Engineering strain for synthesizing L-methionine by using ethylene glycol and application of engineering strain

Abstract

The invention provides an engineering strain for synthesizing L-methionine by using ethylene glycol and application of the engineering strain. The engineering strain comprises an expression plasmid vector or an expression plasmid vector combination, the vector or combination comprises a first group of polynucleotides and a second group of polynucleotides, and the first group of polynucleotides comprises at least one polynucleotide encoding an enzyme promoting ethylene glycol utilization; the second set of polynucleotides comprising at least one polynucleotide encoding an L-methionine synthesis pathway enzyme; and a backbone plasmid capable of autonomously replicating in a host cell. By introducing the ethylene glycol utilization gene, not only is the utilization rate of glucose improved, but also the yield of L-methionine is improved; meanwhile, by overexpressing or strengthening a plurality of L-methionine biosynthetic pathway related genes from different sources and knocking out or weakening L-methionine degradation pathway or transcription inhibition factor related genes, efficient fermentation production of L-methionine is realized.

Description

Technical Field

[0001] This invention relates to the field of engineered bacterial biosynthesis technology, and in particular to an engineered bacterial strain that synthesizes L-methionine using ethylene glycol and its applications. Background Technology

[0002] Ethylene glycol has broad industrial applications, including the manufacture of polyester plastics such as polyethylene terephthalate (PET). Glucose, a common industrial fermentation substrate, plays a role in the metabolic transformation of biomass and the synthesis of high-value chemicals in microbial cell factories, where there is significant overlap between these processes. This limits the efficiency of using glucose to synthesize target products. Ethylene glycol, however, minimizes the interaction between biomass and chemical synthesis pathways, improving substrate utilization. Furthermore, ethylene glycol is relatively economical and can be obtained through the electrochemical conversion of CO2 from the hydrolysis of plastic waste or the hydrogenolysis of cellulose waste. Therefore, it is a potentially abundant and renewable substrate, making it an advantageous choice for microbial production of high-value chemicals.

[0003] Methionine, also known as methionine, exists in two configurations: D-methionine and L-methionine. The L-configuration is an essential amino acid for humans, participating in various aspects of cellular protein synthesis and structural stability, as well as biological growth and development. The main metabolite of L-methionine, S-adenosylmethionine, supplies methyl groups to various important cellular components such as nucleic acids, lipids, and alkaloids. Industrial production of L-methionine primarily relies on chemical synthesis and enzymatic catalysis. However, due to the advantages of microbial synthesis of L-methionine, including low cost, low energy consumption, high yield, strong selectivity, and environmental friendliness, significant research has been conducted in recent years focusing on *Escherichia coli* and *Corynebacterium glutamicum*, systematically optimizing the biosynthetic metabolic network and culture conditions for L-methionine. *Escherichia coli*, with its clear genetic background and rapid growth rate, is widely used in L-methionine biosynthesis research. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an engineered strain for synthesizing L-methionine using ethylene glycol and its application, thereby obtaining a new method for synthesizing L-methionine.

[0005] In *E. coli*, ethylene glycol can be converted to glyoxylic acid, which is then utilized within the *E. coli* metabolic network. Studies have found that the conversion of ethylene glycol to glyoxylic acid requires three enzymes: lactaldehyde reductase, lactaldehyde dehydrogenase, and glycolate oxidase, encoded by the genes fucO, aldA, and glcDEF, respectively. These enzymes catalyze the sequential conversion of ethylene glycol to glycolaldehyde, glycolic acid, and finally glyoxylic acid. 2+ The propylene glycol oxidoreductase FucO, which it depends on, is easily oxidatively inactivated and therefore sensitive to oxygen. Its mutant Fuc... OI6L,L7VIt can improve the oxygen stability of FucO. Furthermore, the NAD-dependent glycerol dehydrogenase encoded by the gldA gene in *E. coli* catalyzes the reaction of glycerol to dihydroxyacetone; the gldA protein subunit requires Zn. 2+ GldA acts as a cofactor in the catalytic reaction. It also exhibits broad substrate specificity, capable of reacting with ethylene glycol to produce glycolaldehyde. Furthermore, inactivating glycolaldehyde reductase YqhD and glyoxylate / hydroxypyruvate reductase YcdW can improve the conversion rate of ethylene glycol.

[0006] A first aspect of the present invention provides an expression plasmid vector or a combination of expression plasmid vectors, the vector or combination comprising: a first group of polynucleotides and a second group of polynucleotides, wherein the first group of polynucleotides comprises at least one polynucleotide encoding an enzyme that promotes ethylene glycol utilization; and the second group of polynucleotides comprises at least one polynucleotide encoding an enzyme of the L-methionine synthesis pathway.

[0007] And the backbone plasmid, which can replicate autonomously in the host cell.

[0008] In a specific embodiment of the present invention, the plasmid vector or vector combination is used to promote the host cell’s utilization of ethylene glycol and / or enhance the expression of L-methionine.

[0009] In a specific embodiment of the present invention, the first group of polynucleotides includes a) a gene encoding glycerol dehydrogenase gldA or a mutant thereof, or lactalaldehyde reductase fucO or a mutant thereof; and / or b) a gene encoding lactalaldehyde dehydrogenase (aldA) or a mutant thereof.

[0010] In specific embodiments of the present invention, the glycerol dehydrogenase is a glycerol dehydrogenase or a propylene glycol / glycerol bifunctional dehydrogenase derived from the genus *Escherichia*, such as AAC43051 derived from *Escherichia coli*, or a glycerol dehydrogenase derived from *Escherichia albertii* such as OSL28309.1; or a glycerol dehydrogenase derived from *Klebsiella pneumoniae* such as ABR79620; or a glycerol dehydrogenase derived from the genus *Shigella* such as *Shigella boydii* or *Shigella dysenteriae* (such as ABB68416.1 or ABB63725.1); or a glycerol dehydrogenase derived from *Streptococcal pneumoniae* such as VTQ29411.1; or a glycerol dehydrogenase derived from *Bacillus stearothermophilus* such as AAA22477; or a glycerol dehydrogenase derived from *Bacillus subtilis*. Glyceryl dehydrogenases (alcohol / choline dehydrogenases) derived from *Pseudomonas subtilis*, such as CAB15083.2, or glyceryl dehydrogenases derived from *Pseudomonas putida*, such as AAC44426, or glyceryl dehydrogenases derived from *Clostridium saccharobutylicum*, such as AAA83520.1, or glyceryl dehydrogenases derived from *Clostridium acetonebutanol*, such as *Clostridium acetonebutanol*. Glyceryl dehydrogenases derived from *Acetobacter acetobutylicum*, such as AAK79593.1, or glyceryl dehydrogenases derived from *Salmonella*, such as CNU06578.1, or alcohol dehydrogenases (glyceryl dehydrogenases) derived from *Corynebacterium glutamicum*, such as SJM54452.1, can also be propylene glycol / glycerol bifunctional dehydrogenases derived from *Parasutterella excrementihominis*, such as MTU07760.1, as well as natural or artificial mutants of the same species. In one embodiment of the present invention, the glyceryl dehydrogenase gldA is derived from *Escherichia coli* or *Klebsiella pneumoniae*.

[0011] Preferably, the glycerol dehydrogenase gldA from *Escherichia coli* has the protein sequence accession number AAC43051 (ec.gldA); preferably, the glycerol dehydrogenase gldA from *Klebsiella pneumoniae* has the protein sequence accession number ABR79620 (kp.gldA); or an enzyme that maintains the above-mentioned enzyme activity and has an amino acid sequence with at least 96%, 97%, or 98%, or at least 99% sequence identity to the amino acid sequence represented by protein accession number AAC43051 or ABR79620.

[0012] In a specific embodiment of the present invention, the lactaldehyde reductase fucO is derived from Escherichia coli (Escherichiacoli).

[0013] Preferably, the lactaldehyde reductase fucO is a protein sequence with accession number AAA23825, or an enzyme that retains the above-mentioned enzyme activity and has an amino acid sequence with at least 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence represented by accession number AAA23825. Preferably, the lactaldehyde reductase fucO mutant is FucO. I6L,L7V .

[0014] In a specific embodiment of the present invention, the second group of polynucleotides encodes at least one enzyme of at least one L-methionine synthesis pathway and / or transport pathway.

[0015] In a specific embodiment of the present invention, the L-methionine synthesis pathway includes the pathway from aspartic acid to L-methionine as described in KEGG entry M00017.

[0016] In a specific embodiment of the present invention, the L-methionine synthesis pathway further includes sulfate transport and / or reductive assimilation pathways.

[0017] In a specific embodiment of the present invention, the reduction and assimilation pathway of the sulfate includes the pathway from sulfate to hydrogen sulfide as described in KEGG entry M00176.

[0018] In a specific embodiment of the present invention, the L-methionine synthesis pathway includes the OAA (oxaloacetic acid) to aspartic acid pathway.

[0019] In a specific embodiment of the present invention, the OAA (oxaloacetic acid) to aspartic acid pathway includes at least one of the OAA (oxaloacetic acid) to aspartic acid pathways in KEGG entries M00170 and M00171.

[0020] In a specific embodiment of the present invention, the L-methionine synthesis pathway further includes at least one of the KEGG entries M00001, M00002 or M00003 that promotes the accumulation of PEP (phosphoenolpyruvate) or OAA.

[0021] In one embodiment of the present invention, the second group of polynucleotides encodes at least one enzyme, including:

[0022] 1) Glucose transport or metabolic pathways and aspartate synthesis pathways, preferably at least one of galactose cotransporter galP, phosphoenolpyruvate synthase ppsA, phosphoenolpyruvate decarboxylase ppc, and aspartate aminotransferase aspC; and / or

[0023] 2) The L-methionine synthesis pathway preferably includes at least one of the following: aspartate kinase lysC, aspartate semialdehyde dehydrogenase asd, homoserine O-succinyltransferase metA, sulfate ABC transporter cysP, sulfate ABC transporter cysT, sulfate ABC transporter cysW, sulfate ABC transporter cysA, 5,10-methylenetetrahydrofolate reductase metF, methionine synthase metH, cystathionine β-lyase metC, cystathionine γ-synthesizer metBL, adenosine sulfate transferase subunit 2cysD, and adenosine sulfate transferase subunit 1cysN.

[0024] 3) L-methionine transport pathway, preferably L-methionine efflux protein yjeH.

[0025] In a specific embodiment of the present invention, the aspartate kinase lysC is a mutant of the enzyme derived from Escherichia coli with protein sequence accession number AAC43118, and the mutation site is T352I.

[0026] In a specific embodiment of the present invention, the homoserine O-succinyltransferase metA is a mutant of the enzyme derived from Escherichia coli with protein sequence accession number CAA32654, and the mutation site is R27CI296P298L.

[0027] In a specific embodiment of the present invention, the polynucleotides in the first and second groups of polynucleotides are homologous or heterologous to the host cell.

[0028] In a specific embodiment of the present invention, the polynucleotides in the first and second groups of polynucleotides contain codon optimizations for the host cell.

[0029] The nucleotide sequences, polynucleotides, and DNA molecules used in this invention are not limited to functional regions and may include at least one of expression repression regions, coding regions, leader sequences, exons, introns, and expression cassettes. Furthermore, the nucleotide sequences or polynucleotides may include double-stranded DNA or single-stranded DNA (i.e., the sense and antisense strands that make up double-stranded DNA) or RNA. Polynucleotides containing a specific polynucleotide sequence may include fragments and / or mutants of that specific polynucleotide sequence. A fragment of a polynucleotide refers to a portion of a polynucleotide that encodes a polypeptide that provides substantially the same function as the polypeptide encoded by the complete polynucleotide sequence. Examples of mutants of a specific polynucleotide sequence include naturally occurring allelic mutants, artificial mutants, and polynucleotide sequences obtained by deleting, substituting, adding, and / or inserting one or more nucleotides into said specific polynucleotide sequence. It should be understood that such fragments and / or mutants of a specific polynucleotide sequence encode a polypeptide that has substantially the same function as the polypeptide encoded by the original specific polynucleotide sequence.

[0030] In a specific embodiment of the present invention, the polynucleotides in the first to second groups of polynucleotides are operatively linked to promoters, which are homologous or heterologous to the host cell.

[0031] A second aspect of the invention provides a transformant that contains one or more of the above-described expression plasmid vectors or combinations thereof in a host cell.

[0032] In this invention, a transformant is a host cell altered by introducing one or more expression plasmid vectors into a host cell, wherein the one or more expression plasmid vectors are the same or different. In some embodiments, the transformant is obtained by introducing the plasmid vector into a host cell that exhibits competence with the expression plasmid vector.

[0033] In a specific embodiment of the present invention, the transformant comprises a first group of polynucleotides and a second group of polynucleotides integrated into the host cell chromosome.

[0034] In this invention, the first and second groups of polynucleotides can be integrated into the host cell chromosome using methods such as plasmid transformation, phage-mediated transformation, and genome editing.

[0035] In a specific embodiment of the present invention, the expression of one or both of the following genes in the transformant is inhibited, weakened or eliminated: glyoxylate / hydroxypyruvate reductase ycdW or / and NADPH-dependent aldehyde reductase yqhD.

[0036] In a further preferred embodiment of the present invention, the pathways for the generation of metabolic byproducts such as succinic acid, lactic acid, ethanol, acetic acid and formic acid, the L-methionine degradation pathway, and the transcriptional repressors of methionine biosynthesis in the transformant are inhibited or blocked.

[0037] In a further preferred embodiment of the present invention, the expression of one, two or more of the following genes in the transformant is inhibited, weakened or eliminated: phosphoacetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvate aldehyde synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavin subunit frdA, homoserine kinase thrB, methionine transcriptional repressor metJ, and threonine transporter rhtA.

[0038] This invention employs gene knockout to inhibit, reduce, or eliminate the expression of the corresponding enzyme. Those skilled in the art will recognize that other methods for inhibiting enzyme expression or reducing / eliminating enzyme activity can also be applied to the construction of genetically engineered strains, such as promoter knockout or replacement, introduction of enzyme inactivation / reduction mutations, deletion (or partial deletion) of essential functional elements or regions for gene transcription / translation, introduction of mutations or nucleic acid sequences that accelerate mRNA degradation to guide enzyme synthesis, introduction of mutations or protein tags that accelerate enzyme degradation / decomposition / inactivation, inhibition of signal activation, RNA interference, gene silencing, CRISPRi, etc. In a specific embodiment of this invention, the expression level of the enzyme is reduced by more than 30% or the activity of the enzyme is reduced by more than 30%.

[0039] In specific embodiments of the present invention, the host strain of the transformant is selected from bacteria or fungi; optionally, the host strain is selected from wild or genetically modified Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces; optionally, the host strain is selected from wild or genetically modified Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; optionally, the host strain is selected from wild or genetically modified Escherichia coli. Escherichia coli cells can be Escherichia coli strains derived from Escherichia coli K12 (e.g., MG1655, W3110, DH10b, DH1, BW2952 and strains derived therefrom) or any Escherichia coli strain or strain derived therefrom of Escherichia coli B.

[0040] A backbone plasmid that can autonomously replicate in a host cell can be any plasmid that can replicate in a host cell. In one embodiment, the expression plasmid vector contains a backbone plasmid that can replicate in *E. coli*. Examples of backbone plasmids include, but are not limited to, backbone plasmids that can replicate in *E. coli* strains, such as pUC (e.g., pUC18 and pUC19 plasmids), pBR322, pSC101, p15a, pACYC, pET, and pSC101 plasmids, and plasmids derived therefrom.

[0041] In a third aspect, the present invention provides a composition comprising the above-described transformant or a culture thereof.

[0042] In a fourth aspect, the present invention provides a method for preparing L-methionine, comprising culturing the above-mentioned transformant in the presence of ethylene glycol to obtain L-methionine; or extracting L-methionine from the above-mentioned culture.

[0043] In one embodiment of the present invention, the above-mentioned transformant is fermented in a medium containing ethylene glycol to obtain L-methionine.

[0044] In one embodiment of the present invention, ethylene glycol accounts for 0.01%-20% (by weight) of the culture medium.

[0045] In one embodiment of the present invention, the culture medium further includes one or more of glucose, yeast powder, M9 salt, magnesium sulfate, calcium chloride, vitamin B1, and calcium carbonate.

[0046] In a fifth aspect, the present invention provides the use of the above-described expression plasmid vector or combination of expression plasmid vectors, transformants or their cultures, or compositions in the preparation of L-methionine.

[0047] In a sixth aspect, the present invention provides a method for increasing the fermentation yield of L-methionine, the method comprising adding ethylene glycol to a culture medium, preferably, the cumulative addition of ethylene glycol being greater than 10 g / L, and / or introducing the aforementioned first group of polynucleotides into a fermentation strain.

[0048] In a seventh aspect, the present invention provides a chassis strain, which is an Escherichia coli strain or a Corynebacterium glutamicum strain, wherein the expression of one or two of the following genes in the chassis strain is inhibited, weakened or eliminated: glyoxylate / hydroxypyruvate reductase ycdW or / and NADPH-dependent aldehyde reductase yqhD.

[0049] In a further preferred embodiment of the present invention, the pathways for the generation of metabolic byproducts succinic acid, lactic acid, ethanol, acetic acid and formic acid in the chassis strain, as well as the pathways for diverting or inhibiting the synthesis of L-methionine, are inhibited or blocked.

[0050] In a further preferred embodiment of the present invention, the expression of one, two or more of the following genes in the chassis strain is inhibited, weakened or eliminated: phosphorylated acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvate aldehyde synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavin subunit frdA, homoserine kinase thrB, methionine transcriptional repressor metJ, and threonine transporter rhtA.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] 1) By introducing an ethylene glycol utilization gene, this invention not only improves glucose utilization but also increases L-methionine production. At the same time, by overexpressing or enhancing genes related to the L-methionine biosynthesis pathway from multiple different sources, and knocking out or weakening genes related to the L-methionine degradation pathway or transcriptional repressor, efficient fermentation production of L-methionine is achieved.

[0053] 2) Compared with traditional chemical synthesis methods, the engineered strain for producing L-methionine provided by this invention has the advantages of high yield, non-toxicity, no pollution, mild conditions, and less environmental pollution. Because it uses food-grade strains for fermentation, it has broad application prospects in the food, pharmaceutical and other industries. Detailed Implementation

[0054] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0055] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0056] Materials and Methods

[0057] LB medium: 10 g / L peptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0058] Fermentation medium: glucose 20g / L, yeast powder 5g / L, M9 salt 11.3g / L, magnesium sulfate 2mM, calcium chloride 0.1mM, ethylene glycol 10g / L, vitamin B1 5mg / L, calcium carbonate 10g / L. The pH of the medium was adjusted to 7.3-7.4 with concentrated ammonia water and sterilized at 115℃ for 15min.

[0059] Shake-flask fermentation method: Single clones of the activated bacterial strain from LB agar plates (containing appropriate concentrations of antibiotics) at 37℃ were inoculated into LB liquid medium (containing appropriate concentrations of antibiotics) and cultured at 37℃ for 12-16 hours at 220 rpm. The overnight LB culture was inoculated at a 1% inoculum into a 250 ml shake flask containing appropriate concentrations of ampicillin, kanamycin, and spectinomycin, along with 25 ml of fermentation medium (containing 10 g / L CaCO3 as a pH stabilizer). After sealing with a breathable membrane, the flask was incubated at 30℃ and 220 rpm. When the OD600 reached 1, IPTG (final concentration 0.2 mM) was added to induce plasmid expression. Fermentation was stopped after 48 hours of incubation, and samples were taken.

[0060] Concentration determination of L-methionine, glucose, and ethylene glycol (EG): All standards used were purchased from Sigma-Aldrich (www.sigmaaldrich.cn). 1 mL of fermentation broth was centrifuged at 10000 r / min for 5 min to remove bacterial cells. The filtrate was filtered through a 0.22 μm filter membrane. After appropriate dilution, the concentration of the above products in the sample was determined by high-performance liquid chromatography (HPLC).

[0061] Determination conditions for glucose and ethylene glycol (EG): High-performance liquid chromatography (HPLC) was performed using a Shimadzu Nexera LC-40 system with a Bio-Rad Aminex HPX-87H column (300×7.8 mm). The column oven was set to 40℃; the detector was a RID (Refractive Index Detector) (set to constant temperature at 40℃). The mobile phase was 5 mM sulfuric acid solution, and the flow rate was 0.6 ml / min.

[0062] L-methionine determination conditions: High performance liquid chromatograph was Shimadzu LC-40D; chromatographic column was Welch Ultimate AQ-C18, 4.6×250mm; column temperature oven was set to 40℃; detector was ultraviolet detector (detector set to constant temperature 40℃), detection wavelength was 338nm; mobile phase was sodium acetate aqueous solution and methanol, gradient elution.

[0063] The information regarding the enzymes involved in this invention is as follows:

[0064]

[0065]

[0066] In the context of this application, the enzymes mentioned include mutants that retain enzyme activity, said mutants having an amino acid sequence that is at least 98% or at least 99% identical to the amino acid sequence represented by the protein accession number in the table.

[0067] The biomaterials constructed in this invention are shown in the table below:

[0068]

[0069]

[0070] Example 1: Construction of L-methionine synthesis chassis strain

[0071] Using the E. coli Keio Knockout Collection 1 Phages were created from the corresponding single-gene knockout strain (Horizon Discovery, CO, USA). Using the P1 phage transfection method, the DNA target gene deletion fragment carrying kanamycin (Kan) resistance was introduced into the starting strain *E. coli* BW25113. The fragment was plated on LB agar plates containing 50 mg / L kanamycin and incubated overnight at 37°C to obtain a Kan-resistant deletion strain. Then, plasmid pCP20 was transformed into the aforementioned Kan-resistant transformants, which were then plated on LB agar plates containing 100 mg / L ampicillin and incubated at 30°C for 24 hours. PCR was used to identify the correct transformants with the Kan-resistant gene removed from the target gene, thus obtaining a non-resistant deletion strain. Finally, single colonies of the correct transformants with the Kan gene removed were streaked onto LB agar plates and incubated at 37 or 42°C to obtain a non-resistant *E. coli* strain with the pCP20 plasmid removed. By repeating this process, a BW25113-based deletion strain, Met00 (ldhAΔptaΔpoxBΔpflBΔmgsAΔfrdAΔadhEΔthrBΔmetJΔrhtA), was constructed. In this strain, the pathways for the generation of metabolic byproducts (succinic acid, lactic acid, ethanol, acetic acid, and formic acid) and the partial degradation pathway of L-methionine were blocked or knocked out to avoid the waste of carbon sources and reduce the degradation of L-methionine.

[0072] Based on the Met00 strain, the yqhD and ycdW genes were knocked out to obtain the Met01 (Met00ΔyqhD), Met02 (Met00ΔycdW), and Met03 (Met00ΔyqhDΔycdW) strains.

[0073] Example 2 Recombinant plasmids pZE-ec.gldA, pZE-fucO, pZE-aldA, pZE-fucO-aldA, pZE-ec.gldA-aldA, pZE-kp.gldA-aldA, pZA-metA R27C-I296S-P298L -cysPTWA-metF-metH and pZS-aspC-lysC T352I Construction of -asd--metC-metBL-cysDN-yjeH

[0074] The kp.gldA gene was cloned from the genome of Klebsiella pneumoniae using PCR; the fucO, ec.gldA, and aldA genes were cloned from the genome of Escherichia coli BW25113, and Gibson assembly was used. 2 The DNA fragments obtained above were combined and ligated into plasmid pZElac in different ways. 3 After the lac promoter was removed, the following recombinant plasmids were obtained (see the Materials and Methods section for combination methods): pZE-ec.gldA, pZE-aldA, pZE-fucO, pZE-ec.gldA-aldA, pZE-kp.gldA-aldA, and pZE-fucO-aldA.

[0075] The gene metA in the genome of E. coli BW25113 was cloned by PCR. R27C I296S P298L The gene sequence includes cysP, cysT, cysW, cysA, metF, and metH. The metA gene was cloned in three segments, with R27C, I296S, and P298L mutations introduced near the segment interface using primers. 4 The obtained clone fragments were ligated to plasmid pZAlac using Gibson assembly. 3 After the lac promoter was removed, the recombinant plasmid pZA-metA was obtained. R27C-I296S-P298L -cysPTWA--metF-metH(pMET01).

[0076] The genes aspC and lysC from the genome of E. coli BW25113 were cloned by PCR. T352I The genes lysC, metC, metBL, cysD, cysN, and yjeH were cloned, with the lysC gene divided into two fragments for cloning. A T352I mutation was introduced near the fragment interface using primers. 5 The obtained clone fragments were ligated to plasmid pZSlac using Gibson assembly. 3 After the lac promoter was removed, the recombinant plasmid pZS-aspC-lysC was obtained. T352I -asd-metC-metBL-cysDN-yjeH(pMET02).

[0077] The above recombinant plasmids were transformed into the Met00-03 strain. For details on the specific plasmid and strain combinations, please refer to the Materials and Methods section.

[0078] Example 3: Shake-flask fermentation of strains Met04, Met05, and Met06

[0079] Using strain Met04 as a control, shake-flask fermentation was performed. The composition of the fermentation medium and fermentation conditions are detailed in the Materials and Methods section. Fermentation data after 48 hours are shown in Table 1.

[0080] Table 1. Data from L-methionine shake-flask fermentation

[0081]

[0082] The results showed that overexpression of the first gene fucO or gldA in the ethylene glycol (EG) metabolic pathway slightly increased the L-methionine production of Met05 and Met06 compared to the control strain (Met04 without overexpression of EG metabolic pathway-related genes).

[0083] Example 4: Shake-flask fermentation of strains Met04, Met07, Met08, and Met09

[0084] Using strain Met04 as a control, shake-flask fermentation was performed. The composition of the fermentation medium and fermentation conditions are detailed in the Materials and Methods section. Fermentation data after 48 hours are shown in Table 2.

[0085] Table 2. Data from L-methionine shake-flask fermentation

[0086]

[0087]

[0088] The results showed that introducing aldA into the gldA / fucO overexpression significantly increased EG consumption; simultaneously, compared with the control strain, the strain overexpressing gldA / fucO-aldA showed increased L-methionine production. Furthermore, isolated overexpression of the aldA gene (Met07) also slightly increased ethylene glycol consumption and L-methionine production.

[0089] Example 5: Effect of deletion of yqhD and / or ycdW genes on L-methionine fermentation

[0090] To explore the role of the EG utilization pathway byproducts YqhD and YcdW, shake-flask fermentation was performed using strain Met09 as a control. The composition of the fermentation medium and fermentation conditions are detailed in the Materials and Methods section. Fermentation data after 48 hours are shown in Table 3.

[0091] Table 3. Data from L-methionine shake-flask fermentation

[0092]

[0093] The results showed that knockout of the yqhD and / or ycdW genes had no significant effect on glucose utilization, but knockout of the yqhD and / or ycdW genes slightly improved the strain's ability to utilize ethylene glycol, while the production of L-methionine also increased.

[0094] Example 6: Effects of different gldA sources on L-methionine fermentation

[0095] Using strain Met12 as a control, strain Met13 carrying the Klebsiella pneumoniae kp.gldA gene was also subjected to shake-flask fermentation. The composition of the fermentation medium and fermentation conditions are detailed in the Materials and Methods section. Fermentation data after 48 hours are shown in Table 4.

[0096] Table 4. Data from L-methionine shake-flask fermentation

[0097]

[0098] The results showed that the activity of the kp.gldA gene from Klebsiella pneumoniae was slightly better than that of ec.gldA from Escherichia coli, resulting in a slight increase in the production of L-methionine.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

[0100] References

[0101] 1. Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.; Datsenko, KA; Tomita, M.; Wanner, BL; Mori, H., Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology 2006,2(1),2006.0008.

[0102] 2.Gibson,D.G.;Young,L.;Chuang,R.-Y.;Venter,J.C.;Hutchison,C.A.;Smith,H.O.,Enzymatic assembly of DNA molecules up to several hundredkilobases.Nature Methods 2009,6(5),343-345.

[0103] 3.Zhang,K.;Li,H.;Cho,K.M.;Liao,J.C.,Expanding metabolism for totalbiosynthesis of the nonnatural amino acid L-homoalanine.Proceedings of theNational Academy of Sciences 2010,107(14),6234-6239.

[0104] 4.Li,Z.;Liu,Q.;Sun,J.;Sun,J.;Li,M.;Zhang,Y.;Deng,A.;Liu,S.;Wen,T.,Multivariate modular metabolic engineering for enhanced l-methioninebiosynthesis in Escherichia coli.Biotechnology for Biofuels and Bioproducts2023,16(1),101.

[0105] 5.Li,Y.;Wei,H.;Wang,T.;Xu,Q.;Zhang,C.;Fan,X.;Ma,Q.;Chen,N.;Xie,X.,Current status on metabolic engineering for the production of l-aspartatefamily amino acids and derivatives.Bioresource Technology 2017,245,1588-1602.

Claims

1. An expression plasmid vector or a combination of expression plasmid vectors, characterized in that: The carrier or combination comprises: a first group of polynucleotides and a second group of polynucleotides, wherein the first group of polynucleotides comprises at least one polynucleotide encoding an enzyme that promotes ethylene glycol utilization; and the second group of polynucleotides comprises at least one polynucleotide encoding an enzyme of the L-methionine synthesis pathway. And the backbone plasmid, which can replicate autonomously in the host cell.

2. The expression plasmid vector or combination of expression plasmid vectors as described in claim 1, characterized in that: The first group of polynucleotides includes a) a gene encoding glycerol dehydrogenase gldA or a mutant thereof, or lactaldehyde reductase fucO or a mutant thereof; and / or b) a gene encoding lactaldehyde dehydrogenase (aldA) or a mutant thereof; Preferably, the glycerol dehydrogenase is a glycerol dehydrogenase or a propylene glycol / glycerol bifunctional dehydrogenase derived from the genus *Escherichia*, more preferably AAC43051 derived from *Escherichia coli*, or a glycerol dehydrogenase derived from *Escherichia albertii*, more preferably OSL28309.1; or a glycerol dehydrogenase derived from *Klebsiella pneumoniae*, more preferably ABR79620; or a glycerol dehydrogenase derived from the genus *Shigella* such as *Shigella boydii* or *Shigella dysenteriae*, more preferably ABB68416.1 or ABB63725.1; or a glycerol dehydrogenase derived from *Streptococcal pneumoniae*, more preferably VTQ29411.1; or a glycerol dehydrogenase derived from *Bacillus stearothermophilus*, more preferably AAA22477; or a glycerol dehydrogenase derived from *Bacillus subtilis*. Glyceryl dehydrogenase from *Pseudomonas subtilis*, more preferably CAB15083.2; or glyceryl dehydrogenase from *Pseudomonas putida*, more preferably AAC44426; or glyceryl dehydrogenase from *Clostridium saccharobutylicum*, more preferably AAA83520.1; or glyceryl dehydrogenase from *Clostridium acetobutylicum*, more preferably AAK79593.1; or glyceryl dehydrogenase from *Salmonella*, more preferably CNU065. 78.1, or an alcohol dehydrogenase derived from Corynebacterium glutamicum, more preferably SJM54452.1, or a propylene glycol / glycerol bifunctional dehydrogenase MTU07760.1 derived from Parasutterella excrementihominis, or a natural or artificial mutant of the same species of enzyme. Preferably, the glycerol dehydrogenase gldA from Escherichia coli has protein sequence accession number AAC43051; preferably, the glycerol dehydrogenase gldA from Klebsiella pneumoniae has protein sequence accession number ABR79620; or an enzyme that maintains the above enzyme activity and has an amino acid sequence with at least 96% or 97% or 98%, or at least 99% sequence identity to the amino acid sequence represented by protein accession number AAC43051 or ABR79620. Preferably, the lactaldehyde reductase fucO is derived from Escherichia coli; Preferably, the lactaldehyde reductase fucO is a protein sequence with accession number AAA23825, or an enzyme that retains the above-mentioned enzyme activity and has an amino acid sequence with at least 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence represented by accession number AAA23825; more preferably, the lactaldehyde reductase fucO mutant is FucO. I6L,L7V .

3. The expression plasmid vector or combination of expression plasmid vectors as described in claim 1, characterized in that: The second group of polynucleotides encodes at least one enzyme in at least one L-methionine synthesis pathway and / or transport pathway; Preferably, the L-methionine synthesis pathway includes the pathway from aspartic acid to L-methionine as described in KEGG entry M00017; Preferably, the L-methionine synthesis pathway further includes sulfate transport and / or reductive assimilation pathways; More preferably, the reduction and assimilation pathway of the sulfate includes the pathway from sulfate to hydrogen sulfide as described in KEGG entry M00176; Preferably, the L-methionine synthesis pathway includes the oxaloacetate to aspartate pathway; More preferably, the oxaloacetate to aspartic acid pathway includes at least one of the oxaloacetate to aspartic acid pathways in KEGG entries M00170 and M00171. Preferably, the L-methionine synthesis pathway further includes at least one of the KEGG entries M00001, M00002 or M00003 that promotes the accumulation of phosphoenolpyruvate or OAA. More preferably, the second group of polynucleotides encodes at least one of the following enzymes: 1) Glucose transport or metabolic pathways and aspartate synthesis pathways, preferably at least one of galactose cotransporter galP, phosphoenolpyruvate synthase ppsA, phosphoenolpyruvate decarboxylase ppc, and aspartate aminotransferase aspC; and / or 2) The L-methionine synthesis pathway preferably includes at least one of the following: aspartate kinase lysC, aspartate semialdehyde dehydrogenase asd, homoserine O-succinyltransferase metA, sulfate ABC transporter cysP, sulfate ABC transporter cysT, sulfate ABC transporter cysW, sulfate ABC transporter cysA, 5,10-methylenetetrahydrofolate reductase metF, methionine synthase metH, cystathionine β-lyase metC, cystathionine γ-synthesizer metBL, adenosine sulfate transferase subunit 2cysD, and adenosine sulfate transferase subunit 1cysN. 3) L-methionine transport pathway, preferably L-methionine efflux protein yjeH.

4. The expression plasmid vector or combination of expression plasmid vectors as described in claim 1, characterized in that: The polynucleotides in the first and second groups of polynucleotides are homologous or heterologous to the host cell; Preferably, the polynucleotides in the first and second groups of polynucleotides contain codons optimized for the host cell; Preferably, the polynucleotides in the first and second groups of polynucleotides are operatively linked to a promoter, which is homologous or heterologous to the host cell.

5. A transformant, characterized in that: The host cell contains one or more expression plasmid vectors or combinations of expression plasmid vectors as described in any one of claims 1-4; or, the transformant contains a first group of polynucleotides and a second group of polynucleotides integrated into the host cell chromosome. Preferably, the expression of one or both of the following genes in the transformant is inhibited, weakened or eliminated: glyoxylate / hydroxypyruvate reductase ycdW or / and NADPH-dependent aldehyde reductase yqhD; Preferably, the pathways for the generation of metabolic byproducts succinic acid, lactic acid, ethanol, acetic acid and formic acid, the L-methionine degradation pathway, and the transcriptional repressor factors for methionine biosynthesis in the transformed organism are inhibited or blocked. Preferably, the expression of one, two or more of the following genes in the transformant is inhibited, weakened or eliminated: phosphorylated acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvate aldehyde synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavin subunit frdA, homoserine kinase thrB, methionine transcriptional repressor metJ, and threonine transporter rhtA.

6. A composition, characterized in that: Includes the transformant or its culture as described in claim 5.

7. A method for preparing L-methionine, characterized in that: This includes culturing the transformant of claim 5 in the presence of ethylene glycol to obtain L-methionine; or extracting L-methionine from the culture of claim 6; Preferably, the above-mentioned transformant is fermented in a medium containing ethylene glycol to obtain L-methionine; Preferably, ethylene glycol accounts for 0.01%-20% of the weight of the culture medium; Preferably, the culture medium further includes one or more of glucose, yeast powder, M9 salt, magnesium sulfate, calcium chloride, vitamin B1, and calcium carbonate.

8. The use of the expression plasmid vector or combination of expression plasmid vectors according to any one of claims 1-4, the transformant according to claim 5, or the composition according to claim 6 in the preparation of L-methionine.

9. A method for increasing the fermentation yield of L-methionine, characterized in that: The method includes adding ethylene glycol to the culture medium, preferably in an aggregate amount greater than 10 g / L, and / or introducing the first group of polynucleotides as described in any one of claims 1-4 into the fermentation strain.

10. A chassis strain, characterized in that: The chassis strain is an Escherichia coli strain or a Corynebacterium glutamicum strain, and the expression of one or two of the following genes in the chassis strain is inhibited, weakened or eliminated: glyoxylate / hydroxypyruvate reductase ycdW or / and NADPH-dependent aldehyde reductase yqhD. Preferably, the pathways for the generation of metabolic byproducts succinic acid, lactic acid, ethanol, acetic acid and formic acid in the chassis strain, as well as the pathways for diverting or inhibiting the synthesis of L-methionine, are inhibited or blocked. Preferably, the expression of one, two or more of the following genes in the chassis strain is inhibited, weakened or eliminated: phosphorylated acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvate aldehyde synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavin subunit frdA, homoserine kinase thrB, methionine transcriptional repressor metJ, and threonine transporter rhtA.

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