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

Abstract

Provided are a microorganism for producing an L-methionine precursor and a method for preparing L-methionine from the L-methionine precursor. Also provided are a method for preparing an L-methionine precursor (O-acetyl-L-homoserine) by means of fermentation, and a method for converting an L-methionine precursor into L-methionine by means of enzymatic conversion. According to the method, a strain capable of producing O-acetyl-L-homoserine with a high yield is constructed by means of genetic engineering, a strain expressing O-acetylhomoserine sulfhydrylase is constructed, and O-acetyl-L-homoserine is catalyzed with a crude enzyme solution of O-acetylhomoserine sulfhydrylase to produce methionine.

Description

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

[0001] This invention belongs to the fields of industrial microbiology and biochemical technology. Specifically, it relates to a microorganism for producing L-methionine precursors and a method for preparing L-methionine from L-methionine precursors. 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, the aminolactone method, condensation hydrolysis, tyrosine hydrolysis, and biological methods. 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, further increasing production costs. In contrast, the biological method produces L-methionine, which can be directly applied in the pharmaceutical, food, and health industries. Fermentation production requires readily available raw materials, has a simple production process, and causes minimal environmental pollution, thus possessing wider application value and greater commercial value. Therefore, the inventors have developed a method for preparing L-methionine, which consists of two steps: fermentation to prepare L-methionine precursor (O-acetyl-L-homoserine) and converting the L-methionine precursor into L-methionine via enzymes. 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 precursors and a method for preparing L-methionine from the L-methionine precursors. The L-methionine precursors described in this invention are produced by fermentation of an L-methionine precursor-producing strain according to this invention. Compared with conventional methods, the method for producing L-methionine of this invention is more environmentally friendly and allows for selective production of L-methionine.

[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 microorganism for producing L-methionine precursors, said microorganism being Escherichia coli, strain number SOAH01Bs.

[0007] In a preferred embodiment of the present invention, the L-methionine precursor is O-acetyl-L-homoserine.

[0008] A second aspect of the present invention provides a method for constructing a microorganism for producing L-methionine precursors as described above, the method comprising the following steps:

[0009] 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.

[0010] Construction of engineered strain SOAH01Bs: The above recombinant vector plasmid pXK-thrA-BsA was introduced into the Escherichia coli mutant ST11C to obtain the recombinant engineered strain, which was named SOAH01Bs.

[0011] In a preferred embodiment of the present invention, the plasmid vector pXA, described in patent 202311317213.2, carries the Escherichia coli aspartate kinase / homoserine dehydrogenase 1 gene thrA (S345F) that relieves feedback inhibition and contains an arabinose inducible promoter.

[0012] In a preferred embodiment of the present invention, the Escherichia coli mutant ST11C is described in patent 202311317213.2, and its genotype is E. coli BW25113ΔptsG::glk,ΔgalR::zglf,ΔompT::ppc,ΔldhA::rhtA,ΔlpxM::rhtB,ΔpflB::asd,ΔpoxB::aspA,ΔiclR,ΔlysA,ΔmetA,ΔthrB,ΔaspC.

[0013] A third aspect of the present invention provides a method for synthesizing L-methionine precursor, comprising fermenting L-methionine precursor produced by microorganisms according to the above-described method for producing L-methionine precursor, or by microorganisms for producing L-methionine precursor constructed according to the above-described method for producing L-methionine precursor.

[0014] In a preferred embodiment of the present invention, the method for synthesizing the L-methionine precursor includes:

[0015] Seed culture: The glycerol engineered strain SOAH01Bs stored at -80℃ was inoculated into LB medium and cultured at 25-42℃ and 100-300rpm for 6-8h to obtain the seed culture;

[0016] Fermentation culture: The obtained seed culture was inoculated into the fermentation medium at an inoculation rate of 5% (V / V). When the initial glucose was consumed, the feed culture medium was started. When the cell density reached an absorbance OD of 600 nm, the culture was fermented. 600 When the concentration is 30, an inducer is added to induce protein expression. Fermentation is stopped when the feed medium is exhausted, and OAH fermentation broth is obtained, which is the L-methionine precursor.

[0017] In a preferred embodiment of the present invention, the fermentation medium comprises: citric acid 1-5 g / L, potassium dihydrogen phosphate 1-20 g / L, nitrogen source 1-5 g / L, polyether defoamer 0.1-1 mL / L, glucose 5-30 g / L, MgSO4·7H2O 0.3-1 g / L, VB1 5-15 mg / L, lysine hydrochloride 0.1-1 g / L, methionine 0.1-1 g / L, threonine 0.1-2 g / L, isoleucine 0.1-1 g / L, trace inorganic salts 1-10 mL / L, and pH 7.0±0.5.

[0018] In a preferred embodiment of the present invention, the nitrogen source is diammonium hydrogen phosphate;

[0019] In a preferred embodiment of the present invention, the composition of the trace inorganic salt I is as follows: EDTA 820-840 mg / L, CoCl2·6H2O 240-250 mg / L, MnCl2·4H2O 1450-1500 mg / L, CuCl2·2H2O 140-150 mg / L, H3BO3 290-300 mg / L, Na2MoO4·2H2O 240-250 mg / L, Zn(CH3COO)2·2H2O 1250-1300 mg / L, and ferric citrate 9-10 g / L.

[0020] In a preferred embodiment of the present invention, the fed-batch culture medium is composed of: glucose 600-650 g / L, MgSO4·7H2O 2.0-2.5 g / L, lysine hydrochloride 3.08-3.10 g / L, methionine 1.2-1.3 g / L, polyether defoamer 2.0-2.5 mL / L, and trace inorganic salt II 10.0-10.5 mL / L.

[0021] In a preferred embodiment of the present invention, the trace inorganic salt II comprises: EDTA 1300-1350 mg / L, CoCl2·6H2O 400-450 mg / L, MnCl2·4H2O 2350-2400 mg / L, CuCl2·2H2O 250-300 mg / L, H3BO3 500-550 mg / L, Na2MoO4·2H2O 400-450 mg / L, Zn(CH3COO)2·2H2O 1600-1650 mg / L, and ferric citrate 4.0-4.5 g / L.

[0022] In a preferred embodiment of the present invention, the inducing agent consists of: L-arabinose 1.0-1.2 g / L and MgSO4·7H2O 1.0-1.2 g / L.

[0023] In a preferred embodiment of the present invention, the fermentation culture conditions are as follows:

[0024] Initial conditions: glucose concentration 20–25 g / L, temperature 35–37 °C, air flow rate 2–3 vvm, stirring speed 300–320 rpm, dissolved oxygen concentration set at 99%–100%.

[0025] Fermentation process conditions: Adjust the air flow rate to 2-3 vvm, control the dissolved oxygen concentration to 30%-35% at all times, and use ammonia water to control the pH to 7.0-7.5 during the fermentation process.

[0026] A fourth aspect of the present invention provides a strain expressing O-acetylhomoserine hydrogen sulfide hydrolase, said strain being Escherichia coli, strain number SMet03CgY.

[0027] A fifth aspect of the present invention provides a method for constructing a strain expressing O-acetylhomoserine hydrogen sulfide hydrolase as described above, the method comprising the following steps:

[0028] Construction of host strain Met03: The E. coli mutant obtained by knocking out the gene metI encoding the methionine transporter, the gene metJ inhibiting methionine synthesis, and the gene metB of endogenous cystathionine γ synthase in E. coli ST11C was named Met03, and its genotype is E. coli ST11CΔmetIΔmetJΔmetB.

[0029] Construction of plasmid pRK-CgY: The O-acetylhomoserine hydrogen sulfide hydrolase gene CgmetY from Corynebacterium glutamicum was inserted between the XhoI and EcoRI sites of plasmid vector pRB1K to obtain a recombinant vector, named pRK-CgY.

[0030] Construction of engineered strain SMet03CgY: The above recombinant vector plasmid pRK-CgY was introduced into the above Escherichia coli mutant Met03 to obtain the recombinant engineered strain, named SMet03CgY.

[0031] In a preferred embodiment of the present invention, the plasmid vector pRB1K is described in patent 202011270812.X, which carries a kanamycin resistance gene and contains an arabinose inducible promoter.

[0032] In a preferred embodiment of the present invention, the *Escherichia coli* ST11C is described in patent 202311317213.2, and its genotype is *E. coli* BW25113ΔptsG::glk,ΔgalR::zglf,ΔompT::ppc,ΔldhA::rhtA,ΔlpxM::rhtB,ΔpflB::asd,ΔpoxB::aspA,ΔiclR,ΔlysA,ΔmetA,ΔthrB,ΔaspC.

[0033] The sixth aspect of the present invention provides a method for synthesizing O-acetylhomoserine hydrogen sulfide hydrolase, which is obtained by fermentation using a strain expressing O-acetylhomoserine hydrogen sulfide hydrolase as described above, or a strain expressing O-acetylhomoserine hydrogen sulfide hydrolase constructed according to the construction method described above.

[0034] In a preferred embodiment of the present invention, the method for synthesizing O-acetylhomoserine hydrogen sulfide hydrolase includes the following steps:

[0035] The SMet03CgY bacterial culture was inoculated at a rate of 1% (V / V) into a shake flask of ZYM self-induction medium. Arabinose and kanamycin were added sequentially, and the culture was induced. The OD was then measured. 600 The absorbance value was determined, and the bacterial concentration was 1×10⁻⁶ when 0.D.600 = 1. 9 Calculate / mL, collect 1×1011 Bacterial cells;

[0036] The above-collected 1×10 11 After initial centrifugation, the precipitate was washed with physiological saline and then resuspended to a concentration of 1×10⁻⁶. 11 / mL of bacterial suspension was obtained, and the bacterial suspension was ultrasonically disrupted. After disruption, the suspension was centrifuged again, and the supernatant was taken as the crude CgmetY enzyme solution, which is O-acetylhomoserine hydrogen sulfide hydrolase.

[0037] In a preferred embodiment of the present invention, the SMet03CgY bacterial solution is obtained by inoculating the engineered strain SMet03CgY glycerol bacteria at an inoculation rate of 1% (V / V) into LB medium containing 50 μg / mL kanamycin, and then culturing it in a shaker at 37°C and 220 rpm for 16 h.

[0038] In a preferred embodiment of the present invention, the arabinose concentration is 0.2% to 0.3% (V / V).

[0039] In a preferred embodiment of the present invention, the concentration of kanamycin is 50-55 μg / mL.

[0040] In a preferred embodiment of the present invention, the induction culture conditions are: temperature 35-37°C, time 16-18h.

[0041] In a preferred embodiment of the present invention, the centrifugation conditions for collecting bacterial cells are: 4000-4500 rpm for 8-10 min.

[0042] In a preferred embodiment of the present invention, the ultrasonic fragmentation conditions are: 1 second of ultrasonication, 2 seconds of pause, 15 to 20 minutes of time, and 30 to 35% power.

[0043] In a preferred embodiment of the present invention, the centrifugation conditions for the ultrasonically broken product are: temperature 4°C, rotation speed 12000 rpm, and time 8-10 min.

[0044] The seventh aspect of the present invention provides a method for synthesizing L-methionine, using OAH fermentation broth synthesized according to the method described above as a substrate and CgmetY crude enzyme solution synthesized according to the method described above as a catalytic enzyme for catalytic synthesis.

[0045] In a preferred embodiment of the present invention, the method for synthesizing L-methionine includes the following steps:

[0046] The supernatant was obtained after centrifuging the OAH fermentation broth.

[0047] The obtained supernatant was mixed with CgmetY crude enzyme solution and sodium methanethiol to obtain the conversion reaction solution;

[0048] The resulting conversion reaction solution was subjected to an enzymatic reaction at 37°C and 200 rpm in a shaker to obtain the final product.

[0049] In a preferred embodiment of the present invention, the centrifugation conditions are: rotation speed 12000 rpm; time 5 min.

[0050] In a preferred embodiment of the present invention, the ratio of the amount of sodium methanethiol added to the molar amount of the OAH fermentation broth is 1:1.

[0051] By employing the above technical solution, the present invention has at least the following advantages:

[0052] This invention constructs a mutant Escherichia coli strain SOAH01Bs that can efficiently produce O-acetyl-L-homoserine, and constructs an O-acetylhomoserine hydrogen sulfide hydrolase expression strain SMet03CgY. The crude enzyme of O-acetylhomoserine hydrogen sulfide hydrolase efficiently catalyzes the production of methionine from O-acetyl-L-homoserine.

[0053] This invention utilizes genetic engineering to obtain an engineered strain that produces high levels of O-acetyl-L-homoserine, and constructs an expression strain for O-acetylhomoserine hydrogen sulfide hydrolase. The crude enzyme of O-acetylhomoserine hydrogen sulfide hydrolase catalyzes the production of methionine from O-acetyl-L-homoserine, resulting in a more environmentally friendly production process and a significant competitive advantage in the market. Continuous optimization and upgrading of the strain and manufacturing process further improve product quality and reduce costs, demonstrating a promising market prospect.

[0054] 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

[0055] Figure 1 shows the physical spectrum of the expression vector pXK-thrA-BsA;

[0056] Figure 2 shows the OAH production results catalyzed by the whole cell of SOAH01Bs strain;

[0057] Figure 3 shows the OAH yield results of SOAH01Bs strain during high-density fermentation for 66 h.

[0058] Figure 4 shows the physical spectrum of the expression vector pRK-CgY;

[0059] Figure 5 shows the SDS-PAGE gel image of crude CgmetY enzyme;

[0060] Figure 6 shows the yield of methionine synthesized by the crude enzyme CgmetY. Detailed Implementation

[0061] 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.

[0062] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0063] Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0064] 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.

[0065] 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.

[0066] Unless otherwise specified, the conversion solution formulation in the following examples is: 50mM glucose, 100mM ammonium chloride, 1×M9 salt, and 2mM MgSO4.

[0067] Example 1: Construction of recombinant plasmid pXK-thrA-BsA synergistically expressing aspartate kinase from Escherichia coli and homoserine-O-acetyltransferase from Bacillus subtilis

[0068] 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.

[0069] 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).

[0070] SEQ ID NO.1:

[0071] The PCR amplification primer sequences are shown in SEQ ID NO.2-3 below:

[0072] SEQ ID NO.2:

[0073] BsmetA-F: 5'-gactcgagaaggagatataatgcctattaatataccaacacacc-3';

[0074] SEQ ID NO.3:

[0075] BsmetA-R: 5'-agaccgagctcaccgaattcttagtcccattcataaggag-3'.

[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 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.

[0078] 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.

[0079] Figure 1 shows the physical map of the constructed expression vector pXK-thrA-BsA.

[0080] Example 2: Construction of a high-yield strain of O-acetyl-L-homoserine

[0081] The method for constructing the Escherichia coli mutant ST11C is described in patent 202311317213.2, which is incorporated herein by reference. The genotype of the Escherichia coli mutant ST11C is E. coli BW25113ΔptsG::glk,ΔgalR::zglf,ΔompT::ppc,ΔldhA::rhtA,ΔlpxM::rhtB,ΔpflB::asd,ΔpoxB::aspA,ΔiclR,ΔlysA,ΔmetA,ΔthrB,ΔaspC.

[0082] The ST11C glycerol mutant of *E. coli* was streaked onto LB agar plates and incubated at 37°C for 16 h. Single colonies of ST11C were picked and placed in shake flasks containing 5 mL of LB medium and incubated at 37°C with shaking until OD (dose elapsed). 600The 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 bacterial 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 bacterial cells were resuspended in 100 μL of solution containing 20 mM CaCl2 and 10% glycerol (V / V) to obtain ST11C competent cells. The recombinant plasmid pXK-thrA-BsA constructed in Example 1 was added to the obtained ST11C competent cells, and the cells were 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. After incubation, centrifuge the bacterial culture at 4000 rpm for 3 minutes, discard most of the supernatant, resuspend the cells in the remaining supernatant, and plate them on LB agar plates containing kanamycin (50 μg / mL). Incubate at 37°C for 16 hours. After incubation, pick single colonies from the plates and inoculate them into 5 mL of LB medium containing kanamycin. Incubate at 37°C on a shaker until OD (dose elapsed). 600 The concentration was 1.6-1.8. Then, 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 SOAH01Bs and stored in an ultra-low temperature freezer at -80℃.

[0083] Example 3: Production of O-acetyl-L-homoserine using engineered strain SOAH01Bs

[0084] (1) Whole-cell catalysis for OAH conversion

[0085] The engineered strain SOAH01Bs glycerol bacterium was inoculated at a rate of 1% (v / v) into LB medium containing kanamycin (50 μg / mL) and cultured in a shaker at 37°C for 16 h. After culture, the bacterial 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 to a final concentration of 50 μg / mL. The culture was then induced at 30°C for 16 h, after which 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.

[0086] The above-collected 2×10 10After centrifuging the bacterial cells at 4000 rpm for 10 min, collect the precipitate, wash twice with 0.85% physiological saline, discard the supernatant, and resuspend the obtained bacterial cells in 1 mL of transformation buffer to obtain a bacterial suspension. Transfer the suspension to a test tube to achieve a final bacterial concentration of 2 × 10⁻⁶. 10 / mL, used for whole-cell transformation to generate OAH fermentation broth. The whole-cell transformation system contains a final concentration of 50mM glucose and 1×M9 salt. Transformation is carried out at 37℃ and 200rpm. Samples are taken for testing after 3h, 6h and 22h of transformation.

[0087] (2) Detection of OAH by high performance liquid chromatography

[0088] Centrifuge the sample at 14000 rpm for 10 min, and collect the supernatant as the test sample. Filter the test sample through a 0.22 μm microporous membrane, perform online amino acid derivatization using o-phthalaldehyde (OPA), and then perform HPLC amino acid detection. The experiment was repeated three times, and the average value was taken.

[0089] 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.

[0090] The high-performance liquid chromatography (HPLC) instrument and detection conditions are as follows:

[0091] 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%.

[0092] A standard curve was established by referring to the peak time and peak area of ​​the standard sample. Then, the OAH content in the sample was calculated based on the peak area of ​​the sample. The results are shown in Figure 2.

[0093] Results: As shown in Figure 2, after 6 hours of transformation, the OAH yield of strain SOAH01Bs was 25.40 mM, with a molar conversion rate of 51%. Based on whole-cell catalysis, it was preliminarily determined that the molar conversion rate of OAH product from strain SOAH01Bs was greater than 50%. The reaction was then scaled up for high-density fermentation to produce OAH.

[0094] (3) High-density fermentation

[0095] The fermentation medium consisted of: citric acid 1.7 g / L, potassium dihydrogen phosphate 14 g / L, diammonium hydrogen phosphate 4 g / L, polyether defoamer 0.5 mL / L, glucose 20 g / L, MgSO4·7H2O 1.2 g / L, vitamin B1 9 mg / L, lysine hydrochloride 1.01 g / L, methionine 0.4 g / L, threonine 1 g / L, isoleucine 0.25 g / L, trace inorganic salts I 10 mL / L, and pH 7.0. The composition of trace inorganic salt I is as follows: EDTA 840 mg / L, CoCl2·6H2O 250 mg / L, MnCl2·4H2O 1500 mg / L, CuCl2·2H2O 150 mg / L, H3BO3 300 mg / L, Na2MoO4·2H2O 250 mg / L, Zn(CH3COO)2·2H2O 1300 mg / L, and ferric citrate 10 g / L.

[0096] The fed-batch culture medium consisted of: glucose 600 g / L, MgSO4·7H2O 2 g / L, lysine hydrochloride 3.08 g / L, methionine 1.2 g / L, polyether defoamer 2 mL / L, and trace inorganic salt II 10 mL / L. Trace inorganic salt II consisted of: EDTA 1300 mg / L, CoCl2·6H2O 400 mg / L, MnCl2·4H2O 2350 mg / L, CuCl2·2H2O 250 mg / L, H3BO3 500 mg / L, Na2MoO4·2H2O 400 mg / L, Zn(CH3COO)2·2H2O 1600 mg / L, and ferric citrate 4 g / L.

[0097] Those skilled in the art can make certain adjustments to the above components according to the actual situation. This embodiment only provides one specific implementation scheme. As an alternative implementation method of this embodiment, the content of the components included in the fermentation culture medium can be replaced with any value within the following range: citric acid 1-5 g / L, potassium dihydrogen phosphate 1-20 g / L, nitrogen source 1-5 g / L, polyether defoamer 0.20-0.25 mL / L, glucose 5-30 g / L, MgSO4·7H2O 0.3-1 g / L, lysine hydrochloride 0.1-1 g / L, methionine 0.1-1 g / L, threonine 0.1-1 g / L, isoleucine 0.1-1 g / L, trace inorganic salts I 1-10 mL / L, pH 7.0±0.5. The composition of trace inorganic salt I is as follows: EDTA 820-840 mg / L, CoCl2·6H2O 240-250 mg / L, MnCl2·4H2O 1450-1500 mg / L, CuCl2·2H2O 140-150 mg / L, H3BO3 290-300 mg / L, Na2MoO4·2H2O 240-250 mg / L, Zn(CH3COO)2·2H2O 1250-1300 mg / L, and ferric citrate 9-10 g / L.

[0098] Seed culture: 100 mL of LB solution was placed in a 250 mL Erlenmeyer flask and sterilized at 121 °C for 20 min. After cooling, the glycerol engineered strain SOAH01Bs, stored at -80 °C, was inoculated. The culture temperature was 37 °C, and the shaking speed was 200 rpm for 6–8 h to obtain the seed culture, which was then used for inoculation of the fermentation medium. Those skilled in the art can adjust the above conditions to a certain extent according to actual conditions without affecting the achievement of the purpose of this invention. This embodiment only provides a specific implementation scheme. As an alternative implementation method, the culture conditions can be replaced with any values ​​within the following range: culture temperature of 25–42 °C, shaking speed of 100–300 rpm.

[0099] Fermentation tank inoculation: In a preferred embodiment, the fermentation medium volume in a 5L fermenter is 2.5L. After sterilization, the above seed liquid is inoculated at an inoculation rate of 5% (V / V), with an initial glucose concentration of 20g / L. The temperature is 37℃, the initial air flux is 2vvm, the stirring speed is 300rpm, and the dissolved oxygen concentration is set to 100%. During cell growth, the air flux is adjusted up to 3vvm, while the stirring speed is correlated with the DO value to ensure that the dissolved oxygen concentration is always greater than 30%. When the initial glucose is consumed, feeding is started. Ammonia is used to control the pH at 7.0 during fermentation. After 16 hours of cultivation, an inducer (1g / L L-arabinose, 1g / L MgSO4·7H2O) is added to induce protein expression. Fermentation is stopped when the feeding medium is exhausted, yielding a fermentation broth containing OAH. The OAH in the fermentation broth can be used as a precursor for L-methionine synthesis. Those skilled in the art can adjust the above conditions to a certain extent according to actual conditions without affecting the achievement of the purpose of this invention. The results are shown in Figure 3.

[0100] Results: As shown in Figure 3, the yield of OAH in the conversion solution reached 120 g / L, and the OAH conversion rate during the entire fermentation stage reached 0.48 g OAH / g glucose.

[0101] Example 4: Construction of recombinant plasmid pRK-CgY expressing O-acetylhomoserine hydrogen sulfide hydrolase from Corynebacterium glutamicum

[0102] The gene sequence of the O-acetylhomoserine hydrogen sulfide hydrolase CgmetY from Corynebacterium glutamicum was obtained from the NCBI database. After codon optimization, the nucleotide sequence was obtained as shown in SEQ ID NO.4, enabling the gene to be expressed in Escherichia coli. The CgmetY gene was amplified by PCR using primer pairs (CgmetY-F and CgmetY-R) containing selective restriction sites XhoI and EcoRI, based on the synthesized nucleotide sequence SEQ ID NO.4.

[0103] SEQ ID NO.4:

[0104] The PCR amplification primer sequences are shown in SEQ ID NO.5-6 below:

[0105] SEQ ID NO.5:

[0106] CgmetY-F: 5'-ccgcgcggcagcctcgagatgccaaagtacgacaat-3';

[0107] SEQ ID NO.6:

[0108] CgmetY-R: 5'-agaccgagctcaccgaattcctagattgcagcaaagccgc-3'.

[0109] 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.

[0110] 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 CgmetY gene, which contains approximately 1.3 kb of XhoI and EcoRI sites.

[0111] The linearized vector of the inducible expression vector pRB1K, containing an 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 plasmid vector pRB1K is described in patent 202011270812.X, which is incorporated herein by reference. The CgmetY gene was ligated into the pRB1K linearized vector using the Gibson method, thereby constructing a recombinant inducible expression vector carrying the CgmetY gene and containing an arabinose inducible promoter, named pRK-CgY. The ligation conditions were 50°C for 1 hour and 4°C for 10 minutes.

[0112] Figure 4 shows the physical map of the constructed expression vector pRK-CgY.

[0113] Example 5: Construction of Escherichia coli mutant Met03

[0114] The E. coli mutant Met03 is an E. coli mutant obtained by knocking out the gene metI encoding the methionine transporter, the methionine synthesis inhibitor gene metJ, and the endogenous cystathionine γ synthase metB gene of E. coli ST11C using CRISPR technology. In this application, it is referred to as Met03, and its genotype is E. coli ST11CΔmetIΔmetJΔmetB.

[0115] The specific construction steps of the E. coli mutant Met03 are as follows:

[0116] (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 ST11C using 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.

[0117] (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-metI-F and pTarget-metI-R, pTarget-metJ-F and pTarget-metJ-R, and pTarget-metB-F and pTarget-metB-R in the presence of the high-fidelity DNA polymerase Phanta Super-Fidelity DNA Polymerase. Fragments of approximately 2100 bp in size were obtained.

[0118] 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-metI, pTarget-metJ, and Target-metB, respectively.

[0119] The primer sequences used are as follows (the underlined sequence is N20): SEQ ID NO.7~13 (in sequential order):

[0120] SEQ ID NO.7:

[0121] pTarget-metI-F: 5'-TCGATGTACCGACAATAACGgttttagagctagaaatag-3';

[0122] SEQ ID NO.8:

[0123] pTarget-metI-R: 5'-CGTTATTGTCGGTACATCGAactagtattatacctagg-3';

[0124] SEQ ID NO.9:

[0125] pTarget-metJ-F: 5'-TAAAGCAAAAAAGAGCGGCGgttttagagctagaaatag-3';

[0126] SEQ ID NO.10:

[0127] pTarget-metJ-R: 5'-CGCCGCTCTTTTTTGCTTTAactagtattatacctagg-3';

[0128] SEQ ID NO.11:

[0129] pTarget-metB-F:5'-TCATGCACGAAATATCTGAAgttttagagctagaaatagc-3';

[0130] SEQ ID NO.12:

[0131] pTarget-metB-R: 5'-TTCAGATATTTCGTGCATGAactagtattatacctaggac-3';

[0132] SEQ ID NO.13:

[0133] pTarget-cexu-F:5'-cgagcgcagcgagtcagt-3'.

[0134] 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.

[0135] 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, to synthesize pTarget-metI, pTarget-metJ, and pTarget-metB plasmid fragments, respectively.

[0136] (3) Amplification of the target fragment: Using the genome of Escherichia coli BW 25113 as a template, PCR amplification was performed using primer pairs metI-up800-F and metI-up800-R, metI-down800-F and metI-down800-R, metJ-up800-F and metJ-up800-R, metJ-down800-F and metJ-down800-R, metB-up800-F and metB-up800-R, and metB-down800-F and metB-down800-R. The fragments were recovered to obtain the upstream 800bp fragment of metI, the downstream 800bp fragment of metI, the upstream 800bp fragment of metJ, the downstream 800bp fragment of metJ, the upstream 800bp fragment of metB, and the downstream 800bp fragment of metB. Using a mixture of 800bp upstream and downstream fragments of metI as a template, PCR amplification was performed using primer pairs metI-up500-F and metI-down500-R, and the ΔmetI targeting fragment of approximately 1000bp was obtained by gel extraction. Similarly, using a mixture of 800bp upstream and downstream fragments of metJ as a template, PCR amplification was performed using primer pairs metJ-up500-F and metJ-down500-R, and the ΔmetJ targeting fragment of approximately 1000bp was obtained by gel extraction. Finally, using a mixture of 800bp upstream and downstream fragments of metB as a template, PCR amplification was performed using primer pairs metB-up500-F and metB-down500-R, and the ΔmetB targeting fragment of approximately 1000bp was obtained by gel extraction.

[0137] The primer sequences used are shown in SEQ ID NO.14~31 (in sequence):

[0138] SEQ ID NO.14:

[0139] metI-up800-F:5'-tcgtcagactgataagcctggacgaatttcttc-3'!

[0140] SEQ ID NO.15:

[0141] metI-up800-R:5'-atgtctgagcgcaagtaacgttcacacacacataaaa-3'!

[0142] SEQ ID NO.16:

[0143] metI-down800-F:5'-ttacttgcgctcagacataacccagtaccctcttactt-3'!

[0144] SEQ ID NO.17:

[0145] metI-down800-R:5'-cgagttgaccaaagctcgccgcc-3'!

[0146] SEQ ID NO.18:

[0147] metJ-up800-F:5'-tggtcgtattaccacgaaaaacagcggccat-3'!

[0148] SEQ ID NO.19:

[0149] metJ-up800-R:5'-atggctgagaataactaagcaaaaagagcggcgc-3'!

[0150] SEQ ID NO.20:

[0151] metJ-down800-F:5'-ttagtattcttcagccatgagattacttaatccctt-3':

[0152] SEQ ID NO.21:

[0153] metJ-down800-R:5'-gtgttatccaccacgctcaccg-3':

[0154] SEQ ID NO.22:

[0155] metB-up800-F:5'-ttgccgttgcgccagttttgc-3'!

[0156] SEQ ID NO.23:

[0157] metB-up800-R:5'-ttaccccttacgcgtcatgtgatgaagttccctggg-3';

[0158] SEQ ID NO.24:

[0159] metB-down800-F:5'-atgacgcgtaaaggggtaaaaatgagtgtgattgcgcaggca-3';

[0160] SEQ ID NO.25:

[0161] metB-down800-R:5'-gggagccgccaggcgcgcca-3';

[0162] SEQ ID NO.26:

[0163] metI-up500-F:5'-aagcccactttttgcagcagcagc-3';

[0164] SEQ ID NO.27:

[0165] metI-down500-R:5'-gacacgttctattctcgaactgctgaaag-3';

[0166] SEQ ID NO.28:

[0167] metJ-up500-F:5'-gtttgcgccagtttttgctgttg-3';

[0168] SEQ ID NO.29:

[0169] metJ-down500-R:5'-gtattagtaagtactgcaccagca-3';

[0170] SEQ ID NO.30:

[0171] metB-up500-F:5'-aggatcagcagcctgtttttcatagttaaacgtc-3';

[0172] SEQ ID NO.31:

[0173] metB-down500-R:5'-ctgcggttgtgcggcgcgtt-3'.

[0174] 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.

[0175] 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.

[0176] (4) Electroporation: 200 ng of pTarget-metI plasmid, 400 ng of the targeting fragment ΔmetI, 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 cultured at 30°C. Positive clones were screened. PCR amplification was performed using primers metI-up800-F and metI-down800-R, and the amplified fragments were sequenced for verification.

[0177] 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.

[0178] The PCR amplification conditions were as follows: 95℃ pre-denaturation for 3 minutes (1 cycle); 95℃ denaturation for 15 seconds, 55℃ annealing for 15 seconds, 72℃ extension for 1-5 minutes (60 seconds / kb) (30 cycles); 72℃ extension for 5 minutes (1 cycle).

[0179] (5) Elimination of pTarget plasmid: Positive clones that were 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. The strains after overnight culture were streaked on LB solid plates containing kanamycin and cultured overnight at 30°C to obtain the E. coli mutant ST11CΔmetI containing the pCas plasmid, named Met01.

[0180] (6) Pick single clones from the plate in step (5) to prepare electrotransfer competent cells, mix them with pTarget-metJ plasmid and ΔmetJ targeting fragment, repeat steps (4)-(5), and use primer pairs metJ-up800-F and metJ-down800-R for sequencing verification to obtain E. coli mutant ST11CΔmetIΔmetJ containing pCas plasmid, named Met02.

[0181] (7) Single clones were picked from the plate containing the pCas plasmid ST11CΔmetIΔmetJ of Escherichia coli mutant obtained in step 6, and electroporation competent cells were prepared. The cells were mixed with pTarget-metB plasmid and ΔmetB targeting fragment. Steps (4)-(5) were repeated, and the cells were sequenced and verified with primer pairs metB-up800-F and metB-down800-R to obtain the ST11CΔmetIΔmetJΔmetB of Escherichia coli mutant containing the pCas plasmid, named Met03.

[0182] (8) Elimination of pCas plasmid: The E. coli mutant ST11CΔmetIΔmetJΔmetB (Met03), which was verified by sequencing to contain 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 ST11CΔmetIΔmetJΔmetB, abbreviated as Met03.

[0183] Example 6: Construction of pRK-CgY expression strain SMet03CgY

[0184] The *E. coli* mutant Met03 glycerol strain constructed in Example 5 was streaked onto LB agar plates and incubated at 37°C for 16 h. Single colonies of Met03 were picked and placed in shake flasks containing 5 mL of LB medium and incubated at 37°C until OD (dose elapsed). 600The concentration was 0.5-0.6. The cultured bacterial solution 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 bacterial 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 bacterial cells were resuspended in 100 μL of solution containing 20 mM CaCl2 and 10% glycerol (V / V) to obtain Met03 competent cells. The recombinant plasmid pRK-CgY constructed in Example 4 was added to the Met03 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 culture 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 (50 μg / mL). Incubate at 37°C for 16 hours. After incubation, pick single colonies from the agar plates and inoculate them into 5 mL of LB medium containing kanamycin. Incubate at 37°C on a shaker until OD (dose elapsed). 600 The concentration was 1.6-1.8. Then, 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 SMet03CgY and stored in an ultra-low temperature freezer at -80℃.

[0185] Example 7: Preparation of methionine from OAH catalyzed by crude CgmetY enzyme

[0186] (1) Preparation of crude CgmetY enzyme:

[0187] The engineered strain SMet03CgY glycerol bacterium was inoculated at a rate of 1% (v / v) into LB medium containing kanamycin (kanamycin concentration of 50 μg / mL) and cultured at 37℃ and 220 rpm for 16 h to obtain SMet03CgY bacterial suspension. The SMet03CgY bacterial suspension was then 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 to a final concentration of 50 μg / mL. The culture was induced at 37℃ for 16 h, and the OD was measured. 600 Light absorbance, and collected 1×10 11 Bacterial cells, according to OD 600 When the concentration is 1, the bacterial concentration is 1×10⁻⁶. 9 / mL calculation.

[0188] The above-collected 1×10 11After centrifuging the bacterial cells at 4000 rpm for 10 min, collect the precipitate, wash twice with 0.85% physiological saline, discard the supernatant, and resuspend the bacterial suspension in 1 mL of 1×PBS buffer to achieve a final bacterial concentration of 1×10⁻⁶. 11 The bacterial suspension was transferred to a 1.5 mL EP tube and sonicated (1 s sonication, 2 s pause, total sonication time 15 min, sonication power 30%). After sonication, the mixture was centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was collected as the crude CgmetY enzyme solution. To detect CgmetY protein expression, 5 μL of 5× loading buffer was added to 20 μL of supernatant, and the sample was boiled in water for 10 min. After cooling to room temperature, it was centrifuged at 12000 rpm for 2 min and then analyzed by SDS-PAGE. The sample loading volume was 10 μL, and the protein marker loading volume was 5 μL.

[0189] Figure 5 shows the SDS-PAGE gel image of the crude CgmetY enzyme. As shown in Figure 5, the CgmetY protein was successfully expressed with a molecular weight of 47 kDa.

[0190] (2) CgmetY crude enzyme catalyzes the production of methionine from OAH fermentation broth:

[0191] The OAH fermentation broth obtained in step 3 of Example 2 was collected and centrifuged at 12000 rpm for 5 min, and the supernatant was collected. In a 10 mL EP tube, 900 μL of the OAH fermentation supernatant, 50 μL of the CgmetY crude enzyme solution obtained in step 1, and sodium methanethiol were added sequentially to prepare the conversion reaction solution. The ratio of sodium methanethiol added to the molar amount of OAH was 1:1. The 10 mL EP tube was placed in a shaker at 37℃ and 200 rpm for enzyme reaction. Samples were taken after 0 h, 3 h, and 19 h of reaction for HPLC analysis. The results are shown in Figure 6.

[0192] Results: As shown in Figure 6, after 19 hours of enzyme reaction, OAH was completely consumed, and the enzyme reaction was complete. At this point, the methionine yield was 187.90 mM, with a molar conversion rate of 85.29%. Liquid chromatography analysis of the reaction solution showed a methionine purity of 88.11%.

[0193] 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 an L-methionine precursor, characterized by, The L-methionine precursor is O-acetyl-L-homoserine, the microorganism is Escherichia coli, strain number SOAH01Bs, 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 engineered strain SOAH01Bs: The above recombinant vector plasmid pXK-thrA-BsA was introduced into the Escherichia coli mutant ST11C to obtain the recombinant engineered strain, named SOAH01Bs.

2. The construction method of claim 1, wherein, 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 genotype of the E. coli mutant ST11C is E. coli BW25113ΔptsG::glk,ΔgalR::zglf,ΔompT::ppc,ΔldhA::rhtA,ΔlpxM::rhtB,ΔpflB::asd,ΔpoxB::aspA,ΔiclR,ΔlysA,ΔmetA,ΔthrB,ΔaspC.

3. A method of synthesizing an L-methionine precursor, characterized by, The precursor of L-methionine is obtained 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 precursor includes: Seed culture: The glycerol engineered strain SOAH01Bs stored at -80℃ was inoculated into LB medium and cultured at 25-42℃ and 100-300rpm for 6-8h to obtain the seed culture; Fermentation culture: the obtained seed liquid was inoculated into fermentation medium at an inoculation amount of 5%-10% (V / V) for fermentation culture. When the initial glucose was consumed, the feeding medium was started to be added. When the cell density reached O.D. 600 30, an inducer was added to induce protein expression. When the feeding medium was consumed, the fermentation was ended, and a fermentation liquid containing OAH was obtained.

4. The method of claim 3, wherein, The fermentation medium consists of: citric acid 1-5 g / L, potassium dihydrogen phosphate 1-20 g / L, nitrogen source 1-5 g / L, polyether defoamer 0.1-1 mL / L, glucose 5-30 g / L, MgSO4·7H2O 0.3-1 g / L, VB1 5-15 mg / L, lysine hydrochloride 0.1-1 g / L, methionine 0.1-1 g / L, threonine 0.1-2 g / L, isoleucine 0.1-1 g / L, trace inorganic salts I 1-10 mL / L, pH 7.0±0.5; The nitrogen source is diammonium hydrogen phosphate; The composition of the trace inorganic salt I is as follows: EDTA 820-840 mg / L, CoCl2·6H2O 240-250 mg / L, MnCl2·4H2O 1450-1500 mg / L, CuCl2·2H2O 140-150 mg / L, H3BO3 290-300 mg / L, Na2MoO4·2H2O 240-250 mg / L, Zn(CH3COO)2·2H2O 1250-1300 mg / L, and ferric citrate 9-10 g / L.

5. The method of claim 3, wherein, The fed-batch culture medium consists of: glucose 600–650 g / L, MgSO4·7H2O 2.0–2.5 g / L, lysine hydrochloride 3.08–3.10 g / L, methionine 1.2–1.3 g / L, polyether defoamer 2.0–2.5 mL / L, and trace inorganic salt II 10.0–10.5 mL / L. The composition of the trace inorganic salt II is as follows: EDTA 1300-1350 mg / L, CoCl2·6H2O 400-450 mg / L, MnCl2·4H2O 2350-2400 mg / L, CuCl2·2H2O 250-300 mg / L, H3BO3 500-550 mg / L, Na2MoO4·2H2O 400-450 mg / L, Zn(CH3COO)2·2H2O 1600-1650 mg / L, and ferric citrate 4.0-4.5 g / L; The inducer consists of: L-arabinose 1.0–1.5 g / L and MgSO4·7H2O 1.0–1.5 g / L.

6. The method of claim 3, wherein, The fermentation conditions are as follows: Initial conditions: glucose concentration of 20-25 g / L, temperature of 35-37℃, air flow rate of 2-3 vvm, stirring speed of 300-320 rpm, and dissolved oxygen concentration of 99%-100%. Fermentation process conditions: Adjust the air flow rate to 2-3 vvm, control the dissolved oxygen concentration to 30%-35% at all times, and use ammonia water to control the pH to 7.0-7.5 during the fermentation process.

7. A method for constructing a strain expressing O-acetylhomoserine sulfhydrylase, characterized by, The strain is *Escherichia coli*, strain number SMet03CgY, and the construction method includes the following steps: Construction of host strain Met03: The E. coli mutant obtained by knocking out the gene metI encoding the methionine transporter, the gene metJ inhibiting methionine synthesis, and the gene metB of endogenous cystathionine γ synthase in E. coli ST11C was named Met03, and its genotype is E. coli ST11C ΔmetI ΔmetJ ΔmetB. Construction of plasmid pRK-CgY: The O-acetylhomoserine hydrogen sulfide hydrolase gene CgmetY from Corynebacterium glutamicum was inserted between the XhoI and EcoRI sites of plasmid vector pRB1K to obtain a recombinant vector, named pRK-CgY. Construction of engineered strain SMet03CgY: The above recombinant vector plasmid pRK-CgY was introduced into the above Escherichia coli mutant Met03 to obtain the recombinant engineered strain, named SMet03CgY; The plasmid vector pRB1K carries a kanamycin resistance gene and contains an arabinose inducible promoter.

8. A method of synthesizing O-acetylhomoserine sulfhydrylase, characterized by, The enzyme is prepared by fermentation using a strain expressing O-acetylhomoserine hydrogen sulfide hydrolase constructed according to the method described in claim 7. The method for synthesizing O-acetylhomoserine hydrogen sulfide hydrolase includes the following steps: The SMet03CgY bacterial liquid was inoculated into a ZYM self-induction medium in a shaking flask at an inoculation amount of 1% (V / V), arabinose and kanamycin were added in sequence, induction culture was performed, and O.D. was measured. 600 The light absorption value was calculated according to the formula: when O.D.600=1, the bacterial concentration was 1×10 9 / mL, 1×10 11 bacterial bodies were collected. The collected 1 x 10 11 The bacterial cells were centrifuged, the precipitate was washed with normal saline, and then resuspended to a concentration of 1 x 10 11 / mL to obtain a bacterial suspension. The bacterial suspension was subjected to ultrasonic disruption. After the disruption was completed, the supernatant was obtained by centrifugation again, and the supernatant was used as a CgmetY crude enzyme solution, i.e., an O-acetylhomoserine sulfhydrylase crude enzyme solution.

9. The method of claim 8, wherein, The concentration of arabinose is 0.2% to 0.3% (V / V); The concentration of kanamycin is 50–55 μg / mL; The induction culture conditions were: temperature 37℃, time 16-18h.

10. The method of claim 8, wherein, The centrifugation conditions for collecting the bacterial cells were: 4000-4500 rpm for 8-10 min. The ultrasonic fragmentation conditions are: 1 second of ultrasound, 2 seconds of pause, duration of 15-20 minutes, and power of 30-35%. The centrifugation conditions for the ultrasonically broken products were: temperature 4℃, rotation speed 12000rpm, and time 8-10min.

11. A method of synthesizing L-methionine, characterized by, Using the OAH fermentation broth synthesized according to claims 3-6 as a substrate, and the crude CgmetY enzyme solution synthesized according to any one of claims 8-10 as a catalytic enzyme, the method for synthesizing L-methionine includes the following steps: After centrifugation, the supernatant containing OAH was obtained; The obtained supernatant containing OAH was mixed with crude CgmetY enzyme solution and sodium methanethiol to obtain the conversion reaction solution; The resulting conversion reaction solution was subjected to an enzyme reaction at 35–37°C and 200–220 rpm in a shaker to obtain the final product. The centrifugation conditions were: 12,000 rpm; 5 min. The ratio of the amount of sodium methanethiol added to the molar amount of the OAH fermentation broth is 1:1.

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