S-adenosylmethionine synthase mutant and use thereof

Abstract

Disclosed are an S-adenosylmethionine synthase mutant and use thereof. The present invention belongs to the technical field of bioengineering. The mutant comprises one or more mutations at one or more of positions 21, 25, 106, 195, and 311 within the region corresponding to positions 1 to 384 of the amino acid sequence of SEQ ID NO: 1. Further, the amino acid sequence of the mutant is set forth in any one of SEQ ID NOs: 2-6. The S-adenosylmethionine synthase mutant has high enzyme activity. Moreover, a more efficient SAM yeast production strain is constructed. The prepared S-adenosylmethionine has a higher titer, a higher substrate conversion rate, and lower production costs, showing better industrial application prospects.

Description

An S-adenosylmethionine synthase mutant and its application Technical Field

[0001] This invention belongs to the field of bioengineering technology, and relates to an S-adenosylmethionine synthase mutant, as well as a method for preparing S-adenosylmethionine using the above mutant. Background Technology

[0002] S-Adenosylmethionine (SAM) is an important metabolic intermediate widely distributed in organisms. In cells, it primarily acts as a methyl donor, participating in various biochemical reactions such as transmethylation, transsulfation, and transaminopropylation. Clinically, SAM is mainly used to treat diseases such as alcoholic liver disease, depression, and rheumatoid arthritis. SAM has been approved by the FDA as a health supplement, therefore, its market demand is expected to increase as people's living standards improve, indicating significant market potential. Furthermore, SAM can also serve as an intermediate for 1,4-butanedisulfonic acid SAM, which is used to treat pre-cirrhotic and post-cirrhotic intrahepatic cholestasis.

[0003] The main methods for synthesizing SAM include chemical synthesis, microbial fermentation, and enzymatic conversion. Chemical synthesis faces challenges such as high cost, low yield, and low purity; enzymatic conversion faces problems such as high ATP cost, low enzyme activity, and difficulty in scale-up. Currently, microbial fermentation is the primary method for SAM production. In vivo, SAM is synthesized from L-methionine and adenosine triphosphate (ATP) as substrates under the catalysis of S-adenosylmethionine synthase, which is the key enzyme in SAM preparation.

[0004] Zhejiang University of Technology, in its CN114134056A publication, disclosed a high-SAM-producing *Saccharomyces cerevisiae* strain obtained through UV mutagenesis and multiple rounds of mutagenesis screening using cordycepin and aminooxyacetic acid as resistance screening agents. The strain achieved a SAM titer of 1.21 g / L in shake-flask fermentation and 5.73 g / L in a fermenter after 72 hours. Furthermore, metabolic engineering of *Saccharomyces cerevisiae* is an effective method to improve SAM fermentation titer. Shandong Jincheng Biopharmaceutical Co., Ltd. and Zhejiang University, in their CN105483153B publication, disclosed the knockout of the adenosine decarboxylase gene SPE2 and the glycogen branched-chain enzyme gene GLC3 in *Saccharomyces cerevisiae*. The resulting mutant strain achieved a SAM titer of 10.71 g / L after 50 hours of fermentation in a fermenter. However, these studies all involve modifications to the *Saccharomyces cerevisiae* strain itself and do not involve research on the key enzyme S-adenosylmethionine synthase.

[0005] In their paper (Characterization and designing of an SAM riboswitch to establish a high-throughput screening platform for SAM overproduction in Saccharomyces cerevisiae, Biotechnol Bioeng. 2023; 1-16), Lu Wenyu's research group at Tianjin University obtained mutants SAM2 (S203F, W164R) and SAM2 (T251S, Y285F) with higher titers than the wild type through SAM2 mutation and high-throughput screening in Saccharomyces cerevisiae. The corresponding strains are M18 and M20, with titers of 179.33 mg / L and 221.95 mg / L, respectively. By site-directed mutagenesis, four mutant SAM2 (S203F, W164R, T251S, Y285F) from M18 and M20 were combined to obtain strain BSM3, with a titer of 470.56 mg / L. Simultaneously, through molecular dynamics calculations and saturation mutagenesis experiments, it was found that S365R and N150S could increase the titer. Introducing these two mutations into SAM2(S203F,W164R) and SAM2(T251S,Y285F), respectively, yielded strains BSM4 and BSM5. Introducing both S365R and N150S mutations into SAM2(S203F,W164R,T251S,Y285F) simultaneously yielded strain BSM6. The titers of strains BSM4, BSM5, and BSM6 were 520.42 mg / L, 484.97 mg / L, and 454.32 mg / L, respectively. Multiple-copy integration of the rDNA site of strain BSM6 yielded strain BSM7, with a titer of 613.52 mg / L. Knockout of the GLC3 and SAH1 genes in strain BSM7 yielded strain BSM8, with a titer of 656.58 mg / L. By optimizing the fermentation medium (such as the concentrations of L-methionine, ferrous sulfate, and dipotassium hydrogen phosphate), the highest titer of BSM8 was 1.25 g / L. The experimental method described above is extremely cumbersome, yet the fermentation titer remains low, reaching only 1.25 g / L at its highest. Therefore, obtaining a more efficient S-adenosylmethionine synthase mutant and constructing a more efficient SAM-producing strain for industrial SAM production remains essential. Summary of the Invention

[0006] Based on previous research, this invention provides a mutant of S-adenosylmethionine synthase. By performing site-directed mutagenesis on S-adenosylmethionine synthase, a mutant S-adenosylmethionine synthase was obtained. This mutant exhibits high enzyme activity, and a more efficient SAM (Saccharomyces cerevisiae) production strain was constructed. The resulting S-adenosylmethionine has a higher potency and substrate conversion rate, saving costs and making it more suitable for industrial production.

[0007] In a first aspect, the present invention provides an S-adenosylmethionine synthase mutant, wherein the S-adenosylmethionine synthase mutant has one or more site mutations, including positions 21, 25, 106, 195, and 311, in the amino acid sequence corresponding to positions 1 to 384 of SEQ ID NO:1, preferably a single site mutation.

[0008] Preferably, the S-adenosylmethionine synthase mutant has one or more of the following mutations in positions 1 to 384 of the amino acid sequence corresponding to SEQ ID NO: 1: K21A, Q25R, I106V, S195R, I311A.

[0009] Preferably, the amino acid sequence of the S-adenosylmethionine synthase mutant is shown in any one of SEQ ID NO:2-6.

[0010] Preferably, the wild type of the S-adenosylmethionine synthase mutant is derived from Saccharomyces cerevisiae, more preferably Saccharomyces cerevisiae CEN.PK, and even more preferably Saccharomyces cerevisiae CEN.PK 2-1C.

[0011] In a second aspect, the present invention provides a coding gene encoding the S-adenosylmethionine synthase mutant of the present invention. Preferably, the nucleotide sequence of the coding gene is shown in any one of SEQ ID NO: 8-12.

[0012] In a third aspect, the present invention provides a recombinant vector containing the encoding gene of the above-described S-adenosylmethionine synthase mutant.

[0013] In a fourth aspect, the present invention provides a recombinant bacterium containing the coding gene of the above-mentioned S-adenosylmethionine synthase mutant or a recombinant vector.

[0014] Preferably, the recombinant strain of the present invention is *Saccharomyces cerevisiae*, more preferably *Saccharomyces cerevisiae* CEN.PK, and even more preferably *Saccharomyces cerevisiae* CEN.PK 2-1C; it comprises a recombinant vector containing a gene encoding an S-adenosylmethionine synthase mutant, the backbone plasmid of the recombinant vector being 307-Hyg, the amino acid sequence of the S-adenosylmethionine synthase mutant being as shown in any one of SEQ ID NO: 2-6, and even more preferably, the nucleotide sequence encoding the S-adenosylmethionine synthase mutant being as shown in any one of SEQ ID NO: 8-12.

[0015] In a fifth aspect, the present invention provides a method for constructing recombinant bacteria, the method comprising the following steps:

[0016] ① Construction of recombinant vector: Using SAM2-F and SAM2-R as primers, the SAM2 gene on the genome of Saccharomyces cerevisiae CEN.PK was amplified; using TEF-R and CYC1-F as primers and plasmid 307-Hyg as template, a linearized 307-Hyg plasmid was amplified. The linearized 307-Hyg plasmid was digested and recovered using the restriction endonuclease DpnI, ligated to the SAM2 gene, and sequenced to verify, resulting in the recombinant plasmid 307-Hyg-SAM2. SAM2-F, SAM2-R, TEF-R, and CYC1-F are shown in SEQ ID NO:14-17, 307-Hyg is shown in SEQ ID NO:13, and the SAM2 gene is shown in SEQ ID NO:7.

[0017] ② Site-directed mutagenesis: Using the recombinant plasmid 307-Hyg-SAM2 obtained in step ① as a template, site-directed mutagenesis was performed using primers K21A-F / K21A-R, Q25R-F / Q25R-R, I106V-F / I106V-R, S195R-F / S195R-R, or I311A-F / I311A-R, respectively, to sequentially obtain plasmids containing SEQ ID. The recombinant plasmids 307-Hyg-SAM2K21A, 307-Hyg-SAM2Q25R, 307-Hyg-SAM2I106V, 307-Hyg-SAM2S195R, and 307-Hyg-SAM2I311A of the SAM2 mutant gene shown in NO:8-12, and K21A-F, K21A-R, Q25R-F, Q25R-R, I106V-F, I106V-R, S195R-F, S195R-R, I311A-F, and I311A-R are shown in SEQ ID NO:18-27, respectively;

[0018] ③ Yeast transformation: The recombinant plasmids 307-Hyg-SAM2K21A, 307-Hyg-SAM2Q25R, 307-Hyg-SAM2I106V, 307-Hyg-SAM2S195R, and 307-Hyg-SAM2I311A obtained in step ② were introduced into competent cells of Saccharomyces cerevisiae to obtain recombinant bacteria expressing the S-adenosylmethionine synthase mutant.

[0019] In a sixth aspect, the present invention provides the application of the above-mentioned mutants, recombinant vectors, and recombinant bacteria in the production of S-adenosylmethionine.

[0020] In a seventh aspect, the present invention provides a method for producing S-adenosylmethionine by fermentation, the method comprising fermenting and culturing the above-mentioned recombinant bacteria to obtain S-adenosylmethionine.

[0021] In one embodiment, the method involves culturing the recombinant bacteria in a shake-flask fermentation medium to prepare S-adenosylmethionine; preferably, the method uses methionine as a substrate.

[0022] Preferably, the shake flask fermentation medium has the following formulation: carbon source 5-20 g / L, nitrogen source 5-25 g / L, DL-methionine 5-20 g / L, ammonium sulfate 1-5 g / L, dipotassium hydrogen phosphate 1-10 g / L, potassium dihydrogen phosphate 1-10 g / L, manganese sulfate 0.1-1 g / L, zinc sulfate 0.1-1 g / L, magnesium sulfate 0.1-1 g / L, and calcium chloride 0.1-1 g / L; more preferably, the carbon source is selected from glucose, sucrose, or fructose, and the nitrogen source is selected from one or more of yeast extract, peptone, and corn steep liquor.

[0023] In another embodiment, the method involves fermenting the above-mentioned recombinant bacteria in a fermenter fermentation medium to prepare S-adenosylmethionine.

[0024] Preferably, the fermentation medium in the fermenter is formulated as follows: carbon source 5-20 g / L, nitrogen source 5-25 g / L, ammonium sulfate 1-5 g / L, dipotassium hydrogen phosphate 1-10 g / L, potassium dihydrogen phosphate 1-10 g / L, manganese sulfate 0.1-1 g / L, zinc sulfate 0.1-1 g / L, magnesium sulfate 0.1-1 g / L, calcium chloride 0.1-1 g / L, and 0.01-0.1% of a foaming agent; more preferably, the carbon source is selected from glucose, sucrose, or fructose, and the nitrogen source is selected from one or more of yeast extract, peptone, and corn steep liquor.

[0025] Preferably, during fermentation in the fermenter, when the initial glucose is depleted and the ethanol concentration in the fermentation broth is below 1 g / L, glucose is added to maintain the glucose concentration at 1-5 g / L. When the culture reaches the mid-logarithmic growth stage (cultural concentration of 15%-35%), DL-methionine is added. More preferably, DL-methionine is added every 1-3 hours, with each addition being 0.1%-0.5% (the mass-volume concentration of the added DL-methionine relative to the real-time fermentation broth, in g / 100 mL), for 6-24 times.

[0026] Compared with existing technologies, the present invention has the following beneficial effects: The present invention obtains a new S-adenosylmethionine synthase mutant with high enzyme activity. The enzyme activity of this mutant is 100% higher than that of the wild type. Based on the S-adenosylmethionine synthase mutant of the present invention, a more efficient SAM-producing Saccharomyces cerevisiae strain was constructed, and the S-adenosylmethionine produced has a higher potency (the potency in shake-flask fermentation reaches 3115 mg / L, which is nearly 5 times that of the wild type, and the potency in a 50L fermenter is as high as 22 g / L); the substrate conversion rate is higher. The S-adenosylmethionine synthase mutant prepared by the present invention has higher catalytic activity, and this mutant can be transformed into Saccharomyces cerevisiae to construct recombinant bacteria to produce SAM. Unlike Escherichia coli, which requires the addition of IPTG to induce protein expression, IPTG is very expensive and difficult to scale up. Moreover, plasmid expression is used instead of integration into the genome, and antibiotics are required during culture, which further increases the cost. The technical solution of the present invention has a lower cost and better industrial application prospects. Attached Figure Description

[0027] Figure 1 shows the spectrum of plasmid 307-Hyg used in Example 1 of this invention.

[0028] Figure 2 shows the spectrum of the recombinant plasmid 307-Hyg-SAM2I311A constructed in Example 1 of this invention. Detailed Implementation

[0029] The following detailed description of specific embodiments of the present invention is provided to facilitate a further understanding of the invention. These embodiments are for illustrative purposes only and are not intended to limit the invention in any way. Any modifications made by those skilled in the art to the implementation of the invention based on the teachings of this specification will fall within the scope of the claims.

[0030] Unless otherwise specified, the reagents and instruments used in the following examples are all conventional reagents and instruments in the art and can be obtained commercially; the methods used are all conventional methods, and those skilled in the art can implement the examples and obtain the corresponding results without any doubt based on the description.

[0031] Experimental materials and reagents:

[0032] LB medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L (solid medium with 20 g / L agar powder added). For plasmid resistance screening, ampicillin (Amp) was added at a concentration of 100 mg / L, and kanamycin at a concentration of 50 mg / L.

[0033] YPD medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L (solid medium with 20 g / L agar powder added). Hygromycin concentration is 200 mg / L for plasmid resistance screening.

[0034] Yeast genomic DNA rapid extraction kit: Shanghai Sangon Biotech Co., Ltd., catalog number: B518227-0050.

[0035] Plasmid extraction kit: Axygen, catalog number: AP-MN-P-250.

[0036] PCR Clean Recovery Kit: Axygen, Catalog No.: AP-PCR-250.

[0037] Restriction endonuclease Dpn I: Takara Bio Engineering (Dalian) Co., Ltd., Product No.: 1235A.

[0038] Restriction endonuclease Sph I: Takara Bio Engineering (Dalian) Co., Ltd., Product No.: 1246A.

[0039] PrimeStar HSDNA Polymerase: Takara Bio Engineering (Dalian) Co., Ltd., Product No.: R010A.

[0040] Seamless Cloning Kit: Shanghai Sangon Biotech Co., Ltd., Catalog No.: B632219-0040.

[0041] The Saccharomyces cerevisiae CEN.PK 2-1C strain was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0042] The PCR reaction system used in this embodiment of the invention consisted of: 1 μL each of primers, 1 μL template, 10 μL 5x PCR buffer, 4 μL dNTPs, 1 μL PrimeStar HSDNA polymerase, and 32 μL ddH2O. The reaction conditions were: 98℃ for 3 min; 98℃ for 30 s; 55℃ for 30 s; 72℃ for 1 min. The cycle number was 30, with a final cycle at 72℃ for 5 min. After PCR, 5 μL was subjected to agarose gel electrophoresis.

[0043] In this invention, the "%" indicating content represents the mass-volume ratio, g / 100mL.

[0044] In this invention, the fermentation potency is detected by high performance liquid chromatography.

[0045] The DL-methionine powder used in this embodiment of the invention comprises equal parts of D-methionine and L-methionine.

[0046] Experimental methods not specified in the embodiments of this invention shall be performed in accordance with "Molecular Cloning: A Laboratory Manual (Third Edition)" (J. Sambrook, DW. Russell, translated by Huang Peitang, Science Press, August 2002).

[0047] Example 1: Construction of recombinant plasmids expressing SAM2 mutant genes containing S-adenosylmethionine synthase (SAM2K21A; SAM2Q25R; SAM2I106V; SAM2S195R; SAM2I311A).

[0048] 1. Construct a recombinant plasmid expressing the wild-type S-adenosylmethionine synthase gene SAM2.

[0049] Genomic DNA was extracted from *Saccharomyces cerevisiae* CEN.PK 2-1C using a yeast genomic DNA rapid extraction kit. Using SAM2-F and SAM2-R (SEQ ID NO: 14-15, see Table 1) as primers and *Saccharomyces cerevisiae* CEN.PK 2-1C genomic DNA as a template, PCR was performed to amplify the SAM2 gene. The SAM2 gene (GenBank ID: AY693186.1, nucleotide sequence SEQ ID NO: 7, amino acid sequence SEQ ID NO: 1) was confirmed by agarose gel electrophoresis and recovered using a PCR clean recovery kit. Using the 307-Hyg plasmid (SEQ ID NO: 13, its structure is shown in Figure 1) synthesized by GenScript as a template and TEF-R and CYC1-F (SEQ ID NO: 16-17, see Table 1) as primers, the linearized 307-Hyg plasmid was amplified by PCR. The plasmid was digested with the restriction endonuclease DpnI and recovered using a PCR clean recovery kit to obtain the linearized 307-Hyg plasmid.

[0050] The SAM2 gene and the linearized 307-Hyg plasmid were ligated using a seamless cloning kit from Shanghai Sangon Biotech Co., Ltd., and the ligation product was transformed into *E. coli* DH5α competent cells. The heat shock transformation method was as follows: 10 μL of the ligation product was added to 100 μL of DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and then incubated on ice for 2 min. 900 μL of LB medium was added, and the cells were incubated at 37°C and 200 rpm for 1 hour. After centrifugation at 5000 rpm for 5 min, a portion of the supernatant was removed, and the remaining 200 μL of liquid was resuspended and plated on LB agar plates containing 100 mg / L Amp. The obtained positive transformants were inoculated into LB liquid medium containing 100 mg / L Amp and cultured overnight. The plasmid was then extracted using a plasmid extraction kit and sent to Suzhou Genewiz Co., Ltd. for sequencing verification, yielding the recombinant plasmid 307-Hyg-SAM2.

[0051] 2) Construction of recombinant plasmids containing S-adenosylmethionine synthase mutant genes (SAM2K21A; SAM2Q25R; SAM2I106V; SAM2S195R; SAM2I311A)

[0052] a) Design primers

[0053] The nucleotide sequences of the primers are shown in Table 1. The primers were synthesized by Zhejiang Youkang Biotechnology Co., Ltd.

[0054] Table 1. Nucleotide sequences of primers

[0055] b) Site-directed mutation

[0056] Using recombinant plasmid 307-Hyg-SAM2 as a template, PCR amplification was performed using the upstream and downstream primers corresponding to each point mutation (K21A-F / K21A-R, Q25R-F / Q25R-R, I106V-F / I106V-R, S195R-F / S195R-R, I311A-F / I311A-R, sequences shown in SEQ ID NO:18-19,20-21,22-23,24-25,26-27, respectively). After verification by agarose gel electrophoresis, the PCR products were digested with DpnI (37℃, 1 hour) to remove the template plasmid, and then transformed into *E. coli* DH5α competent cells using the heat shock method. Positive transformants were inoculated into LB liquid medium containing 100 mg / L Amp and cultured overnight. Plasmids were then extracted and sequenced to confirm that the mutation sites had occurred and that plasmids contained the corresponding mutant genes (SEQ ID NO: 8-12). The corresponding mutants were based on single-point site-directed mutations of K21A, Q25R, I106V, S195R, and I311A in SAM2 (SEQ ID NO: 1), and were denoted as mutants SAM2K21A, SAM2Q25R, SAM2I106V, SAM2S195R, and SAM2I311A, respectively. Specifically, sequencing revealed that the base mutations in the SAM2K21A mutant were 61A>G, 62A>C, and 63G>T. The changes in the amino acid codons were that "5'-AAG-3'" at positions 61-63 was changed to "5'-GCT-3'", and the amino acid at position 21 was changed from lysine to alanine. The SAM2Q25R mutant has base mutations of 73C>A and 74A>G, with the amino acid codon change from "5'-CAA-3'" at positions 73-75 to "5'-AGA-3'", and the amino acid at position 25 changing from glutamic acid to arginine. The SAM2I106V mutant has base mutations of 316A>G and 318C>T, with the amino acid codon change from "5'-ATC-3'" at positions 316-318 to "5'-GTT-3'", and the amino acid at position 106 changing from isoleucine to valine. The SAM2S195R mutant has base mutations of 583T>A, 584C>G, and 585T>A, with the amino acid codon changes: "5'-TCT-3'" at positions 583-585 becomes "5'-AGA-3'", and the amino acid at position 195 changes from serine to arginine. The SAM2I311A mutant has base mutations of 931A>G and 932T>C, with the amino acid codon changes: "5'-ATT-3'" at positions 931-933 becomes "5'-GCT-3'", and the amino acid at position 311 changes from isoleucine to alanine.The corresponding mutants SAM2K21A, SAM2Q25R, SAM2I106V, SAM2S195R, and SAM2I311A correspond to the amino acid sequences SEQ ID NO:2-6 and the nucleotide sequences SEQ ID NO:8-12. The constructed recombinant plasmids are 307-Hyg-SAM2K21A, 307-Hyg-SAM2Q25R, 307-Hyg-SAM2I106V, 307-Hyg-SAM2S195R, and 307-Hyg-SAM2I311A, respectively. The structure of the constructed recombinant plasmid 307-Hyg-SAM2I311A is shown in Figure 2.

[0057] c) Yeast conversion

[0058] The Saccharomyces cerevisiae transformation method used in this invention is the lithium acetate transformation method, and the specific steps are as follows: Saccharomyces cerevisiae CEN.PK 2-1C is inoculated into 3 mL of YPD liquid culture medium and cultured overnight at 30°C and 220 rpm. 1 mL of culture medium is centrifuged at 10,000 rpm for 3 min to collect the cells. The cells are resuspended in 0.1 M lithium acetate, centrifuged at 10,000 rpm for 3 min, the supernatant is removed, and the cells are washed once more with 0.1 M lithium acetate. Resuspend the bacterial cells in 50 μL of 0.1M lithium acetate solution, then add 240 μL of PEG3350 solution, 36 μL of 2M lithium acetate solution, 10 μL of salmon sperm DNA, and 1 μg of SphI-digested recombinant plasmid 307-Hyg (37℃, SphI digestion for 1 hour). After vortexing to mix, heat shock at 42℃ for 90 min, shaking every 30 min. After heat shock, 1 mL of YPD liquid medium was added, and the mixture was incubated at 30°C and 220 rpm for 2 hours. After centrifugation, the supernatant was removed, and the cells were resuspended in 1 mL of sterile water and washed twice by centrifugation. Finally, the cells were resuspended in 200 μL of sterile water and spread onto YPD plates containing 200 mg / L hygromycin B. The plates were incubated at 30°C for 2-3 days. Positive recombinant strains of *Saccharomyces cerevisiae* CEN.PK 2-1C (307-Hyg-SAM2), CEN.PK 2-1C (307-Hyg-SAM2K21A), CEN.PK 2-1C (307-Hyg-SAM2Q25R), CEN.PK 2-1C (307-Hyg-SAM2I106V), CEN.PK 2-1C (307-Hyg-SAM2S195R), and CEN.PK 2-1C were obtained. 2-1C(307-Hyg-SAM2I311A).

[0059] d) Expression and enzyme activity assay of SAM2 and its mutants in Escherichia coli

[0060] To determine the enzyme activity of SAM2 and its mutants, the SAM2 and mutant genes were expressed in *E. coli*. The specific steps were as follows: Using the pet28a plasmid as a template, the PCR fragments of the pet28a plasmid were amplified using primers pet28a-F (SEQ ID NO:28) and pet28a-R (SEQ ID NO:29). Using 307-Hyg-SAM2, 307-Hyg-SAM2K21A, 307-Hyg-SAM2Q25R, 307-Hyg-SAM2I106V, 307-Hyg-SAM2S195R, and 307-Hyg-SAM2I311A as templates, and primers SAM2-pet-F (SEQ ID NO:30) and SAM2-pet-R (SEQ ID NO:31), the PCR fragments of the SAM2 and mutant genes were obtained. The PCR fragments of the pet28a plasmid and the SAM2 and its mutant genes were digested and recovered using Dpn I. The pet28a plasmid PCR fragment was seamlessly cloned with the SAM2 and mutant gene PCR fragments, respectively, and transformed into E. coli BL21(DE3) competent cells. Positive clones were cultured overnight in LB medium, and plasmids were extracted and sequenced. The correctly sequenced plasmids were labeled as pet28a-SAM2, pet28a-SAM2K21A, pet28a-SAM2-SAM2Q25R, pet28a-SAM2I106V, pet28a-SAM2S195R, and pet28a-SAM2I311A, respectively.

[0061] Correctly sequenced E. coli BL21(DE3) clones were inoculated into 3 mL of LB medium supplemented with 50 mg / L kanamycin and incubated at 37°C and 220 rpm for 8–12 h. The clones were then transferred to 50 mL of LB medium supplemented with 50 mg / L kanamycin. The bacterial concentration was determined by OD0.05. 600 When the concentration reaches 0.6-0.8, add 0.1 mM IPTG and continue culturing at 30℃ and 160 rpm for 12 h. Collect the bacterial cells by centrifugation at 10000×g for 5 min at 4℃, and resuspend the cells in 100 mM Tris-HCl buffer to achieve a concentration of 200 g / L. Place the resuspended cells in an ice bath for sonication and centrifugation at 12000×g for 5 min to collect the supernatant.

[0062] Enzyme activity assay: The total reaction system of 500 μL contained: 5 mM L-methionine, 5 mM ATP, 150 mM KCl, 20 mM MgCl2, and 100 μL cell lysate supernatant. The reaction was carried out at 37℃ and 220 rpm for 20 min. After the reaction, the supernatant was collected by centrifugation, and the SAM content was detected by HPLC. The HPLC detection conditions were: SHIM-pack CLC-ODS column (6×150 mm, 5 μm, Shimadzu Corporation), column temperature 30℃, detection wavelength 260 nm, and mobile phase consisting of ammonium formate / sodium octane sulfonate buffer, methanol, and acetonitrile. The buffer was prepared by dissolving 12.6 g of ammonium formate and 1 g of sodium octane sulfonate in 1 L of purified water and adjusting the pH to 2.8 with formic acid. The mobile phase ratio was: ammonium formate / sodium octane sulfonate buffer: methanol: acetonitrile = 750:250:5 (v:v:v). One enzyme activity unit (U) is defined as the amount of enzyme that produces 1 μmol of SAM per minute. The results of the enzyme activity experiment are shown in Table 2.

[0063] Table 2 Enzyme activity of SAM2 and its mutants

[0064] As shown in Table 2, the enzyme activity of the mutants was increased to varying degrees compared with the wild-type SAM2, with the enzyme activity of mutant I311A increasing by 100%.

[0065] Example 2: Shake-flask fermentation of recombinant bacteria

[0066] ① The positive recombinant strains of Saccharomyces cerevisiae CEN.PK 2-1C(307-Hyg-SAM2) (hereinafter referred to as SAM2 wild type in Table 3), Saccharomyces cerevisiae CEN.PK 2-1C(307-Hyg-SAM2K21A) (hereinafter referred to as SAM2K21A in Table 3, the same below), Saccharomyces cerevisiae CEN.PK 2-1C(307-Hyg-SAM2Q25R), Saccharomyces cerevisiae CEN.PK 2-1C(307-Hyg-SAM2I106V), Saccharomyces cerevisiae CEN.PK 2-1C(307-Hyg-SAM2S195R) and Saccharomyces cerevisiae CEN.PK 2-1C(307-Hyg-SAM2I311A) prepared in Example 1 were inoculated into 3 mL of YPD medium and cultured at 30℃ and 220 rpm for 20 hours to obtain the culture medium.

[0067] ② Transfer 0.5 mL of the culture medium to a shake flask containing 20 mL of fermentation medium for fermentation. The fermentation medium formula is as follows: sucrose 50 g / L, yeast extract 8 g / L, DL-methionine 10 g / L, ammonium sulfate 3 g / L, dipotassium hydrogen phosphate 4 g / L, potassium dihydrogen phosphate 8 g / L, manganese sulfate 0.1 g / L, zinc sulfate 0.1 g / L, magnesium sulfate 0.3 g / L, and calcium chloride 0.1 g / L. Fermentate at 30℃ and 220 rpm.

[0068] ③ SAM potency detection: After 72 hours of fermentation culture, 1 mL of fermentation broth was taken, concentrated sulfuric acid was added, heated at 90℃ for 30 min, the supernatant was collected by centrifugation, filtered, and the SAM content was detected by HPLC. The shake-flask fermentation potency of wild-type and mutant SAM2 is shown in Table 3.

[0069] Table 3. Shake-flask fermentation potency of SAM2 wild-type and mutant strains

[0070] As shown in Table 3 above, the fermentation titer of the SAM2 mutant strain was significantly higher than that of the wild-type SAM2, with the highest titer of the SAM2 mutant reaching 3115 mg / L, which is nearly 5 times that of the wild-type.

[0071] Example 3: Recombinant bacterial fermentation tank culture

[0072] The positive recombinant strains of *Saccharomyces cerevisiae* CEN.PK 2-1C (307-Hyg-SAM2) and *Saccharomyces cerevisiae* CEN.PK 2-1C (307-Hyg-SAM2I311A) prepared in Example 1 were inoculated into YPD slants and cultured statically at 28°C for 3 days. The slants were then transferred to 300 mL of YPD liquid medium and cultured overnight at 28°C and 220 rpm for 18-20 h. All samples were then transferred to 2.7 L YPD liquid medium and cultured for another 18-20 h. Finally, all samples were inoculated into a 50 L fermenter containing 27 L of fermentation medium. The fermentation medium formulation was: glucose 10 g / L, yeast extract 15 g / L, ammonium sulfate 3 g / L, dipotassium hydrogen phosphate 4 g / L, potassium dihydrogen phosphate 8 g / L, manganese sulfate 0.1 g / L, zinc sulfate 0.1 g / L, magnesium sulfate 0.3 g / L, calcium chloride 0.1 g / L, and foaming agent 0.05%. The feed medium was a 500 g / L glucose solution. Fermentation temperature was controlled at 30℃, aeration rate at 1500 L / h, stirring speed at 200-600 rpm, and ammonia was added to maintain the pH at around 5. When the initial glucose was depleted and the ethanol concentration in the fermentation broth was below 1 g / L, glucose was added again, maintaining a glucose concentration of 1-5 g / L. When the culture reached mid-logarithmic growth (20% cell concentration), DL-methionine powder was added, 140 g each time, every two hours, for a total of 7 additions. SAM titer was monitored until it stopped increasing, at which point fermentation was terminated (fermentation broth volume approximately 40 L). The strain expressing the wild-type SAM2 achieved a final SAM titer of 10.6 g / L, with a substrate conversion rate of 32.5% based on L-methionine. The strain expressing the SAM2I311A mutant gene achieved a final SAM titer of 22 g / L, with a substrate conversion rate of 67.5% based on L-methionine. The formula for calculating substrate conversion rate is: Substrate conversion rate = SAM titer × final fermentation broth volume / (1 / 2 × mass of DL-methionine added each time × number of additions × SAM molecular weight / Met molecular weight) × 100%

[0073] Example 4: Recombinant bacterial fermentation tank culture

[0074] The prepared positive recombinant strains, *Saccharomyces cerevisiae* CEN.PK 2-1C (307-Hyg-SAM2) and CEN.PK 2-1C (307-Hyg-SAM2I311A), were inoculated into YPD slants and incubated statically at 28°C for 3 days. The slants were then transferred to 300 mL of YPD liquid medium and incubated overnight at 28°C and 220 rpm for 18-20 h. The entire culture was then transferred to 2.7 L of YPD liquid medium and incubated for another 18-20 h. Finally, the entire culture was inoculated into a 50 L fermenter containing 27 L of fermentation medium. The fermentation medium formula was: 6 g / L sucrose, 20 g / L corn steep liquor, 1 g / L ammonium sulfate, 8 g / L dipotassium hydrogen phosphate, 5 g / L potassium dihydrogen phosphate, 0.8 g / L manganese sulfate, 0.7 g / L zinc sulfate, 0.8 g / L magnesium sulfate, 0.3 g / L calcium chloride, and 0.05% foaming agent. The feed medium was a 500 g / L glucose solution. Fermentation temperature was controlled at 30℃, aeration rate at 1500 L / h, stirring speed at 200-600 rpm, and ammonia was added to maintain the pH at around 5. When the initial glucose was depleted and the ethanol concentration in the fermentation broth was below 1 g / L, glucose was added again, maintaining a glucose concentration of 1-5 g / L. When the culture reached mid-logarithmic growth (25% cell concentration), DL-methionine powder was added, 160 g each time, every two hours, for a total of 6 additions. SAM titer was monitored until it stopped increasing, at which point fermentation was terminated (fermentation broth volume approximately 35 L). The strain expressing the wild-type SAM2 achieved a final SAM titer of 9.3 g / L, with a substrate conversion rate of 25.4% based on L-methionine. The strain expressing the SAM2I311A mutant gene achieved a final SAM titer of 23 g / L, with a substrate conversion rate of 62.9% based on L-methionine.

[0075] sequence list

[0076] SEQ ID NO:1 SAM2 amino acid sequence

[0077] SEQ ID NO:2 SAM2K21A mutant amino acid sequence

[0078] SEQ ID NO:3 SAM2Q25R mutant amino acid sequence

[0079] SEQ ID NO:4 SAM2I106V mutant amino acid sequence

[0080] SEQ ID NO:5 SAM2S195R mutant amino acid sequence

[0081] SEQ ID NO:6 SAM2I311A mutant amino acid sequence

[0082] SEQ ID NO:7 SAM2 nucleotide sequence

[0083] SEQ ID NO:8 SAM2K21A mutant nucleotide sequence

[0084] SEQ ID NO:9 SAM2Q25R mutant nucleotide sequence

[0085] SEQ ID NO:10 SAM2I106V mutant nucleotide sequence

[0086] SEQ ID NO:11 SAM2S195R mutant nucleotide sequence

[0087] SEQ ID NO:12 SAM2I311A mutant nucleotide sequence

[0088] SEQ ID NO:13 plasmid 307-Hyg sequence

Claims

1. An S-adenosylmethionine synthase mutant, characterized in that, The S-adenosylmethionine synthase mutant has one or more site mutations, including positions 21, 25, 106, 195, and 311, in the amino acid sequence corresponding to positions 1 to 384 of SEQ ID NO:1; preferably, the S-adenosylmethionine synthase mutant has one or more of the following mutations in the amino acid sequence corresponding to positions 1 to 384 of SEQ ID NO:1: K21A, Q25R, I106V, S195R, and I311A.

2. The S-adenosylmethionine synthase mutant as described in claim 1, characterized in that, The amino acid sequence of the S-adenosylmethionine synthase mutant is shown in any one of SEQ ID NO:2-6.

3. A coding gene encoding a mutant of S-adenosylmethionine synthase as described in claim 1 or 2, preferably, the nucleotide sequence of the coding gene is shown in any one of SEQ ID NO: 8-12.

4. A recombinant vector, characterized in that, The recombinant vector contains the encoding gene of the S-adenosylmethionine synthase mutant as described in claim 3.

5. A recombinant bacterium, characterized in that, The recombinant bacteria contain the encoding gene of the S-adenosylmethionine synthase mutant as described in claim 3 or the recombinant vector as described in claim 4.

6. The recombinant bacteria as described in claim 5, characterized in that, The recombinant strain is *Saccharomyces cerevisiae*, preferably *Saccharomyces cerevisiae* CEN.PK, and more preferably *Saccharomyces cerevisiae* CEN.PK 2-1C; it contains a recombinant vector encoding a gene for an S-adenosylmethionine synthase mutant, the backbone plasmid of the recombinant vector is 307-Hyg, the amino acid sequence of the S-adenosylmethionine synthase mutant is shown in any one of SEQ ID NO: 2-6, and even more preferably, the nucleotide sequence encoding the S-adenosylmethionine synthase mutant is shown in any one of SEQ ID NO: 8-12.

7. A method for constructing the recombinant bacteria as described in claim 5 or 6, the method comprising the following steps: ① Construction of recombinant vector: Using SAM2-F and SAM2-R as primers, the SAM2 gene on the genome of Saccharomyces cerevisiae CEN.PK was amplified; using TEF-R and CYC1-F as primers and plasmid 307-Hyg as template, a linearized 307-Hyg plasmid was amplified. The linearized 307-Hyg plasmid was digested and recovered using the restriction endonuclease DpnI, ligated to the SAM2 gene, and sequenced to verify, resulting in the recombinant plasmid 307-Hyg-SAM2. SAM2-F, SAM2-R, TEF-R, and CYC1-F are shown in SEQ ID NO:14-17, 307-Hyg is shown in SEQ ID NO:13, and the SAM2 gene is shown in SEQ ID NO:

7. ② Site-directed mutagenesis: Using the recombinant plasmid 307-Hyg-SAM2 obtained in step ① as a template, site-directed mutagenesis was performed using primers K21A-F / K21A-R, Q25R-F / Q25R-R, I106V-F / I106V-R, S195R-F / S195R-R, or I311A-F / I311A-R, respectively, to sequentially obtain plasmids containing SEQ ID. The recombinant plasmids 307-Hyg-SAM2K21A, 307-Hyg-SAM2Q25R, 307-Hyg-SAM2I106V, 307-Hyg-SAM2S195R, and 307-Hyg-SAM2I311A of the SAM2 mutant gene shown in NO:8-12, and K21A-F, K21A-R, Q25R-F, Q25R-R, I106V-F, I106V-R, S195R-F, S195R-R, I311A-F, and I311A-R are shown in SEQ ID NO:18-27, respectively; ③ Yeast transformation: The recombinant plasmids 307-Hyg-SAM2K21A, 307-Hyg-SAM2Q25R, 307-Hyg-SAM2I106V, 307-Hyg-SAM2S195R, and 307-Hyg-SAM2I311A obtained in step ② were introduced into competent cells of Saccharomyces cerevisiae to obtain recombinant bacteria expressing the S-adenosylmethionine synthase mutant.

8. The use of the mutant as described in claim 1 or 2, the recombinant vector as described in claim 4, and the recombinant bacteria as described in claim 5 or 6 in the production of S-adenosylmethionine.

9. A method for producing S-adenosylmethionine, wherein the method comprises culturing the recombinant bacteria described in claim 5 or 6 in a shake-flask fermentation medium to prepare S-adenosylmethionine, preferably, the shake-flask fermentation medium has the following formulation: carbon source 5-20 g / L, nitrogen source 5-25 g / L, DL-methionine 5-20 g / L, ammonium sulfate 1-5 g / L, dipotassium hydrogen phosphate 1-10 g / L, potassium dihydrogen phosphate 1-10 g / L, manganese sulfate 0.1-1 g / L, zinc sulfate 0.1-1 g / L, magnesium sulfate 0.1-1 g / L, calcium chloride 0.1-1 g / L, more preferably, the carbon source is selected from glucose, sucrose or fructose, and the nitrogen source is selected from one or more of yeast extract, peptone, and corn steep liquor.

10. A method for producing S-adenosylmethionine, wherein the method comprises fermenting the recombinant bacteria of claim 5 or 6 in a fermenter fermentation medium to prepare S-adenosylmethionine, preferably, the fermenter fermentation medium has the following formulation: carbon source 5-20 g / L, nitrogen source 5-25 g / L, ammonium sulfate 1-5 g / L, dipotassium hydrogen phosphate 1-10 g / L, potassium dihydrogen phosphate 1-10 g / L, manganese sulfate 0.1-1 g / L, zinc sulfate 0.1-1 g / L, magnesium sulfate 0.1-1 g / L, calcium chloride 0.1-1 g / L, and 0.01-0.1% bubbly precipitant; more preferably, the carbon source is selected from glucose, sucrose, or fructose, and the nitrogen source is selected from one or more of yeast extract, peptone, and corn steep liquor.

11. The method as described in claim 10, characterized in that, During fermentation in the fermenter, when the initial glucose is depleted and the concentration of ethanol in the fermentation broth is below 1 g / L, glucose is added to maintain the glucose concentration at 1-5 g / L. When the culture reaches the mid-logarithmic growth stage, DL-methionine is added. Preferably, DL-methionine is added every 1-3 hours, with each addition being 0.1%-0.5% of DL-methionine, for 6-24 times.

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