Microorganism for producing l-methionine and method for preparing l-methionine

Abstract

Provided are a microorganism for producing L-methionine and a method for preparing L-methionine by means of fermentation using the microorganism. By means of genetic engineering, thrA, metA and metZ genes required for the production of L-methionine are co-expressed in an Escherichia coli mutant to construct a strain capable of producing L-methionine with a high yield, and L-methionine is directly produced in one step by means of a biological fermentation method.

Description

Microorganisms that produce L-methionine and methods for preparing L-methionine Technical Field

[0001] This invention belongs to the field of industrial microbiology and biochemical technology. Specifically, it relates to a microorganism for producing L-methionine and a method for preparing L-methionine. Background Technology

[0002] Methionine is one of the essential amino acids for the human body and is widely used as an animal feed and food additive, as well as a component of aqueous solutions and other raw materials in pharmaceutical products. Methionine is a precursor to choline (lecithin) and creatine, and a raw material for the synthesis of cysteine ​​and taurine, and also acts as a sulfur donor. S-adenosyl-L-methionine, derived from L-methionine, acts as a methyl donor in the body and is involved in the synthesis of various neurotransmitters in the brain. Furthermore, methionine and / or S-adenosyl-L-methionine (SAM) can prevent lipid accumulation in the liver and arteries and effectively treat depression, inflammation, liver disease, and muscle pain. In the feed industry, the demand for methionine is high; adding a small amount of methionine to livestock and poultry diets can effectively improve protein utilization. Methionine deficiency in livestock and poultry manifests as poor development, weight loss, impaired liver and kidney function, muscle atrophy, and deterioration of coat quality.

[0003] Currently, methionine is produced through several methods, including the malonic ester method, aminolactone method, condensation hydrolysis method, tyrosine hydrolysis method, and biological method. Chemical synthesis methods, such as the malonic ester method, require strong acids, strong alkalis, and toxic substances like acrolein and cyanide, generating large amounts of wastewater, waste liquid, and waste residue, resulting in significant environmental pollution and high production costs. The product of chemical synthesis is DL-methionine, which can only be used as a feed additive. When the methionine concentration in feed is low, D-methionine's relative efficacy is lower than L-methionine. Since humans cannot absorb D-methionine, obtaining pure L-methionine requires further chemical or enzymatic separation of DL-methionine, which undoubtedly increases production costs further. In contrast, the biological method produces L-methionine, which can be directly applied in the pharmaceutical, food, and health industries. Bio-fermentation offers advantages such as readily available raw materials, a simple production process, and low environmental pollution, thus possessing wider application value and greater commercial value. For example, patent CN103397057A describes a method for synthesizing L-methionine, which consists of two steps: 1) preparing an L-methionine precursor-producing strain and preparing the L-methionine precursor, namely O-acetylhomoserine or O-succinylhomoserine, through fermentation; 2) preparing L-methionine and organic acids through an enzymatic reaction with the L-methionine precursor. While the above method can produce L-methionine, it suffers from numerous production steps and high operating costs. Therefore, this invention develops a highly efficient method for preparing L-methionine. This method uses genetic engineering to co-express the key catalytic enzymes required for L-methionine production in *E. coli* mutants to construct a high-yield L-methionine strain, and then directly produces L-methionine through bio-fermentation. This invention not only efficiently produces L-methionine but also features a green and environmentally friendly production process, simple operation, reduced production costs, and a significant competitive advantage in the market. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and provide a microorganism for producing L-methionine and a method for preparing L-methionine. Compared with traditional methods, the method for producing L-methionine in this invention is more environmentally friendly, simpler to operate, reduces production costs, and has a more obvious competitive advantage in the market.

[0005] The objective of this invention and the technical problem it solves are achieved by the following technical solutions.

[0006] The first aspect of the present invention provides a method for constructing a microorganism for producing L-methionine, wherein the microorganism is *Escherichia coli*, strain number SMet05BsA-CsZ, and the construction method includes the following steps:

[0007] Construction of plasmid pXK-thrA-BsA: The homoserine-O-acetyltransferase gene BsmetA from Bacillus subtilis was inserted between the XhoI and EcoRI sites of plasmid vector pXA to obtain a recombinant vector, named pXK-thrA-BsA.

[0008] Construction of plasmid pBAD-CsZ: The O-acetylhomoserine hydrogen sulfide hydrolase gene CsmetZ from Rhodotorula globulus was inserted between the XhoI and EcoRI sites of the plasmid vector pBAD-HisB to obtain the recombinant vector, named pBAD-CsZ.

[0009] Construction of host strain Met05: The E. coli mutant obtained by knocking out the rhtA and rhtB genes encoding threonine and homoserine transporters inserted into the genome of mutant E. coli ST11 was named Met05.

[0010] Construction of engineered strain SMet05BsA-CsZ: The above recombinant vector plasmids pXK-thrA-BsA and pBAD-CsZ were introduced into the host strain Met05 to obtain the recombinant engineered strain, named SMet05BsA-CsZ.

[0011] In some embodiments, the plasmid vector pXA carries the E. coli aspartate kinase / homoserine dehydrogenase 1 gene thrA (S345F) that relieves feedback inhibition and contains an arabinose inducible promoter.

[0012] In some embodiments, the mutant Escherichia coli ST11 is described in patent 202011270812.X, with the genotype E. coli BW25113ΔptsG::glk,ΔgalR::zglf,ΔompT::ppc,ΔldhA::rhtA,ΔlpxM::rhtB,ΔpflB::asd,ΔpoxB::aspA,ΔiclR,ΔlysA,ΔmetA,ΔthrB.

[0013] In some embodiments, the mutant Escherichia coli Met05 genotype is E. coli BW25113ΔptsG::glk,ΔgalR::zglf,ΔompT::ppc,ΔldhA,ΔlpxM,ΔpflB::asd,ΔpoxB::aspA,ΔiclR,ΔlysA,ΔmetA,ΔthrB.

[0014] A second aspect of the present invention provides a method for synthesizing L-methionine, wherein the L-methionine is produced by fermentation using a microorganism for producing L-methionine constructed according to the construction method described above, the method comprising:

[0015] Seed culture: The engineered strain SMet05BsA-CsZ glycerol bacteria was inoculated into LB medium containing kanamycin and ampicillin and cultured on a shaker. Seed culture was obtained after the culture was completed.

[0016] Fermentation culture: The seed culture was transferred to a shake flask of ZYM self-induction medium, arabinose was added, followed by kanamycin and ampicillin. The culture was induced and cultured. The bacterial cells were collected after the culture was completed.

[0017] Catalytic production of methionine: The collected bacterial cells were centrifuged, and the resulting precipitated bacterial cells were washed and resuspended in conversion solution to obtain a bacterial suspension. The obtained bacterial suspension was subjected to a catalytic reaction in a shaker to obtain the final product methionine.

[0018] In some embodiments, during the seed culture, the engineered strain SMet05BsA-CsZ glycerol bacterium is inoculated at an inoculation rate of 1-5% by volume.

[0019] In some embodiments, during the seed culture, the final concentrations of kanamycin and ampicillin in the LB medium are each 50–55 μg / mL.

[0020] In some embodiments, the shaking culture conditions in the seed culture are: temperature of 35-37°C and time of 16-18 hours.

[0021] In some embodiments, the final concentration of arabinose in the fermentation culture is 0.2-0.3% by volume.

[0022] In some embodiments, the final concentrations of kanamycin and ampicillin during the fermentation culture are each 50–55 μg / mL.

[0023] In some embodiments, the induction culture conditions during fermentation are: a temperature of 28–30°C and a time of 16–18 h.

[0024] In some embodiments, during the fermentation culture, OD is measured after the culture is completed. 600 Light absorbance values ​​were collected, and 2–4 × 10⁻⁴ ppm were collected. 10 Bacterial cells, according to OD 600 When the concentration is 1, the bacterial concentration is 1×10⁻⁶. 9 / mL calculation.

[0025] In some embodiments, the centrifugation conditions for the catalytic methionine production are: a rotation speed of 4000–4500 rpm and a time of 8–10 min.

[0026] In some embodiments, the catalytic methionine production process includes a conversion solution containing 50-100 mM glucose, 50-100 mM sodium methanethiol, 100-200 mM ammonium chloride, 2-4 mM magnesium sulfate, and 1×M9 salt at a final concentration.

[0027] In some embodiments, the 1×M9 salt contains 47.75 mM disodium hydrogen phosphate, 22.04 mM potassium dihydrogen phosphate, 8.56 mM sodium chloride, and 18.70 mM ammonium chloride at a final concentration.

[0028] In some embodiments, the concentration of the bacterial suspension in the catalytic methionine production is 2 × 10⁻⁶. 10 / mL.

[0029] In some embodiments, the catalytic reaction conditions for the catalytic production of methionine are: a temperature of 35–37°C and a rotation speed of 200–220 rpm.

[0030] By employing the above technical solution, the present invention has at least the following advantages:

[0031] This invention constructs a mutant Escherichia coli strain SMet05BsA-CsZ capable of efficiently producing methionine, enabling direct one-step L-methionine production via bio-fermentation. Compared to existing technologies, this invention's production process is more environmentally friendly, simpler to operate, and reduces production costs, resulting in a significant competitive advantage in the market. Through continuous optimization and upgrading of the strain and manufacturing process, this invention yields a higher quality and lower cost L-methionine product, possessing great market potential.

[0032] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0033] Figure 1 shows the physical spectrum of the expression vector pXK-thrA-BsA;

[0034] Figure 2 shows the physical spectrum of the expression vector pBAD-CsZ;

[0035] Figure 3 shows the standard curve for methionine.

[0036] Figure 4 shows the L-methionine production results of strain SMet05BsA-CsZ. Detailed Implementation

[0037] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0039] Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] Unless otherwise specified, the LB medium formulation in the following examples is: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride.

[0041] Unless otherwise specified, the ZYM self-induction culture medium formulation in the following examples is: 100mL A + 2mL B + 2mL C + 200μL D + 100μL E (all are mass percentage concentrations); wherein, A. ZY: 1% tryptone, 0.5% yeast extract; B. 50×M: 1.25M Na2HPO4, 1.25M KH2PO4, 2.5M NH4Cl and 0.25M Na2SO4; C. 50×5052: 25% glycerol, 2.5% glucose, 10% L-arabinose; D. 500×MgSO4: 1M MgSO4; E. 1000× Trace elements: 50mM FeCl3, 20mM CaCl2, 10mM MnCl2, 10mM ZnSO4, 2mM each of CoCl2, NiCl2, Na2Mo4, Na2SeO3 and H2BO3.

[0042] Unless otherwise specified, the conversion solution formulation in the following examples is: 50 mM glucose, 50 mM sodium methanethiol, 100 mM ammonium chloride, 1×M9 salt, and 2 mM MgSO4, wherein the 1×M9 salt contains 47.75 mM disodium hydrogen phosphate, 22.04 mM potassium dihydrogen phosphate, 8.56 mM sodium chloride, and 18.70 mM ammonium chloride.

[0043] Example 1: Construction of recombinant plasmid pXK-thrA-BsA synergistically expressing aspartate kinase from Escherichia coli and homoserine-O-acetyltransferase from Bacillus subtilis

[0044] For the cloning of the BsmetA gene, genomic DNA of Bacillus subtilis subsp. subtilis str. 168 was used as a template, and the BsmetA gene encoding Bacillus subtilis homoserine-O-acetyltransferase (metA) was amplified by PCR. The base sequence of the BsmetA gene was obtained from the NCBI GenBank database and is represented as SEQ ID NO. 1.

[0045] Based on the aforementioned base sequence, using primer pairs (BsmetA-F and BsmetA-R) containing selective restriction sites XhoI and EcoRI, and with Bacillus subtilis genomic DNA as a template, the ORF from ATG to TAA was amplified by PCR in the presence of the high-fidelity DNA polymerase Phanta Super-Fidelity DNA Polymerase (Nanjing Novizan Biotechnology Co., Ltd., product catalog P501).

[0046] SEQ ID NO.1:

[0047] The PCR amplification primer sequences are shown in SEQ ID NO.2-3 below:

[0048] SEQ ID NO.2:

[0049] SEQ ID NO.3:

[0050] The PCR amplification system consisted of: 1 μL template, 2 μL forward primer, 2 μL reverse primer, 1 μL dNTP Mix, 1 μL Phanta Super-Fidelity DNA Polymerase, 10 μL 5×SF Buffer, and 33 μL deionized water.

[0051] The PCR amplification conditions were: 30 cycles of denaturation at 95℃ for 30 seconds, annealing at 60℃ for 30 seconds, and extension at 72℃ for 2 minutes, to synthesize the approximately 1kb BsmetA gene containing XhoI and EcoRI sites.

[0052] The pXA plasmid was digested with restriction enzymes XhoI and EcoRI to obtain its linearized vector. The digestion conditions were 37°C for 2 hours and 65°C for 10 minutes. The pXA plasmid construction method is described in patent 202210853986.1, which is a recombinant inducible expression vector carrying the E. coli aspartate kinase / homoserine dehydrogenase 1 gene thrA (S345F) that relieves feedback inhibition and contains an arabinose inducible promoter, which is incorporated herein by reference. The BsmetA gene was ligated into a pXA linearized vector using the Gibson method (Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, 3rd, Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 2009, 6:343-345.), thereby constructing a recombinant inducible expression vector carrying the thrA and BsmetA genes and containing an arabinose inducible promoter, named pXK-thrA-BsA. The ligation conditions were incubation at 50°C for 1 hour and at 4°C for 10 minutes.

[0053] Figure 1 shows the genetic map of the constructed expression vector pXK-thrA-BsA.

[0054] Example 2: Construction of recombinant plasmid pBAD-CsZ

[0055] The gene sequence of CsmetZ, an O-acetylhomoserine hydrogen sulfide hydrolase from *Rhodotorula gravidarum*, was obtained from the NCBI database. Codon optimization yielded the nucleotide sequence shown in SEQ ID NO.4, enabling gene expression in *E. coli*. The CsmetZ gene was amplified by PCR using primer pairs (CsmetZ-F and CsmetZ-R) containing selective restriction sites XhoI and EcoRI, based on the synthesized nucleotide sequence SEQ ID NO.4.

[0056] SEQ ID NO.4:

[0057] The PCR amplification primer sequences are shown in SEQ ID NO.5-6 below:

[0058] SEQ ID NO.5:

[0059] SEQ ID NO.6:

[0060] The PCR amplification system consisted of: 1 μL template, 2 μL forward primer, 2 μL reverse primer, 1 μL dNTP Mix, 1 μL Phanta Super-Fidelity DNA Polymerase, 10 μL 5×SF Buffer, and 33 μL deionized water.

[0061] The PCR amplification reaction conditions were: 30 cycles of denaturation at 95℃ for 30 seconds, annealing at 60℃ for 30 seconds, and extension at 72℃ for 2 minutes, thereby synthesizing the CsmetZ gene, which contains approximately 1.2 kb of XhoI and EcoRI sites.

[0062] The linearized vector of the inducible expression vector pBAD-HisB containing the arabinose inducible promoter was obtained by digesting it with the restriction enzymes XhoI and EcoRI. The digestion conditions were 37°C for 2 hours and 65°C for 10 minutes. The CsmetZ gene was ligated into the pBAD-HisB linearized vector using the Gibson method, thereby constructing a recombinant inducible expression vector carrying the CsmetZ gene and containing the arabinose inducible promoter, named pBAD-CsZ. The ligation conditions were 50°C for 1 hour and 4°C for 10 minutes.

[0063] Figure 2 shows the genetic map of the constructed expression vector pBAD-CsZ.

[0064] Example 3: Construction of Escherichia coli mutant Met05

[0065] The E. coli mutant Met05 is an E. coli mutant obtained by knocking out the rhtA and rhtB genes encoding threonine and homoserine transporters inserted into the genome of mutant E. coli ST11 using CRISPR technology. Its genotype is E. coli BW25113ΔptsG::glk,ΔgalR::zglf,ΔompT::ppc,ΔldhA,ΔlpxM,ΔpflB::asd,ΔpoxB::aspA,ΔiclR,ΔlysA,ΔmetA,ΔthrB.

[0066] The mutant *E. coli* ST11 is described in patent 202011270812.X, with the genotype *E. coli* BW25113ΔptsG::glk,ΔgalR::zglf,ΔompT::ppc,ΔldhA::rhtA,ΔlpxM::rhtB,ΔpflB::asd,ΔpoxB::aspA,ΔiclR,ΔlysA,ΔmetA,ΔthrB. The specific construction steps for the *E. coli* mutant Met05 are as follows:

[0067] (1) Preparation of electrocompetent cells: pCas plasmid (Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coligenome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81:2506-2514.) was transformed into Escherichia coli ST11 by chemical transformation. Positive clones were screened by culturing on LB agar plates containing kanamycin (kanamycin concentration of 50 μg / mL) at 30°C. Positive clones were inoculated into LB liquid medium containing 2 g / L arabinose and cultured at 30°C until OD. 600 After adjusting the concentration to 0.6–0.8, electrocompetent cells were prepared.

[0068] (2) Construction of pTarget plasmid: Using the website https: / / crispy.secondarymetabolites.org, the N20 knockout site was selected, and primers were designed to construct the pTarget plasmid. Using pTargetF (Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81:2506-2514.) as a template, PCR amplification was performed using primer pairs pTarget-AF and pTarget-AR, pTarget-BF and pTarget-BR in the presence of the high-fidelity DNA polymerase Phanta Super-Fidelity DNA Polymerase, respectively, and fragments of approximately 2100 bp were obtained.

[0069] After reacting with DpnI methyltransferase for approximately 3 hours, *E. coli* Fast-T1 competent cells (Nanjing Novizan Biotechnology Co., Ltd., product catalog C505) were directly transformed using chemical transformation. Positive clones were screened on LB agar plates containing streptomycin (streptomycin concentration 50 μg / mL) and verified by sequencing using primer pTarget-cexu-F. After correct sequencing, the clones were named pTarget-A and pTarget-B, respectively.

[0070] The primer sequences used are as follows (the underlined sequence is N20) SEQ ID NO.7~11:

[0071] SEQ ID NO.7:

[0072] SEQ ID NO.8:

[0073] SEQ ID NO.9:

[0074] SEQ ID NO.10:

[0075] SEQ ID NO.11:

[0076] The PCR amplification system consisted of: 1 μL template, 2 μL forward primer, 2 μL reverse primer, 1 μL dNTP Mix, 1 μL Phanta Super-Fidelity DNA Polymerase, 10 μL 5×SF Buffer, and 33 μL deionized water.

[0077] The PCR amplification reaction conditions were: 30 cycles of denaturation at 95℃ for 30 seconds, annealing at 60℃ for 30 seconds, and extension at 72℃ for 3 minutes.

[0078] (3) Amplification of the target fragment: Using the genome of *E. coli* BW 25113 as a template, PCR amplification was performed using primer pairs ldhA-up800-F and ldhA-up800-R, ldhA-down800-F and ldhA-down800-R, lpxM-up800-F and lpxM-up800-R, and lpxM-down800-F and lpxM-down800-R. The fragments were recovered to obtain 800bp upstream fragment, 800bp downstream fragment, 800bp upstream fragment, and 800bp downstream fragment of lpxM, respectively. Using a mixture of the 800bp upstream and 800bp downstream fragments of ldhA as a template, PCR amplification was performed using primer pairs ldhA-up500-F and ldhA-down500-R, and the ΔldhA target fragment of approximately 1000bp was recovered by gel electrophoresis. Using a mixture of 800bp upstream and 800bp downstream fragments of lpxM as a template, PCR amplification was performed using primer pairs lpxM-up500-F and lpxM-down500-R, and the ΔlpxM targeting fragment of approximately 1000bp was obtained by gel recovery.

[0079] The primer sequences used are shown in SEQ ID NO.12~23 below:

[0080] SEQ ID NO.12:

[0081] SEQ ID NO.13:

[0082] SEQ ID NO.14:

[0083] SEQ ID NO.15:

[0084] SEQ ID NO.16:

[0085] SEQ ID NO.17:

[0086] SEQ ID NO.18:

[0087] SEQ ID NO.19:

[0088] SEQ ID NO.20:

[0089] SEQ ID NO.21:

[0090] SEQ ID NO.22:

[0091] SEQ ID NO.23:

[0092] The PCR amplification system consisted of: 1 μL template, 2 μL forward primer, 2 μL reverse primer, 1 μL dNTP Mix, 1 μL Phanta Super-Fidelity DNA Polymerase, 10 μL 5×SF Buffer, and 33 μL deionized water.

[0093] The PCR amplification conditions were: 30 cycles of denaturation at 95℃ for 30 seconds, annealing at 60℃ for 30 seconds, and extension at 72℃ for 2 minutes, to synthesize the target fragments.

[0094] (4) Electroporation: 200 ng of pTarget-A plasmid, 400 ng of the targeting fragment ΔldhA, and 100 μL of the electroporation competent cells prepared in step 1 were mixed and placed in a 2 mm electroporation cuvette. Electroporation was performed at 2.5 kV, followed by the addition of 1 mL of LB liquid medium. After recovery at 30°C, the mixture was plated on LB plates containing kanamycin and streptomycin (kanamycin concentration 50 μg / mL, streptomycin concentration 50 μg / mL) and incubated at 30°C. Positive clones were screened. PCR amplification was performed using primers ldhA-up800-F and ldhA-down800-R, and the amplified fragments were sequenced for verification.

[0095] The PCR amplification system consisted of: 5 μL Green Taq Mix (Nanjing Novizan Biotechnology Co., Ltd., product catalog P131), 0.5 μL each of primers (10 uM), 3.5 μL distilled water, and 0.5 μL template bacterial culture, for a total volume of 10 μL. The PCR amplification conditions were: 95℃ pre-denaturation for 3 minutes (1 cycle); 95℃ denaturation for 15 seconds, 55℃ annealing for 15 seconds, and 72℃ extension for 1-5 minutes (60 seconds / kb) (30 cycles); and 72℃ extension for 5 minutes (1 cycle).

[0096] (5) Elimination of pTarget plasmid: Positive clones verified by sequencing were inoculated into LB liquid medium containing 0.1 mM IPTG and kanamycin and cultured overnight at 30°C to eliminate the pTarget plasmid. After overnight culture, the strain was streaked on LB solid plates containing kanamycin and cultured overnight at 30°C to obtain the E. coli mutant containing the pCas plasmid, named Met04.

[0097] (6) Pick single clones from the plate in step (5) to prepare electrotransfer competent cells, mix them with pTarget-B plasmid and ΔlpxM targeting fragment, repeat steps (4)-(5), and use primer pairs lpxM-up800-F and lpxM-down800-R for sequencing verification to obtain E. coli mutant containing pCas plasmid, named Met05.

[0098] (7) Elimination of pCas plasmid: The correctly sequenced E. coli mutant Met05 containing the pCas plasmid was inoculated into LB liquid medium and cultured overnight at 42°C to eliminate the pCas plasmid. The strain after overnight culture was streaked on LB solid plates and cultured overnight at 42°C to obtain the plasmid-free E. coli mutant E. coli BW25113ΔptsG::glk,ΔgalR::zglf,ΔompT::ppc,ΔldhA,ΔlpxM,ΔpflB::asd,ΔpoxB::aspA,ΔiclR,ΔlysA,ΔmetA,ΔthrB, abbreviated as Met05.

[0099] Example 4: Construction of strain SMet05BsA-CsZ co-expressing pXK-thrA-BsA and pBAD-CsZ

[0100] The *E. coli* mutant Met05 glycerol strain constructed in Example 3 was streaked onto LB agar plates and incubated at 37°C for 16 h. Single colonies of Met05 were picked and placed in shake flasks containing 5 mL of LB medium and incubated at 37°C until OD (dose elapsed). 600 The concentration was 0.5-0.6. The bacterial culture was transferred to a sterile 10 mL centrifuge tube and incubated on ice for 10 minutes, then centrifuged at 4°C and 4000 rpm for 10 minutes. The supernatant was discarded, and the cells were resuspended in 5 mL of pre-chilled solution containing 80 mM CaCl2 and 20 mM MgCl2, then incubated on ice for 30 minutes. After the ice bath, the cells were centrifuged at 4°C and 4000 rpm for 10 minutes. The supernatant was discarded, and the cells were resuspended in 100 μL of solution containing 20 mM CaCl2 and 10% glycerol (V / V) to obtain Met05 competent cells. The recombinant plasmid pXK-thrA-BsA constructed in Example 1 and the recombinant plasmid pBAD-CsZ constructed in Example 2 were added to the Met05 competent cells and incubated on ice for 30 minutes. After the ice bath, incubate in a 42°C water bath for 1 minute, then transfer to ice for 2 minutes. Add 600 μL of LB medium and incubate at 37°C on a shaker for 1 hour. Centrifuge the bacterial suspension at 4000 rpm for 2 minutes, discard most of the supernatant, resuspend the cells in the remaining supernatant, and spread on LB agar plates containing kanamycin and ampicillin (50 μg / mL). Incubate at 37°C for 18 hours. Pick single colonies from the plates and inoculate them into 5 mL of LB medium containing kanamycin and ampicillin. Incubate at 37°C on a shaker until OD (dose elapsed). 600 The concentration was 1.6-1.8. An equal volume of 50% glycerol (V / V) was added and mixed well. The mixture was then dispensed into preservation tubes at 1 mL / tube. The resulting cloned strain was named SMet05BsA-CsZ and stored in an ultra-low temperature freezer at -80℃.

[0101] Example 5: Production of L-methionine using engineered strain SMet05BsA-CsZ

[0102] (1) Whole-cell catalysis of L-methionine conversion

[0103] The engineered strain SMet05BsA-CsZ glycerol bacterium obtained in Example 4 was inoculated at a 1% (V / V) inoculum into LB medium containing kanamycin and ampicillin (concentration of kanamycin and ampicillin: 50 μg / mL), and cultured in a shaker at 37°C for 18 h to obtain a seed culture. The seed culture was transferred to a shake flask containing 20 mL of ZYM self-induction medium, and arabinose was added to a final concentration of 0.2% (V / V), followed by kanamycin and ampicillin to a final concentration of 50 μg / mL. The culture was induced at 30°C for 16 h, and the OD was measured. 600 Light absorbance, and collected 2×10 10 Bacterial cells, according to OD 600 When the concentration is 1, the bacterial concentration is 1×10⁻⁶. 9 / mL calculation.

[0104] The above-collected 2×10 10 The bacterial cells were centrifuged at 4000 rpm for 10 min, and the precipitate was collected. The precipitate was washed twice with 0.85% physiological saline, centrifuged again, and the supernatant was discarded to obtain the precipitated bacterial cells. The obtained bacterial cells were resuspended in 1 mL of transformation buffer to obtain a bacterial suspension, which was then transferred to test tubes to achieve a final bacterial concentration of 2 × 10⁻⁶. 10 / mL. The test tube containing the bacterial suspension was placed at 37℃ and 200rpm for conversion to obtain a fermentation broth containing L-methionine.

[0105] (2) Detection of methionine by high performance liquid chromatography

[0106] The fermentation broth sample containing L-methionine obtained above was centrifuged at 14000 rpm for 10 min, and the supernatant was collected as the sample to be tested. Before detection, the sample was filtered through a 0.22 μm microporous membrane, and online amino acid derivatization was performed using o-phthalaldehyde (OPA). HPLC amino acid detection was then performed. The experiment was repeated three times, and the average value of the results was taken.

[0107] The online derivatization syringe program is as follows: ① Draw 2.5 μL of borate buffer from vial 1; ② Draw 0.5 μL of the sample to be tested; ③ Mix 3 μL "in air" at maximum speed, twice; ④ Wait 0.5 minutes; ⑤ Draw 0 μL of cleaning solution from vial 2 (rinse the needle with water from the uncapped vial); ⑥ Draw 0.5 μL of OPA solution from vial 3; ⑦ Mix 3 μL "in air" at maximum speed, six times; ⑧ Draw 0 μL of cleaning solution from vial 2 (rinse the needle with water from the uncapped vial); ⑨ Inject the sample.

[0108] The high-performance liquid chromatography (HPLC) instrument and detection conditions are as follows:

[0109] HPLC qualitative analysis of amino acids: An Agilent 1260 Infinity II instrument was used, with an Agilent Eclipse AAA column (4.6 mm × 150 mm, 5 μm). The mobile phase was A: 0.04 M Na₂HPO₄; B: acetonitrile-methanol-water mixture (9:9:2, v / v). The mobile phase was filtered through a 0.45 μm aqueous / organic filter and degassed by sonication for 20 min until no obvious bubbles remained. The flow rate was 2 mL / min, the column temperature was 40℃, the injection volume was 1 μL, the detection wavelength was 338 nm, and the data acquisition time was 35 min. Gradient elution was used, with the following program (pump B): 1–2.5 min, 12%; 2.5–13 min, 16–36%; 13–28 min, 38–100%; 28–35 min, 10%.

[0110] A standard curve was constructed by referring to the peak time and peak area of ​​the standard sample, as shown in Figure 3. The methionine content in the sample was calculated based on the peak area, and the results are shown in Figure 4.

[0111] Results: As shown in Figure 4, after 22 hours of transformation, the methionine yield produced by strain SMet05BsA-CsZ was 30.67 mM, with a molar conversion rate of 61.34%. Liquid chromatography analysis showed that the purity of methionine in the fermentation broth was 88.11%.

[0112] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for constructing a microorganism producing L-methionine, characterized by, The microorganism is *Escherichia coli*, strain number SMet05BsA-CsZ, and the construction method includes the following steps: Construction of plasmid pXK-thrA-BsA: The homoserine-O-acetyltransferase gene BsmetA from Bacillus subtilis was inserted between the XhoI and EcoRI sites of plasmid vector pXA to obtain a recombinant vector, named pXK-thrA-BsA. Construction of plasmid pBAD-CsZ: The O-acetylhomoserine hydrogen sulfide hydrolase gene CsmetZ from Rhodotorula globulus was inserted between the XhoI and EcoRI sites of the plasmid vector pBAD-HisB to obtain the recombinant vector, named pBAD-CsZ. Construction of host strain Met05: The E. coli mutant obtained by knocking out the rhtA and rhtB genes encoding threonine and homoserine transporters inserted into the genome of mutant E. coli ST11 was named Met05. Construction of engineered strain SMet05BsA-CsZ: The above recombinant vector plasmids pXK-thrA-BsA and pBAD-CsZ were introduced into the host strain Met05 to obtain the recombinant engineered strain, named SMet05BsA-CsZ.

2. The construction method according to claim 1, characterized in that, The plasmid vector pXA carries the E. coli aspartate kinase / homoserine dehydrogenase 1 gene thrA (S345F) that relieves feedback inhibition and contains an arabinose inducible promoter. The mutant Escherichia coli ST11 genotype is E. coli BW25113ΔptsG::glk,ΔgalR::zglf,ΔompT::ppc,ΔldhA::rhtA,ΔlpxM::rhtB,ΔpflB::asd,ΔpoxB::aspA,ΔiclR,ΔlysA,ΔmetA,ΔthrB; The mutant Escherichia coli Met05 genotype is E. coli BW25113ΔptsG::glk,ΔgalR::zglf,ΔompT::ppc,ΔldhA,ΔlpxM,ΔpflB::asd,ΔpoxB::aspA,ΔiclR,ΔlysA,ΔmetA,ΔthrB.

3. A method of synthesizing L-methionine, characterized by, L-methionine is produced by fermentation using microorganisms that produce L-methionine constructed according to the construction method described in claim 1 or 2, wherein the method for synthesizing L-methionine includes: Seed culture: The engineered strain SMet05BsA-CsZ glycerol bacteria was inoculated into LB medium containing kanamycin and ampicillin and cultured on a shaker. Seed culture was obtained after the culture was completed. Fermentation culture: The seed culture was transferred to a shake flask of ZYM self-induction medium, arabinose was added, followed by kanamycin and ampicillin. The culture was induced and cultured. The bacterial cells were collected after the culture was completed. Catalytic production of methionine: The collected bacterial cells were centrifuged, and the resulting precipitated bacterial cells were washed and resuspended in conversion solution to obtain a bacterial suspension. The obtained bacterial suspension was subjected to a catalytic reaction in a shaker to obtain the final product methionine.

4. The method of claim 3, wherein, In the seed culture, the engineered strain SMet05BsA-CsZ glycerol bacterium was inoculated at an inoculation rate of 1-5% by volume. The final concentrations of kanamycin and ampicillin in the LB medium were each 50–55 μg / mL.

5. The method of claim 3, wherein, In the seed culture, the shaking incubator conditions are: temperature 35-37℃, time 16-18h.

6. The method of claim 3, wherein, In the fermentation culture, the final concentration of arabinose is 0.2-0.3% by volume; the final concentrations of kanamycin and ampicillin are each 50-55 μg / mL.

7. The method of claim 3, wherein, In the fermentation culture, the induction culture conditions are: temperature 28-30℃, time 16-18h.

8. The method of claim 3, wherein, In the catalytic methionine production process, the centrifugation conditions are: rotation speed 4000-4500 rpm, time 8-10 min.

9. The method according to claim 3, characterized in that, In the catalytic methionine production, the conversion solution contains 50-100 mM glucose, 50-100 mM sodium methanethiol, 100-200 mM ammonium chloride, 2-4 mM magnesium sulfate, and 1×M9 salt at a final concentration. The 1×M9 salt contains 47.75 mM disodium hydrogen phosphate, 22.04 mM potassium dihydrogen phosphate, 8.56 mM sodium chloride, and 18.70 mM ammonium chloride at a final concentration.

10. The method according to claim 3, characterized in that, In the catalytic production of methionine, the catalytic reaction conditions are: temperature of 35-37°C and rotation speed of 200-220 rpm.

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