Sulfoxide synthase and its use in the preparation of ergothioneine or intermediates thereof

Abstract

The application discloses a sulfoxide synthase and application thereof in preparation of ergothioneine or an intermediate thereof. The sulfoxide synthase has an amino acid sequence as shown in SEQ ID NO: 3 or a sequence identity of at least 99% with SEQ ID NO: 3. The application first truncates and modifies a gene for synthesizing a sulfoxide synthase protein, retains sulfoxide synthase activity of the sulfoxide synthase protein, and improves catalytic activity of the sulfoxide synthase protein. The sulfoxide synthase protein after truncation and modification is modified by a gene mutation method, a mutant with further improved catalytic activity is screened, and ergothioneine yield of the mutant sulfoxide synthase can reach 392 mg / L.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a sulfoxide synthase and its application in the preparation of ergothionein or its intermediates. Background Technology

[0002] Ergothioneine (hereinafter referred to as "ERG") is a unique sulfur-containing amino acid that is widely present in various tissues and organs of the human body. However, the human body cannot synthesize ergothioneine and mainly obtains it from food. Due to its unique chemical structure, ergothioneine has high stability and strong antioxidant function, and can be used as a health product, food preservative, and protective agent against various diseases. At present, ergothioneine can be synthesized by chemical methods or extracted from natural products such as mushrooms, but both methods have disadvantages such as low yield, many impurities, and high cost. With the discovery of multiple microbial synthesis pathways for ergothioneine, it is expected that ergothioneine can be produced in large quantities through microbial fermentation or in vitro enzymatic reactions. Current research mainly includes (1) finding microorganisms that naturally synthesize ergothioneine and (2) directly introducing or combining ergothioneine synthesis pathways from different sources and using various microbial host bacteria (such as Escherichia coli, Aspergillus oryzae, Saccharomyces cerevisiae, etc.) to produce ergothioneine. For example, CN113186107A provides an ergothioneine-producing bacterium, *Panus conchatus*, which reduces fermentation costs through fermentation process control and optimization, but the yield is low, reaching 140.89 mg / L after 96 hours of fermentation. Patent CN110358719A cloned the egtABCDE gene cluster from different sources, achieving heterologous expression of ergothioneine in *Escherichia coli* and *Bacillus subtilis*, respectively. However, the egtABCDE synthesis pathway involves multiple steps, and ergothioneine may compete with glutathione synthesis. Furthermore, it suffers from long fermentation times and low conversion rates of precursor amino acids such as histidine. CN112251392A provides a high-yielding ergothioneine-producing genetically engineered bacterium through gene modification and host bacterium modification, achieving a yield of 105.7 mg / L after 30 hours of shake-flask fermentation. However, this operation is complex, integrating multiple exogenous genes into the *E. coli* genome, which may impose a certain metabolic burden on the host bacterium. Due to the low yield of naturally synthesized ergothioneine by microorganisms and the low activity of naturally occurring enzymes, there is currently a lack of strains that can efficiently produce ergothioneine. The natural ergothioneine synthesis pathway suffers from drawbacks such as low yield and low activity of related enzymes. Summary of the Invention

[0003] The NcEgt1 protein (i.e., sulfoxide synthase) derived from Neurospora crassa is involved in the catalysis of ergothionein formation, but its activity is low. The purpose of this invention is to modify the NcEgt1 protein through genetic engineering and obtain mutants with enhanced activity through screening.

[0004] To address the issues of the numerous steps involved in the egtABCDE synthesis pathway and its competition with glutathione synthesis, this invention utilizes *Escherichia coli* as the host bacterium to construct a genetically engineered bacterium containing a non-natural ergothioneine synthesis pathway. This pathway comprises three enzymes: EgtD (histidine methyltransferase) from *Mycobacterium smegmatis*, NcEgt1 from *Neurospora crassa*, and EgtE (CS bond cleavage enzyme), also from *Mycobacterium smegmatis*. These enzymes are responsible for catalyzing the trimethylation of histidine to generate compound I, catalyzing the reaction of cysteine ​​and compound I to generate compound II, and cleaving the CS bond in compound II to generate ergothioneine (e.g., lysine). Figure 1 (As shown). This pathway is shorter than the egtABCDE pathway, requiring only three enzymatic catalytic reactions. Furthermore, the NcEgt1 enzyme in this pathway uses cysteine ​​and histidine betaine as substrates, eliminating the competition between ergothioneine and glutathione synthesis (Hu W, Song H, Her AS, et al. Bioinformatic and biochemical characterizations of CS bond formation and cleavage enzymes in the fungus Neurospora crassa ergothioneine biosynthetic pathway.[J].OrganicLetters,2014,16(20):5382-5).

[0005] The reaction catalyzed by NcEgt1 is a crucial step in the ergothioneine synthesis pathway. However, this natural enzyme suffers from large molecular weight and low activity, making its catalytic reaction the rate-limiting step in the pathway. To improve the catalytic activity of the NcEgt1 protein, this invention provides a sulfoxide synthase and its application in the preparation of ergothioneine or its intermediates. The inventors modified the NcEgt1 protein using methods such as truncation, random mutation, and combinatorial mutation, and obtained mutants with enhanced activity through screening. The mutants with enhanced activity, in conjunction with EgtD and EgtE, further increased the yield of ergothioneine.

[0006] The first aspect of the present invention provides a sulfoxide synthase having an amino acid sequence as shown in SEQ ID NO:3 or having at least 99% sequence identity with SEQ ID NO:3.

[0007] In this invention, the positions of the first two amino acids M and G in SEQ ID NO:3 are defined as -2 and -1, and the position of the third amino acid M is defined as 1.

[0008] The amino acid sequence preferably differs from SEQ ID NO:3 by containing one or more amino acid residues selected from the following:

[0009] G-1R / S; T27S; Q34R; Q66R / H; K69R; K72I; A74V; A79T; K83D; E86G; D107Y / A; L153Q; L170P; Q195H; A201S; S215N; Q238H; D259 V; N270S; G276S; Q293R; S297G; V311M; A316S; L317R; S324N / C; G330C; T368S; A395E; F424Y; V440I; L445I; P469I / T; P509S;

[0010] It also has an activity of at least sulfoxide synthase as shown in the amino acid sequence of SEQ ID NO:3.

[0011] The above differences can be achieved by mutations in SEQ ID NO:3 or by mutations in other sequences, as long as the final result shows that its amino acid sequence differs from that of SEQ ID NO:3.

[0012] For example, in these differences, G-1R / S means that G at the -1 site is eventually replaced by R or S.

[0013] In a preferred embodiment, the amino acid sequence differs from SEQ ID NO:3 by comprising amino acid residues selected from any of the following groups:

[0014] G-1S and D107A;

[0015] Q66H and S324C; or,

[0016] D107Y and V311M; or,

[0017] A79T and S297G; or,

[0018] L170P and Q195H; or,

[0019] K83D and S215N; or,

[0020] D259V and V440I; or,

[0021] T27S and A74V; or,

[0022] Q238H and Q293R; or,

[0023] K69R and T368S, P509S; or,

[0024] G276S, G330C, and A395E; or,

[0025] E86G, A316S, and L317R; or,

[0026] Q34R, K72I, N270S, and F424Y; or,

[0027] S324N and L445I; or,

[0028] D107A and P469T; or,

[0029] Q66R and A201S; or,

[0030] L445I and P469I; or,

[0031] K83D, D107A, and S215N; or,

[0032] Q66R, D107Y, and V311M; or,

[0033] G-1S, D107A, and P469T; or,

[0034] G-1R, T27S, and A74V; or,

[0035] Q66H, E86G, A316S, and L317R; or,

[0036] G276S, G330C, A395E, and P469T; or,

[0037] Q66H, A79T, S297G, and S324C; or,

[0038] L170P, Q195H, D259V, and V440I; or,

[0039] K69R, D259V, T368S, V440I, and P509S; or,

[0040] L153Q, L170P, Q195H, Q238H, and Q293R; or,

[0041] T27S, Q34R, K72I, A74V, N270S, and F424Y; or,

[0042] G-1S, L153Q, L170P, Q195H, Q238H and Q293R.

[0043] Another aspect of the present invention provides a fusion protein comprising sulfoxide synthase as described in the present invention, as well as histidine methyltransferase and CS bond cleavage enzyme.

[0044] The amino acid sequence of the histidine methyltransferase is preferably as shown in SEQ ID NO:4, or the amino acid sequence of the CS bond lyase is preferably as shown in SEQ ID NO:6.

[0045] The fusion protein preferably comprises, from the N-terminus to the C-terminus, the sulfoxide synthase, the histidine methyltransferase, and the CS bond cleavage enzyme.

[0046] Another aspect of the present invention provides an isolated nucleic acid that encodes a sulfoxide synthase as described in the present invention or a fusion protein as described in the present invention.

[0047] The nucleotide sequence of the nucleic acid is preferably as shown in SEQ ID NO:2.

[0048] Another aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the present invention.

[0049] The backbone plasmid of the recombinant expression vector is preferably pETDuet-1.

[0050] The nucleotide sequence encoding the histidine methyltransferase is preferably as shown in SEQ ID NO:5, or the nucleic acid sequence encoding the CS bond lyase is preferably as shown in SEQ ID NO:7.

[0051] Another aspect of the present invention provides a transformant comprising a nucleic acid as described in the present invention or a recombinant expression vector as described in the present invention.

[0052] The host cell for the transformant is preferably Escherichia coli, such as E. coli BL21(DE3).

[0053] Another aspect of the present invention provides a method for preparing sulfoxide synthase as described in the present invention, the method comprising culturing a transformant as described in the present invention to obtain the sulfoxide synthase.

[0054] Another aspect of the present invention provides a method for preparing compound II, the method comprising the steps of: preparing compound II using a sulfoxide synthase as described in the present invention or a transformant as described in the present invention;

[0055]

[0056] In a preferred embodiment, the method involves culturing the transformant in a fermentation medium with shaking, followed by the addition of isopropyl thiogalactoside to induce the expression of compound II.

[0057] The fermentation medium preferably contains Fe. 2+ and cysteine;

[0058] In a preferred embodiment, the method involves reacting cysteine ​​and Fe... 2+ In the presence of oxygen and the sulfoxide synthase, it reacts with histidine trimethyl inner salt to obtain compound II.

[0059] In a preferred embodiment, the sulfoxide synthase exists as a bacterial cell in the supernatant after cell lysis.

[0060] In a preferred embodiment, the mass-to-volume ratio of the histidine trimethyl inner salt to the supernatant is 3-15 mg / ml, for example, 9.9 mg / ml.

[0061] In a preferred embodiment, the cysteine ​​and Fe 2+ The molar ratio of the histidine trimethyl inner salt is (1-3):1:(1-2), for example 5:2:2.5.

[0062] In a preferred embodiment, the reaction is carried out at a rotation speed of 200-250 rpm, for example 220 rpm, and a temperature of 20°C-30°C, for example 25°C.

[0063] In a preferred embodiment, the reaction is carried out at a pH of 6-8, for example, pH 7.

[0064] Another aspect of the present invention provides a method for preparing ergothioneine, which includes the step of preparing compound II as described in the present invention, and further includes the step of preparing compound II into ergothioneine.

[0065] Another aspect of the present invention provides the use of sulfoxide synthases or transformants as described in the present invention in the preparation of ergothioneine or intermediates thereof, such as compound II.

[0066] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0067] The reagents and raw materials used in this invention are all commercially available.

[0068] The positive and progressive effects of this invention are as follows:

[0069] 1) For the first time, the gene that synthesizes the NcEgt1 protein was truncated and modified, preserving its sulfoxide synthase activity and improving its catalytic activity.

[0070] 2) By modifying the truncated NcEgt1 protein through gene mutation and other means, mutants with further enhanced catalytic activity were screened, and the mutant sulfoxide synthase could produce 392 mg / L when preparing ergothionein. Attached Figure Description

[0071] Figure 1 The present invention illustrates the ergothioneine biosynthetic pathway constructed in this invention, wherein Histidine is histidine, Hercynine is histidine trimethyl inner salt (i.e., (αS)-α-Carboxy-N,N,N-trimethyl-1H-imidazole-4-ethanaminium hydroxide, inner salt), Methione is methionine, SAM is S-adenosylmethionine, Cysteine ​​is cysteine, Hercynylcysteine ​​sulfoxide is (2S)-3-(2-{[(2R)-2-amino-2-carboxyethyl]sulfinyl}-1H-imidazol-5-yl)-2-(trimethylazine cation) propionate, and Ergothioneine is ergothioneine.

[0072] Figure 2 The plasmid map containing the ergothioneine synthesis pathway is displayed.

[0073] Figure 3 This is a domain of the NcEgt1 protein.

[0074] Figure 4 Plasmid map for expressing the truncated NcEgt1 protein.

[0075] Figure 5 The images show the in vitro experimental liquid chromatography detection chromatograms (arrows indicate product peaks); where A is the in vitro reaction chromatogram catalyzed by W bacteria, and B is the in vitro reaction chromatogram catalyzed by W1 bacteria.

[0076] Figure 6 The images show the liquid chromatography detection spectra of ergothioneine (arrows indicate ergothioneine); where A is the liquid chromatography detection spectra of ERG in Example 7, and B is the liquid chromatography detection spectra of ERG in Example 8. Detailed Implementation

[0077] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0078] The strains, plasmids, and reagents used in this invention

[0079] The *E. coli* DH5α and *E. coli* BL21(DE3) used in this invention were purchased from Kangwei Century Co., Ltd., and were used for gene cloning and protein expression, respectively. The DNA gel extraction and purification kit and plasmid extraction kit used in this invention were purchased from Sangon Biotech (Shanghai) Co., Ltd., all restriction endonucleases and DNA polymerases were purchased from Takara, and the homologous recombination kit was purchased from Vazyme.

[0080] Plasmid construction method: The target fragment was obtained by PCR, the plasmid was digested with enzymes, and the DNA was recovered by gel extraction to obtain the target fragment and the vector fragment. After homologous recombination, the fragments were transformed into competent E. coli DH5α or BL21(DE3) cells and plated with resistant plates.

[0081] LB medium (g / L): tryptone 10.0, yeast extract 5.0, NaCl 10.0, deionized water 1L, pH 7.0.

[0082] Fermentation medium formulation (g / L): yeast extract (24.0), soybean peptone (12.0), sodium chloride (3.0), glycerol (5.0), dipotassium hydrogen phosphate (2.0), magnesium sulfate heptahydrate (0.5), cysteine ​​hydrochloride (1.0), histidine (1.0), methionine (1.0), triferroamine citrate (0.06), 1L deionized water, pH 7.0.

[0083] Fermentation broth treatment method: Take 2 mL of fermentation broth, incubate in a 100℃ water bath for 5 min, centrifuge at 12000 rpm for 5 min, take the supernatant, filter it through a 0.22 μm filter membrane, and then perform HPLC analysis.

[0084] HPLC conditions: Ultimate Hilic Silica column, 5 μm, 4.6 × 250 mm; mobile phase: water:acetonitrile = 20:80 (v / v); column temperature: 30℃; detection wavelength: 254 nm; flow rate: 1.0 mL / min; injection volume: 20 μL.

[0085] In vitro reaction HPLC detection conditions: chromatographic column was Cosmosil PBr column (250 mm L × 4.6 mm ID, 5 μm; Nacalai Tesque, Inc., Kyoto, Japan); mobile phase was 0.1% formic acid in water; column temperature was 35℃; detection wavelength was 250 nm; flow rate was 1.0 mL / min; injection volume was 20 μL.

[0086] Example 1: Construction of engineered ergothionein and ergothioneinase bacteria

[0087] The genes encoding histidine methyltransferase (EgtD) from *Mycobacterium smegmatis*, the gene encoding PLP-dependent CS bond cleavage enzyme (EgtE), and the gene encoding NcEgt1 enzyme from *Neurospora crassa* were commissioned to a commercial company for whole-genome synthesis and cloned into the pETDuet-1 vector to obtain the plasmid pETDuet-1-ncEgt1-egtD-egtE (e.g., ...). Figure 2 (As shown). The plasmid was transformed into E. coli BL21(DE3) competent cells, plated on ampicillin-resistant plates (50 μg / mL), and incubated overnight at 37°C. Positive transformation colonies were picked, cultured in LB blotting, and the plasmid was extracted and sequenced for verification, yielding recombinant genetically engineered bacteria S containing the ergothionein synthesis pathway. Following the same procedure, the gene sequence of ergothioneinase (accession number BAM63550.1) from Burkholderia sp. HME13 was ligated into the vector pACYCDuet-1, and the ergothioneinase supernatant was prepared using the same method.

[0088] Example 2: Truncated Modification of NcEgt1 Enzyme

[0089] The NcEgt1 protein contains 878 amino acids (SEQ ID NO:1) and has two domains—a methyltransferase domain and a CS bond synthase domain (e.g., ...). Figure 3 (As shown). The inventors discovered that the reaction catalyzed by the NcEgt1 protein to form compound II from cysteine ​​is the rate-limiting step in the ergothioneine synthesis pathway. To facilitate subsequent enzyme modification, we first truncated the gene. Primers were designed at the junction of the two domains, and the truncated ncEgt1 gene (referred to as...) was obtained by PCR. T ncEgt1 (nucleotide sequence shown in SEQ ID NO:2) was digested with HindIII and NcoI in plasmid pETDuet-1, and then the ncEgt1 fragment was recombined using homologous recombination. T The ncEgt1 gene fragment was inserted into the HindIII and NcoI restriction sites of the pETDuet-1 vector, respectively (e.g., Figure 4 As shown, the bacteria were transformed into *E. coli* BL21(DE3) competent cells, plated on 50 μg / mL ampicillin-containing plates, and incubated overnight at 37°C. Positive transformation colonies were picked, cultured in LB broth, and plasmids were extracted and sequenced for verification, yielding strains W (containing the ncEgt1 gene) and W1 (containing...). T (ncEgt1 gene).

[0090] Example 3 In vitro catalytic reaction

[0091] Ergothioneine biosynthesis pathway such as Figure 1 As shown.

[0092] 10 μL of glycerol culture from strain W was inoculated into 5 mL of LB medium (containing 50 μg / mL ampicillin) and cultured at 37°C and 220 rpm for 12 h. Then, the seed culture was transferred at a 1.5% inoculation rate to a shake flask containing 100 mL of LB medium (containing 50 μg / mL ampicillin) and cultured at 37°C and 200 rpm for 3 h. Isopropyl thiogalactoside (IPTG) was added to a final concentration of 1 mM, and protein expression was induced at 28°C. Cultured for another 20 h. After fermentation, the cells were obtained by centrifugation at 12000 rpm and 4°C. W1 cells were obtained using the same method. A certain amount of W and W1 cells were diluted to the same concentration with phosphate buffer (pH = 7.0), and the cells were disrupted using an ultrasonic cell disruptor. The supernatant was obtained by centrifugation at 12000 rpm and 4°C. The reaction system was as follows: 2 mL reaction mixture, 100 mM HEPES (pH 7.5), 5 mM Cys·HCl, 2 mM FeSO4·7H2O, 2.5 mM HER (Hercynine, histidine trimethylolpropionate), and 100 μL of bacterial cell lysis supernatant. The reaction was carried out at 25℃ and 220 rpm under aerobic conditions. The reaction was terminated after 30 min. The reaction solution was diluted with 0.1% formic acid solution and analyzed by high-performance liquid chromatography (HPLC). Figure 5 (As shown). Under the same reaction conditions, the catalytic ability of W1 cells was 27 times that of W cells.

[0093] Table 1

[0094] W SEQ ID NO:1 100％ W1 SEQ ID NO:3 2700％

[0095] Example 4 T Construction and screening of NcEgt1 enzyme random mutation library

[0096] The plasmid pETDuet-1-ncEgt1-egtD-egtE was digested with HindIII / NcoI to obtain a vector without the ncEgt1 fragment. The vector was then ligated using enzyme ligation. T The ncEgt1 fragment was inserted into the HindIII / NcoI restriction site of plasmid pETDuet-1-ncEgt1-egtD-egtE. The resulting strain was transformed into competent E. coli BL21(DE3) cells and cultured overnight at 37°C on LB agar containing 50 μg / mL ampicillin. Positive clones were picked, sequenced, and the resulting strain was named S1.

[0097] by T Using the ncEgt1 gene as a template, a random point mutation kit was used ( II. Site-Directed Mutagenesis Kit) was used for error-prone PCR, and the PCR fragment was recovered by gel extraction. The PCR fragment was inserted into the HindIII / NcoI restriction site of plasmid pETDuet-1-ncEgt1-egtD-egtE via homologous recombination, and then transformed into *E. coli* BL21(DE3) competent cells and cultured overnight at 37°C on LB solid medium containing 50 μg / mL ampicillin. Single colonies of the mutant were selected and cultured overnight at 37°C and 200 rpm in 96-well plates containing 400 μL of LB liquid medium (containing 50 μg / mL ampicillin) per well. Then, 10 μL of seed culture was transferred to 96-well deep-plates containing 600 μL of fermentation medium (containing 50 μg / mL ampicillin) per well and cultured at 37°C and 200 rpm for 3 h. Then, isopropyl thiogalactoside (IPTG) was added to a final concentration of 1 mM, and the temperature was lowered to 28°C to induce mutant expression. The culture was continued for 20 h. After fermentation, the 96-well plates were centrifuged at 4000g for 30 min at 4°C. The supernatant was diluted 5-fold with 0.1 M phosphate buffer, and 190 μL was transferred to each well of the plate. The initial absorbance of the fermentation broth was measured at 311 nm. Then, 10 μL of ergothionease supernatant (containing 10 U of ergothionease in 0.1 M phosphate buffer, pH 7.0, where U is defined as the amount of enzyme required to catalyze the conversion of 1 mmol of substrate in 1 min) was added (the preparation method of this enzyme supernatant is the same as in Example 3). After reacting at room temperature for 10 min, a stop value was measured at 311 nm. The corresponding ergothionein content was calculated based on the standard curve. Mutants with high ergothionein content were selected for sequencing and shake-flask verification. The screening results are shown in Table 2.

[0098] Table 2

[0099]

[0100]

[0101] Note: The positions of the first two amino acids M and G in SEQ ID NO:3 are defined as -2 and -1.

[0102] Example 5 T Construction and screening of NcEgt1 enzyme combinatorial mutant libraries

[0103] This embodiment involves selecting corresponding mutation sites for combined mutation based on the random mutation in Example 4.

[0104] Based on the results of random mutations, corresponding primers were designed to... TUsing the ncEgt1 gene as a template, overlap PCR was performed using PrimeSTARGXL DNA Polymerase (TaKaRa, Japan). The PCR fragment was recovered via gel electrophoresis and inserted into the HindⅢ / NcoⅠ site of plasmid pETDuet-1-ncEgt1-egtD-egtE via homologous recombination. The resulting fragment was then transformed into *E. coli* BL21(DE3) and cultured overnight at 37°C on LB agar containing 50 μg / mL ampicillin. Positive clones were picked and cultured overnight at 37°C and 200 rpm in 5 mL LB agar. Sequencing verification was performed, followed by shake-flask fermentation. ERG content was detected using high-performance liquid chromatography (HPLC). The screening results are shown in Table 3.

[0105] Table 3

[0106]

[0107]

[0108] Note: The positions of the first two amino acids M and G in SEQ ID NO:3 are defined as -2 and -1.

[0109] Example 6: Production of Ergothioneine by Fermentation using S strain S

[0110] 10 μl of glycerol-containing strain S was inoculated into 5 mL of LB medium (containing 50 μg / mL ampicillin) and cultured at 37°C and 200 rpm for 12 h. Then, the seed culture was transferred at an inoculum volume of 1.5% to a shake flask containing 20 mL of fermentation medium (containing 50 μg / mL ampicillin) and cultured at 37°C and 200 rpm for 3 h. Isopropyl thiogalactoside (IPTG) was added to a final concentration of 1 mM, and mutant expression was induced at 28°C. Cultured for another 20 h. After fermentation, the fermentation broth was treated according to the previous sample processing method, and high-performance liquid chromatography (HPLC) analysis was performed. The ergothioneine content was 40.0 mg / L.

[0111] Example 7: Production of Ergothioneine by Fermentation using S1 Strains

[0112] 10 μl of glycerol bacteria (S1 strain) was inoculated into 5 mL LB medium (containing 100 μg / mL ampicillin) and cultured at 37℃ and 200 rpm for 12 h. Then, the seed culture was transferred at an inoculum volume of 1.5% to a shake flask containing 20 mL of fermentation medium (containing 50 μg / mL ampicillin) and cultured at 37℃ and 200 rpm for 3 h. Isopropyl thiogalactoside (IPTG) was added to a final concentration of 1 mM, and mutant expression was induced at 28℃. Cultured for another 20 h. After fermentation, the fermentation broth was treated according to the previous sample treatment method, and the concentration was determined by high-performance liquid chromatography to be 54.9 mg / L (see...). Figure 6 A in the middle.

[0113] Example 8: Fermentation production of ergothionein using a mutant strain containing the gene corresponding to mutation 25.

[0114] 10 μl of glycerol bacteria (containing the mutant strain with the mutant 25 sequence) was inoculated into 5 mL LB medium (containing 50 μg / mL ampicillin) and cultured at 37℃ and 200 rpm for 12 h. Then, the seed culture was transferred at a 1.5% inoculum to a shake flask containing 20 mL of fermentation medium (containing 50 μg / mL ampicillin) and cultured at 37℃ and 200 rpm for 3 h. Isopropyl thiogalactoside (IPTG) was added to a final concentration of 1 mM, and mutant expression was induced at 28℃. Cultured for another 20 h. After fermentation, the fermentation broth was processed according to the previous sample treatment method and analyzed by high-performance liquid chromatography (HPLC). The ergothioneine content was 392 mg / L (see...). Figure 6 (B in the middle). SEQUENCE LISTING <110> Shanghai Pharmaceutical Industry Research Institute Co., Ltd. China National Pharmaceutical Industry Research Institute Co., Ltd. <120> sulfoxide synthase and its application in the preparation of ergothioneine or its intermediates <130> P210110037C <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 878 <212> PRT <213> Artificial Sequence <220> <223> NcEgt1 amino acid sequence <400> 1 Met Gly Met Pro Ser Ala Glu Ser Met Thr Pro Ser Ser Ala Leu Gly 1 5 10 15 Gln Leu Lys Ala Thr Gly Gln His Val Leu Ser Lys Leu Gln Gln Gln 20 25 30 Thr Ser Asn Ala Asp Ile Ile Asp Ile Arg Arg Val Ala Val Glu Ile 35 40 45 Asn Leu Lys Thr Glu Ile Thr Ser Met Phe Arg Pro Lys Asp Gly Pro 50 55 60 Arg Gln Leu Pro Thr Leu Leu Leu Tyr Asn Glu Arg Gly Leu Gln Leu 65 70 75 80 Phe Glu Arg Ile Thr Tyr Leu Glu Glu Tyr Tyr Leu Thr Asn Asp Glu 85 90 95 Ile Lys Ile Leu Thr Lys His Ala Thr Glu Met Ala Ser Phe Ile Pro 100 105 110 Ser Gly Ala Met Ile Ile Glu Leu Gly Ser Gly Asn Leu Arg Lys Val 115 120 125 Asn Leu Leu Leu Glu Ala Leu Asp Asn Ala Gly Lys Ala Ile Asp Tyr 130 135 140 Tyr Ala Leu Asp Leu Ser Arg Glu Glu Leu Glu Arg Thr Leu Ala Gln 145 150 155 160 Val Pro Ser Tyr Lys His Val Lys Cys His Gly Leu Leu Gly Thr Tyr 165 170 175 Asp Asp Gly Arg Asp Trp Leu Lys Ala Pro Glu Asn Ile Asn Lys Gln 180 185 190 Lys Cys Ile Leu His Leu Gly Ser Ser Ile Gly Asn Phe Asn Arg Ser 195 200 205 Asp Ala Ala Thr Phe Leu Lys Gly Phe Thr Asp Val Leu Gly Pro Asn 210 215 220 Asp Lys Met Leu Ile Gly Val Asp Ala Cys Asn Asp Pro Ala Arg Val 225 230 235 240 Tyr His Ala Tyr Asn Asp Lys Val Gly Ile Thr His Glu Phe Ile Leu 245 250 255 Asn Gly Leu Arg Asn Ala Asn Glu Ile Ile Gly Glu Thr Ala Phe Ile 260 265 270 Glu Gly Asp Trp Arg Val Ile Gly Glu Tyr Val Tyr Asp Glu Glu Gly 275 280 285 Gly Arg His Gln Ala Phe Tyr Ala Pro Thr Arg Asp Thr Met Val Met 290 295 300 Gly Glu Leu Ile Arg Ser His Asp Arg Ile Gln Ile Glu Gln Ser Leu 305 310 315 320 Lys Tyr Ser Lys Glu Glu Ser Glu Arg Leu Trp Ser Thr Ala Gly Leu 325 330 335 Glu Gln Val Ser Glu Trp Thr Tyr Gly Asn Glu Tyr Gly Leu His Leu 340 345 350 Leu Ala Lys Ser Arg Met Ser Phe Ser Leu Ile Pro Ser Val Tyr Ala 355 360 365 Arg Ser Ala Leu Pro Thr Leu Asp Asp Trp Glu Ala Leu Trp Ala Thr 370 375 380 Trp Asp Val Val Thr Arg Gln Met Leu Pro Gln Glu Glu Leu Leu Glu 385 390 395 400 Lys Pro Ile Lys Leu Arg Asn Ala Cys Ile Phe Tyr Leu Gly His Ile 405 410 415 Pro Thr Phe Leu Asp Ile Gln Leu Thr Lys Thr Thr Lys Gln Ala Pro 420 425 430 Ser Glu Pro Ala His Phe Cys Lys Ile Phe Glu Arg Gly Ile Asp Pro 435 440 445 Asp Val Asp Asn Pro Glu Leu Cys His Ala His Ser Glu Ile Pro Asp 450 455 460 Glu Trp Pro Pro Val Glu Glu Ile Leu Thr Tyr Gln Glu Thr Val Arg 465 470 475 480 Ser Arg Leu Arg Gly Leu Tyr Ala His Gly Ile Ala Asn Ile Pro Arg 485 490 495 Asn Val Gly Arg Ala Ile Trp Val Gly Phe Glu His Glu Leu Met His 500 505 510 Ile Glu Thr Leu Leu Tyr Met Met Leu Gln Ser Asp Lys Thr Leu Ile 515 520 525 Pro Thr His Ile Pro Arg Pro Asp Phe Asp Lys Leu Ala Arg Lys Ala 530 535 540 Glu Ser Glu Arg Val Pro Asn Gln Trp Phe Lys Ile Pro Ala Gln Glu 545 550 555 560 Ile Thr Ile Gly Leu Asp Asp Pro Glu Asp Gly Ser Asp Ile Asn Lys 565 570 575 His Tyr Gly Trp Asp Asn Glu Lys Pro Pro Arg Arg Val Gln Val Ala 580 585 590 Ala Phe Gln Ala Gln Gly Arg Pro Ile Thr Asn Glu Glu Tyr Ala Gln 595 600 605 Tyr Leu Leu Glu Lys Asn Ile Asp Lys Leu Pro Ala Ser Trp Ala Arg 610 615 620 Leu Asp Asn Glu Asn Ile Ser Asn Gly Thr Thr Asn Ser Val Ser Gly 625 630 635 640 His His Ser Asn Arg Thr Ser Lys Gln Gln Leu Pro Ser Ser Phe Leu 645 650 655 Glu Lys Thr Ala Val Arg Thr Val Tyr Gly Leu Val Pro Leu Lys His 660 665 670 Ala Leu Asp Trp Pro Val Phe Ala Ser Tyr Asp Glu Leu Ala Gly Cys 675 680 685 Ala Ala Tyr Met Gly Gly Arg Ile Pro Thr Phe Glu Glu Thr Arg Ser 690 695 700 Ile Tyr Ala Tyr Ala Asp Ala Leu Lys Lys Lys Lys Glu Ala Glu Arg 705 710 715 720 Gln Leu Gly Arg Thr Val Pro Ala Val Asn Ala His Leu Thr Asn Asn 725 730 735 Gly Val Glu Ile Thr Pro Pro Ser Ser Pro Ser Ser Glu Thr Pro Ala 740 745 750 Glu Ser Ser Ser Pro Ser Asp Ser Asn Thr Thr Leu Ile Thr Thr Glu 755 760 765 Asp Leu Phe Ser Asp Leu Asp Gly Ala Asn Val Gly Phe His Asn Trp 770 775 780 His Pro Met Pro Ile Thr Ser Lys Gly Asn Thr Leu Val Gly Gln Gly 785 790 795 800 Glu Leu Gly Gly Val Trp Glu Trp Thr Ser Ser Val Leu Arg Lys Trp 805 810 815 Glu Gly Phe Glu Pro Met Glu Leu Tyr Pro Gly Tyr Thr Ala Asp Phe 820 825 830 Phe Asp Glu Lys His Asn Ile Val Leu Gly Gly Ser Trp Ala Thr His 835 840 845 Pro Arg Ile Ala Gly Arg Lys Ser Phe Val Asn Trp Tyr Gln Arg Asn 850 855 860 Tyr Pro Tyr Ala Trp Val Gly Ala Arg Val Val Arg Asp Leu 865 870 875 <210> 2 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> TNcEgt1 <400> 2 atgggcatga gctttagcct gattccgagc gtttatgcgc gtagcgcgct gccgaccctg 60 gatgattggg aggcgctgtg ggcgacctgg gatgtggtta cccgtcaaat gctgccgcag 120 gaagagctgc tggaaaagcc gatcaaactg cgtaacgcgt gcatcttcta tctgggccac 180 attccgacct ttctggacat ccaactgacc aagaccacca aacaagcgcc gagcgaaccg 240 gcgcacttct gcaaaatctt tgagcgtggt attgacccgg atgttgacaa cccggagctg 300 tgccacgcgc acagcgaaat tccggacgag tggccgccag tggaggaaat cctgacctac 360 caagaaaccg ttcgtagccg tctgcgtggt ctgtatgcgc acggcatcgc gaacattccg 420 cgtaacgtgg gtcgtgcgat ttgggttggc ttcgagcacg aactgatgca catcgagacc 480 ctgctgtaca tgatgctgca gagcgataag accctgatcc cgacccacat tccgcgtccg 540 gatttcgaca agctggcgcg taagcggag agcgaacgtg tgccgaacca atggtttaaa 600 attccggcgc aggaaatcac cattggtctg gacgatccgg aggatggcag cgacatcaac 660 aagcactacg gctgggacaa cgaaaaaccg ccgcgtcgtg tgcaagttgc ggcgttccaa 720 gcgcagggtc gtccgattac caacgagaa tacgcgcagt atctgctgga gaagaacatc 780 gataaactgc cggcgagctg ggcgcgtctg gaacgaaa acatcagcaa cggcaccacc 840 aacagcgtta gcggccacca cagcaaccgt accagcaagc aacagctgcc gagcagcttt 900 ctggagaaaa ccgcggtgcg taccgtttat ggtctggtgc cgctgaagca tgcgctggat 960 tggccggttt tcgcgagcta cgacgaactg gcgggttgcg cggcgtatat gggtggccgt 1020 attccgacct ttgaggaaac ccgtagcatc tacgcgtatg cggatgcgct gaaaaaag 1080 aaagaggcgg aacgccaact gggtcgtacc gtgccggcgg ttaacgcgca cctgaccaac 1140 aacggtgttg agatcacccc gccgagcagc ccgagcagcg aaccccggc ggagagcagc 1200 agcccgagcg atagcaacac caccctgatt accaccgaag acctgttcag cgatctggac 1260 ggtgcgaacg tgggctttca caactggcac ccgatgccga tcaccagcaa gggtaacacc 1320 ctggttggtc agggcgaact gggtggcgtg tgggagtgga ccagcagcgt tctgcgtaaaa 1380 tgggagggct tcgaaccgat ggagctgtac ccgggttata ccgcggattt ctttgacgag 1440 aagcacaaca ttgttctggg tggcagctgg gcgacccacc cgcgtattgc gggtcgtaag 1500 agcttcgtga actggtatca gcgtaactac ccgtatgcgt gggttggtgc gcgtgtggtt 1560 cgtgacctgt aa 1572 <210> 3 <211> 523 <212> PRT <213> Artificial Sequence <220> <223> TNcEgt1 <400> 3 Master Gly Master Sister Phe Sister Leu Ile Pro Sister Val Tyr Ala Arg Sir Ala 1 5 10 15 Leu Pro Thr Leu Asp Asp Trp Glu Ala Leu Trp Ala Thr Trp Asp Val 20 25 30 Val Thr Arg Gln Met Leu Pro Gln Glu Glu Leu Leu Glu Lys Pro Ile 35 40 45 Lys Leu Arg Asn Ala Cys Ile Phe Tyr Leu Gly His Ile Pro Thr Phe 50 55 60 Leu Asp Ile Gln Leu Thr Lys Thr Thr Lys Gln Ala Pro Ser Glu Pro 65 70 75 80 Ala His Phe Cys Lys Ile Phe Glu Arg Gly Ile Asp Pro Asp Val Asp 85 90 95 Asn Pro Glu Leu Cys His Ala His Ser Glu Ile Pro Asp Glu Trp Pro 100 105 110 Pro Val Glu Glu Ile Leu Thr Tyr Gln Glu Thr Val Arg Ser Arg Leu 115 120 125 Arg Gly Leu Tyr Ala His Gly Ile Ala Asn Ile Pro Arg Asn Val Gly 130 135 140 Arg Ala Ile Trp Val Gly Phe Glu His Glu Leu Met His Ile Glu Thr 145 150 155 160 Leu Leu Tyr Met Met Leu Gln Ser Asp Lys Thr Leu Ile Pro Thr His 165 170 175 Ile Pro Arg Pro Asp Phe Asp Lys Leu Ala Arg Lys Ala Glu Ser Glu 180 185 190 Arg Val Pro Asn Gln Trp Phe Lys Ile Pro Ala Gln Glu Ile Thr Ile 195 200 205 Gly Leu Asp Asp Pro Glu Asp Gly Ser Asp Ile Asn Lys His Tyr Gly 210 215 220 Trp Asp Asn Glu Lys Pro Pro Arg Arg Val Gln Val Ala Ala Phe Gln 225 230 235 240 Ala Gln Gly Arg Pro Ile Thr Asn Glu Glu Tyr Ala Gln Tyr Leu Leu 245 250 255 Glu Lys Asn Ile Asp Lys Leu Pro Ala Ser Trp Ala Arg Leu Asp Asn 260 265 270 Glu Asn Ile Ser Asn Gly Thr Thr Asn Ser Val Ser Gly His His Ser 275 280 285 Asn Arg Thr Ser Lys Gln Gln Leu Pro Ser Ser Phe Leu Glu Lys Thr 290 295 300 Ala Val Arg Thr Val Tyr Gly Leu Val Pro Leu Lys His Ala Leu Asp 305 310 315 320 Trp Pro Val Phe Ala Ser Tyr Asp Glu Leu Ala Gly Cys Ala Ala Tyr 325 330 335 Met Gly Gly Arg Ile Pro Thr Phe Glu Glu Thr Arg Ser Ile Tyr Ala 340 345 350 Tyr Ala Asp Ala Leu Lys Lys Lys Lys Glu Ala Glu Arg Gln Leu Gly 355 360 365 Arg Thr Val Pro Ala Val Asn Ala His Leu Thr Asn Asn Gly Val Glu 370 375 380 Ile Thr Pro Pro Ser Ser Pro Ser Ser Glu Thr Pro Ala Glu Ser Ser 385 390 395 400 Ser Pro Ser Asp Ser Asn Thr Thr Leu Ile Thr Thr Glu Asp Leu Phe 405 410 415 Ser Asp Leu Asp Gly Ala Asn Val Gly Phe His Asn Trp His Pro Met 420 425 430 Pro Ile Thr Ser Lys Gly Asn Thr Leu Val Gly Gln Gly Glu Leu Gly 435 440 445 Gly Val Trp Glu Trp Thr Ser Ser Val Leu Arg Lys Trp Glu Gly Phe 450 455 460 Glu Pro Met Glu Leu Tyr Pro Gly Tyr Thr Ala Asp Phe Phe Asp Glu 465 470 475 480 Lys His Asn Ile Val Leu Gly Gly Ser Trp Ala Thr His Pro Arg Ile 485 490 495 Ala Gly Arg Lys Ser Phe Val Asn Trp Tyr Gln Arg Asn Tyr Pro Tyr 500 505 510 Ala Trp Val Gly Ala Arg Val Val Arg Asp Leu 515 520 <210> 4 <211> 321 <212> PRT <213> Artificial Sequence <220> <223> EgtD amino acid sequence <400> 4 Met Thr Leu Ser Leu Ala Asn Tyr Leu Ala Ala Asp Ser Ala Ala Glu 1 5 10 15 Ala Leu Arg Arg Asp Val Arg Ala Gly Leu Thr Ala Ala Pro Lys Ser 20 25 30 Leu Pro Pro Lys Trp Phe Tyr Asp Ala Val Gly Ser Asp Leu Phe Asp 35 40 45 Gln Ile Thr Arg Leu Pro Glu Tyr Tyr Pro Thr Arg Thr Glu Ala Gln 50 55 60 Ile Leu Arg Thr Arg Ser Ala Glu Ile Ile Ala Ala Ala Gly Ala Asp 65 70 75 80 Thr Leu Val Glu Leu Gly Ser Gly Thr Ser Glu Lys Thr Arg Met Leu 85 90 95 Leu Asp Ala Met Arg Asp Ala Glu Leu Leu Arg Arg Phe Ile Pro Phe 100 105 110 Asp Val Asp Ala Gly Val Leu Arg Ser Ala Gly Ala Ala Ile Gly Ala 115 120 125 Glu Tyr Pro Gly Ile Glu Ile Asp Ala Val Cys Gly Asp Phe Glu Glu 130 135 140 His Leu Gly Lys Ile Pro His Val Gly Arg Arg Leu Val Val Phe Leu 145 150 155 160 Gly Ser Thr Ile Gly Asn Leu Thr Pro Ala Pro Arg Ala Glu Phe Leu 165 170 175 Ser Thr Leu Ala Asp Thr Leu Gln Pro Gly Asp Ser Leu Leu Leu Gly 180 185 190 Thr Asp Leu Val Lys Asp Thr Gly Arg Leu Val Arg Ala Tyr Asp Asp 195 200 205 Ala Ala Gly Val Thr Ala Ala Phe Asn Arg Asn Val Leu Ala Val Val 210 215 220 Asn Arg Glu Leu Ser Ala Asp Phe Asp Leu Asp Ala Phe Glu His Val 225 230 235 240 Ala Lys Trp Asn Ser Asp Glu Glu Arg Ile Glu Met Trp Leu Arg Ala 245 250 255 Arg Thr Ala Gln His Val Arg Val Ala Ala Leu Asp Leu Glu Val Asp 260 265 270 Phe Ala Ala Gly Glu Glu Met Leu Thr Glu Val Ser Cys Lys Phe Arg 275 280 285 Pro Glu Asn Val Val Ala Glu Leu Ala Glu Ala Gly Leu Arg Gln Thr 290 295 300 His Trp Trp Thr Asp Pro Ala Gly Asp Phe Gly Leu Ser Leu Ala Val 305 310 315 320 Arg <210> 5 <211> 966 <212> DNA <213> Artificial Sequence <220> <223> EgtD nucleotide sequence <400> 5 atgaccctga gcctggcgaa ctacctggcg gcggatagcg cggcggaggc gctgcgtcgt 60 gatgtgcgtg cgggtctgac cgcggcgccg aagagcctgc cgccgaaatg gttctatgat 120 gcggttggca gcgacctgtt cgaccagatc acccgtctgc cggagtacta tccgacccgt 180 accgaagcgc aaattctgcg tacccgtagc gcggagatca ttgcggcggc gggtgcggac 240 accctggtgg agctgggtag cggcaccagc gaaaagaccc gtatgctgct ggatgcgatg 300 cgtgacgcgg aactgctgcg tcgtttcatc ccgtttgacg tggatgcggg tgttctgcgt 360 agcgcgggtg cggcgattgg tgcggagtac ccgggtatcg aaattgatgc ggtgtgcggc 420 gacttcgagg aacacctggg caagatcccg cacgttggcc gtcgtctggt ggttttcctg 480 ggtagcacca ttggtaacct gaccccggcg ccgcgtgcgg aatttctgag caccctggcg 540 gataccctgc aaccgggtga cagcctgctg ctgggcaccg atctggtgaa agacaccggt 600 cgtctggttc gtgcgtatga cgatgcggcg ggcgtgaccg cggcgtttaa ccgtaacgtt 660 ctggcggtgg ttaaccgtga gctgagcgcg gacttcgatc tggacgcgtt tgaacacgtg 720 gcgaagtgga acagcgacga ggaacgtatc gagatgtggc tgcgtgcgcg taccgcgcag 780 catgtgcgtg ttgcggcgct ggatctggaa gtggacttcg cggcgggcga ggaaatgctg 840 accgaggttt cttgcaaatt tcgtccggaa aacgttgttg cggagctggc ggaagcgggt 900 ctgcgtcaaa cccactggtg gaccgatccg gcgggtgact ttggtctgag cctggcggtt 960 cgttaa 966 <210> 6 <211> 372 <212> PRT <213> Artificial Sequence <220> <223> EgtE amino acid sequence <400> 6 Met Val Met Leu Ala Gln Gln Trp Arg Asp Ala Arg Pro Lys Val Ala 1 5 10 15 Gly Leu His Leu Asp Ser Gly Ala Cys Ser Arg Gln Ser Phe Ala Val 20 25 30 Ile Asp Ala Thr Thr Ala His Ala Arg His Glu Ala Glu Val Gly Gly 35 40 45 Tyr Val Ala Ala Glu Ala Ala Thr Pro Ala Leu Asp Ala Gly Arg Ala 50 55 60 Ala Val Ala Ser Leu Ile Gly Phe Ala Ala Ser Asp Val Val Tyr Thr 65 70 75 80 Ser Gly Ser Asn His Ala Ile Asp Leu Leu Leu Ser Ser Trp Pro Gly 85 90 95 Lys Arg Thr Leu Ala Cys Leu Pro Gly Glu Tyr Gly Pro Asn Leu Ser 100 105 110 Ala Met Ala Ala Asn Gly Phe Gln Val Arg Ala Leu Pro Val Asp Asp 115 120 125 Asp Gly Arg Val Leu Val Asp Glu Ala Ser His Glu Leu Ser Ala His 130 135 140 Pro Val Ala Leu Val His Leu Thr Ala Leu Ala Ser His Arg Gly Ile 145 150 155 160 Ala Gln Pro Ala Ala Glu Leu Val Glu Ala Cys His Asn Ala Gly Ile 165 170 175 Pro Val Val Ile Asp Ala Ala Gln Ala Leu Gly His Leu Asp Cys Asn 180 185 190 Val Gly Ala Asp Ala Val Tyr Ser Ser Ser Arg Lys Trp Leu Ala Gly 195 200 205 Pro Arg Gly Val Gly Val Leu Ala Val Arg Pro Glu Leu Ala Glu Arg 210 215 220 Leo Gln Pro Arg Ile Pro Pro Ser Asp Trp Pro Ile Pro Met Ser Val 225 230 235 240 Leu Glu Lys Leu Glu Leu Gly Glu His Asn Ala Ala Ala Arg Val Gly 245 250 255 Phe Ser Val Ala Val Gly Glu His Leu Ala Ala Gly Pro Thr Ala Val 260 265 270 Arg Glu Arg Leu Ala Glu Val Gly Arg Leu Ser Arg Gln Val Leu Ala 275 280 285 Glu Val Asp Gly Trp Arg Val Val Glu Pro Val Asp Gln Pro Thr Ala 290 295 300 Ile Thr Thr Leu Glu Ser Thr Asp Gly Ala Asp Pro Ala Ser Val Arg 305 310 315 320 Ser Trp Leu Ile Ala Glu Arg Gly Ile Val Thr Thr Ala Cys Glu Leu 325 330 335 Ala Arg Ala Pro Phe Glu Met Arg Thr Pro Val Leu Arg Ile Ser Pro 340 345 350 His Val Asp Val Thr Val Asp Glu Leu Glu Gln Phe Ala Ala Ala Leu 355 360 365 Arg Glu Ala Pro 370 <210> 7 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> EgtE nucleotide sequence <400> 7 atggttatgc tggcgcagca atggcgtgat gcgcgtccga aagtggcggg tctgcatctg 60 gacagcggtg cgtgcagccg tcagagcttc gcggttatcg atgcgaccac cgcgcatgcg 120 cgtcatgagg cggaagttgg tggctacgtg gcggcggaag cggcgacccc ggcgctggat 180 gcgggtcgtg cggcggtggc gagcctgatc ggttttgcgg cgagcgatgt ggtttacacc 240 agcggcagca accacgcgat tgacctgctg ctgagcagct ggccgggtaa acgtaccctg 300 gcgtgcctgc cgggcgagta tggtccgaac ctgagcgcga tggcggcgaa cggcttccaa 360 gttcgtgcgc tgccggtgga cgatgacggt cgtgtgctgg ttgatgaagc gagccatgag 420 ctgagcgcgc acccggttgc gctggtgcac ctgaccgcgc tggcgagcca tcgtggtatt 480 gcgcaaccgg cggcggagct ggttgaagcg tgccacaacg cgggtatccc ggtggttatt 540 gatgcggcgc aagcgctggg tcacctggat tgcaacgttg gtgcggacgc ggtgtacagc 600 agcagccgta aatggctggc gggtccgcgt ggtgtgggcg ttctggcggt tcgtccggag 660 ctggcggaac gtctgcaacc gcgtatcccg ccgagcgatt ggccgattcc gatgagcgtt 720 ctggagaaac tggaactggg cgagcacaac gcggcggcgc gtgtgggttt tagcgtggcg 780 gttggtgaac acctggcggc gggtccgacc gcggttcgtg aacgtctggc ggaagtgggc 840 cgtctgagcc gtcaggttct ggcggaagtg gatggttggc gtgtggttga gccggttgac 900 caaccgaccg cgatcaccac cctggaaagc accgatggtg cggacccggc gagcgttcgt 960 agctggctga tcgcggagcg tggtattgtt accaccgcgt gcgaactggc gcgtgcgccg 1020 tttgagatgc gtaccccggt gctgcgtatt agcccgcacg tggatgttac cgtggacgag 1080 ctggaacagt tcgcggcggc gctgcgtgag gcgccgtaa 1119

Claims

1. A sulfoxide synthase, characterized in that, The amino acid sequence of the sulfoxide synthase is shown in SEQ ID NO: 3; or, The amino acid sequence of the sulfoxide synthase differs from that of SEQ ID NO: 3 by containing amino acid residues selected from any of the following groups: G-1R; D107A; Q66R; L153Q; S324N; L445I; A201S; P469I; P469T; G-1S and D107A; Q66H and S324C; D107Y and V311M; A79T and S297G; L170P and Q195H; K83D and S215N; D259V and V440I; T27S and A74V; Q238H and Q293R; K69R and T368S, P509S; G276S, G330C and A395E; E86G, A316S, and L317R; Q34R, K72I, N270S, and F424Y; S324N and L445I; D107A and P469T; Q66R and A201S; L445I and P469I; K83D, D107A and S215N; Q66R, D107Y, and V311M; G-1S, D107A, and P469T; G-1R, T27S and A74V; Q66H, E86G, A316S, and L317R; G276S, G330C, A395E, and P469T; Q66H, A79T, S297G, and S324C; L170P, Q195H, D259V and V440I; K69R, D259V, T368S, V440I and P509S; L153Q, L170P, Q195H, Q238H and Q293R; T27S, Q34R, K72I, A74V, N270S, and F424Y; and, G-1S, L153Q, L170P, Q195H, Q238H and Q293R; Specifically, the positions of the first two amino acids M and G in SEQ ID NO: 3 are defined as -2 and -1, respectively, and the position of the third amino acid M is defined as 1.

2. A fusion protein, characterized in that, The fusion protein includes the sulfoxide synthase as described in claim 1, as well as histidine methyltransferase and CS bond cleavage enzyme.

3. The fusion protein as described in claim 2, characterized in that, The amino acid sequence of the histidine methyltransferase is shown in SEQ ID NO: 4, or the amino acid sequence of the CS bond cleavage enzyme is shown in SEQ ID NO: 6; and / or, The fusion protein comprises, from N-terminus to C-terminus, the sulfoxide synthase, the histidine methyltransferase, and the CS bond cleavage enzyme.

4. An isolated nucleic acid, characterized in that, The nucleic acid encodes the sulfoxide synthase as described in claim 1 or the fusion protein as described in claim 2 or 3.

5. The nucleic acid as described in claim 4, characterized in that, The nucleotide sequence of the nucleic acid is shown in SEQ ID NO:

2.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 4 or 5.

7. The recombinant expression vector as described in claim 6, characterized in that, The backbone plasmid of the recombinant expression vector is pETDuet-1, and / or the nucleotide sequence encoding the histidine methyltransferase is shown in SEQ ID NO: 5 or the nucleic acid sequence encoding the CS bond lyase is shown in SEQ ID NO:

7.

8. A transformant, characterized in that, It comprises the nucleic acid as described in claim 4 or 5 or the recombinant expression vector as described in claim 6 or 7; the transformant is a non-animal variety or a non-plant variety.

9. The transformant as described in claim 8, characterized in that, The host cell for the transformed organism was *Escherichia coli* (Escherichia coli). Escherichia coli ).

10. The transformant as described in claim 9, characterized in that, The host cell of the transformed organism is E. coli BL21 (DE3).

11. A method for preparing the sulfoxide synthase as described in claim 1, characterized in that, The method includes culturing the transformant as described in any one of claims 8-10 to obtain the sulfoxide synthase.

12. A method for preparing compound II, characterized in that, The method comprises the following steps: preparing compound II using the sulfoxide synthase as described in claim 1 or the transformant as described in any one of claims 8-10; the structural formula of compound II is as follows: 。 13. The method as described in claim 12, characterized in that, The method satisfies any one of the following sets: (1) The transformant was cultured in a fermentation medium with shaking, and then isopropyl thiogalactoside was added to induce the expression of compound II; (2) In cysteine, Fe 2+ In the presence of oxygen and the sulfoxide synthase, it reacts with histidine trimethyl inner salt to obtain compound II.

14. The method as described in claim 13, characterized in that, The fermentation medium contains Fe 2+ And cysteine.

15. The method as described in claim 13, characterized in that, In the reaction: The sulfoxide synthase is present in the supernatant after cell lysis; And / or, the mass-to-volume ratio of the histidine trimethylammonium salt to the supernatant is 3-15 mg / ml; And / or, the cysteine, Fe 2+ The molar ratio of the histidine trimethyl inner salt is (1-3):1:(1-2); And / or, the reaction is carried out at a rotation speed of 200-250 rpm and a temperature of 20℃-30℃; And / or, the reaction is carried out under conditions of pH 6-8.

16. A method for preparing ergothioneine, comprising the step of preparing compound II as described in any one of claims 12-15, further comprising the step of preparing compound II as ergothioneine.

17. The use of the sulfoxide synthase as described in claim 1 or the transformant as described in any one of claims 8-10 in the preparation of ergothionein or an intermediate thereof.

18. The application as described in claim 17, characterized in that, The intermediate is compound II, and the structural formula of compound II is as follows: 。

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