δ ilvG :: P trc - pntAB Δ yghX :: P trc - pntAB / pTrc99a- metA fbr ).

[0051] 9) P trc - pntAB Integrate separately into yjiP gene locus by E . coli Using the W3110 genome as a template, according to yjiP Design upstream homologous arm primers based on gene (Gene ID: 38094982) sequence. yjiP -1 and yjiP -2 and downstream homologous arm primers yjiP -3 and yjiP -4, obtained through PCR amplification yjiP Upstream and downstream homologous arms of the gene. Additionally... E . coli Using the W3110 genome as a template, according to pntAB Gene sequence design primers pntAB -F and pntAB -R, where P trc Promoter sequence design in primers yjiP -2 and pntAB In -F, the corresponding fragments are obtained through PCR amplification, and then fusion PCR is performed using each fragment as a template to obtain... P trc - pntAB Integrate the fragment. Introduce primer N20- yjiP -F and N20- yjiP The DNA fragment formed after -R annealing was ligated to plasmid pGRB to construct plasmid pGRB- yjiP Plasmid pGRB- yjiP and P trc - pntAB The integrated fragments were co-electroplated into H7 electrocompetent cells containing pREDCas9, and positive transformants were obtained through screening. After plasmid elimination, strain H8 was obtained. E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Δ yeeP :: P trc - aspC Δ ygaY :: P trc - sucABΔ metI :: P trc - yjeH Δ ilvG :: P trc - pntAB Δ yghX :: P trc - pntAB Δ yjiP :: P trc - pntAB The plasmid pTrc99a- obtained in step 1 was used... metA fbr Transformed into strain H8, resulting in engineered strain W8 ( E . coli W3110 H0 Δ metJ Δ ycdN ::P trc - aspA Δ yes :: P trc - aspC Δ weather :: P trc - sucAB Δ meth :: P trc - yjeH Δ ilvG :: P trc - pntAB Δ yghX :: P trc - pntAB Δ yjiP :: P trc - pntAB / pTrc99a- Meta fbr ).

[0052] 10) The specific steps are the same as steps 1) to 9), the difference being that the plasmid in step 1) is adjusted to: pTrc99a- Meta fbr - yjeH Following steps 1) to 9) (omitting step 6), strain W9 was prepared, with the following genotype: ( E . coli W3110 H0 Δ MetJ Δ ycdN ::P trc - aspA Δ yes :: P trc - aspC Δ weather :: P trc - sucAB Δ ilvG :: P trc - pntAB Δ yghX :: P trc - pntAB Δ yjiP :: P trc - pntAB / pTrc99a- Meta fbr - yjeH ).

[0053] Example 2: Production of OSH by Shake Flask Fermentation Using Genetically Engineered Bacteria OSH was prepared by shake-flask fermentation using the genetically engineered strain W0-W9 obtained in Example 1.

[0054] The specific steps are as follows: (1) Seed culture The bacterial strain stored at -80℃ was streaked onto an agar slant and incubated at 37℃ for 12 h, and then passaged once. Subsequently, a loopful of the agar slant was picked up and inoculated into a 500 mL round-bottom Erlenmeyer flask containing 30 mL of seed culture medium. The flask was sealed with nine layers of gauze and incubated at 37℃ and 220 r / min for 10–12 h to obtain the seed culture.

[0055] The slant culture medium consisted of 10 g / L NaCl, 5 g / L yeast extract, and 10 g / L peptone.

[0056] The seed culture medium consisted of: 30 g / L glucose, 10 g / L yeast extract, 4 g / L (NH4)2SO4, 3 g / L KH2PO4, 2 g / L MgSO4·7H2O, 2 g / L sodium citrate, 30 mg / L FeSO4·7H2O, and 0.5 mg / L LNG. H and 0.5 mg / LV B1 The rest is water, with a pH of 6.5-6.7.

[0057] (2) Fermentation culture The seed culture was inoculated at a rate of 15% (v / v) into a 500 mL baffled Erlenmeyer flask containing 30 mL of fermentation medium. The flask was sealed with nine layers of gauze and fermented at 37°C and 220 r / min. During fermentation, phenol red was used as an indicator, and the pH was maintained at 6.5-6.7 by adding 25% ammonia. When the glucose in the medium was depleted, 60% (m / v) glucose solution was added to maintain fermentation. The fermentation cycle was 48 h.

[0058] The fermentation medium consisted of: 10 g / L glucose, 5 g / L yeast extract, 5 g / L (NH4)2SO4, 3 g / L KH2PO4, 2 g / L MgSO4·7H2O, 2 g / L sodium citrate, 30 mg / L FeSO4·7H2O, and 0.5 mg / L LNG. H 0.5mg / LV B1 It contains 8 mg / L phenol red (indicator), with the remainder being water, and a pH of 6.5-6.7. After 48 hours of shake-flask fermentation, the results of shake-flask fermentation for each OSH genetically engineered strain are as follows: Figure 1 As shown in Figure 3 and Table 3: Table 3: Yield of different strains

[0059] The results showed that the OSH yield in the fermentation broth of strain W8 was 42.12 g / L. No other amino acids or organic acid byproducts were detected.

[0060] Example 3: Production of OSH by fermentation of genetically engineered strain W8 in a 5 L fermenter The specific steps are as follows: (1) Seed culture Add an appropriate amount of sterile water to the slant, elute the W8 cells prepared in Example 1 using an inoculation loop to form a bacterial suspension, and then inoculate the bacterial suspension into a seed culture medium for cultivation. The seed culture conditions are: culture temperature 37℃, initial aeration rate 2 L / min, initial stirring speed 200 r / min, pH of the culture medium controlled at 6.5-6.7 by automatic addition of 25% ammonia water, and dissolved oxygen controlled at 25-35% by adjusting the stirring speed and aeration rate. When OD... 600 When the concentration reaches 20, seed culture is obtained and used to inoculate the fermentation medium.

[0061] The seed culture medium consisted of: 30 g / L glucose, 10 g / L yeast extract, 4 g / L (NH4)2SO4, 3 g / L KH2PO4, 2 g / L MgSO4·7H2O, 2 g / L sodium citrate, 30 mg / L FeSO4·7H2O, and 0.5 mg / L LV. H and 0.5 mg / LV B1 The rest is water, with a pH of 6.5-6.7.

[0062] (2) Fermentation culture The seed culture was inoculated into the fermentation medium at an inoculation rate of 15% (v / v) for fermentation culture. The fermentation conditions were as follows: culture temperature 37℃, pH of the medium was controlled at 6.5-6.7 by automatically adding 25% ammonia water, dissolved oxygen was controlled at 25-35% by adjusting the stirring rate and aeration, and the glucose residual sugar concentration in the fermentation broth was controlled at 1-10 g / L by batch feeding. The fermentation time was 60-72 h.

[0063] The fermentation medium consisted of: 10 g / L glucose, 5 g / L yeast extract, 5 g / L (NH4)2SO4, 3 g / L KH2PO4, 2 g / L MgSO4·7H2O, 2 g / L sodium citrate, 30 mg / L FeSO4·7H2O, 10 mg / L MnSO4·H2O, and 0.5 mg / L L V. H and 0.5 mg / LV B1 The rest is water, with a pH of 6.5-6.7.

[0064] The fermentation process employs a fed-batch culture method. The fed-batch culture medium consists of: 10 g / L yeast extract, 10 g / L peptone, 10 g / L KH₂PO₄, 5 g / L MgSO₄·7H₂O, 5 g / L citric acid, 15 mg / L FeSO₄·7H₂O, and 1 mg / L LV. H and 1 mg / LV B1 The fed-batch medium was started 20 hours after the start of fermentation.

[0065] The results show: After 60 h of fermentation in a 5 L fermenter, the OSH yield reached 137.4 g / L, with a sugar-acid conversion rate of 50.5% and a yield of 2.29 g / L / h. No other amino acids or organic acid byproducts were detected. The feed-batch fermentation curve of strain W8 in a 5 L fermenter is shown below. Figure 2 As shown.

[0066] Obviously, the above embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, various equivalent substitutions or modifications can be made to the present invention without departing from the spirit and substance of the invention, and all such equivalents or modifications should fall within the scope of protection of the present invention.

Claims

1. A recombinant *Escherichia coli* strain that increases the yield of O-succinyl-L-homoserine, characterized in that, The recombinant *E. coli* strain was based on *E. coli* that produces L-homoserine, with the repressor protein coding gene knocked out in the genome. metJ It also expressed homoserine O-succinyltransferase, aspartate aminotransferase, aspartate aminotransferase, 2-ketoglutarate decarboxylase, dihydrolipoamide succinyltransferase, OSH transporter and pyridine nucleotide transhydrogenase derived from Escherichia coli.

2. The recombinant Escherichia coli according to claim 1, characterized in that, The amino acid sequence of the homoserine O-succinyl transferase is shown in SEQ ID NO.8, the amino acid sequence of the aspartate amino acid lyase is shown in SEQ ID NO.9, the amino acid sequence of the aspartate transaminase is shown in SEQ ID NO.10, the amino acid sequence of the 2-ketoglutarate decarboxylase is shown in SEQ ID NO.11, the amino acid sequence of the dihydrolipoamide succinyl transferase is shown in SEQ ID NO.12, the amino acid sequence of the OSH transporter is shown in SEQ ID NO.13, and the amino acid sequence of the pyridine nucleotide transhydrogenase is shown in SEQ ID NO.14-15.

3. The recombinant Escherichia coli according to claim 2, characterized in that, The gene encoding the homoserine O-succinyltransferase metA fbr The nucleotide sequence is shown in SEQ ID NO.2, and the gene encoding the aspartate aminopyase is... aspA The nucleotide sequence is shown in SEQ ID NO.3, and the gene encoding the aspartate transaminase is... aspC The nucleotide sequence is shown in SEQ ID NO.4, which encodes the genes for 2-ketoglutarate decarboxylase and dihydrolipoic acid succinyltransferase. sucAB The nucleotide sequence is shown in SEQ ID NO.5, and the gene encoding the OSH transporter protein is... yjeH The nucleotide sequence is shown in SEQ ID NO.6, and the gene encoding the pyridine nucleotide transhydrogenase is... pntAB The nucleotide sequence is shown in SEQ ID NO.

7.

4. The recombinant Escherichia coli according to claim 3, characterized in that, The aspartate aminotransferase, aspartate transaminase, 2-ketoglutarate decarboxylase, dihydrolipoamide succinyltransferase, OSH transporter, and pyridine nucleotide transhydrogenase all employ promoters. P trc Regulation is used to express; Preferably, the aspA Integration ycdN Gene loci, the aspC Integration yeeP Gene loci, the sucAB Integration ygaY Gene loci, the yjeH Integration metI Gene loci, the pntAB Integrate separately into ilvG , yghX and / or yjiP Gene loci; Preferably, the promoter P trc The nucleotide sequence is shown in SEQ ID NO.1; Preferably, metJ Gene, ycdN Gene, yeeP Gene, ygaY Gene, metI Gene, ilvG Gene, yghX Gene, yjiP The gene IDs on NCBI are: 948435, 948956, 946524, 2847696, 944894, 2847699, 2847694, and 38094982.

5. A method for increasing the yield of O-succinyl-L-homoserine from Escherichia coli, characterized in that, The method is as follows: Starting with L-homoserine-producing *Escherichia coli* strains, the gene encoding the repressor protein in the genome was knocked out. metJ It also expressed homoserine O-succinyl transferase, aspartate aminotransferase, aspartate aminotransferase, 2-ketoglutarate decarboxylase, dihydrolipoic acid succinyl transferase, OSH transporter, and pyridine nucleotide transhydrogenase derived from Escherichia coli. Preferably, the amino acid sequence of the homoserine O-succinyl transferase is shown in SEQ ID NO.8, the amino acid sequence of the aspartate amino acid lyase is shown in SEQ ID NO.9, the amino acid sequence of the aspartate transaminase is shown in SEQ ID NO.10, the amino acid sequence of the 2-ketoglutarate decarboxylase is shown in SEQ ID NO.11, the amino acid sequence of the dihydrolipoamide succinyl transferase is shown in SEQ ID NO.12, the amino acid sequence of the OSH transporter is shown in SEQ ID NO.13, and the amino acid sequence of the pyridine nucleotide transhydrogenase is shown in SEQ ID NO.14-15.

6. The method according to claim 5, characterized in that, The gene encoding the homoserine O-succinyltransferase metA fbr The nucleotide sequence is shown in SEQ ID NO.2, and the gene encoding the aspartate aminopyase is... aspA The nucleotide sequence is shown in SEQ ID NO.3, and the gene encoding the aspartate transaminase is... aspC The nucleotide sequence is shown in SEQ ID NO.4, which encodes the genes for 2-ketoglutarate decarboxylase and dihydrolipoamide succinyltransferase. sucAB The nucleotide sequence is shown in SEQ ID NO.5, and the gene encoding the OSH transporter protein is... yjeH The nucleotide sequence is shown in SEQ ID NO. 6, and the gene encoding the pyridine nucleotide transhydrogenase is... pntAB The nucleotide sequence is shown in SEQ ID NO.7; Preferably, the aspartate aminotransferase, aspartate transaminase, 2-ketoglutarate decarboxylase, dihydrolipoamide succinyltransferase, OSH transporter, and pyridine nucleotide transhydrogenase all employ promoters. P trc Regulation is used to express; Preferably, the aspA Integration ycdN Gene loci, the aspC Integration yeeP Gene loci, the sucAB Integration ygaY Gene loci, the yjeH Integration metI Gene loci, the pntAB Integrate separately into ilvG , yghX and / or yjiP Gene loci; Preferably, the promoter P trc The nucleotide sequence is shown in SEQ ID NO.1; Preferably, metJ Gene, ycdN Gene, yeeP Gene, ygaY Gene, metI Gene, ilvG Gene, yghX Gene, yjiP The gene IDs on NCBI are: 948435, 948956, 946524, 2847696, 944894, 2847699, 2847694, and 38094982.

7. A method for preparing O-succinyl-L-homoserine, characterized in that, The method involves fermenting the recombinant Escherichia coli according to any one of claims 1 to 4 to prepare O-succinyl-L-homoserine.

8. The method according to claim 7, characterized in that, The method involves activating the recombinant Escherichia coli and inoculating it into a seed culture medium to obtain a seed liquid; then inoculating the seed liquid into a fermentation culture medium for fermentation to prepare O-succinyl-L-homoserine.

9. The method according to claim 7 or 8, characterized in that, The recombinant Escherichia coli was activated and then inoculated into a seed culture medium for cultivation at a temperature of 35-37 ℃ and a pH of 6.5-6.

7. Preferably, the fermentation culture temperature is 35-37 ℃, the pH is 6.5-6.7, the post-induction culture temperature is 30-32 ℃, the pH is 6.5-6.7, and the glucose concentration is controlled at 1-10 g / L; when OD 600 When the concentration reaches 20, add the inducing agent IPTG to make the final concentration 0.5 mM; Preferably, the fermentation medium comprises the following components: 10-20 g / L glucose, 5-10 g / L yeast extract, 1-5 g / L (NH4)2SO4, 1-3 g / L KH2PO4, 1-2 g / L MgSO4·7H2O, 1-2 g / L citric acid or citrate, 20-30 mg / L FeSO4·7H2O, and 0.5-1.0 mg / L L... H and 0.5~1.0 mg / LV B1 ; Preferably, the fermentation process can also be fed using a fed-batch culture medium, which comprises the following components: 5-10 g / L yeast extract, 1-5 g / L (NH4)2SO4, 1-3 g / L KH2PO4, 1-2 g / L MgSO4·7H2O, 1-2 g / L citric acid or citrate, 20-30 mg / L FeSO4·7H2O, and 0.5-1.0 mg / L LV. H and 0.5~1.0 mg / LV B1 ; Preferably, the fed-batch culture medium is added 14-20 hours after the start of fermentation during the fermentation process.

10. The use of the recombinant Escherichia coli according to any one of claims 1 to 4 in the preparation of O-succinyl-L-homoserine or products containing O-succinyl-L-homoserine.

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