Methyl-lyase subunit mpEgtB mutants and use thereof

Abstract

The application discloses a methyl bacillus subtilis sulfoxide synthase MpEgtB mutant and application thereof, and belongs to the technical field of bioengineering. The application provides a methyl bacillus subutilis-derived sulfoxide synthase MpEgtB mutant capable of efficiently preparing ergothioneine and a modification method of the mutant, and the mutant protein is used to catalyze histidine trimethyl inner salt, L-Cys is used as a sulfur donor to directly convert the histidine trimethyl inner salt into histidine trimethyl inner salt-based cysteine sulfoxide, and the obtained mutant is coupled with Mycobacterium smegmatis-derived MsEgtD and MsEgtE to prepare L-EGT by using L-His and L-Met as substrates. The optimal mutant strain is used as a biological catalyst to perform whole-cell conversion, a continuous feeding strategy is adopted in a 5L fermenter, and the conversion is performed for 48h, so that the yield of L-EGT reaches 8.32g / L. The production process is simple, has few impurities, and has an important industrial application prospect.

Description

Methylobacterium sulfoxide synthase MpEgtB mutant and its application Technical Field

[0001] This invention relates to the MpEgtB mutant of methylbacillus sulfoxide synthase and its applications, belonging to the field of bioengineering technology. Background Technology

[0002] L-ergothioneine (L-EGT) is a histidine-derived thiol compound isolated from the fungus *Claviceps purpurea* over a century ago. Due to its unique thione structure and high redox potential, EGT is not easily autoxidized under physiological pH conditions, exhibiting higher stability and antioxidant activity than glutathione. It can participate in maintaining the redox state within cells, regulating energy, and preventing cancer and cardiovascular diseases. However, research shows that the human body cannot synthesize ergothioneine and must obtain it from foods such as mushrooms and red beans. Currently, the market demand for EGT as a dietary supplement and cosmetic additive is growing rapidly, and naturally sourced EGT cannot meet the demand.

[0003] The main methods for preparing EGT include natural biological extraction, chemical synthesis, and biosynthesis. Extraction methods are limited by available sources and difficult to scale up for industrial production; chemical synthesis is challenging and product safety is difficult to guarantee; biological methods offer advantages such as safety, environmental friendliness, easily scalable production, and sustainable production, making them the primary direction for EGT preparation. Aerobic and anaerobic biosynthetic pathways for EGT have been reported, including those in prokaryotes (represented by mycobacteria), eukaryotes (represented by Neurospora crassa), and anaerobic bacteria (represented by Chlorobium limicola). Guided by these synthetic pathways, EGT was prepared by fermentation using unengineered natural edible fungi, such as enoki mushrooms (Flammulinavelutipes), oyster mushrooms (Pleurotus ostreatus), and shiitake mushrooms (Lentinus edodes). By establishing a deep fermentation control strategy for accumulating EGT, the accumulation of EGT in shake-flask fermentation broth exceeded 500 mg / L (Mei Baoliang et al., 2015). EGT-producing engineered bacteria were constructed through heterologous expression of ergothionein biosynthetic enzyme, and the EGT synthesis efficiency was further improved through strategies such as modification and optimization of the precursor amino acid synthesis pathway. Patent (CN 113234652 A) discloses a method of combining the EGT synthesis gene egtBCDE with the heterologous ergothioneine synthesis pathway enzyme egt1, while simultaneously expressing the isoenzyme egtA of the E. coli gene gshA, to obtain an EGT yield of 125.8 mg / L in engineered strain E1 A1; further, the histidine, methionine, and cysteine ​​metabolism of engineered strain E1 A1 was modified to increase the EGT yield of engineered strain to 244.97 mg / L, and after 108 hours of batch feeding fermentation in a 3L fermenter, the EGT yield obtained was 1102.96 mg / L.

[0004] However, there are few reports on the modification of key enzymes in the EGT synthesis pathway. Osawa R et al. (J Agric Food Chem. 2018 Feb 7; 66(5):1191-1196) proposed that the EgtB enzyme from the prokaryotic Mycobacterium smegmatis is the rate-limiting enzyme for EGT synthesis. Modifying key enzymes in the ergothioneine synthesis pathway, including EgtB enzyme, to improve their catalytic performance is expected to enhance the biosynthesis level of EGT and provide a reference for the industrial application of enzymatic synthesis of EGT. Summary of the Invention

[0005] This invention provides a mutant MpEgtB of sulfoxide synthase from *Methylobacterium umpseudosasicola* capable of efficiently preparing L-EGT, and a method for modifying it. The mutant protein catalyzes the direct conversion of histidine trimethyl inner salt (HER) to histidine trimethyl inner salt cysteine ​​sulfoxide (Cys-HER) using L-Cys as a sulfur donor. Furthermore, the obtained mutant is coupled with SAM (S-adenosylmethionine)-dependent histidine methyltransferase MsEgtD and PLP-binding CS lyase MsEgtE from *Mycobacterium smegmatis*, using L-His and L-Met as substrates to prepare L-EGT.

[0006] This invention provides a mutant of sulfoxide synthase MpEgtB derived from M. pseudoosasicola. The mutant is obtained by mutating one or more of the amino acids 106, 173 and 409, starting with the amino acid sequence of sulfoxide synthase MpEgtB as shown in SEQ ID NO.1.

[0007] In one embodiment of the present invention, the nucleotide sequence encoding the parent enzyme of the sulfoxide synthase MpEgtB is shown in SEQ ID NO.2.

[0008] In one embodiment of the present invention, the mutant, relative to the MpEgtB parent, has its tyrosine residue at position 106 mutated to phenylalanine to obtain the mutant MpEgtB. Y106F .

[0009] In one embodiment of the present invention, the mutant is obtained by mutating leucine at position 173 to threonine relative to the MpEgtB parent, thus obtaining the mutant MpEgtB. L173T .

[0010] In one embodiment of the present invention, the mutant, relative to the MpEgtB parent, has its glutamine at position 409 mutated to arginine to obtain the mutant MpEgtB. Q409R .

[0011] In one embodiment of the present invention, the mutant, relative to the MpEgtB parent, has its tyrosine at position 106 mutated to phenylalanine and its leucine at position 173 mutated to threonine to obtain the mutant MpEgtB. Y106F / L173T .

[0012] In one embodiment of the present invention, the mutant, relative to the MpEgtB parent, has its tyrosine at position 106 mutated to phenylalanine and its glutamine at position 409 mutated to arginine to obtain the mutant MpEgtB. Y106F / Q409R .

[0013] In one embodiment of the present invention, the mutant, relative to the MpEgtB parent, has its leucine at position 173 mutated to threonine and its glutamine at position 409 mutated to arginine to obtain the mutant MpEgtB. L173T / Q409R .

[0014] In one embodiment of the present invention, the mutant, relative to the MpEgtB parent, has its tyrosine at position 106 mutated to phenylalanine, its leucine at position 173 mutated to threonine, and its glutamine at position 409 mutated to arginine, to obtain the mutant MpEgtB. Y106F / L173T / Q409R .

[0015] In one embodiment of the present invention, the mutant MpEgtB Y106F MpEgtB L173T MpEgtB Q409R MpEgtB Y106F / L173T MpEgtB Y106F / Q409R MpEgtB L173T / Q409R MpEgtB Y106F / L173T / Q409R The amino acid sequences are shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, and SEQ ID NO.15, respectively.

[0016] The present invention also provides a gene encoding the mutant.

[0017] In one embodiment of the present invention, the mutant MpEgtB is encoded. Y106F MpEgtB L173T MpEgtB Q409R MpEgtB Y106F / L173T MpEgtB Y106F / Q409R MpEgtB L173T / Q409R MpEgtB Y106F / L173T / Q409R The nucleotide sequences of the genes are shown in SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, and SEQ ID NO.16, respectively.

[0018] This invention provides a method for obtaining the MpEgtB mutant, the method comprising the following steps:

[0019] (1) Using a vector carrying the MpEgtB gene of sulfoxide synthase from M. pseudoosasicola as shown in SEQ ID NO.2 as a template, site-directed mutagenesis was performed; a plasmid vector containing the mutant was constructed.

[0020] (2) Transform the plasmid vector constructed in step (1) into host cells and select positive clones;

[0021] (3) Select the positive clones prepared in step (2) for fermentation culture and purify MpEgtB.

[0022] The present invention also provides a recombinant vector carrying the said gene.

[0023] In one embodiment of the present invention, the recombinant vector is pRSFDuet-1 or pACYC-Duet-1 as the expression vector.

[0024] In one embodiment of the present invention, the vector is a vector containing the MpEgtB mutant encoding MpEgtB. Y106F / L173T / Q409R A recombinant vector for the gene; the recombinant vector is a vector containing the MpEgtB mutant shown in SEQ ID NO.16. Y106F / L173T / Q409R The gene was integrated into the expression vector pRSFDuet-1, thus obtaining the recombinant expression vector pRSFDuet-MpEgtB.

[0025] The present invention also provides microbial cells that express the above-mentioned mutants or carry genes encoding the above-mentioned mutants or carry the above-mentioned recombinant vectors.

[0026] In one embodiment of the present invention, the microbial cells use bacteria and fungi as expression hosts.

[0027] In one embodiment of the present invention, the microbial cell is Escherichia coli, including but not limited to Escherichia coli BL21(DE3).

[0028] The present invention also provides a genetically engineered bacterium that simultaneously overexpresses the above-mentioned Methylbacillus sulfoxide synthase MpEgtB mutant, histidine-dependent methyltransferase MsEgtD, and PLP-binding CS lyase MsEgtE.

[0029] In one embodiment of the present invention, the nucleotide sequence encoding MsEgtD is shown in SEQ ID NO.17, and the nucleotide sequence encoding MsEgtE is shown in SEQ ID NO.18.

[0030] In one embodiment of the present invention, the genetically engineered bacteria uses Escherichia coli as the expression host.

[0031] In one embodiment of the present invention, the genetically engineered bacterium uses E. coli BL21(DE3) as the expression host.

[0032] In one embodiment of the present invention, the pACYC-Duet-1 vector was used to overexpress the histidine-dependent methyltransferase MsEgtD and the PLP-binding CS lyase MsEgtE, and the pRSF-Duet-1 vector was used to overexpress the MpEgtB mutant.

[0033] In one embodiment of the present invention, the vector contains a co-expression vector encoding the MsEgtD gene and the MsEgtE gene; the recombinant vector is obtained by integrating the MsEgtD gene as shown in SEQ ID NO.17 and the MsEgtE gene as shown in SEQ ID NO.18 into the expression vector pACYC-Duet-1, thereby obtaining the co-expression vector pACYC-Duet-EgtDE.

[0034] The present invention also provides a recombinant Escherichia coli BL21-EgtBDE containing the recombinant vector pRSFDuet-MpEgtB and the co-expression vector pACYC-Duet-EgtDE.

[0035] The present invention also provides a method for preparing ergothioneine, wherein the method comprises using the above-mentioned genetically engineered bacteria or recombinant cells as catalysts to catalyze L-histidine and L-methionine to produce ergothioneine.

[0036] In one embodiment of the present invention, the method is to inoculate Escherichia coli BL21-EgtBDE into a fermenter at an inoculation rate of 2-10%, culture for 3 hours, add IPTG at a final concentration of 0.5 mmol / L for induction, the induction temperature is 30°C, the induction time is 24 hours, after fermentation is completed, the bacterial cells are collected by centrifugation at 8000 rpm, and stored at -20°C for later use.

[0037] In one embodiment of the present invention, the method is as follows: using L-His, L-Cys and L-Met as reaction substrates, and BL21-EgtBDE cells as catalysts, after reacting for 2-3 hours, L-His and L-Met are continuously added to the transformation system, and the reaction is carried out for 24-72 hours under the conditions of 100 mM Tris-HCl buffer, pH 7.0-9.0, and 20-35°C.

[0038] The present invention also provides the application of the MpEgtB mutant, the gene, the recombinant vector, the recombinant cell, the genetically engineered bacteria, or the recombinant Escherichia coli BL21-EgtBDE in the preparation of products containing L-EGT, or products produced using L-EGT as a raw material.

[0039] Beneficial effects

[0040] (1) This invention constructs a sulfoxide synthase MpEgtB mutant derived from *M. pseudoosasicola* for the catalytic production of L-EGT. The specific enzyme activity of the mutant of this invention is 4.32 U mg. -1 protein) and k cat / K m (0.93) was 31 times and 103 times higher than the control, respectively, which improved the production capacity per unit catalyst and effectively reduced the production cost.

[0041] (2) The dual-plasmid expression strain described in this invention can be obtained in large quantities through culture without cell disruption and can be directly used for transformation reactions, making the operation simple. In a 5L reaction system, using recombinant Escherichia coli BL21-EgtBDE as a whole-cell catalyst, the yield of L-EGT can reach 8.32 g / L by continuously adding L-His and L-Met, which accelerates the industrialization process of L-EGT production by enzymatic conversion. Attached Figure Description

[0042] Figure 1: L-EGT biosynthetic pathway.

[0043] Figure 2: Purified recombinant MpEgtB and MpEgtB Y106F / L173T / Q409R SDS-PAGE analysis; where M: Marker; Con: Crude extract of recombinant strain E. coli BL21-MpEgtB; Lane 1: Purified MpEgtB enzyme protein; Lane 2: Purified MpEgtB Y106F / L173T / Q409R Enzyme protein.

[0044] Figure 3: Wild-type MpEgtB and mutant MpEgtB Y106F / L173T / Q409R Molecular dynamics simulations; where (A) represents: wild-type MpEgtB and mutant MpEgtB calculated based on molecular dynamics simulations. Y106F / L173T / Q409R (A) represents the RMSD values ​​of wild-type MpEgtB and mutant MpEgtB, calculated based on molecular dynamics simulations. Y106F / L173T / Q409R The RMSF value of the residues.

[0045] Figure 4: Optimal mutant strain MpEgtB Y106F / L173T / Q409R L-EGT production was achieved through continuous feeding conversion on a 5L fermenter. Detailed Implementation

[0046] The MpEgtB gene involved in this invention is derived from *Methylobacteriumpseudosasicola*, and its nucleotide sequence is shown in SEQ ID NO.2. The pRSFDuet-1 and pACYC-Duet-1 plasmids were purchased from Novagen (Madison, WI, USA), and restriction endonucleases, primeSTAR, etc., were purchased from TaKaRa (Dalian, China). Standards were purchased from SIGMA. All MpEgtB mutants were obtained through molecular modification, and all other reagents were commercially available.

[0047] The culture media involved in the following examples are as follows:

[0048] Prepare LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and sterilize at 121°C for 20 min.

[0049] Fermentation medium was prepared as follows: tryptone 12 g / L, yeast extract 24 g / L, glucose 10 g / L, glycerol 4 mL / L, KH2PO4 2.31 g / L, K2HPO4 12.31 g / L, MgSO4 0.6 g / L, and CaCl2 0.02 g / L.

[0050] The detection methods involved in the following embodiments are as follows:

[0051] Enzyme activity assay method:

[0052] The enzyme activity of MpEgtB was determined by HPLC.

[0053] The enzyme activity of 1 unit of MpEgtB is defined as the amount of enzyme (U) required to generate 1 μmol of histidine trimethyl lactone cysteine ​​sulfoxide (Cys-HER) product. Enzyme activity can be calculated by measuring the content of Cys-HER.

[0054] Enzyme activity is defined as the number of enzyme activity units per milligram of protein (U / mg protein).

[0055]

[0056] Detection of Cys-HER content:

[0057] Sample preparation for HPLC determination of Cys-HER content: Take 1 mL of the converted solution, centrifuge at 12000 rpm for 10 min, take the supernatant, dilute it, filter it through a 0.45 μm filter membrane, and use the filtrate for liquid chromatography analysis.

[0058] HPLC determination of Cys-HER content: Dionex high-performance liquid chromatograph (equipped with UV-Vis detector), using a Daicel array. A CR-I(+) (150×3mm, 5μm) column was used. The mobile phase was dilute sulfuric acid (perchloric acid:acetonitrile = 9:1, pH adjusted to 1.5 with perchloric acid). The mobile phase was filtered through a 0.22μm filter membrane and degassed by sonication. The flow rate was 0.2mL / min, the column temperature was 25℃, and the detection was performed at a UV detection wavelength of 245nm.

[0059] Example 1: Construction and screening of single mutation mutants

[0060] The specific steps are as follows:

[0061] (1) Preparation of pRSFDuet-MpEgtB

[0062] The MpEgtB gene involved in this patent is derived from Methylobacterium pseudosasicola. Based on the MpEgtB gene sequence from M. pseudosasicola published in the NCBI database, Suzhou Genewise Biotechnology Co., Ltd. was commissioned to synthesize the gene, optimize the codons, and ligate it into the vector pRSFDuet-1. The resulting recombinant vector is pRSFDuet-MpEgtB. The nucleotide sequence of the MpEgtB gene is shown in SEQ ID NO.2, and the amino acid sequence of the sulfoxide synthase MpEgtB encoded by the gene is shown in SEQ ID NO.1.

[0063] (2) Construction of single mutants:

[0064] Design MpEgtB Y106F MpEgtB L173T and MpEgtB Q409R The primers for the mutation sites are shown in Table 1. Using pRSFDuet-MpEgtB prepared in step (1) as a template, mutants were constructed by whole plasmid PCR.

[0065] Table 1: Primer sequences for single mutant mutations

[0066]

[0067] Construct the PCR amplification system: 0.5 μL PrimSTAR enzyme, 10 μL 5×PrimeSTAR Buffer, 4 μL dNTPs, 1 μL each of the two primers for each mutation site, and template (CgDAPDH as shown in SEQ ID NO.2). BC6214 μL of water and 32.5 μL of water were added. The reaction conditions were: ① 94℃ for 3 min; ② 98℃ for 10 s; ③ 55℃ for 30 s; ④ 72℃ for 3 min; ⑤ Repeat steps ② to ④ 29 times; ⑥ 72℃ for 5 min; ⑦ Keep warm at 12℃.

[0068] (3) The above reaction system was incubated at 37℃ for 3h to digest the plasmid template (the digestion system was: DpnI 0.5μL, the above reaction PCR product 45μL, 10×T Buffer 5μL). After digestion, the digestion product was introduced into Escherichia coli BL21 competent cells by chemical transformation. The specific steps of chemical transformation were as follows: (a) 10μl of homologous recombination product was introduced into 100μl of BL21 competent cells; (b) Ice bath for 15-30min; (c) Heat shock in a 42℃ water bath for 90s, and then quickly placed in ice for 3-5min; (d) 800μl of antibiotic-free LB medium was added and mixed, and cultured at 37℃ and 200rpm for 1h; (e) The cells were collected by centrifugation at 5000rpm for 2min; (f) The supernatant was removed, and the remaining 100-200μl was mixed by pipetting and spread onto a plate containing 0.05mg / mL kanamycin resistance, and cultured at 37℃ for about 12h. (g) Select a single clone and incubate it in LB containing 0.05 mg / mL kanamycin. After incubation at 37°C for 12 h at 200 rpm, send it to the company for sequencing. The one with correct sequencing is the positive transformant.

[0069] (4) The mutant strain with correct sequencing was inoculated into LB seed medium and cultured at 200 rpm and 37°C for about 10 days. Then, it was inoculated into shake flask fermentation medium at a 5% inoculation rate and cultured at 200 rpm and 37°C until OD. 600 =Approximately 0.8, add IPTG to a final concentration of 0.5 mM for induction, and incubate at 200 rpm and 25℃ for 14 h. Collect the induced bacterial culture by centrifugation at 6000 rpm for 10 min, and prepare products containing MpEgtB. Y106F MpEgtB L173T and MpEgtB Q409R The wet fungal body.

[0070] (5) The conversion conditions were as follows: 1 mL of reaction system contained 10 mg of wet bacterial cells, 100 mM 2-(4-(2-hydroxyethyl)piperazine) ethanesulfonic acid (HEPES) (pH 8.0) buffer, 100 mM NaCl, 1 mM HER, 1 mM Cys, 2 mM ascorbic acid, and 2 mM trichloroethyl phosphate, and the reaction was carried out at 30 °C for 2 hours.

[0071] The yield of Cys-HER was determined by HPLC after the conversion process, and the specific enzyme activity was calculated. The results are shown in Table 2.

[0072] Table 2: Results of shake-flask screening of single mutants

[0073]

[0074] The results showed that the mutant MpEgtB Y106F The best results are achieved this way.

[0075] Example 2: Construction and screening of double and triple mutants

[0076] The specific steps are as follows:

[0077] (1) Construction of double mutants:

[0078] In mutant MpEgtB Y106F Based on this, with MpEgtB Y106F Using the template L173T-F and L173T-R from Table 1 as the mutation primers, a double mutant was constructed by whole-plasmid PCR, yielding the double mutant MpEgtB. Y106F / L173T ; with MpEgtB Y106F Using the mutation primers Q409R-F and Q409R-R from Table 1 as templates, a double mutant was constructed by whole-plasmid PCR, yielding the double mutant MpEgtB. Y106F / Q409R ; with MpEgtB L173T Using the mutation primers Q409R-F and Q409R-R from Table 1 as templates, a double mutant was constructed by whole-plasmid PCR, yielding the double mutant MpEgtB. L173T / Q409R .

[0079] For specific implementation details, please refer to steps (1) to (2) in Example 1 to prepare a recombinant strain containing three double-mutant mutants: BL21 / pRSFDuet-MpEgtB Y106F / L173T BL21 / pRSFDuet-MpEgtB Y106F / Q409R and BL21 / pRSFDuet-MpEgtB L173T / Q409R .

[0080] (2) Construction of the triple mutant:

[0081] In mutant MpEgtB Y106F / L173T Based on this, using the mutation primers Q409R-F and Q409R-R in Table 1, a triple mutant was constructed by whole-plasmid PCR. For specific implementation details, please refer to Example 1. The resulting recombinant strain containing the triple mutant was: BL21 / pRSFDuet-MpEgtB. Y106F / L173T / Q409R .

[0082] Example 3: Expression and purification method of mutant enzyme

[0083] The specific steps are as follows:

[0084] (1) The positive transformants of the mutant recombinant strains prepared in Example 2 were inoculated into LB medium and cultured at 37°C until OD600. 600 The enzyme expression was induced by adding 0.5 mM IPTG at a final concentration of 0.6–0.8, the induction temperature was 25 °C, and the induction time was 14 h, resulting in the fermentation broth.

[0085] (2) The prepared fermentation broth was centrifuged at 4℃, 6000 rpm for 10 min to collect the bacterial cells. 10 mL of binding solution A (20 mM Tris, 20 mM imidazole, 1% glycerol, pH adjusted to 8.5 with HCl) was added to fully resuspend the bacterial cells. The centrifuge tubes were then placed in an ice bath and placed in a high-pressure homogenizer. The homogenization conditions were 850 MPa for 60 s. The resulting homogenate was centrifuged at low temperature and high speed at 4℃, 11000 rpm for 15 min to obtain the crude enzyme solution. The solution was filtered through a 0.22 μm microporous membrane for later use.

[0086] (3) Protein purification was performed using an AKTA protein purification system. First, the system was washed with 20% ethanol at a flow rate of 5 mL / min for 15 min. Then, the system was equilibrated with binding buffer A at a flow rate of 5 mL / min for 15 min. After loading the column, the column was equilibrated with binding buffer A at a flow rate of 3 mL / min for 30 min. After the system baseline stabilized, the sample was loaded at a flow rate of 3 mL / min. After loading, contaminating proteins were washed with binding buffer A until baseline equilibration. After baseline equilibration, elution was performed using elution buffer B (20 mM Tris, 500 mM imidazole, pH adjusted to 8.5 with HCl). The eluent with the absorption peak was collected to obtain the target protein that had reached electrophoretic purity, as shown in Figure 2.

[0087] (4) Wild-type parental enzyme MpEgtB and mutant MpEgtB were obtained respectively. Y106F MpEgtB Y106F / L173T MpEgtB Y106F / Q409R MpEgtB L173T / Q409R and MpEgtB Y106F / L173T / Q409R The specific activity and kinetic parameters of the above enzymes were determined at 30℃. cat / K m The initial rate of Cys-HER generated by measuring different concentrations of HER at 30°C was calculated.

[0088] The reaction system for enzyme activity determination: A 200 μL reaction system contained an appropriate amount of purified *Cys-HER* synthase, 100 mM 2-(4-(2-hydroxyethyl)piperazine)ethanesulfonic acid (HEPES) (pH 8.0) buffer, 100 mM NaCl, 1 mM HER, 1 mM Cys, 2 mM ascorbic acid, and 2 mM trichloroethyl phosphate. The reaction was carried out at 30℃ for 2 min. After centrifugation, the supernatant was diluted and the amount of trimethylammonium cysteine ​​sulfoxide (Cys-HER) produced was determined by HPLC, and the enzyme activity was calculated. One enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol of Cys-HER per minute at 30℃. The results are shown in Table 3.

[0089] Table 3: Kinetic parameters of the parental MpEgtB enzyme and its mutants

[0090]

[0091] The results showed that all mutants had increased specific enzyme activity compared to the original enzyme MpEgtB, with MpEgtB showing the highest activity. Y106F / L173T / Q409R 4.32 U mg -1 Protein, compared to 0.14 U mg of MpEgtB. -1 The protein level increased 31-fold. Consistent with this, the k values ​​of each mutant... cat / K m Both were improved compared to MpEgtB. MpEgtB Y106F / L173T / Q409R k cat / K m It is 1.12, which is 120 times that of MpEgtB (0.014).

[0092] Example 4: Molecular dynamics simulation of the MpEgtB parental enzyme and mutant

[0093] Molecular dynamics simulations of the MpEgtB parental enzyme and mutant were performed using Amber 18 with the standard Amber14SB force field and a simulation time of 100 ns. The simulation results were analyzed using Amber's built-in tools.

[0094] The results are shown in Figure 3. The results indicate that, compared to the parental enzyme MpEgtB, MpEgtB... Y106F / L173T / Q409R The root mean square deviation (RMSD) of the alpha-C atoms in the whole system showed a moderate decrease, indicating that the overall stability of the protein was improved after the MpEgtB mutation of these three key residues.

[0095] In addition, MpEgtB Y106F / L173T / Q409RThe RMSF values ​​near positions 106, 173, and 409 were significantly reduced, indicating a significant decrease in residue flexibility in these regions compared to MpEgtB. The structural changes in the MpEgtB mutant compared to the parent enzyme suggest a possible reason for its improved enzyme activity and catalytic efficiency.

[0096] Example 5: Construction of co-expression strains of MpEgtB, MsEgtD, and MsEgtE

[0097] The specific steps are as follows:

[0098] (1) Construction of expression carrier

[0099] The mutant MpEgtB of SEQ ID NO.16 is used. Y106F / L173T / Q409R The gene was double-copy integrated into the expression vector pRSFDuet-1, resulting in the recombinant expression vector pRSFDuet-MpEgtB. Y106F / L173T / Q409R .

[0100] The MsEgtD gene shown in SEQ ID NO.17 and the MsEgtE gene shown in SEQ ID NO.18 were integrated into the expression vector pACYC-Duet-1 to obtain the co-expression vector pACYC-Duet-EgtDE. The fragment and vector were ligated by one-step homologous recombination. The specific primers are shown in Table 4. The underlined parts are the restriction enzyme sites, and the italicized parts are the RBS sequences.

[0101] Table 4: Primer Sequences

[0102]

[0103] Two recombinant plasmids were introduced into the same Escherichia coli BL21(DE3) competent cell. For specific transformation steps, please refer to Example 1, which describes the chemical transformation method used to introduce the plasmids into Escherichia coli BL21 competent cells, ultimately constructing the recombinant strain.

[0104] BL21(DE3) / pRSFDuet-MpEgtB Y106F / L173T / Q409R / pACYC-Duet-EgtDE; named as: BL21-EgtBDE strain.

[0105] Example 6: Optimizing the conversion system for producing L-EGT

[0106] 1. Preparation of whole-cell catalysts:

[0107] The BL21-EgtBDE single colony prepared in Example 5, which grew on LB plates containing both ampicillin and kanamycin resistance, was inoculated into LB plates containing 0.05 mg / mL kanamycin / ampicillin resistance and cultured at 200 rpm and 37°C for 10 h to prepare seed culture;

[0108] The prepared seed culture was inoculated into the fermentation medium at a volume ratio of 3% (v / v) and cultured at 200 rpm and 37°C until OD reached. 600 When the concentration of the drug was 0.8, IPTG was added to a final concentration of 0.5 mmol / L for induction. The induction temperature was 30℃, and the cells were collected by centrifugation at 8000 rpm for 8 min after 24 h of induction.

[0109] 2. Optimization of the transformation system

[0110] (1) Effect of different pH values ​​of the conversion system on L-EGT concentration

[0111] To a 10 mL 100 mM Tris-HCl reaction system, add a final concentration of 20 g / L of recombinant strain, 50 mM L-His, 150 mM L-Met, 50 mM L-Cys, 200 mM ascorbic acid, 200 mM trichloroethyl phosphate, and 200 mM NaCl. Adjust the pH to 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively, and transform for 24 h at 25 °C and 200 rpm.

[0112] After the reaction was complete, a portion of the conversion solution was centrifuged at 12,000 × g for 15 min. The supernatant was filtered through a 0.22 μm microfiltration membrane and then analyzed by HPLC. The results are shown in Table 5.

[0113] Table 5: Results of pH Optimization for Conversion

[0114]

[0115] The results showed that slightly alkaline conditions were more suitable for the synthesis of L-EGT, and the highest concentration of L-EGT reached 2.23 g / L when the pH was controlled at 8.0.

[0116] (2) Effect of different conversion temperatures on L-EGT concentration

[0117] A 10 mL reaction system containing 100 mM Tris-HCl was added with a final concentration of 20 g / L of recombinant bacterial strain, 50 mM L-His, 150 mM L-Met, 50 mM L-Cys, 200 mM ascorbic acid, 200 mM TCEP, and 200 mM NaCl. The pH was adjusted to 8.5, and the transformation was carried out at temperatures of 20℃, 25℃, 30℃, and 35℃, and a rotation speed of 200 rpm for 24 h. The results are shown in Table 6.

[0118] Table 6: Results of Conversion Temperature Optimization

[0119]

[0120] The results showed that when the temperature was 30℃, 2.52 g / L L-EGT could be generated after 24 h of reaction.

[0121] Example 7: Optimal mutant strain MpEgtB Y106F / L173T / Q409R Feed-in batch conversion was carried out in a 5L fermenter.

[0122] The specific steps are as follows:

[0123] (1) Recombinant bacterial culture:

[0124] The BL21-EgtBDE single colony prepared in Example 5, which grew on LB plates containing both ampicillin and kanamycin resistance, was inoculated into LB plates containing 0.05 mg / mL kanamycin / ampicillin resistance and cultured at 200 rpm and 37°C for 10 h to prepare seed culture;

[0125] The prepared seed culture was inoculated into the fermentation medium at a volume ratio of 3% (v / v) and cultured at 200 rpm and 37°C until OD reached. 600 When the concentration of the drug was 0.8, IPTG was added to a final concentration of 0.5 mmol / L for induction. The induction temperature was 30℃, and the cells were collected by centrifugation at 8000 rpm for 8 min after 24 h of induction.

[0126] (2) 5L fermenter conversion (1L reaction system):

[0127] The whole-cell catalyst obtained in step (1) was suspended in 100 mM pH 8.0 Tris-HCl buffer to obtain wet cells;

[0128] The initial feed consisted of 20 g / L wet cells, 50 mM L-His, 150 mM L-Met, 50 mM L-Cys, 200 mM ascorbic acid, 200 mM TCEP, and 200 mM NaCl. After reacting for 2 hours, L-His, L-Met, L-Cys, and glycerol were continuously added to the reaction system, with a total feed of 5 M L-His, 15 M L-Met, 5 M L-Cys, and 50 mL glycerol. Transformation was carried out at 30 °C, with the pH maintained around 8.0 using 2 M NaOH. The stirring speed was 600 rpm, and the reaction lasted for 52 hours. Samples were taken every 4 hours to detect the L-EGT content.

[0129] According to the L-EGT yield determination, the results are shown in Figure 4. The highest L-EGT yield reached 8.32 g / L after 48 h of conversion.

[0130] Comparative Example 1

[0131] For specific implementation methods, please refer to Examples 5 and 6, the difference being that the mutant MpEgtB is used. Y106F / L173T / Q409R The parental enzyme MpEgtB was replaced. Fermentation and transformation experiments were conducted according to the method in Example 6. At a pH of 8.5 and a temperature of 30°C, after 24 hours of reaction, a portion of the transformation solution was centrifuged at 12,000 × g for 15 min. The supernatant was filtered through a 0.22 μm microfiltration membrane and analyzed by HPLC. The HPLC chromatogram results showed that the L-EGT yield of the recombinant *E. coli* BL21(DE3) / pRSFDuet-MpEgtB / pACYC-Duet-EgtDE expressing the original enzyme was 0.54 g / L.

[0132] 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 various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A mutant of MpEgtB, a methylbacterium sulfoxide synthase, characterized in that, The mutant is formed by mutating the tyrosine residue at position 106 of the methylbacillus sulfoxide synthase, as shown in SEQ ID NO.1, to phenylalanine.

2. A mutant of MpEgtB, a methylbacterium sulfoxide synthase, characterized in that, The mutant is any one of (a) to (c): (a) mutating tyrosine at position 106 of MpEgtB, the amino acid sequence of which is shown in SEQ ID NO.1, to phenylalanine, and simultaneously mutating leucine at position 173 to threonine; (b) mutating tyrosine at position 106 of MpEgtB, the amino acid sequence of which is shown in SEQ ID NO.1, to phenylalanine, and simultaneously mutating glutamine at position 409 to arginine; (c) mutating tyrosine at position 106 of MpEgtB, the amino acid sequence of which is shown in SEQ ID NO.1, to phenylalanine, and simultaneously mutating leucine at position 173 to threonine, and simultaneously mutating glutamine at position 409 to arginine.

3. A gene encoding the MpEgtB mutant of methylbacillus sulfoxide synthase according to any one of claims 1 to 2.

4. A recombinant vector carrying the gene of claim 3.

5. The recombinant vector according to claim 4, characterized in that, The recombinant vector is pACYC-Duet-1 or pRSF-Duet-1 as the expression vector.

6. Genetically engineered bacteria expressing the MpEgtB mutant of methylbacillus sulfoxide synthase according to any one of claims 1 to 2, or carrying the gene according to claim 3, or containing the recombinant vector according to any one of claims 4 to 5.

7. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria overexpress the MpEgtB mutant of methylbacillus sulfoxide synthase, the histidine-dependent methyltransferase MsEgtD, and the PLP-binding CS lyase MsEgtE as described in claim 1 or 2; the nucleotide sequence encoding MsEgtD is shown in SEQ ID NO.17, and the nucleotide sequence encoding MsEgtE is shown in SEQ ID NO.

18.

8. The genetically engineered bacteria according to claim 7, characterized in that, The genetically engineered bacteria used Escherichia coli BL21(DE3) as the expression host.

9. The genetically engineered bacterium according to claim 8, characterized in that, The dependent histidine methyltransferase MsEgtD and the PLP-binding CS lyase MsEgtE were overexpressed using the pACYC-Duet-1 vector, and the MpEgtB mutant was overexpressed using the pRSF-Duet-1 vector.

10. A method for preparing ergothioneine, characterized in that, Ergothionein was prepared by fermentation using the genetically engineered bacteria described in any one of claims 7 to 9 as a catalyst.

11. The use of the MpEgtB mutant of methylbacillus sulfoxide synthase according to claim 1, or the gene according to claim 3, or the recombinant vector according to claim 4 or 5, or the genetically engineered bacteria according to any one of claims 7 to 9, or the method according to claim 10 in the preparation of products containing ergothioneine.

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