Engineering strain for efficiently producing L-methionine as well as construction method and application of engineering strain

Abstract

The invention discloses an engineering strain for efficiently producing L-methionine as well as a construction method and application of the engineering strain, and belongs to the technical field of gene engineering. The engineering strain overexpresses the following genes: a metA * gene, a yjaH gene, a metC gene, a cysEM201R gene, a serA * gene, a cysDN gene, a cysB gene, a pck gene, a zwf gene, a crp gene, a csgD gene, a prsD128A gene, a spoT gene, a RuBisCO gene, a groEL-groES gene and a metB * gene; expressing a prK gene; according to the engineering strain, the following genes are deleted on a genome: a metJ gene, a lacI gene, an rhtA gene, a metQIN gene, a pykF gene, a pykA gene, a purR gene, a sucCD gene and an aceA gene. The engineering strain disclosed by the invention can be used for efficiently producing L-methionine.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to an engineering strain for efficiently producing L-methionine, a construction method and an application thereof. Background Art

[0002] L-methionine, also known as L-methionine (L-Met), is an essential amino acid in humans and animals. L-methionine deficiency can lead to a variety of conditions, including toxemia, muscle paralysis, depression, and schizophrenia. Currently, L-methionine production methods include chemical synthesis, enzymatic conversion, and fermentation. Chemical synthesis produces a racemic mixture of DL-methionine, and obtaining pure L-methionine requires additional processing steps such as enzymatic conversion, extraction, and chromatography. Alternatively, L-methionine precursors, O-succinyl-L-homoserine or O-acetyl-L-homoserine, can be produced through fermentation and then enzymatically converted to pure L-methionine. However, this process is costly, requires high raw material purity, and is technically complex, making it suitable only for specialized applications in the medical and pharmaceutical fields. Fermentation processes utilizing natural resources offer a new alternative for producing pure L-methionine. However, due to the complex biosynthetic pathway of L-methionine, developing a commercially competitive fermentation production process is difficult.

[0003] The production of L-methionine by metabolically engineered Escherichia coli has attracted widespread attention due to its environmental friendliness, low material costs, and strong sustainability. However, the existing technology for producing L-methionine by E. coli fermentation results in low L-methionine yields due to factors such as excessive energy consumption and carbon source loss. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide an engineered strain for efficiently producing L-methionine, and a construction method and application thereof, to overcome the problem of low yield when using Escherichia coli fermentation to produce L-methionine in the prior art.

[0005] In a first aspect, the present invention provides an engineered strain for efficiently producing L-methionine, wherein the engineered strain overexpresses the following genes:

[0006] metA* gene, yjeH gene, metC gene, cysE M201R gene, serA* gene, cysDN gene, cysB gene, pck gene, zwf gene, crp gene, csgD gene, prs D128A gene, spoT gene, RuBisCO gene, groEL-groES gene, metB* gene.

[0007] The engineered strain expresses the prK gene.

[0008] The engineered strain has the following genes missing from its genome:

[0009] metJ gene, lacI gene, rhtA gene, metQIN gene, pykF gene, pykA gene, purR gene, sucCD gene, aceA gene.

[0010] The metA* gene is a homoserine O-succinyltransferase gene, the yjeH gene is an L-Met transporter gene, the metC gene is a cystathionine β-lyase gene, and the cysE M201R The gene is serine O-acetyltransferase gene, the serA* gene is 3-phosphoglycerate dehydrogenase gene, the cysDN gene is sulfate adenylyltransferase gene, the cysB gene is cysteine regulatory protein gene, the pck gene is phosphoenolpyruvate carboxykinase gene, the zwf gene is glucose-6-phosphate dehydrogenase gene, the crp gene is a global transcription factor gene, the csgD gene is a regulatory protein gene, and the prs D128A The gene is phosphoribosyl pyrophosphate synthase gene, spoT gene is a bifunctional enzyme gene, prK gene is phosphoribosyl kinase gene, RuBisCO gene is ribulose-1,5-bisphosphate carboxylase / oxidase gene, groEL-groES genes are molecular chaperone protein genes, metB* gene is cystathionine γ-synthase gene, metJ gene is L-methionine biosynthesis gene transcription repressor gene, lacI gene is Lac repressor protein gene, rhtA gene is homoserine transporter gene, metQIN gene is L-Met inner membrane transporter gene, pykF gene is pyruvate kinase gene, pykA gene is pyruvate kinase gene, purR gene is DNA binding transcription repressor gene, sucCD gene is succinyl-CoA synthase gene, aceA gene is isocitrate lyase gene.

[0011] Compared with the prior art, the engineered strain of the present invention overexpresses and deletes specific genes in the genome, thereby enabling the engineered strain to synthesize L-methionine from scratch using glucose as a carbon source, effectively improving the efficiency of the engineered strain in producing L-methionine.

[0012] First, the engineered strain provided by the present invention enhances L-methionine (L-Met) terminal synthesis. L-homoserine-O-succinyltransferase, encoded by the metA gene, is the first key enzyme in the L-methionine terminal synthesis module. It is subject to feedback inhibition by L-Met and S-adenosylmethionine (SAM) and is extremely sensitive to heat. The present invention introduces point mutations into the metA gene, mutating the threonine at position 242 to alanine, reducing the temperature sensitivity of L-homoserine-O-succinyltransferase. It also mutates the isoleucine at position 296 to serine and the arginine at position 27 to cysteine, eliminating the feedback inhibition of L-Met and S-adenosylmethionine (SAM) on L-homoserine-O-succinyltransferase. The engineered strain of the present invention has a genome deletion of the metJ gene encoding the transcriptional repressor of L-methionine biosynthesis genes, thereby relieving the inhibition of the transcriptional repressor on genes related to L-methionine biosynthesis (metA, metB, metC, etc.), while simultaneously strengthening the metabolism of L-homoserine reacting with succinyl-CoA to produce O-succinyl homoserine and enhancing the expression of the metA* mutant gene. The engineered strain of the present invention has a genome deletion of the lacI gene, relieving the inhibitory effect of the lacI repressor protein on the Ptrc promoter and further strengthening the metabolism of L-homoserine reacting with succinyl-CoA to produce O-succinyl homoserine. The engineered strain of the present invention has a genome deletion of the rhtA gene and overexpresses the yjeH gene, weakening the transport of the precursor L-homoserine to the extracellular space while strengthening the transport of L-Met to the extracellular space. The present invention knocks out the metQIN gene in the engineered strain to block the transport of extracellular methionine into the intracellular space.

[0013] Second, the engineered strain of the present invention couples the central carbon metabolism module with the L-methionine terminal synthesis module. The cystathionine-β-lyase encoded by the metC gene can catalyze cystathionine to produce cysteine, and at the same time catalyze cysteine to produce pyruvate. The pyruvate kinase encoded by pykF and pykA catalyzes phosphoenolpyruvate to produce pyruvate. Both the central carbon metabolism module and the L-methionine terminal synthesis module can generate pyruvate. The metabolic coupling strategy is used to delete pyruvate kinase, so that the central carbon metabolism is coupled with the L-methionine terminal synthesis. The engineered strain of the present invention deletes the pykF and pykA genes in the genome and overexpresses the metC gene, which increases the synthesis of L-methionine precursors and couples the synthesis of L-methionine with cell growth.

[0014] Third, the engineered strain of the present invention enhances the synthesis of L-cysteine. The synthesis module of L-cysteine involves carbon metabolism and sulfur metabolism. In terms of carbon metabolism, phosphoglycerate dehydrogenase encoded by the serA gene and L-serine acetyltransferase encoded by the cysE gene are two key rate-limiting enzymes, which are feedback inhibited by L-serine and L-cysteine respectively. The present invention mutates the methionine at position 201 of the cysE gene to arginine to obtain the mutant gene cysE M201R , relieving the feedback inhibition of L-cysteine. The histidine at position 344 of the serA gene is mutated to alanine, and the asparagine at position 346 is mutated to alanine to obtain the mutant gene serA*, which relieves the feedback inhibition of L-serine. The engineered strain of the present invention overexpresses cysE M201R , increasing the synthesis of O-acetylserine and relieving the feedback inhibition of L-cysteine. By overexpressing serA*, the synthesis of 3-phosphohydroxypyruvate is increased and the feedback inhibition of L-serine is relieved. In terms of sulfur metabolism, sulfate is often used as a sulfur source, which can only be assimilated through the sulfate assimilation pathway. Since the cysD gene and cysN gene are key genes in sulfur metabolism and are adjacent to each other on the genome, the assimilation of sulfate is enhanced by overexpressing the cysDN gene. The cysteine regulatory protein encoded by the cysB gene is activated after binding to thiosulfate, promoting the transcription of the sulfate transporter gene cysP and the cysteine synthase gene cysK. Therefore, by overexpressing the cysB gene, the assimilation of sulfate is further enhanced.

[0015] Fourth, the engineered strain of the present invention enhances the supply of ATP and NADPH. Since sulfur metabolism consumes a large amount of ATP and NADPH, it is necessary to enhance the supply of intracellular ATP and NADPH. By overexpressing the pck gene, the intracellular ATP supply is enhanced. By overexpressing the zwf gene, the metabolism of glucose-6-phosphate into 6-phosphogluconolactone is enhanced, thereby enhancing the intracellular NADPH supply. By overexpressing the global transcription factor gene crp, the positive regulation of the key genes zwf and gnd in the pentose phosphate pathway is enhanced, the carbon metabolism flow is optimized to the NADPH generation pathway, and sufficient reducing power is provided for the sulfur metabolism pathway of the bacteria, while enhancing the supply of the precursor aspartic acid.

[0016] Fifth, the engineered strain of the present invention enhances methyl supply. The biosynthesis of L-methionine requires 5,10-methylenetetrahydrofolate to provide methyl groups. Enhancing the biosynthesis of 5,10-methylenetetrahydrofolate can promote the synthesis of L-methionine. The regulatory protein gene csgD can positively control the regeneration of 5,10-methylenetetrahydrofolate. The repressor protein encoded by the purR gene represses the regulatory protein gene csgD and the phosphoribosyl pyrophosphate synthetase gene prs. D128AThe present invention mutates the 128th aspartic acid of phosphoribosylpyrophosphate synthetase gene prs to alanine to obtain a mutant gene prs D128A , relieves the feedback inhibition of ATP and ADP on phosphoribosyl pyrophosphate synthase, and controls the synthesis of 5-phosphoribosyl-1-pyrophosphate (PRPP), the precursor of folic acid. D128A , and lacks the DNA-binding transcription repressor purR, which promotes the regeneration of 5,10-methylenetetrahydrofolate, provides sufficient methyl groups for the synthesis of L-methionine, and promotes the synthesis of L-methionine.

[0017] Sixth, the engineered strain of the present invention enhances carbon source conversion regulation. The bifunctional enzyme encoded by the spoT gene has the activity of synthesizing and hydrolyzing (p)ppGpp (guanylate pentaphosphate and tetraphosphate). Overexpression of the spoT gene can enhance the hydrolysis of (p)ppGpp. Low levels of (p)ppGpp can reduce the inhibition of ribosomal RNA, promote protein expression, especially the expression of glucose transporters, and enhance glucose uptake and metabolism. Therefore, overexpressing the spoT gene in the engineered strain enhances glucose uptake and metabolism.

[0018] Seventh, the engineered strain of the present invention establishes a heterotrophic RuBisCO basal bypass, enabling in situ CO2 fixation and increased L-methionine production. The present invention expresses the ribosyltransferase gene prK to enhance the metabolism of ribulose-5-phosphate to ribulose-1,5-bisphosphate; overexpresses the ribulose-1,5-bisphosphate carboxylase / oxidase gene RuBisCO to enhance the metabolism of ribulose-1,5-bisphosphate and CO2 to triphosphoglycerate; overexpresses the molecular chaperone protein genes groEL-groES to promote the functional expression of RuBisCO; and overexpresses the phosphoenolpyruvate carboxykinase gene pck to enhance the metabolism of phosphoenolpyruvate to oxaloacetate, thereby increasing ATP supply, enhancing CO2 fixation capacity, and increasing biomass.

[0019] Eighth, the engineered strain of the present invention couples the central carbon metabolism module with the L-methionine front-end synthesis module. In the reaction mediated by metA*-metB*, succinyl-CoA is ultimately converted into succinic acid, which is consistent with the reaction catalyzed by succinyl-CoA synthase (encoded by sucCD) in the TCA cycle and isocitrate lyase (encoded by aceA) in the glyoxylate cycle. If the conversion of succinyl-CoA to succinic acid is blocked by deleting sucCD and aceA, more succinyl-CoA will enter the L-methionine synthesis pathway mediated by metA*-metB*, thereby promoting its synthesis. At the same time, the enhanced metA*-metB*-dependent pathway will release more succinic acid to compensate for the cell growth requirements of the TCA and glyoxylate cycles. In order to enhance the O-succinyl-L-homoserine capturing ability and catalytic activity of cystathionine gamma-synthase, the present invention makes the following mutations in the cystathionine gamma-synthase gene metB: the 5th amino acid is mutated from glutamine (Q) to arginine (R), the 29th amino acid is mutated from leucine (L) to histidine (H), the 69th amino acid is mutated from glycine (G) to aspartic acid (D), the 87th amino acid is mutated from phenylalanine (F) to isoleucine (I), the 136th amino acid is mutated from glutamic acid (E) to glycine (G), the 148th amino acid is mutated from asparagine (N) to tyrosine (Y), the 273rd amino acid is mutated from lysine (K) to glycine (G), and the 346th amino acid is mutated from alanine (A) to threonine (T), thereby obtaining a mutant gene metB*. The engineered strain of the present invention lacks the sucCD gene and the aceA gene in the genome and overexpresses the cystathionine gamma-synthase gene metB*, thereby filling the gaps in the TCA and glyoxylate cycles and coordinating L-methionine production with cell growth.

[0020] Furthermore, the nucleotide sequence of the metA* gene is shown in SEQ ID NO. 1;

[0021] The nucleotide sequence of the yjeH gene is shown in SEQ ID NO. 2;

[0022] The nucleotide sequence of the metC gene is shown in SEQ ID NO. 3;

[0023] cysE M201R The nucleotide sequence of the gene is shown in SEQ ID NO.4;

[0024] The nucleotide sequence of the serA* gene is shown in SEQ ID NO. 5;

[0025] The nucleotide sequence of the cysDN gene is shown in SEQ ID NO.6;

[0026] The nucleotide sequence of the cysB gene is shown in SEQ ID NO.7;

[0027] The nucleotide sequence of the pck gene is shown in SEQ ID NO.8;

[0028] The nucleotide sequence of the zwf gene is shown in SEQ ID NO.9;

[0029] The nucleotide sequence of the crp gene is shown in SEQ ID NO.10;

[0030] The nucleotide sequence of the csgD gene is shown in SEQ ID NO. 11;

[0031] prs D128A The nucleotide sequence of the gene is shown in SEQ ID NO.12;

[0032] The nucleotide sequence of the spoT gene is shown in SEQ ID NO. 13;

[0033] The nucleotide sequence of the prK gene is shown in SEQ ID NO. 14;

[0034] The nucleotide sequence of the RuBisCO gene is shown in SEQ ID NO. 15;

[0035] The nucleotide sequence of the groEL-groES gene is shown in SEQ ID NO. 16;

[0036] The nucleotide sequence of the metB* gene is shown in SEQ ID NO. 17;

[0037] The nucleotide sequence of the metJ gene is shown in SEQ ID NO. 18;

[0038] The nucleotide sequence of the lacI gene is shown in SEQ ID NO. 19;

[0039] The nucleotide sequence of the rhtA gene is shown in SEQ ID NO. 20;

[0040] The nucleotide sequence of the metQIN gene is shown in SEQ ID NO. 21;

[0041] The nucleotide sequence of the pykF gene is shown in SEQ ID NO. 22;

[0042] The nucleotide sequence of the pykA gene is shown in SEQ ID NO. 23;

[0043] The nucleotide sequence of the purR gene is shown in SEQ ID NO. 24;

[0044] The nucleotide sequence of the sucCD gene is shown in SEQ ID NO. 25;

[0045] The nucleotide sequence of the aceA gene is shown in SEQ ID NO.26.

[0046] Furthermore, metA* gene, yjeH gene, metC gene, cysEM201R gene, serA* gene, cysDN gene, cysB gene, pck gene, zwf gene, crp gene, csgD gene, prs D128A The genes, spoT, RuBisCO, groEL-groES and metB* are all controlled by the Ptrc promoter.

[0047] The expression of the prK gene can be controlled by either no promoter or the Plac promoter.

[0048] Furthermore, the nucleotide sequence of the Ptrc promoter is shown in SEQ ID NO.27, and the nucleotide sequence of the Ptrc terminator is shown in SEQ ID NO.28.

[0049] Furthermore, the metA* gene is located at the metJ gene locus and the lacI gene locus;

[0050] The yjeH gene is located at the rhtA gene locus;

[0051] The metC gene is located at the pykF gene locus and the pykA gene locus;

[0052] cysE M201R The gene is located at the ygaY pseudogene locus;

[0053] The serA* gene is located at the yjgX pseudogene locus;

[0054] The cysDN gene is located at the gapC pseudogene locus;

[0055] The cysB gene is located in the rph pseudogene locus;

[0056] The pck gene is located at the mbhA pseudogene locus and the yghE pseudogene locus;

[0057] The zwf gene is located at the yciQ pseudogene locus;

[0058] The crp gene is located at the yjiT pseudogene locus;

[0059] The csgD gene is located at the purR gene locus;

[0060] prs D128A The gene is located in the yeel pseudogene locus;

[0061] The spoT gene is located at the yeeP pseudogene locus;

[0062] The prK gene is located at the ylbE pseudogene locus;

[0063] The RuBisCO gene is located at the yjiP pseudogene locus;

[0064] The groEL-groES gene is located in the yghX pseudogene locus;

[0065] The metB* gene is located at the sucCD gene locus and the aceA gene locus.

[0066] In a second aspect, the present invention provides a method for constructing an engineered strain for efficiently producing L-methionine, comprising the following steps:

[0067] The metA* gene was overexpressed using the Ptrc promoter at the metJ gene locus and the lacI gene locus, respectively;

[0068] The yjeH gene was overexpressed using the Ptrc promoter at the rhtA gene locus;

[0069] Knockout of metQIN gene;

[0070] The metC gene was overexpressed at the pykF gene locus and the pykA gene locus using the Ptrc promoter;

[0071] Overexpression of cysE at the ygaY pseudogene locus using the Ptrc promoter M201R Gene;

[0072] The serA* gene was overexpressed using the Ptrc promoter at the yjgX pseudogene locus;

[0073] The cysDN gene was overexpressed using the Ptrc promoter at the gapC pseudogene locus;

[0074] The cysB gene was overexpressed using the Ptrc promoter at the rph pseudogene locus;

[0075] The pck gene was overexpressed using the Ptrc promoter at the mbhA pseudogene locus and the yghE pseudogene locus, respectively;

[0076] The zwf gene was overexpressed using the Ptrc promoter at the yciQ pseudogene locus;

[0077] The crp gene was overexpressed using the Ptrc promoter at the yjiT pseudogene locus;

[0078] The csgD gene was overexpressed using the Ptrc promoter at the purR gene locus;

[0079] Overexpression of prs at the yeel pseudogene locus using the Ptrc promoter D128A Gene;

[0080] The spoT gene was overexpressed using the Ptrc promoter at the yeeP pseudogene locus;

[0081] The prK gene is expressed at the ylbE pseudogene locus;

[0082] The RuBisCO gene was overexpressed using the Ptrc promoter at the yjiP pseudogene locus;

[0083] The groEL-groES gene was overexpressed using the Ptrc promoter at the yghX pseudogene locus;

[0084] The metB* gene was overexpressed at the sucCD gene locus and the aceA gene locus using the Ptrc promoter.

[0085] Compared with the prior art, the technical effect of the method for constructing an engineered strain provided by the present invention is the same as the technical effect of the engineered strain for efficiently producing L-methionine, and will not be described in detail here.

[0086] Furthermore, the engineering strain uses Escherichia coli as a starting strain; the Escherichia coli is E. coli K-12W3110.

[0087] In a third aspect, the present invention provides the use of the above-mentioned engineered strain in producing L-methionine.

[0088] Compared with the prior art, the engineered strain of the present invention utilizes glucose as a substrate when producing L-methionine and directly synthesizes L-methionine from scratch, resulting in an L-methionine yield of up to 28.6 g / L and an L-methionine conversion rate of up to 26%. No metabolic by-products are generated, the strain is highly stable, and has excellent industrial application value.

[0089] Further, producing L-methionine comprises the following steps:

[0090] The engineered strain was inoculated onto an activated slant, cultured at 30-37°C for 10-14 hours, and passaged 1-2 times to obtain an activated engineered strain;

[0091] The activated engineered strain was transferred to the seed culture medium and cultured at a temperature of 30-37°C, a pH of 6.8-7.1, and a dissolved oxygen value of 25-35% until the OD value of the culture medium reached 0. 600 When the temperature reaches 10-15, the seed solution is obtained;

[0092] The seed liquid is inoculated into the fermentation medium at an inoculum amount of 5% to 10% by volume, and fermentation is carried out under the conditions of a culture temperature of 30 to 37°C, a pH of 6.8 to 7.1, and a dissolved oxygen value of 25 to 35%. A feeding medium containing glucose is fed, and the glucose concentration in the fermentation liquid is controlled to be ≤1 g / L until the OD value of the fermentation liquid begins to decrease. The fermentation is terminated to obtain L-methionine. The fermentation time can generally be referenced to 68 to 72 hours.

[0093] Furthermore, the culture medium is initially fed at a rate of 3.5 to 5 g / L / h, and then the flow acceleration is increased by 1.5 to 2.5 g / L / h every 4 hours, and finally the flow acceleration reaches 7 to 9 g / L / h until the fermentation is completed. DETAILED DESCRIPTION

[0094] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0095] It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0096] In the present invention, CRISPR / Cas9-mediated gene editing was used to edit the genome of E. coli K-12W3110 (see Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21). CRISPR / Cas9 is a precise and efficient new gene-targeted modification technology. The two plasmids used in this method are pGRB and pREDCas9. The pREDCas9 plasmid is a temperature-sensitive plasmid that carries a gRNA plasmid elimination system, the Red recombination system of λ phage, and the Cas9 protein expression system. It has spectinomycin resistance (working concentration: 100 mg / L) and an optimal culture temperature of 32°C. The pGRB plasmid uses pUC18 as a backbone, contains the promoter J23100, the gRNA-Cas9 binding region sequence, and the terminator sequence. It has ampicillin resistance (working concentration: 100 mg / L) and an optimal culture temperature of 37°C.

[0097] Example 1

[0098] Use the Ptrc promoter to control the overexpression of the metA* gene at the metJ gene locus. The specific steps are as follows:

[0099] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the metJ gene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers metJ-US (SEQ ID NO.29), metJ-UA (SEQ ID NO.30), metJ-DS (SEQ ID NO.31), and metJ-DA (SEQ ID NO.32).

[0100] Using the endogenous metA* gene carrying a mutation point synthesized by Anshengda Biotechnology Co., Ltd. in Escherichia coli as a template and metA*-S (SEQ ID NO.33) and metA*-A (SEQ ID NO.34) as primers, the Ptrc-metA* gene fragment was amplified by HS enzyme PCR.

[0101] The Ptrc promoter was designed into the antisense primer for the upstream homology arm of the metJ gene and the sense primer for the metA* gene; the Ptrc terminator was designed into the antisense primer for the metA* gene and the sense primer for the downstream homology arm of the metJ gene. The nucleotide sequence of the Ptrc terminator is shown in SEQ ID NO. 28.

[0102] Then, using the upstream homology arm, downstream homology arm and Ptrc-metA* gene fragment of the above-mentioned metJ gene as templates, metJ-US and metJ-DA as primers, and HS enzyme, the metJ::Ptrc-metA* gene integration fragment was obtained by overlapping PCR. The gene integration fragment consists of the metJ upstream homology arm, the Ptrc-metA* target gene and the metJ downstream homology arm.

[0103] Using pGRB-metJ-S (SEQ ID NO.35) and pGRB-metJ-A (SEQ ID NO.36) as primers, a DNA fragment containing the metJ target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-metJ was extracted from them.

[0104] The metJ::Ptrc-metA* gene integration fragment and the pGRB-metJ plasmid obtained in the above steps were electroporated into the E. coli K-12W3110 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met1-1.

[0105] Example 2

[0106] The Ptrc promoter was used to control the overexpression of the metA* gene at the lacI gene locus. The specific steps are as follows:

[0107] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the lacI gene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers lacI-US (SEQ ID NO.37), lacI-UA (SEQ ID NO.38), lacI-DS (SEQ ID NO.39), and lacI-DA (SEQ ID NO.40), respectively.

[0108] Using the endogenous metA* gene carrying a mutation point synthesized by Anshengda Biotechnology Co., Ltd. in Escherichia coli as a template and metA*-S (SEQ ID NO.33) and metA*-A (SEQ ID NO.34) as primers, the Ptrc-metA* gene fragment was amplified by HS enzyme PCR.

[0109] Among them, the Ptrc promoter is designed in the antisense strand primer of the upstream homologous arm of the lacI gene and the positive strand primer of the metA* gene; the Ptrc terminator is designed in the antisense strand primer of the metA* gene and the positive strand primer of the downstream homologous arm of the lacI gene.

[0110] Then, using the upstream homology arm, downstream homology arm and Ptrc-metA* gene fragment of the above-mentioned lacI gene as templates, lacI-US and lacI-DA as primers, and HS enzyme, overlapping PCR was used to obtain the lacI::Ptrc-metA* gene integration fragment, which consists of the lacI upstream homology arm, the Ptrc-metA* target gene and the lacI downstream homology arm.

[0111] Using pGRB-lacI-S (SEQ ID NO.41) and pGRB-lacI-A (SEQ ID NO.42) as primers, a DNA fragment containing the lacI target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, the fragment was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-lacI was extracted from them.

[0112] The lacI::Ptrc-metA* gene integration fragment obtained in the above steps and the pGRB-lacI plasmid were electroporated into the E. coli Met1-1 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met1-2.

[0113] Example 3

[0114] The Ptrc promoter was used to control the overexpression of the yjeH gene at the rhtA gene locus. The specific steps are as follows:

[0115] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the rhtA gene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers rhtA-US (SEQ ID NO.43), rhtA-UA (SEQ ID NO.44), rhtA-DS (SEQ ID NO.45), and rhtA-DA (SEQ ID NO.46).

[0116] Using the E. coli K-12W3110 genome as a template and yjeH-S (SEQ ID NO.47) and yjeH-A (SEQ ID NO.48) as primers, the Ptrc-yjeH gene fragment was amplified by HS enzyme PCR.

[0117] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the rhtA gene and the sense primer of the yjeH gene; the Ptrc terminator was designed in the antisense primer of the yjeH gene and the sense primer of the downstream homologous arm of the rhtA gene.

[0118] Using the upstream homology arm, downstream homology arm and Ptrc-yjeH gene fragment of the above-mentioned rhtA gene as templates, rhtA-US and rhtA-DA as primers, and HS enzyme, an overlapping PCR method was used to obtain the rhtA::Ptrc-yjeH gene integration fragment, which consists of the rhtA upstream homology arm, the Ptrc-yjeH target gene and the rhtA downstream homology arm.

[0119] Using pGRB-rhtA-S (SEQ ID NO. 49) and pGRB-rhtA-A (SEQ ID NO. 50) as primers, a DNA fragment containing the rhtA target sequence was constructed by PCR annealing procedure. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells. Positive transformants were screened and plasmid pGRB-rhtA was extracted from them.

[0120] The yciQ::Ptrc-yjeH gene integration fragment obtained in the above steps and the pGRB-rhtA plasmid were electroporated into the E. coli Met1-2 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met1-3.

[0121] Example 4

[0122] Knockout the metQIN gene. The specific steps are as follows:

[0123] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the metQIN gene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers metQIN-US (SEQ ID NO.51), metQIN-UA (SEQ ID NO.52), metQIN-DS (SEQ ID NO.53), and metQIN-DA (SEQ ID NO.54).

[0124] Using the upstream homology arm and downstream homology arm of the above-mentioned metQIN gene as templates, metQIN-US and metQIN-DA as primers, and HS enzyme, an overlapping PCR method was used to obtain a gene integration fragment, which consists of the metQIN upstream homology arm and the metQIN downstream homology arm.

[0125] Using pGRB-metQIN-S (SEQ ID NO. 55) and pGRB-metQIN-A (SEQ ID NO. 56) as primers, a DNA fragment containing the metQIN gene target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, the fragment was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-metQIN was extracted from them.

[0126] The gene integration fragment obtained in the above steps and the pGRB-metQIN plasmid were electroporated into the E. coli Met1-3 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met2.

[0127] Example 5

[0128] The Ptrc promoter was used to control the overexpression of the metC gene at the pykF gene locus. The specific steps are as follows:

[0129] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the pykF gene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers pykF-US (SEQ ID NO.57), pykF-UA (SEQ ID NO.58), pykF-DS (SEQ ID NO.59), and pykF-DA (SEQ ID NO.60).

[0130] Using the E. coli K-12W3110 genome as a template and metC-S (SEQ ID NO.61) and metC-A (SEQ ID NO.62) as primers, the Ptrc-metC target gene fragment was amplified by HS enzyme PCR.

[0131] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the pykF gene and the positive chain primer of the metC gene; the Ptrc terminator was designed in the antisense primer of the metC gene and the positive chain primer of the downstream homologous arm of the pykF gene.

[0132] Using the upstream homology arm, downstream homology arm and Ptrc-metC target gene fragment of the above-mentioned pykF gene as templates, pykF-US and pykF-DA as primers, and HS enzyme, the pykF::Ptrc-metC gene integration fragment was obtained by overlapping PCR. The gene integration fragment consists of the pykF upstream homology arm, the Ptrc-metC target gene and the pykF downstream homology arm.

[0133] Using pGRB-pykF-S (SEQ ID NO.63) and pGRB-pykF-A (SEQ ID NO.64) as primers, a DNA fragment containing the pykF target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-pykF was extracted from them.

[0134] The pykF::Ptrc-metC gene integration fragment obtained in the above steps and the pGRB-pykF plasmid were electroporated into the E. coli Met2 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met2-1.

[0135] Example 6

[0136] The Ptrc promoter was used to control the overexpression of the metC gene at the pykA gene locus. The specific steps are as follows:

[0137] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the pykA gene were amplified by HS enzyme PCR using the upstream and downstream homology arms primers pykA-US (SEQ ID NO.65), pykA-UA (SEQ ID NO.66), pykA-DS (SEQ ID NO.67), and pykA-DA (SEQ ID NO.68), respectively.

[0138] Using the E. coli K-12W3110 genome as a template and metC-S (SEQ ID NO.61) and metC-A (SEQ ID NO.62) as primers, the Ptrc-metC target gene fragment was amplified by HS enzyme PCR.

[0139] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the pykA gene and the positive chain primer of the metC gene; the Ptrc terminator was designed in the antisense primer of the metC gene and the positive chain primer of the downstream homologous arm of the pykA gene.

[0140] Then, using the above-mentioned pykA upstream homology arm, downstream homology arm and Ptrc-metC target gene fragment as templates, pykA-US and pykA-DA as primers, and HS enzyme, the pykA::Ptrc-metC gene integration fragment was obtained by overlapping PCR. The gene integration fragment consists of the pykA upstream homology arm, the Ptrc-metC target gene and the pykA downstream homology arm.

[0141] Using pGRB-pykA-S (SEQ ID NO. 69) and pGRB-pykA-A (SEQ ID NO. 70) as primers, a DNA fragment containing the pykA target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, the fragment was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-pykA was extracted from them.

[0142] The pykA::Ptrc-metC gene integration fragment obtained in the above steps and the pGRB-pykA plasmid were electroporated into the E. coli Met2-1 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met3.

[0143] Example 7

[0144] Control of cysE by the Ptrc promoter at the ygaY pseudogene locus M201RGene overexpression, the specific steps are as follows:

[0145] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the ygaY pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers ygaY-US (SEQ ID NO.71), ygaY-UA (SEQ ID NO.72), ygaY-DS (SEQ ID NO.73), and ygaY-DA (SEQ ID NO.74).

[0146] The cysE endogenous to Escherichia coli carrying mutation point synthesized by Anshengda Biotechnology Co., Ltd. M201R gene as a template, using cysE M201R -S (SEQ ID NO. 75), cysE M201R -A (SEQ ID NO.76) was used as primer, and Ptrc-cysE was obtained by PCR amplification with HS enzyme. M201R Target gene fragment.

[0147] Among them, the Ptrc promoter was designed in the antisense strand primer of the upstream homology arm of the ygaY pseudogene and the cysE M201R The positive strand primer of the gene; the Ptrc terminator is designed in cysE M201R The antisense primer of the gene and the sense primer of the downstream homology arm of the ygaY pseudogene were used.

[0148] Then, the upstream homology arm, downstream homology arm and Ptrc-cysE of ygaY were used. M201R The target gene fragment was used as a template, ygaY-US and ygaY-DA were used as primers, and HS enzyme was used to obtain ygaY::Ptrc-cysE by overlapping PCR. M201R The gene integration fragment consists of a ygaY upstream homology arm, a Ptrc-cysEM201R target gene and a ygaY downstream homology arm.

[0149] Using pGRB-ygaY-S (SEQ ID NO. 77) and pGRB-ygaY-A (SEQ ID NO. 78) as primers, a DNA fragment containing the ygaY target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, the fragment was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-ygaY was extracted from them.

[0150] The ygaY::Ptrc-cysE obtained in the above steps M201RThe gene integration fragment and the pGRB-ygaY plasmid were electroporated into the E. coli Met3 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met3-1.

[0151] Example 8

[0152] The Ptrc promoter was used to control the overexpression of the serA* gene at the yjgX pseudogene locus. The specific steps are as follows:

[0153] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the yjgX pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers yjgX-US (SEQ ID NO.79), yjgX-UA (SEQ ID NO.80), yjgX-DS (SEQ ID NO.81), and yjgX-DA (SEQ ID NO.82).

[0154] Using the endogenous Escherichia coli serA* gene carrying a mutation point synthesized by Anshengda Biotechnology Co., Ltd. as a template, serA*-S (SEQ ID NO.83) and serA*-A (SEQ ID NO.84) as primers, the Ptrc-serA* target gene fragment was amplified by HS enzyme PCR.

[0155] Among them, the Ptrc promoter was designed in the antisense strand primer of the upstream homologous arm of the yjgX pseudogene and the positive strand primer of the serA* gene; the Ptrc terminator was designed in the antisense strand primer of the serA* gene and the positive strand primer of the downstream homologous arm of the yjgX pseudogene.

[0156] Then, using the above-mentioned upstream homology arm, downstream homology arm and Ptrc-serA* target gene fragment of yjgX as templates, yjgX-US and yjgX-DA as primers, and HS enzyme, the yjgX::Ptrc-serA* gene integration fragment was obtained by overlapping PCR. The gene integration fragment consists of the yjgX upstream homology arm, the Ptrc-serA* target gene and the yjgX downstream homology arm.

[0157] Using pGRB-yjgX-S (SEQ ID NO.85) and pGRB-yjgX-A (SEQ ID NO.86) as primers, a DNA fragment containing the yjgX target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-yjgX was extracted from them.

[0158] The yjgX::Ptrc-serA* gene integration fragment obtained in the above steps and the pGRB-yjgX plasmid were electroporated into the E. coli Met3-1 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met3-2.

[0159] Example 9

[0160] The Ptrc promoter was used to control the overexpression of the cysDN gene at the gapC pseudogene locus. The specific steps are as follows:

[0161] Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of the gapC pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers gapC-US (SEQ ID NO.87), gapC-UA (SEQ ID NO.88), gapC-DS (SEQ ID NO.89), and gapC-DA (SEQID NO.90).

[0162] Using the E. coli W3110 genome as a template and cysDN-S (SEQ ID NO.91) and cysDN-A (SEQ ID NO.92) as primers, the Ptrc-cysDN target gene fragment was amplified by HS enzyme PCR.

[0163] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the gapC pseudogene and the sense primer of the cysDN gene; the Ptrc terminator was designed in the antisense primer of the cysDN gene and the sense primer of the downstream homologous arm of the gapC pseudogene.

[0164] Then, using the above-mentioned gapC upstream homology arm, downstream homology arm and Ptrc-cysDN target gene fragment as templates, gapC-US and gapC-DA as primers, and HS enzyme, overlapping PCR was used to obtain the gapC::Ptrc-cysDN gene integration fragment, which consists of the gapC upstream homology arm, the Ptrc-cysDN target gene and the gapC downstream homology arm.

[0165] Using pGRB-gapC-S (SEQ ID NO.93) and pGRB-gapC-A (SEQ ID NO.94) as primers, a DNA fragment containing the gapC target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-gapC was extracted from them.

[0166] The gapC::Ptrc-cysDN gene integration fragment obtained in the above step and the pGRB-gapC plasmid were electroporated into the E. coli Met3-2 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met3-3.

[0167] Example 10

[0168] The Ptrc promoter was used to control the overexpression of the cysB gene at the rph pseudogene locus. The specific steps are as follows:

[0169] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the rph pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers rph-US (SEQ ID NO.95), rph-UA (SEQ ID NO.96), rph-DS (SEQ ID NO.97), and rph-DA (SEQID NO.98).

[0170] Using the E. coli K-12W3110 genome as a template and cysB-S (SEQ ID NO.99) and cysB-A (SEQ ID NO.100) as primers, the Ptrc-cysB target gene fragment was amplified by HS enzyme PCR.

[0171] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the rph pseudogene and the positive chain primer of the cysB gene; the Ptrc terminator was designed in the antisense primer of the cysB gene and the positive chain primer of the downstream homologous arm of the rph pseudogene.

[0172] Then, using the upstream homology arm, downstream homology arm and Ptrc-cysB target gene fragment of the above-mentioned rph as templates, rph-US and rph-DA as primers, and HS enzyme, the rph::Ptrc-cysB gene integration fragment was obtained by overlapping PCR. The gene integration fragment consists of the rph upstream homology arm, the Ptrc-cysB target gene and the rph downstream homology arm.

[0173] Using pGRB-rph-S (SEQ ID NO. 101) and pGRB-rph-A (SEQ ID NO. 102) as primers, a DNA fragment containing the rph target sequence was constructed by PCR annealing. After recombination with the linearized pGRB vector, the fragment was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-rph was extracted from them.

[0174] The rph::Ptrc-cysB gene integration fragment obtained in the above step and the pGRB-rph plasmid were electroporated into the E. coli Met3-3 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met4.

[0175] Example 11

[0176] The Ptrc promoter was used to control the overexpression of the pck gene at the mbhA pseudogene locus. The specific steps are as follows:

[0177] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of mbhA pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arms primers mbhA-US (SEQ ID NO.103), mbhA-UA (SEQ ID NO.104), mbhA-DS (SEQ ID NO.105), and mbhA-DA (SEQ ID NO.106), respectively.

[0178] Using the E. coli K-12W3110 genome as a template and pck-S (SEQ ID NO.107) and pck-A (SEQ ID NO.108) as primers, the Ptrc-pck target gene fragment was amplified by HS enzyme PCR.

[0179] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the mbhA pseudogene and the positive chain primer of the pck gene; the Ptrc terminator was designed in the antisense primer of the pck gene and the positive chain primer of the downstream homologous arm of the mbhA pseudogene.

[0180] Then, using the above-mentioned mbhA upstream homology arm, downstream homology arm and Ptrc-pck target gene fragment as templates, mbhA-US and mbhA-DA as primers, and HS enzyme, the mbhA::Ptrc-pck gene integration fragment was obtained by overlapping PCR. The gene integration fragment consists of the mbhA upstream homology arm, the Ptrc-pck target gene and the mbhA downstream homology arm.

[0181] Using pGRB-mbhA-S (SEQ ID NO. 109) and pGRB-mbhA-A (SEQ ID NO. 110) as primers, a DNA fragment containing the mbhA target sequence was constructed by PCR annealing. After recombination with the linearized pGRB vector, the fragment was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-mbhA was extracted from them.

[0182] The mbhA::Ptrc-pck gene integration fragment obtained in the above steps and the pGRB-mbhA plasmid were electroporated into the E. coli Met4 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met4-1.

[0183] Example 12

[0184] The Ptrc promoter was used to control the overexpression of the zwf gene at the yciQ pseudogene locus. The specific steps are as follows:

[0185] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of yciQ were amplified by HS enzyme PCR using the upstream and downstream homology arm primers yciQ-US (SEQ ID NO.111), yciQ-UA (SEQ ID NO.112), yciQ-DS (SEQ ID NO.113), and yciQ-DA (SEQ ID NO.114) of the yciQ pseudogene, respectively.

[0186] Using the E. coli K-12W3110 genome as a template and zwf-S (SEQ ID NO.115) and zwf-A (SEQ ID NO.116) as primers, the Ptrc-zwf target gene fragment was amplified by HS enzyme PCR.

[0187] Among them, the Ptrc promoter was designed in the antisense chain primer of the upstream homologous arm of the yciQ pseudogene and the positive chain primer of the zwf gene; the Ptrc terminator was designed in the antisense chain primer of the zwf gene and the positive chain primer of the downstream homologous arm of the yciQ pseudogene.

[0188] Then, using the above-mentioned yciQ upstream homology arm, downstream homology arm and Ptrc-zwf target gene fragment as templates, yciQ-US and yciQ-DA as primers, and HS enzyme, the yciQ::Ptrc-zwf gene integration fragment was obtained by overlapping PCR. The gene integration fragment consists of the yciQ upstream homology arm, the Ptrc-zwf target gene and the yciQ downstream homology arm.

[0189] Using pGRB-yciQ-S (SEQ ID NO.117) and pGRB-yciQ-A (SEQ ID NO.118) as primers, a DNA fragment containing the yciQ target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain the plasmid pGRB-yciQ.

[0190] The yciQ::Ptrc-zwf gene integration fragment obtained in the above steps and the pGRB-yciQ plasmid were electroporated into the E. coli Met4-1 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met4-2.

[0191] Example 13

[0192] The Ptrc promoter was used to control the overexpression of the crp gene at the yjiT pseudogene locus. The specific steps are as follows:

[0193] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the yjiT pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arms primers yjiT-US (SEQ ID NO.119), yjiT-UA (SEQ ID NO.120), yjiT-DS (SEQ ID NO.121), and yjiT-DA (SEQ ID NO.122), respectively.

[0194] Using the E. coli K-12W3110 genome as a template and crp-S (SEQ ID NO.123) and crp-A (SEQ ID NO.124) as primers, the Ptrc-crp target gene fragment was amplified by HS enzyme PCR.

[0195] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the yjiT pseudogene and the positive chain primer of the crp gene; the Ptrc terminator was designed in the antisense primer of the crp gene and the positive chain primer of the downstream homologous arm of the yjiT pseudogene.

[0196] Then, using the above-mentioned yjiT upstream homology arm, downstream homology arm and Ptrc-crp target gene fragment as templates, yjiT-US and yjiT-DA as primers, and HS enzyme, the yjiT::Ptrc-crp gene integration fragment was obtained by overlapping PCR. The gene integration fragment consists of the yjiT upstream homology arm, the Ptrc-crp target gene and the yjiT downstream homology arm.

[0197] Using pGRB-yjiT-S (SEQ ID NO.125) and pGRB-yjiT-A (SEQ ID NO.126) as primers, a DNA fragment containing the yjiT target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-yjiT was extracted from them.

[0198] The yjiT::Ptrc-crp gene integration fragment obtained in the above steps and the pGRB-yjiT plasmid were electroporated into the E. coli Met4-2 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met5.

[0199] Example 14

[0200] The Ptrc promoter was used to control the overexpression of the csgD gene at the purR gene locus. The specific steps are as follows:

[0201] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the purR gene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers purR-US (SEQ ID NO.127), purR-UA (SEQ ID NO.128), purR-DS (SEQ ID NO.129), and purR-DA (SEQ ID NO.130), respectively.

[0202] Using the E. coli K-12W3110 genome as a template and csgD-S (SEQ ID NO.131) and csgD-A (SEQ ID NO.132) as primers, the Ptrc-csgD target gene fragment was amplified by HS enzyme PCR.

[0203] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the purR gene and the sense primer of the csgD gene; the Ptrc terminator was designed in the antisense primer of the csgD gene and the sense primer of the downstream homologous arm of the purR gene.

[0204] Then, using the above-mentioned purR upstream homology arm, downstream homology arm and Ptrc-csgD target gene fragment as templates, purR-US and purR-DA as primers, and HS enzyme, overlapping PCR was used to obtain the purR::Ptrc-csgD gene integration fragment, which consists of the purR upstream homology arm, the Ptrc-csgD target gene and the purR downstream homology arm.

[0205] Using pGRB-purR-S (SEQ ID NO.133) and pGRB-purR-A (SEQ ID NO.134) as primers, a DNA fragment containing the purR target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, the fragment was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-purR was extracted from them.

[0206] The purR::Ptrc-csgD gene integration fragment obtained in the above steps and the pGRB-purR plasmid were electroporated into the E. coli Met5 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met5-1.

[0207] Example 15

[0208] Control of prs using the Ptrc promoter at the yeel pseudogene locus D128A Gene overexpression, the specific steps are as follows:

[0209] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the yeel pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arms primers yeel-US (SEQ ID NO.135), yeel-UA (SEQ ID NO.136), yeel-DS (SEQ ID NO.137), and yeel-DA (SEQ ID NO.138), respectively.

[0210] The prs carrying mutation point was synthesized by Anshengda Biotechnology Co., Ltd. D128A Gene as template, using prs D128A -S (SEQ ID NO. 139), prs D128A -A (SEQ ID NO.140) was used as primer, and Ptrc-prs was obtained by HS enzyme PCR amplification. D128A Target gene fragment.

[0211] Among them, the Ptrc promoter was designed in the antisense strand primer of the upstream homology arm of the yeel pseudogene and the prs D128A The positive strand primer of the gene; the Ptrc terminator is designed in the prs D128A The antisense primer of the gene and the sense primer of the downstream homology arm of the yeel pseudogene.

[0212] Then use the upstream homology arm, downstream homology arm and Ptrc-prs D128A The target gene fragment was used as a template, yeel-US and yeel-DA were used as primers, and HS enzyme was used to obtain yeel::Ptrc-prs by overlapping PCR. D128A Gene integration fragment, which consists of yeel upstream homology arm, Ptrc-prs D128A It consists of the target gene and the downstream homology arm of yeel.

[0213] Using pGRB-yeel-S (SEQ ID NO.141) and pGRB-yeel-A (SEQ ID NO.142) as primers, a DNA fragment containing the yeel target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, the fragment was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-yeel was extracted from them.

[0214] The yeel::Ptrc-prs obtained in the above steps D128A The gene integration fragment and the pGRB-yeel plasmid were electroporated into the E. coli Met5-1 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met6.

[0215] Example 16

[0216] The Ptrc promoter was used to control the overexpression of the spoT gene at the yeeP pseudogene locus. The specific steps are as follows:

[0217] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the yeeP pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers yeeP-US (SEQ ID NO.143), yeeP-UA (SEQ ID NO.144), yeeP-DS (SEQ ID NO.145), and yeeP-DA (SEQ ID NO.146).

[0218] Using the E. coli K-12W3110 genome as a template and spoT-S (SEQ ID NO.147) and spoT-A (SEQ ID NO.148) as primers, the Ptrc-spoT target gene fragment was amplified by HS enzyme PCR.

[0219] Among them, the Ptrc promoter was designed in the antisense chain primer of the upstream homologous arm of the yeeP pseudogene and the positive chain primer of the spoT gene; the Ptrc terminator was designed in the antisense chain primer of the spoT gene and the positive chain primer of the downstream homologous arm of the yeeP pseudogene.

[0220] Then, using the upstream homology arm, downstream homology arm and Ptrc-spoT target gene fragment of the above-mentioned yeeP pseudogene as templates, yeeP-US and yeeP-DA as primers, and HS enzyme, the overlapping PCR method was used to obtain the yeeP::Ptrc-spoT gene integration fragment, which consists of the yeeP upstream homology arm, Ptrc-spoT and yeeP downstream homology arm.

[0221] Using pGRB-yeeP-S (SEQ ID NO.149) and pGRB-yeeP-A (SEQ ID NO.150) as primers, a DNA fragment containing the yeeP target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, the fragment was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-yeeP was extracted from them.

[0222] The yeeP::Ptrc-spoT gene integration fragment obtained in the above steps and the pGRB-yeep plasmid were electroporated into the E. coli Met6 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met7.

[0223] Example 17

[0224] The prK gene is expressed at the ylbE pseudogene locus as follows:

[0225] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the ylbE pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers ylbE-US (SEQ ID NO.151), ylbE-UA (SEQ ID NO.152), ylbE-DS (SEQ ID NO.153), and ylbE-DA (SEQ ID NO.154).

[0226] The PprK target gene fragment was amplified by HS enzyme PCR using the cyanobacterium (Synechocystis sp. PCC 6803) genome as a template and prK-S (SEQ ID NO. 155) and prK-A (SEQ ID NO. 156) as primers.

[0227] Then, using the upstream homology arm, downstream homology arm and PprK target gene fragment of the above-mentioned ylbE pseudogene as templates, ylbE-US and ylbE-DA as primers, and HS enzyme, overlapping PCR was used to obtain the ylbE::PprK gene integration fragment, which consists of the ylbE upstream homology arm, prK gene fragment and ylbE downstream homology arm.

[0228] Using pGRB-ylbE-S (SEQ ID NO. 157) and pGRB-ylbE-A (SEQ ID NO. 158) as primers, a DNA fragment containing the ylbE target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, the fragment was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-ylbE was extracted from them.

[0229] The ylbE::PprK gene integration fragment obtained in the above steps and the pGRB-ylbE plasmid were electroporated into the E. coli Met7 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met7-1.

[0230] Example 18

[0231] The Ptrc promoter was used to control the overexpression of the RuBisCO gene at the yjiP pseudogene locus. The specific steps are as follows:

[0232] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the yjiP pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers yjiP-US (SEQ ID NO.159), yjiP-UA (SEQ ID NO.160), yjiP-DS (SEQ ID NO.161), and yjiP-DA (SEQ ID NO.162).

[0233] The genome of cyanobacteria (Synechocystis sp. PCC 6803) was used as a template and RuBisCO-S (SEQ ID NO. 163) and RuBisCO-A (SEQ ID NO. 164) were used as primers to obtain the Ptrc-RuBisCO target gene fragment by PCR amplification with HS enzyme.

[0234] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the pseudogene yjiP and the positive chain primer of the RuBisCO gene; the Ptrc terminator was designed in the antisense primer of the RuBisCO gene and the positive chain primer of the downstream homologous arm of the pseudogene yjiP.

[0235] Then, using the upstream homology arm, downstream homology arm and Ptrc-RuBisCO target gene fragment of the above-mentioned yjiP pseudogene as templates, yjiP-US and yjiP-DA as primers, and HS enzyme, the yjiP::Ptrc-RuBisCO gene integration fragment was obtained by overlapping PCR. The gene integration fragment consists of the yjiP upstream homology arm, the Ptrc-RuBisCO target gene and the yjiP downstream homology arm.

[0236] Using pGRB-yjiP-S (SEQ ID NO.165) and pGRB-yjiP-A (SEQ ID NO.166) as primers, a DNA fragment containing the yjiP target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-yjiP was extracted from them.

[0237] The yjiP::Ptrc-RuBisCO gene integration fragment obtained in the above steps and the pGRB-yjiP plasmid were electroporated into the E. coli Met7-1 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met7-2.

[0238] Example 19

[0239] The Ptrc promoter was used to control the overexpression of the GroEL-GroES genes at the yghX pseudogene locus. The specific steps are as follows:

[0240] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the yghX pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers yghX-US (SEQ ID NO.167), yghX-UA (SEQ ID NO.168), yghX-DS (SEQ ID NO.169), and yghX-DA (SEQ ID NO.170), respectively.

[0241] Using the E. coli K-12W3110 genome as a template and GroEL-GroES-S (SEQ ID NO.171) and GroEL-GroES-A (SEQ ID NO.172) as primers, the Ptrc-GroEL-GroES target gene fragment was amplified by HS enzyme PCR.

[0242] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the pseudogene yghX and the positive chain primer of the GroEL-GroES gene; the Ptrc terminator was designed in the antisense primer of the GroEL-GroES gene and the positive chain primer of the downstream homologous arm of the pseudogene yghX.

[0243] Then, using the upstream homology arm, downstream homology arm and Ptrc-GroEL-GroE target gene fragment of the above-mentioned yghX pseudogene as templates, yghX-US and yghX-DA as primers, and HS enzyme, overlapping PCR was used to obtain the yghX::Ptrc-GroEL-GroE gene integration fragment, which consists of the yghX upstream homology arm, the Ptrc-GroEL-GroES target gene and the yghX downstream homology arm.

[0244] Using pGRB-yghX-S (SEQ ID NO. 173) and pGRB-yghX-A (SEQ ID NO. 174) as primers, a DNA fragment containing the yghX target sequence was constructed by PCR annealing. After recombination with the linearized pGRB vector, the fragment was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-yghX was extracted from them.

[0245] The yghX::Ptrc-GroEL-GroES gene integration fragment obtained in the above step and the pGRB-yghX plasmid were electroporated into the E. coli Met7-2 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met7-3.

[0246] Example 20

[0247] The Ptrc promoter was used to control the overexpression of the pck gene at the yghE pseudogene locus. The specific steps are as follows:

[0248] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the yghE pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers yghE-US (SEQ ID NO.175), yghE-UA (SEQ ID NO.176), yghE-DS (SEQ ID NO.177), and yghE-DA (SEQ ID NO.178).

[0249] Using the E. coli K-12W3110 genome as a template and pck-S (SEQ ID NO.107) and pck-A (SEQ ID NO.108) as primers, the Ptrc-pck target gene fragment was amplified by HS enzyme PCR.

[0250] Among them, the Ptrc promoter was designed in the antisense chain primer of the upstream homologous arm of the pseudogene yghE and the positive chain primer of the pck gene; the Ptrc terminator was designed in the antisense chain primer of the pck gene and the positive chain primer of the downstream homologous arm of the pseudogene yghE.

[0251] Then, using the upstream homology arm, downstream homology arm and Ptrc-pck target gene fragment of the above-mentioned yghE pseudogene as templates, yghE-US and yghE-DA as primers, and HS enzyme, the overlapping PCR method was used to obtain the yghE::Ptrc-pck gene integration fragment, which consists of the yghE upstream homology arm, the Ptrc-pck target gene and the yghE downstream homology arm.

[0252] Using pGRB-yghE-S (SEQ ID NO. 179) and pGRB-yghE-A (SEQ ID NO. 180) as primers, a DNA fragment containing the yghE target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, the fragment was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-yghE was extracted from them.

[0253] The yghE::Ptrc-pck gene integration fragment obtained in the above steps and the pGRB-yghE plasmid were electroporated into the E. coli Met7-3 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met8.

[0254] Example 21

[0255] The Ptrc promoter was used to control the overexpression of the metB* gene at the sucCD gene locus. The specific steps are as follows:

[0256] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the sucCD gene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers sucCD-US (SEQ ID NO.181), sucCD-UA (SEQ ID NO.182), sucCD-DS (SEQ ID NO.183), and sucCD-DA (SEQ ID NO.184).

[0257] Using the metB* gene synthesized by Anshengda Biotechnology Co., Ltd. as a template and metB*-S (SEQ ID NO.185) and metB*-A (SEQ ID NO.186) as primers, the Ptrc-metB* target gene fragment was amplified by HS enzyme PCR.

[0258] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homology arm of the pseudogene sucCD and the positive chain primer of the metB* gene; the Ptrc terminator was designed in the antisense primer of the metB* gene and the positive chain primer of the downstream homology arm of the pseudogene sucCD.

[0259] Then, using the upstream homology arm, downstream homology arm and Ptrc-metB* target gene fragment of the above-mentioned sucCD gene as templates, sucCD-US and sucCD-DA as primers, and HS enzyme, overlapping PCR was used to obtain the sucCD::Ptrc-metB* gene integration fragment, which consists of the sucCD upstream homology arm, the Ptrc-metB* target gene and the sucCD downstream homology arm.

[0260] Using pGRB-sucCD-S (SEQ ID NO. 187) and pGRB-sucCD-A (SEQ ID NO. 188) as primers, a DNA fragment containing the sucCD target sequence was constructed by PCR annealing. After recombining with the linearized pGRB vector, the fragment was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-sucCD was extracted from them.

[0261] The sucCD::Ptrc-metB* gene integration fragment obtained in the above steps and the pGRB-sucCD plasmid were electroporated into the E. coli Met8 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met8-1.

[0262] Example 22

[0263] The Ptrc promoter was used to control the overexpression of the metB* gene at the aceA gene locus. The specific steps are as follows:

[0264] Using the E. coli K-12W3110 genome as a template, the upstream and downstream homology arms of the aceA gene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers aceA-US (SEQ ID NO.189), aceA-UA (SEQ ID NO.190), aceA-DS (SEQ ID NO.191), and aceA-DA (SEQ ID NO.192).

[0265] Using the metB* gene synthesized by Anshengda Biotechnology Co., Ltd. as a template and metB*-S (SEQ ID NO.185) and metB*-A (SEQ ID NO.186) as primers, the Ptrc-metB* target gene fragment was amplified by HS enzyme PCR.

[0266] Among them, the Ptrc promoter was designed in the antisense chain primer of the upstream homologous arm of the pseudogene aceA and the positive chain primer of the metB* gene; the Ptrc terminator was designed in the antisense chain primer of the metB* gene and the positive chain primer of the downstream homologous arm of the pseudogene aceA.

[0267] Then, using the upstream homology arm, downstream homology arm and Ptrc-metB* target gene fragment of the above-mentioned aceA gene as templates, aceA-US and aceA-DA as primers, and HS enzyme, the aceA::Ptrc-metB* gene integration fragment was obtained by overlapping PCR. The gene integration fragment consists of the aceA upstream homology arm, the Ptrc-metB* target gene and the aceA downstream homology arm.

[0268] Using pGRB-aceA-S (SEQ ID NO. 193) and pGRB-aceA-A (SEQ ID NO. 194) as primers, a DNA fragment containing the aceA target sequence was constructed by PCR annealing procedure. After recombining with the linearized pGRB vector, the fragment was transformed into E. coli DH5α competent cells. Positive transformants were screened and the plasmid pGRB-aceA was extracted from them.

[0269] The aceA::Ptrc-metB* gene integration fragment obtained in the above steps and the pGRB-aceA plasmid were electroporated into the E. coli Met8-1 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Met9.

[0270] Example 23

[0271] The strain E. coli Met9 obtained in Example 22 was used to ferment and produce L-methionine, and the specific steps were as follows:

[0272] S1. Seed Activation: Streak E. coli Met9 onto a non-resistant activation slant, incubate at 37°C for 12 hours, and subculture twice to obtain activated cells. Elute the activated cells from the slant with sterile distilled water and transfer them to a 5-L fermenter filled with seed medium to initiate seed culture. The final volume of the seed medium is 2 L.

[0273] S2. Seed culture: Set the culture temperature to 37°C, automatically add 25% ammonia solution to maintain pH 7, and adjust the stirring speed or ventilation volume to maintain the dissolved oxygen value at 30%. When the OD 600 After 15 minutes, the seed solution was obtained.

[0274] S3. Fermentation culture: The seed liquid is inoculated into a fermentation tank at an inoculum volume ratio of 10% for fermentation culture, so that the final volume of the fermentation medium in the fermentation tank is 2L, the culture temperature is controlled at 37°C, and the culture pH value is maintained at 7 by automatically feeding a 25% volume fraction of ammonia solution. The culture dissolved oxygen value is maintained at 30% by adjusting the stirring speed or ventilation volume. A fed-batch medium containing glucose is fed, and the glucose concentration in the fermentation liquid is controlled to be ≤1g / L until the OD value of the fermentation liquid begins to decrease, and the fermentation is terminated. The fed-batch medium is initially fed at a rate of 4g / L / h, and the flow acceleration is then increased by 2g / L / h every 4 hours, eventually reaching a flow acceleration of 8g / L / h, until the fermentation process is completed.

[0275] Example 24

[0276] The strain E. coli Met9 obtained in Example 22 was used to ferment and produce L-methionine, and the specific steps were as follows:

[0277] S1. Seed Activation: Streak E. coli Met9 onto a non-resistant activation slant, incubate at 30°C for 10 hours, and subculture once to obtain activated cells. Elute the activated cells from the slant with sterile distilled water and transfer them to a 5 L fermenter filled with seed medium to initiate seed culture. The final volume of the seed medium is 2 L.

[0278] S2. Seed culture: Set the culture temperature to 30°C, automatically add 25% ammonia solution to maintain the pH value at 6.8, and adjust the stirring speed or ventilation volume to maintain the dissolved oxygen value at 25%. When the OD value of the culture solution reaches 600 After 10 minutes, the seed solution was obtained.

[0279] S3. Fermentation culture: The seed liquid is inoculated into a fermentation tank at an inoculum size of 5% by volume for fermentation culture, so that the final volume of the fermentation medium in the fermentation tank is 2 L, the culture temperature is controlled at 30°C, and the culture pH value is maintained at 6.8 by automatically feeding a 25% volume fraction of ammonia solution. The culture dissolved oxygen value is maintained at 25% by adjusting the stirring speed or ventilation volume. A fed-batch medium containing glucose is fed, and the glucose concentration in the fermentation liquid is controlled to be ≤1 g / L until the OD value of the fermentation liquid begins to decrease, and the fermentation is terminated. The fed-batch medium is initially fed at a rate of 3.5 g / L / h, and then the flow acceleration is increased by 2.5 g / L / h every 4 hours, and finally the flow acceleration reaches 8.5 g / L / h until the fermentation process is completed.

[0280] Example 25

[0281] The strain E. coli Met9 obtained in Example 22 was used to ferment and produce L-methionine, and the specific steps were as follows:

[0282] S1. Seed Activation: Streak E. coli Met9 onto a non-resistant activation slant, incubate at 35°C for 14 hours, and subculture twice to obtain activated cells. Elute the activated cells from the slant with sterile distilled water and transfer them to a 5 L fermenter filled with seed medium to initiate seed culture. The final volume of seed medium is 2 L.

[0283] S2. Seed culture: Set the culture temperature to 35°C, automatically add 25% ammonia solution to maintain the pH value at 7.1, and adjust the stirring speed or ventilation volume to maintain the dissolved oxygen value at 35%. 600 After 12 minutes, the seed solution is obtained.

[0284] S3. Fermentation culture: The seed liquid is inoculated into a fermentation tank at an inoculum volume ratio of 7% for fermentation culture, so that the final volume of the fermentation medium in the fermentation tank is 2L, the culture temperature is controlled at 35°C, and the culture pH value is maintained at 7.1 by automatically feeding a 25% volume fraction of ammonia solution. The culture dissolved oxygen value is maintained at 35% by adjusting the stirring speed or ventilation volume. A fed-batch medium containing glucose is fed, and the glucose concentration in the fermentation liquid is controlled to be ≤1g / L until the OD value of the fermentation liquid begins to decrease, and the fermentation is terminated. The fed-batch medium is initially fed at a rate of 5g / L / h, and then the flow acceleration is increased by 1.5g / L / h every 4 hours, and finally the flow acceleration reaches 8g / L / h until the fermentation process is completed.

[0285] In the fermentation production of L-methionine in Examples 23 to 25 above, the seed culture medium used was: 30 g / L glucose, 5 g / L yeast, 3 g / L peptone, 1 g / L (NH4)2SO4, 2 g / L KH2PO4, 1 g / L MgSO4·7H2O, and the remainder water.

[0286] The fermentation medium used in the fermentation culture was: glucose 10 g / L, (NH4)2SO4 8 g / L, KH2PO4 3 g / L, MgSO4·7H2O 1 g / L, (NH4)2S2O3 5.6 g / L, CaCO3 3 g / L, yeast extract 2 g / L, refined corn steep liquor (powder) 1 g / L, vitamin B 12 0.01g / L, pyridoxal phosphate (PLP) 2mg / L, trace element solution 5mL / L, and the rest is water.

[0287] The composition of the trace element solution is: FeSO4·7H2O 6g / L, CaCl2 1.35g / L, ZnSO4·7H2O 0.8g / L, MnSO4·4H2O 1.5g / L, CuSO4·5H2O 0.15g / L, (NH4)6Mo7O 24 ·4H2O 0.2g / L, H3BO3 0.1g / L, CoCl2·6H2O 0.25g / L, HCl with a volume fraction of 35% 10mL / L.

[0288] The fed-batch medium used in the fermentation culture was: glucose 450 g / L, MgSO4·7H2O 6 g / L, KH2PO4 3 g / L, (NH4)2SO4 33 g / L, (NH4)2S2O3 33.5 g / L.

[0289] Comparative Example 1

[0290] Using E. coli K-12W3110 as the starting strain, an engineered strain was prepared according to the methods of Examples 1 to 3 and 5 to 22, that is, the step of knocking out the metQIN gene in Example 4 was not performed. In Example 5, the pykF::Ptrc-metC gene integration fragment and the pGRB-pykF plasmid were electroporated into the strain E. coli Met1-3 containing the pREDCas9 plasmid. After screening to obtain positive transformants, the plasmid used for gene editing was eliminated to obtain the strain E. coli Met2-1′. The strain E. coli Met2-1′ was constructed into an engineered strain according to the methods of Examples 6 to 22, and the engineered strain was used to produce L-methionine according to the method of Example 23.

[0291] Comparative Example 2

[0292] Using E. coli K-12W3110 as the starting strain, the engineered strain was prepared according to the methods of Examples 1 to 6 and 8 to 22, i.e., the method of Example 7 was omitted to overexpress cysE at the ygaY pseudogene site. M201R In the step of gene editing, the yjgX::Ptrc-serA* gene integration fragment and the pGRB-yjgX plasmid were electroporated into the strain E. coli Met3 containing the pREDCas9 plasmid in Example 8. After screening to obtain positive transformants, the plasmid used for gene editing was eliminated to obtain the strain E. coli Met3-2′. The strain E. coli Met3-2′ was constructed into an engineered strain according to the methods of Examples 9 to 22, and the engineered strain was used to produce L-methionine according to the method of Example 23.

[0293] Comparative Example 3

[0294] Using E. coli K-12W3110 as the starting strain, an engineered strain was prepared according to the methods of Examples 1 to 9 and 11 to 22, that is, the step of overexpressing the cysB gene at the rph pseudogene site in Example 10 was not performed. In Example 11, the mbhA::Ptrc-pck gene integration fragment and the pGRB-mbhA plasmid were electroporated into the strain E. coli Met3-3 containing the pREDCas9 plasmid. After screening to obtain positive transformants, the plasmid used for gene editing was eliminated to obtain the strain E. coli Met4-1′. The strain E. coli Met4-1′ was constructed into an engineered strain according to the methods of Examples 12 to 22, and the engineered strain was used to produce L-methionine according to the method of Example 23.

[0295] Comparative Example 4

[0296] Using E. coli K-12W3110 as the starting strain, an engineered strain was prepared according to the methods of Examples 1 to 10 and 12 to 22, that is, the step of overexpressing the pck gene at the mbhA pseudogene site in Example 11 was not performed. In Example 12, the yciQ::Ptrc-zwf gene integration fragment and the pGRB-yciQ plasmid were electroporated into the strain E. coli Met4 containing the pREDCas9 plasmid. After screening to obtain positive transformants, the plasmid used for gene editing was eliminated to obtain the strain E. coli Met4-2′. The strain E. coli Met4-2′ was constructed into an engineered strain according to the methods of Examples 13 to 22, and the engineered strain was used to produce L-methionine according to the method of Example 23.

[0297] Comparative Example 5

[0298] Using E. coli K-12W3110 as the starting strain, an engineered strain was prepared according to the methods of Examples 1 to 12 and 14 to 22, that is, the step of overexpressing the crp gene at the yjiT pseudogene site in Example 13 was not performed. In Example 14, the purR::Ptrc-csgD gene integration fragment and the pGRB-purR plasmid were electroporated into the strain E. coli Met4-2 containing the pREDCas9 plasmid. After screening to obtain positive transformants, the plasmid used for gene editing was eliminated to obtain the strain E. coli Met5-1′. The strain E. coli Met5-1′ was constructed into an engineered strain according to the methods of Examples 15 to 22, and the engineered strain was used to produce L-methionine according to the method of Example 23.

[0299] Comparative Example 6

[0300] Using E. coli K-12W3110 as the starting strain, the engineered strains were prepared according to the methods of Examples 1 to 13 and 15 to 22, i.e., the step of overexpressing the csgD gene at the purR gene locus in Example 14 was omitted. In Example 15, the yeel::Ptrc-prs D128A The gene integration fragment and the pGRB-yeel plasmid were electroporated into the E. coli Met5 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the plasmid used for gene editing was eliminated to obtain the strain E. coli Met6′. The strain E. coli Met6′ was constructed into an engineered strain according to the methods of Examples 16 to 22, and the engineered strain was used to produce L-methionine according to the method of Example 23.

[0301] Comparative Example 7

[0302] Using E. coli K-12W3110 as the starting strain, the engineered strain was prepared according to the methods of Examples 1 to 14 and 16 to 22, i.e., Example 15 was not performed to overexpress prs at the yeel pseudogene site. D128A In Example 16, the yeeP::Ptrc-spoT gene integration fragment and the pGRB-yeep plasmid were electroporated into the strain E. coliMet5-1 containing the pREDCas9 plasmid. After screening to obtain positive transformants, the plasmid used for gene editing was eliminated to obtain the strain E. coliMet7′. The strain E. coli Met7′ was constructed into an engineered strain according to the methods of Examples 17 to 22, and the engineered strain was used to produce L-methionine according to the method of Example 23.

[0303] Comparative Example 8

[0304] Using E. coli K-12W3110 as the starting strain, an engineered strain was prepared according to the methods of Examples 1 to 15 and 17 to 22, that is, the step of overexpressing the spoT gene at the yeeP pseudogene site in Example 16 was not performed. In Example 17, the ylbE::PprK gene integration fragment and the pGRB-ylbE plasmid were electroporated into the strain E. coli Met6 containing the pREDCas9 plasmid. After screening to obtain positive transformants, the plasmid used for gene editing was eliminated to obtain the strain E. coli Met7-1′. The strain E. coli Met7-1′ was constructed into an engineered strain according to the methods of Examples 18 to 22, and the engineered strain was used to produce L-methionine according to the method of Example 23.

[0305] Comparative Example 9

[0306] Using E. coli K-12W3110 as the starting strain, an engineered strain was prepared according to the methods of Examples 1 to 16 and 18 to 22, i.e., the step of expressing the prK gene at the ylbE pseudogene site in Example 17 was not performed. In Example 18, the yjiP::Ptrc-RuBisCO gene integration fragment and the pGRB-yjiP plasmid were electroporated into the E. coli Met7 strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the plasmid used for gene editing was eliminated to obtain the strain E. coli Met7-2′. The strain E. coli Met7-2′ was used to construct an engineered strain according to the methods of Examples 19 to 22, and the engineered strain was used to produce L-methionine according to the method of Example 23.

[0307] Comparative Example 10

[0308] Using E. coli K-12W3110 as the starting strain, an engineered strain was prepared according to the methods of Examples 1 to 17 and 19 to 22, that is, the step of overexpressing the RuBisCO gene at the yjiP pseudogene site in Example 18 was not performed. In Example 19, the yghX::Ptrc-GroEL-GroES gene integration fragment and the pGRB-yghX plasmid were electroporated into the strain E. coli Met7-1 containing the pREDCas9 plasmid. After screening to obtain positive transformants, the plasmid used for gene editing was eliminated to obtain the strain E. coli Met7-3′. The strain E. coli Met7-3′ was constructed into an engineered strain according to the methods of Examples 20 to 22, and the engineered strain was used to produce L-methionine according to the method of Example 23.

[0309] Comparative Example 11

[0310] Using E. coli K-12W3110 as the starting strain, an engineered strain was prepared according to the methods of Examples 1 to 18 and 20 to 22, that is, the step of overexpressing the GroEL-GroES gene at the yghX pseudogene site in Example 19 was not performed. In Example 20, the yghE::Ptrc-pck gene integration fragment and the pGRB-yghE plasmid were electroporated into the strain E. coliMet7-2 containing the pREDCas9 plasmid. After screening to obtain positive transformants, the plasmid used for gene editing was eliminated to obtain the strain E. coliMet8′. The strain E. coli Met8′ was constructed into an engineered strain according to the methods of Examples 21 to 22, and the engineered strain was used to produce L-methionine according to the method of Example 23.

[0311] Comparative Example 12

[0312] Using E. coli K-12W3110 as the starting strain, an engineered strain was prepared according to the methods of Examples 1 to 19 and 21 to 22, that is, the step of overexpressing the pck gene at the yghE pseudogene site in Example 20 was not performed. In Example 21, the sucCD::Ptrc-metB* gene integration fragment and the pGRB-sucCD plasmid were electroporated into the strain E. coliMet7-3 containing the pREDCas9 plasmid. After screening to obtain positive transformants, the plasmid used for gene editing was eliminated to obtain the strain E. coliMet8-1′. The strain E. coli Met8-1′ was constructed into an engineered strain according to the method of Example 22, and the engineered strain was used to produce L-methionine according to the method of Example 23.

[0313] Comparative Example 13

[0314] Using E. coli K-12W3110 as the starting strain, an engineered strain was prepared according to the methods of Examples 1 to 21, i.e., the step of overexpressing the metB* gene at the aceA gene locus in Example 22 was not performed. The strain E. coliMet8-1 obtained in Example 21 was used to produce L-methionine using the engineered strain according to the method of Example 23.

[0315] Test Case

[0316] The yields of L-methionine in the fermentation broths of Examples 23 to 25 and Comparative Examples 1 to 13 after fermentation were detected, and the conversion rates of glucose to L-methionine were calculated. The results are shown in Table 1.

[0317] Table 1

[0318] L-methionine (g / L) Conversion rate (%) Example 23 28.6 26 Example 24 26.4 25.9 Example 25 28.1 26 Comparative Example 1 25.1 22.8 Comparative Example 2 24.9 22.6 Comparative Example 3 25.6 23.3 Comparative Example 4 25.9 23.5 Comparative Example 5 25.8 23.5 Comparative Example 6 25.6 23.3 Comparative Example 7 26 23.6 Comparative Example 8 26.2 24.3 Comparative Example 9 24.2 22 Comparative Example 10 18.1 20.1 Comparative Example 11 26.2 23.8 Comparative Example 12 25.9 23.5 Comparative Example 13 25.2 22.9

[0319] From the above results, it can be seen that in Comparative Example 1, the metQIN gene was not knocked out, and the L-methionine yield and conversion rate in the fermentation broth decreased, indicating that not expressing the metQIN gene in the engineered strain can increase the L-methionine yield and conversion rate.

[0320] Comparative Example 2 did not overexpress cysE M201R Gene, Comparative Example 3 did not overexpress cysB gene, Comparative Example 5 did not overexpress crp gene, the yield and conversion rate of L-methionine in the fermentation broth decreased, indicating that overexpression of cysE in the engineered strain M201R Gene, cysB gene or crp gene can increase the production and conversion rate of L-methionine.

[0321] In Comparative Example 6, the csgD gene was not overexpressed at the purR gene site, and the yield and conversion rate of L-methionine in the fermentation broth decreased, indicating that not expressing the purR gene in the engineered strain while overexpressing the csgD gene can increase the yield and conversion rate of L-methionine.

[0322] Comparative Example 7 did not overexpress prs D128A The production and conversion rate of L-methionine in the fermentation broth decreased, indicating that the overexpression of prs in the engineered strain was D128A Gene, overexpression of prs D128A Gene expression, prK gene expression, RuBisCO gene overexpression or GroEL-GroES gene overexpression can increase the production and conversion rate of L-methionine.

[0323] In comparative example 4, the pck gene was not overexpressed at the mbhA pseudogene site, and in comparative example 12, the pck gene was not overexpressed at the yghE pseudogene site. The yield and conversion rate of L-methionine in the fermentation broth both decreased, indicating that the simultaneous overexpression of the pck gene at the above two pseudogene sites in the engineered strain can further increase the yield and conversion rate of L-methionine.

[0324] In Comparative Example 13, the metB* gene was not overexpressed at the aceA gene site, and the yield and conversion rate of L-methionine in the fermentation broth decreased, indicating that not expressing the aceA gene in the engineered strain while overexpressing the metB* gene can increase the yield and conversion rate of L-methionine.

[0325] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An engineered strain for efficiently producing L-methionine, characterized in that: The engineered strain overexpresses the following genes: metA* gene, yjeH gene, metC gene, cysE M201R gene, serA* gene, cysDN gene, cysB gene, pck gene, zwf gene, crp gene, csgD gene, prs D128A gene, spoT gene, RuBisCO gene, groEL-groES gene, metB* gene; The engineered strain expresses the prK gene; The engineered strain has the following genes deleted in its genome: metJ gene, lacI gene, rhtA gene, metQIN gene, pykF gene, pykA gene, purR gene, sucCD gene, aceA gene.

2. The engineered strain for efficiently producing L-methionine according to claim 1, characterized in that The nucleotide sequence of the metA* gene is shown in SEQ ID NO.1; The nucleotide sequence of the yjeH gene is shown in SEQ ID NO.2; The nucleotide sequence of the metC gene is shown in SEQ ID NO.3; The cysE M201R The nucleotide sequence of the gene is shown in SEQ ID NO.4; The nucleotide sequence of the serA* gene is shown in SEQ ID NO.5; The nucleotide sequence of the cysDN gene is shown in SEQ ID NO.6; The nucleotide sequence of the cysB gene is shown in SEQ ID NO.7; The nucleotide sequence of the pck gene is shown in SEQ ID NO.8; The nucleotide sequence of the zwf gene is shown in SEQ ID NO.9; The nucleotide sequence of the crp gene is shown in SEQ ID NO.10; The nucleotide sequence of the csgD gene is shown in SEQ ID NO.11; The prs D128A The nucleotide sequence of the gene is shown in SEQ ID NO.12; The nucleotide sequence of the spoT gene is shown in SEQ ID NO.13; The nucleotide sequence of the prK gene is shown in SEQ ID NO.14; The nucleotide sequence of the RuBisCO gene is shown in SEQ ID NO.15; The nucleotide sequence of the groEL-groES gene is shown in SEQ ID NO.16; The nucleotide sequence of the metB* gene is shown in SEQ ID NO.17; The nucleotide sequence of the metJ gene is shown in SEQ ID NO.18; The nucleotide sequence of the lacI gene is shown in SEQ ID NO.19; The nucleotide sequence of the rhtA gene is shown in SEQ ID NO.20; The nucleotide sequence of the metQIN gene is shown in SEQ ID NO.21; The nucleotide sequence of the pykF gene is shown in SEQ ID NO.22; The nucleotide sequence of the pykA gene is shown in SEQ ID NO.23; The nucleotide sequence of the purR gene is shown in SEQ ID NO.24; The nucleotide sequence of the sucCD gene is shown in SEQ ID NO.25; The nucleotide sequence of the aceA gene is shown in SEQ ID NO.

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3. The engineered strain for efficiently producing L-methionine according to claim 1, characterized in that The metA* gene, yjeH gene, metC gene, cysEM201R gene, serA* gene, cysDN gene, cysB gene, pck gene, zwf gene, crp gene, csgD gene, prs D128A The genes, spoT, RuBisCO, groEL-groES and metB* are all controlled by the Ptrc promoter.

4. The engineered strain for efficiently producing L-methionine according to claim 3, characterized in that The nucleotide sequence of the Ptrc promoter is shown in SEQ ID NO.

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5. The engineered strain for efficiently producing L-methionine according to claim 1, characterized in that The metA* gene is located at the metJ gene site and the lacI gene site; The yjeH gene is located at the rhtA gene site; The metC gene is located at the pykF gene site and the pykA gene site; The cysE M201R The gene is located at the ygaY pseudogene locus; The serA* gene is located at the yjgX pseudogene site; The cysDN gene is located at the gapC pseudogene site; The cysB gene is located at the rph pseudogene site; The pck gene is located at the mbhA pseudogene site and the yghE pseudogene site; The zwf gene is located at the yciQ pseudogene site; The crp gene is located at the yjiT pseudogene site; The csgD gene is located at the purR gene site; The prs D128A The gene is located in the yeel pseudogene locus; The spoT gene is located at the yeeP pseudogene site; The prK gene is located at the ylbE pseudogene site; The RuBisCO gene is located at the yjiP pseudogene site; The groEL-groES gene is located at the yghX pseudogene site; The metB* gene is located at the sucCD gene site and the aceA gene site.

6. A method for constructing an engineered strain for efficiently producing L-methionine according to any one of claims 1 to 5, characterized in that: The following steps are involved: The metA* gene was overexpressed using the Ptrc promoter at the metJ gene locus and the lacI gene locus, respectively; The yjeH gene was overexpressed using the Ptrc promoter at the rhtA gene locus; Knockout of metQIN gene; The metC gene was overexpressed at the pykF gene locus and the pykA gene locus using the Ptrc promoter; Overexpression of cysE at the ygaY pseudogene locus using the Ptrc promoter M201R Gene; The serA* gene was overexpressed using the Ptrc promoter at the yjgX pseudogene locus; The cysDN gene was overexpressed using the Ptrc promoter at the gapC pseudogene locus; The cysB gene was overexpressed using the Ptrc promoter at the rph pseudogene locus; The pck gene was overexpressed using the Ptrc promoter at the mbhA pseudogene locus and the yghE pseudogene locus, respectively; The zwf gene was overexpressed using the Ptrc promoter at the yciQ pseudogene locus; The crp gene was overexpressed using the Ptrc promoter at the yjiT pseudogene locus; The csgD gene was overexpressed using the Ptrc promoter at the purR gene locus; Overexpression of prs at the yeel pseudogene locus using the Ptrc promoter D128A Gene; The spoT gene was overexpressed using the Ptrc promoter at the yeeP pseudogene locus; The prK gene is expressed at the ylbE pseudogene locus; The RuBisCO gene was overexpressed using the Ptrc promoter at the yjiP pseudogene locus; The groEL-groES gene was overexpressed using the Ptrc promoter at the yghX pseudogene locus; The metB* gene was overexpressed at the sucCD gene locus and the aceA gene locus using the Ptrc promoter.

7. The construction method according to claim 6, characterized in that The engineering strain uses Escherichia coli as the starting strain; the Escherichia coli is E. coli K-12W3110.

8. Use of the engineered strain according to any one of claims 1 to 5 in producing L-methionine.

9. The use according to claim 8, characterized in that The production of L-methionine comprises the following steps: The engineered strain according to any one of claims 1 to 5 is inoculated onto an activated slant, cultured at 30 to 37° C. for 10 to 14 hours, and passaged 1 to 2 times to obtain an activated engineered strain; The activated engineered strain was transferred to a seed culture medium and cultured at a temperature of 30-37°C, a pH of 6.8-7.1, and a dissolved oxygen value of 25-35% until the OD value of the culture solution reached 0. 600 When the temperature reaches 10-15, the seed solution is obtained; The seed liquid is inoculated into a fermentation medium at an inoculum amount of 5% to 10% by volume, and fermentation culture is carried out under the conditions of a culture temperature of 30 to 37° C., a pH of 6.8 to 7.1, and a dissolved oxygen value of 25 to 35%. A fed-batch medium containing glucose is fed, and the glucose concentration in the fermentation liquid is controlled to be ≤1 g / L until the OD value of the fermentation liquid begins to decrease, thereby terminating the fermentation.

10. The use according to claim 9, characterized in that The feed medium is initially fed at a rate of 3.5-5 g / L / h, and then the flow acceleration is increased by 1.5-2.5 g / L / h every 4 hours, and finally the flow acceleration reaches 7-9 g / L / h until the fermentation is completed.