A fusion protein and its application in preparing L-cysteine

Abstract

The present application relates to the field of biotechnology, in particular to a kind of fusion protein and its application in preparation L-cysteine.The present application provides a kind of fusion protein, the fusion protein contains the alpha subunit of tryptophan synthetase TrpS, the beta subunit of tryptophan synthetase TrpS, has tryptophan synthetase catalytic activity, the present application is through genetic engineering method, under the premise of retaining the space conformation of the alpha subunit of tryptophan synthetase TrpS, beta subunit respectively, TrpB and TrpA are connected as single fusion protein TrpSL ( TrpB- Linker-TrpA ) by Linker, avoids dimer dissociation, and the fusion enzyme after immobilization only needs to be simply cleaned after reaction and can be reused, significantly improves the efficiency of industrial production, and reduces economic cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a fusion protein and application thereof in preparation of L-cysteine. BACKGROUND

[0002] L-cysteine and L-cystine, as important sulfur-containing amino acids, are widely demanded in the pharmaceutical, food and feed industries. The traditional chemical hydrolysis method has the problems of serious pollution and low yield, while the enzyme catalytic synthesis technology in the biological method has become a research hotspot due to its mild conditions and strong specificity. Tryptophan synthase (TrpS) has become a key tool enzyme due to its ability to catalyze the synthesis of L-cysteine from L-serine and a sulfur donor (such as sodium hydrosulfide).

[0003] The heterodimeric enzyme (TrpA and TrpB) of tryptophan synthase TrpS needs to act synergistically in the catalytic reaction, but the dimer in the free state has poor stability, and TrpA or TrpB is easily detached due to physical shearing force in the washing step after resin immobilization, making it difficult to realize multiple rounds of reaction.

[0004] Therefore, it is urgent to develop a tryptophan synthase TrpS variant that can complete multiple rounds of reaction after resin immobilization. SUMMARY

[0005] The present application aims to at least solve one of the technical problems in the related art. To this end, one object of the present application is to provide a fusion protein containing an alpha subunit of tryptophan synthase TrpS, a beta subunit of tryptophan synthase TrpS, and having tryptophan synthase catalytic activity. The present application connects TrpB and TrpA into a single fusion protein TrpSL (TrpB-Linker-TrpA) by genetic engineering method under the premise of retaining the spatial conformation of each of the alpha subunit and the beta subunit of tryptophan synthase TrpS, avoiding dissociation of the dimer, and enabling the fusion enzyme after immobilization to be reused after only a simple washing after reaction, thereby significantly improving the efficiency of industrial production and reducing economic costs. TrpB-Linker-TrpA

[0006] To this end, the present application provides a fusion protein in a first aspect. According to an embodiment of the present application, the fusion protein includes an alpha subunit of tryptophan synthase TrpS, a beta subunit of tryptophan synthase TrpS, and a connecting peptide.

[0007] The amino acid sequence of the connecting peptide is shown in SEQ ID NO: 20-22.

[0008] SEQ ID NO: 20

[0009] ​AEAAAKEAAAKAAVLEYLTAEILELAAAVLEYLTAEILELAGGAAVLEYLTAEILELAAAVLEYLTAEILELA

[0010] SEQ ID NO: 21

[0011] ASAKMLHEMQRKNEQGGGGAAVLEYLTAEILELAAAVLEYLTAEILELAGGGASAKMLHEMQRKNEQ

[0012] SEQ ID NO: 22

[0013] ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK

[0014] The present application discloses a fusion protein, an isolated nucleic acid, an expression vector and a method for producing tryptophan. Enterobacteriaceae The present application discloses a fusion protein, an isolated nucleic acid, an expression vector and a method for producing tryptophan.

[0015] According to an embodiment of the present application, the C-terminal of the beta subunit of the tryptophan synthase TrpS is connected to the alpha subunit of the tryptophan synthase TrpS through the connecting peptide.

[0016] According to an embodiment of the present application, the amino acid sequence of the alpha subunit of the tryptophan synthase TrpS is shown as SEQ ID NO: 1.

[0017] The amino acid sequence of the beta subunit of the tryptophan synthase TrpS is shown as SEQ ID NO: 2.

[0018] The present application discloses a fusion protein, an isolated nucleic acid, an expression vector and a method for producing tryptophan.

[0019] The present application discloses a fusion protein, an isolated nucleic acid, an expression vector and a method for producing tryptophan.

[0020] The fourth aspect of the present application provides a host cell. According to embodiments of the present application, the host cell comprises the expression vector of the third aspect.

[0021] The fifth aspect of the present application provides use of the fusion protein of the first aspect, the isolated nucleic acid of the second aspect, the expression vector of the third aspect, or the host cell of the fourth aspect in the preparation of L-cysteine or L-cystine.

[0022] The sixth aspect of the present application provides a method for immobilizing tryptophan synthase. According to embodiments of the present application, the method comprises:

[0023] (1) ion exchange equilibration of a resin to obtain an equilibrated resin;

[0024] (2) contacting the equilibrated resin with the fusion protein of the first aspect, and / or a fusion protein expressed by at least one of the isolated nucleic acid of the second aspect, the expression vector of the third aspect, or the host cell of the fourth aspect, so as to immobilize the fusion protein on the resin.

[0025] According to embodiments of the present application, in step (2), when the amino acid sequence of the linker peptide in the fusion protein is as shown in SEQ ID NO: 20, the resin is ES-103B resin.

[0026] According to embodiments of the present application, in step (2), when the amino acid sequence of the linker peptide in the fusion protein is as shown in SEQ ID NO: 21, the resin is LX-109S resin.

[0027] According to embodiments of the present application, in step (2), when the amino acid sequence of the linker peptide in the fusion protein is as shown in SEQ ID NO: 22, the resin is ES-103B resin.

[0028] The seventh aspect of the present application provides a tryptophan synthase immobilized resin column. According to embodiments of the present application, the tryptophan synthase immobilized resin column comprises:

[0029] a resin column and a fusion protein immobilized thereon,

[0030] the fusion protein is selected from the fusion protein of the first aspect, and / or a fusion protein expressed by at least one of the isolated nucleic acid of the second aspect, the expression vector of the third aspect, or the host cell of the fourth aspect.

[0031] According to embodiments of the present application, when the amino acid sequence of the linker peptide in the fusion protein is as shown in SEQ ID NO: 20, the resin is ES-103B resin.

[0032] According to an embodiment of the present application, when the amino acid sequence of the linker peptide in the fusion protein is as shown in SEQ ID NO: 21, the resin is LX-109S resin.

[0033] According to an embodiment of the present application, when the amino acid sequence of the linker peptide in the fusion protein is as shown in SEQ ID NO: 22, the resin is ES-103B resin.

[0034] The eighth aspect of the present application provides a method for preparing L-cysteine. According to an embodiment of the present application, the method comprises:

[0035] The immobilized enzyme obtained by the method for immobilizing tryptophan synthase of the sixth aspect and / or the tryptophan synthase immobilized resin column of the seventh aspect catalyzes the synthesis of L-cysteine by taking L-serine and a sulfur donor as substrates.

[0036] The ninth aspect of the present application provides a method for preparing L-cystine. According to an embodiment of the present application, the method comprises:

[0037] S1: The immobilized enzyme obtained by the method for immobilizing tryptophan synthase of the sixth aspect and / or the tryptophan synthase immobilized resin column of the seventh aspect catalyzes the synthesis of L-cysteine by taking L-serine and a sulfur donor as substrates;

[0038] S2: Taking the L-cysteine synthesized in step S1 as a substrate, an oxidation reaction is performed so as to synthesize L-cystine.

[0039] The present application successfully constructs a tryptophan synthase fusion based on Linker connection, which can be used for the synthesis of L-cysteine, and ingeniously solves the problem that TrpB and TrpA in tryptophan synthase cannot complete multiple rounds of reactions after resin immobilization, which opens up ideas for the immobilization reaction of double-enzyme or even multi-enzyme complexes, and significantly improves the efficiency and economy of industrial production.

[0040] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter in the description. BRIEF DESCRIPTION OF DRAWINGS

[0041] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the accompanying drawings, in which:

[0042] Figure 1 A plasmid map showing that TrpA is constructed into an E. coli double expression vector pETDuet-1 with two promoters is shown;

[0043] Figure 2 A plasmid map showing the construction of TrpB into E. coli dual expression vector pETDuet-1 with two promoters is shown;

[0044] Figure 3 A protein three-dimensional spatial structure obtained by protein modeling of the sequence of TrpS through RoseTTA Fold2 new artificial intelligence software tool online software is shown;

[0045] Figure 4 SDS-PAGE gel electrophoresis results of supernatant and precipitate obtained after culture of mutant LK1-LK10 genetically engineered bacteria are shown. DETAILED DESCRIPTION

[0046] Embodiments of the present application are described in detail below. The embodiments described below are examples only and are not intended to limit the present application, which is defined by the claims.

[0047] It should be noted that the terms "first", "second" are used only for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0048] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The ranges or values should be construed to be approximations that allow for significant variation. Within the ranges or values, endpoints are provided as a separate point for each endpoint, and each separate point can be independently combined with other endpoints or points to form a new range or value. These new ranges or values are also specifically disclosed herein.

[0049] In order to facilitate the understanding of the present application, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this application belongs.

[0050] In this document, the terms "comprising" or "including" are open-ended, that is, they mean including, but not limited to, the recited items.

[0051] In this document, the terms "optionally", "optional" or "optional" generally mean that the event or circumstance subsequently described can or can not occur, and the description includes situations in which the event or circumstance occurs, as well as situations in which it does not occur.

[0052] According to a specific embodiment of the present application, the present application provides a fusion protein comprising an α subunit of tryptophan synthetase TrpS, a β subunit of tryptophan synthetase TrpS and a connecting peptide,

[0053] The amino acid sequence of the connecting peptide is shown in SEQ ID NO: 20-22.

[0054] It should be noted that in the fusion protein provided by the present application, the α subunit of tryptophan synthetase TrpS and the β subunit of tryptophan synthetase TrpS are connected by the connecting peptide shown in SEQ ID NO: 20-22, and the connection mode is not particularly limited, for example, the N- or C-terminus of the α subunit of tryptophan synthetase TrpS can be connected to the C- or N-terminus of the β subunit of tryptophan synthetase TrpS by the connecting peptide, or the N- or C-terminus of the β subunit of tryptophan synthetase TrpS can be connected to the C- or N-terminus of the α subunit of tryptophan synthetase TrpS by the connecting peptide. According to a preferred embodiment of the present application, the C-terminus of the β subunit of tryptophan synthetase TrpS is connected to the α subunit of tryptophan synthetase TrpS by the connecting peptide.

[0055] It should be noted that the amino acid sequences of the α subunit and the β subunit of tryptophan synthetase TrpS are not particularly limited, including but not limited to tryptophan synthetase TrpS derived from Enterobacteriaceae , which consists of an α subunit and a β subunit, wherein the amino acid sequence of the α subunit TrpA (GenBank: WP_000443067.1) is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the β subunit TrpB (GenBank: WP_000209520.1) is shown in SEQ ID NO: 2 in the sequence listing. Alternatively, the amino acid sequences of the α subunit and the β subunit of tryptophan synthetase TrpS can also be derivatives of tryptophan synthetase TrpS derived from Enterobacteriaceae , which have 80% or more or 90% or more homology with the amino acid sequence of tryptophan synthetase TrpS, and these derivatives of tryptophan synthetase maintain the catalytic ability and enzyme activity of tryptophan synthetase itself, only the amino acid sequence is slightly different from the amino acid sequence of the wild-type tryptophan synthetase TrpS. The above-mentioned tryptophan synthetase TrpS derived from Enterobacteriaceae and its derivatives are all included in the scope of the wild-type tryptophan synthetase described in the present application.

[0056] The present application provides a fusion protein comprising an α subunit of tryptophan synthetase TrpS, a β subunit of tryptophan synthetase TrpS and a connecting peptide, EnterobacteriaceaeThe tryptophan synthetase TrpS (amino acid sequences of the alpha subunit TrpA and the beta subunit TrpB are shown as SEQ ID NO: 1 and 2, respectively) is connected by a specific linker, which avoids the dissociation of the dimer under the premise of ensuring the enzyme activity. The immobilized fusion enzyme can be reused for multiple rounds after simple cleaning, which significantly improves the efficiency of industrial production and reduces the economic cost.

[0057] According to a specific embodiment of the present application, the amino acid sequence of the alpha subunit of the tryptophan synthetase TrpS is shown as SEQ ID NO: 1.

[0058] The amino acid sequence of the beta subunit of the tryptophan synthetase TrpS is shown as SEQ ID NO: 2.

[0059] According to a specific embodiment of the present application, the present application provides a nucleic acid sequence encoding the fusion protein.

[0060] According to a specific embodiment of the present application, the present application provides a recombinant vector containing the nucleic acid sequence encoding the fusion protein, and a genetically engineered bacterium containing the nucleic acid sequence encoding the fusion protein. Specifically, the vector can be any of various expression vectors, including but not limited to any of pET expression vector, pCW expression vector, pUC expression vector, or pPIC9k expression vector. The host cell of the genetically engineered bacterium can be any suitable host cell, including but not limited to Escherichia coli, Bacillus subtilis, Streptomyces, or Pichia pastoris.

[0061] According to a specific embodiment of the present application, the present application also relates to the use of the fusion protein, the isolated nucleic acid, the expression vector, and the host cell in the preparation of L-cysteine or L-cystine.

[0062] According to a specific embodiment of the present application, the present application provides a method for immobilizing tryptophan synthetase, comprising:

[0063] (1) ion exchange equilibrium of the resin to obtain the equilibrium resin;

[0064] (2) contacting the equilibrium resin with the fusion protein described above, and / or the fusion protein expressed by at least one of the isolated nucleic acid, the expression vector, and the host cell described above, so as to immobilize the fusion protein on the resin.

[0065] The cleaning solution used in the ion exchange equilibrium process is not particularly limited, for example, it can be PBK solution, PBS solution, etc. All types of cleaning solutions available in the art that can perform ion exchange equilibrium are covered within the scope of the present application.

[0066] According to a specific embodiment of the present application, in step (2), when the amino acid sequence of the linker peptide in the fusion protein is as shown in SEQ ID NO: 20, the resin is ES-103B resin.

[0067] According to a specific embodiment of the present application, in step (2), when the amino acid sequence of the linker peptide in the fusion protein is as shown in SEQ ID NO: 21, the resin is LX-109S resin.

[0068] According to a specific embodiment of the present application, in step (2), when the amino acid sequence of the linker peptide in the fusion protein is as shown in SEQ ID NO: 22, the resin is ES-103B resin.

[0069] According to a specific embodiment of the present application, the present application provides a tryptophan synthetase immobilized resin column, comprising:

[0070] a resin column and a fusion protein immobilized thereon,

[0071] the fusion protein is selected from the fusion proteins described above, and / or expressed by at least one of the nucleic acids described above, the expression vectors described above, the host cells described above.

[0072] According to a specific embodiment of the present application, when the amino acid sequence of the linker peptide in the fusion protein is as shown in SEQ ID NO: 20, the resin is ES-103B resin; when the amino acid sequence of the linker peptide in the fusion protein is as shown in SEQ ID NO: 21, the resin is LX-109S resin; and when the amino acid sequence of the linker peptide in the fusion protein is as shown in SEQ ID NO: 22, the resin is ES-103B resin.

[0073] According to a specific embodiment of the present application, the present application provides a method for preparing L-cysteine, comprising:

[0074] the immobilized enzyme obtained by the method for immobilizing tryptophan synthetase described above and / or the tryptophan synthetase immobilized resin column described above, catalyzing the synthesis of L-cysteine by using L-serine and a sulfur donor (such as sodium sulfide) as substrates.

[0075] It should be noted that there is no particular restriction on the conditions for preparing L-cysteine using tryptophan synthetase, and the catalytic conditions commonly used in the art for enzyme catalysis can be used. However, regardless of whether optimized conditions or conventional reaction conditions are used, the fusion protein of the present application can perform multiple rounds of catalytic reactions compared to the wild-type tryptophan synthetase that has not been modified, thereby ingeniously solving the problem that TrpB and TrpA in tryptophan synthetase cannot complete multiple rounds of reactions after resin immobilization, and opening up ideas for the immobilization reaction of double-enzyme or even multi-enzyme complexes, thereby significantly improving the efficiency and economy of industrial production.

[0076] According to a specific embodiment of the present application, the present application provides a method for preparing L-cystine, comprising:

[0077] S1: obtaining an immobilized enzyme by the method for immobilizing tryptophan synthetase described above and / or using the tryptophan synthetase immobilized resin column described above, and catalyzing the synthesis of L-cysteine using L-serine and a sulfur donor (such as sodium sulfide) as substrates;

[0078] S2: using the L-cysteine synthesized in step S1 as a substrate, and performing an oxidation reaction to synthesize L-cystine.

[0079] It should be noted that there is no particular restriction on the conditions for preparing L-cysteine using tryptophan synthetase, and the catalytic conditions commonly used in the art for enzyme catalysis can be used. However, regardless of whether optimized conditions or conventional reaction conditions are used, the fusion protein of the present application can perform multiple rounds of catalytic reactions compared to the wild-type tryptophan synthetase that has not been modified, thereby ingeniously solving the problem that TrpB and TrpA in tryptophan synthetase cannot complete multiple rounds of reactions after resin immobilization, and opening up ideas for the immobilization reaction of double-enzyme or even multi-enzyme complexes.

[0080] The solutions of the present disclosure will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. If a specific technique or condition is not specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained from the market.

[0081] Materials and methods

[0082] 1) Materials: Plasmid pET28a was purchased from Changsha Aibeiwei Biotechnology Co., Ltd.; Restriction endonucleases, high-fidelity enzyme premix, one-step rapid cloning kit were purchased from Shanghai Yixing Biological Technology Co., Ltd.; Synthetic primers, E. coli BL21 (DE3) competent cells, DNA marker, plasmid extraction kit, DNA gel recovery and purification kit, kanamycin sulfate, ampicillin, isopropyl-β-d-thiogalactopyranoside, L-cysteine, L-cystine standard were purchased from Shanghai Shenguo Biological Engineering Co., Ltd.; HisSep Ni-NTA Agarose Resin (His-tag protein agarose purification resin) was purchased from Shanghai Yixing Biological Technology Co., Ltd., centrifugal ultrafiltration tube (10KDa) was purchased from Millipore company, LX-107S and other immobilized enzyme carrier resins were purchased from Xi'an Lanxiao New Material Technology Co., Ltd.; Chemical reagents were analytical pure from National Pharmaceutical Group. The plasmid extraction operation steps refer to the plasmid extraction kit instructions; the DNA gel recovery operation steps refer to the DNA gel recovery and purification kit instructions; the DNA fragment ligation operation steps refer to the one-step rapid cloning kit instructions;

[0083] 2) LB medium (g / L): 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of sodium chloride, sterilized at 121℃ for 20 min;

[0084] 3) Tryptophan synthase TrpS substrate reaction solution: 200 mM L-serine, 240 mM sodium hydrosulfide, 0.1 mM pyridoxal phosphate (PLP), pH 8.2;

[0085] 4) Enzyme purification and immobilization buffer:

[0086] Binding Buffer: 50 mM PBK buffer (pH 7.8);

[0087] Wash Buffer: 50 mM PBK buffer (pH 7.8), 30 mM imidazole.

[0088] Elution Buffer: 50 mM PBK buffer (pH 7.8), 300 mM imidazole.

[0089] 5) Enzyme activity determination: accurately take the supernatant of ultrasonic broken bacteria liquid, add the substrate reaction solution preheated to 35℃, so that the final concentration of bacteria is 20 mg / mL, then stir in the water bath at 35℃ for 30 min, then add equal volume of 20% trichloroacetic acid solution to terminate the reaction, shake well and centrifuge, take the supernatant for HPLC analysis, and use L-cysteine and L-cystine standards as controls;

[0090] 6) HPLC analysis method: Thermo Fisher Scientific ODS-2 Hypersil™ (4.6 × 250 mm, 5 µm); flow rate 1.0 mL / min; detection wavelength 210 nm; TrpS enzyme reaction mobile phase: methanol: 0.05 mol / L potassium dihydrogen phosphate + 0.005 mol / L sodium decane sulfonate (pH 2.30) = 23:77;

[0091] 7) Definition of enzyme activity unit: Under the conditions of pH 8.2 and temperature 35℃, the amount of enzyme required to produce 1 micromole (μmol) of L-cysteine ​​per unit time is defined as 1 U.

[0092] Example 1: Construction of wild-type tryptophan synthase TrpS genetically engineered bacteria

[0093] (1) Wild type trpS Gene synthesis

[0094] Based on publicly available information from the NCBI database, the following sources were selected. Enterobacteriaceae The tryptophan synthase TrpS is composed of an α subunit and a β subunit. The amino acid sequence of the α subunit TrpA (GenBank: WP_000443067.1) is shown in SEQ ID NO. 1 of the sequence listing, and the amino acid sequence of the β subunit TrpB (GenBank: WP_000209520.1) is shown in SEQ ID NO. 2 of the sequence listing. (To facilitate subsequent purification, 6 x His tags were added to the C-terminus of TrpA and TrpB, respectively.) Based on the codon bias of E. coli, the gene coding sequence was codon optimized. BamHI and HindIII restriction sites were designed and added to both ends of the coding sequence. The sequences were then sent to a biotechnology company for artificial synthesis, yielding TrpA (SEQ ID NO: 3) and TrpB (SEQ ID NO: 4), respectively.

[0095] The amino acid sequence of the α subunit TrpA of tryptophan synthase is shown in SEQ ID NO: 1:

[0096] MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIIDTLIEAGADALELGIPFSDPLAD GPTIQNATLRAFAAGVTPAQCFEMLALIRQKHPTIPIGLLMYANLVFNKGIDEFYAQCEK VGVDSVLVADVPVEESAPFRQAALRHNVAPIFICPPNADDDLLRQIASYGRGYTYLLS RAGVTGAENRAALPLNHLVAKLKEYNAAPPLQGFGISAPDQVKAAIDAGAAGAISGSAIV KIIEQHINEPEKMLAALKVFVQPMKAATRSHHHHHH

[0097] Amino acid sequence of the tryptophan synthetase beta subunit TrpB, as set forth in SEQ ID NO: 2:

[0098] MTTLLNPYFGEFGGMYVPQILMPALRQLEEAFVSAQKDPEFQAQFNDLLKNYAGRPTAL TKCQNITAGTNTTLYLKREDLLHGGAHKTNQVLGQALLAKRMGKTEIIAETGAGQHGVAS ALASALLGLKCRIYMGAKDVERQSPNVFRMRLMGAEVIPVHSGSATLKDACNEALRDWS GSYETAHYMLGTAAGPHPYPTIVREFQRMIGEETKAQILEREGRLPDAVIACVGGGSNA IGMFADFINETNVGLIGVEPGGHGIETGEHGAPLKHGRVGIYFGMKAPMMQTEDGQIEES TISISGLDFPSVGPQHAYLNSTGRADYVSITDDEALEAFKTLCLHEGIIPALESSHALAH ALKMMRENPDKEQLLVVNLSGRGDKDIFTVHDILKARGEIHHHHHH

[0099] Amino acid sequence of the tryptophan synthetase beta subunit TrpB, as set forth in SEQ ID NO: 2:

[0100] ggatccatggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcgtcacgctcggtgatccgggcattgagcagtcattgaaaattatcgatacgctaattgaagccggtgctgacgcgctggagttaggtatccccttctccgacccactggcggatggcccgacgattcaaaacgccactctgcgcgcctttgcggcaggtgtgactccggcacaatgttttgaaatgctggcactgattcgccagaaacacccgaccattcccattggcctgttgatgtatgccaatctggtgtttaacaaaggcattgatgagttttatgcccagtgcgaaaaagtcggcgtcgattcggtgctggttgccgatgtgccagttgaagagtccgcgcccttccgccaggccgcgttgcgtcataatgtcgcacctatcttcatctgcccgccaaatgccgatgacgacctgctgcgccagatagcctcttacggtcgtggttacacctatttgctgtcacgagcaggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcgaagctgaaagagtacaacgctgcacctccattgcagggatttggtatttccgccccggatcaggtaaaagcagcgattgatgcaggagctgcgggcgcgatttctggttcggccattgttaaaatcatcgagcaacatattaatgagccagagaaaatgctggcggcactgaaagtttttgtacaaccgatgaaagcggcgacgcgcagttaa aagctt

[0101] The sequence of the codon-optimized TrpB gene coding sequence is shown in SEQ ID NO: 4:

[0102] ggatccaagctt

[0103] (2) Construction of wild-type TrpS-pETDuet-1 vector

[0104] TrpA was constructed into the multiple cloning site 1 of the E. coli dual expression vector pETDuet-1 with two promoters (the plasmid map is shown as Figure 1 Primer for vector construction was designed, and the linearized vector was amplified by PCR with 1-F (5'-ggcagcagccatcaccatcatcaccac-3', as shown in SEQ ID NO: 5) / 1-R (5'- ggtatatctccttcttaaagttaaacaaaatta -3', as shown in SEQ ID NO: 6) as primers, and the purchased vector pETDuet-1 as a template, 2xHieff Canace® Plus PCR Master Mix (With Dye) high-fidelity enzyme premix; the 5' and 3' ends of the gene DNA fragment (SEQ ID NO: 3) were amplified by PCR with 2-F (5'- ttaagaaggagatataccatggaacgctacgaatct-3', as shown in SEQ ID NO: 7) / 2-R (5'- atggtgatggctgctgccttagtggtggtggtggtgatg -3', as shown in SEQ ID NO: 8) as primers, and the artificially synthesized gene DNA fragment (SEQ ID NO: 3) as a template, high-fidelity enzyme premix PCR to amplify the 5' and 3' ends of the gene DNA fragment, respectively, with the same sequence as the two ends of the linearized vector TrpA TrpA The amplified linearized vector (5.4 kb) and the DNA fragment of the gene (0.8 kb) were recovered by 1% agarose gel electrophoresis detection and DNA gel recovery purification kit. The linearized vector and the gene fragment recovered by gel were subjected to homologous recombination by using Hieff Clone® Plus One Step Cloning Kit one-step cloning kit. The recombination system was: pETDuet-1 linearized vector 2.5 μL, the gene fragment 2.5 μL, 2xHieff Clone® Enzyme Premix 5 μL. Reaction at 50°C for 30 min. Subsequently, the plasmid vector TrpA-pETDuet-1 of the wild-type gene was obtained by (3). TrpA TrpA The amplified linearized vector (5.4 kb) and the DNA fragment of the gene (0.8 kb) were recovered by 1% agarose gel electrophoresis detection and DNA gel recovery purification kit. The linearized vector and the gene fragment recovered by gel were subjected to homologous recombination by using Hieff Clone® Plus One Step Cloning Kit one-step cloning kit. The recombination system was: pETDuet-1 linearized vector 2.5 μL, the gene fragment 2.5 μL, 2xHieff Clone® Enzyme Premix 5 μL. Reaction at 50°C for 30 min. Subsequently, the plasmid vector TrpA-pETDuet-1 of the wild-type gene was obtained by (3). TrpA TrpA The amplified linearized vector (5.4 kb) and the DNA fragment of the gene (0.8 kb) were recovered by 1% agarose gel electrophoresis detection and DNA gel recovery purification kit. The linearized vector and the gene fragment recovered by gel were subjected to homologous recombination by using Hieff Clone® Plus One Step Cloning Kit one-step cloning kit. The recombination system was: pETDuet-1 linearized vector 2.5 μL, the gene fragment 2.5 μL, 2xHieff Clone® Enzyme Premix 5 μL. Reaction at 50°C for 30 min. Subsequently, the plasmid vector TrpA-pETDuet-1 of the wild-type gene was obtained by (3).

[0105] Similarly, TrpB was constructed into the multiple cloning site 2 of the E. coli dual expression vector pETDuet-1 with two promoters (the plasmid map is shown as Figure 2 ​​​linearized vector; 4-F (5'-gaaggagatatacatatgacaacattacttaacccctat-3', as shown in SEQ ID NO: 11) / 4-R (5'-tatccaattgagatctgctcagtggtggtggtggtg-3', as shown in SEQ ID NO: 12) as primers, and the artificially synthesized gene DNA fragment (SEQ ID NO: 4) as a template, the high-fidelity enzyme premix PCR was used to amplify the 5' and 3' ends of the gene DNA fragment, respectively, with the 5' and 3' ends of the linearized vector being completely consistent with the sequences of the two ends of the linearized vector. TrpB linearized vector (6.2 kb) and the gene (1.2 kb) DNA fragment were recovered by 1% agarose gel electrophoresis detection and a DNA gel recovery and purification kit. TrpB The linearized vector and the gene fragment were subjected to homologous recombination by using a Hieff Clone® Plus OneStep Cloning Kit one-step rapid cloning kit. The recombination system was: TrpA-pETDuet-1 linearized vector 2.5 μL, the gene fragment 2.5 μL, and 2×HieffClone® Enzyme Premix 5 μL. The reaction was performed at 50°C for 30 min. TrpB TrpB TrpB

[0106] (3) Construction of a wild-type TrpS genetically engineered bacterium

[0107] The homologous recombination product obtained in (2) was transformed into E. coli BL21 (DE3) competent cells by heat shock method, and the transformation product was plated on an LB plate containing 50 μg / mL ampicillin. After overnight culture at 37°C, positive transformants were selected and sent to a sequencing company for sequencing. The transformant with correct sequencing contained a plasmid vector TrpS-pETDuet-1 containing a wild-type TrpS gene and a wild-type TrpA gene, and the transformant was also a wild-type TrpS genetically engineered bacterium. TrpA TrpB

[0108] ​​​​​Example 2: Preparation and expression of tryptophan synthetase TrpSLX genetically engineered bacteria

[0109] (1) LinkerX gene synthesis

[0110] In order to connect the C-terminal of TrpB with the N-terminal of TrpA with Linker, protein modeling was performed on the sequence of TrpS by RoseTTA Fold2 new artificial intelligence software tool online software (http: / / robetta.bakerlab.org / ), and the three-dimensional spatial structure of the protein was obtained as shown in Figure 3 PyMol was used to visualize and process the model. According to literature research and protein design, 10 kinds of Linker were selected, and the gene coding sequence was codon optimized according to the codon bias of E. coli. After designing to increase the BamHI and HindIII enzyme cutting sites at both ends of the coding sequence, it was sent to a biological company for artificial synthesis, and L1-L10 were obtained as shown in Table 1.

[0111] Table 1:

[0112]

[0113] (2) Construction of fusion protein TrpSLX-pET28a vector

[0114] The primer sequences used to design the fusion protein are shown in Table 2:

[0115] Table 2:

[0116]

[0117] The plasmid TrpB-pET28a artificially synthesized from a biological company was used as a template to obtain the recombinant plasmid TrpS-X-pETDuet-1 carrying the mutant of tryptophan synthetase. The linearized vector large fragment (B) carrying TrpB was amplified by PCR using 5-F (5'-gcggcgacgcgcagttaaaagcttgcggccgcactc-3', as SEQ ID NO: 53) 5-R (5'-gatttcccctcgtgcttt-3', as SEQ ID NO: 54) primers and 2xHieff Canace® Plus PCR Master Mix (With Dye) high-fidelity enzyme premix. The small fragment gene (A) carrying TrpA was amplified by PCR using 6-F (5'-atggaacgctacgaatct-3', as SEQ ID NO: 55) / 6-R (5'-gcggcgacgcgcagttaa-3', as SEQ ID NO: 56) primers and TrpA-pET28a plasmid as a template, and 2xHieff Canace® Plus PCR Master Mix (With Dye) high-fidelity enzyme premix. The small fragment gene 1 carrying L1 was amplified by PCR using L1-F / L2-R primers and L1-pET28a plasmid as a template, and 2xHieff Canace® Plus PCR Master Mix (With Dye) high-fidelity enzyme premix. The 5' end of the small fragment gene 1 carried a sequence identical to the 3' end sequence of the large fragment, and the 3' end carried a sequence identical to the 5' end sequence of the small fragment gene 2. The 3' end of the small fragment gene 2 carried a sequence identical to the 5' end sequence of the large fragment. The amplified DNA of the large and small fragments was detected by 1% agarose gel electrophoresis and purified using a DNA gel recovery purification kit. The DNA of the large and small fragments was subjected to homologous recombination using Hieff Clone® Plus One Step Cloning Kit one-step cloning kit. The recombination system was: 1 μL of DNA of the large fragment (B), 2 μL of DNA of the small fragment (A), 2 μL of DNA of the small fragment 1, and 5 μL of 2xHieff Clone® Enzyme Premix. The reaction was performed at 50°C for 30 min.

[0118] The obtained homologous recombination product was transformed into E. coli BL21 (DE3) competent cells by heat shock method, the transformation product was plated on LB plate containing 50 μg / mL kanamycin sulfate, and after overnight culture at 37°C, positive transformants were selected and sent to Shanghai SunGene Biotech Co., Ltd. for sequencing. The transformant with correct sequencing, i.e., the plasmid vector TrpSL1-pET28a containing the TrpB-L1-TrpA gene, was the TrpB-L1-TrpA genetic engineering bacteria, numbered as LK1. The same way was used to construct the engineering bacteria LK2 with TrpB-L2-TrpA, LK3 with TrpB-L3-TrpA, LK4 with TrpB-L4-TrpA, LK5 with TrpB-L5-TrpA, LK6 with TrpB-L6-TrpA, LK7 with TrpB-L7-TrpA, LK8 with TrpB-L8-TrpA, LK9 with TrpB-L9-TrpA, and LK10 with TrpB-L10-TrpA.

[0119] The mutant LK1-LK10 genetic engineering bacteria with correct sequencing were inoculated into test tubes containing 4 mL LB medium containing 50 μg / mL kanamycin sulfate, and after overnight culture at 37°C and 200 rpm, they were transferred into 50 mL LB medium, and cultured at 37°C and 200 rpm until the OD 600 was 0.6-0.8. IPTG was added to a final concentration of 0.2 mM, and the culture was further incubated at 20°C and 200 rpm for 18 h. The fermentation broth was centrifuged at 9000 rpm for 5 min, and the supernatant was removed. The bacterial pellet was resuspended in water to a concentration of 100 mg / mL, and ultrasonically broken for 10 min. The pellet was removed by centrifugation, and the supernatant was subjected to SDS-PAGE gel electrophoresis analysis. The results are shown in Figure 4 , indicating that these proteins were normally expressed.

[0120] Example 3: Determination of enzyme activity of fusion protein TrpSLX

[0121] 100 μL of enzyme solution was added to a centrifuge tube containing 900 μL of substrate solution (containing 200 mM L-serine, 240 mM sodium hydrosulfide, and 0.1 mM pyridoxal phosphate (PLP) in water), and placed at 35°C for 30 min of reaction. After the reaction was completed, an equal volume of 20% trichloroacetic acid solution was added to terminate the reaction, and after shaking and centrifugation, the supernatant was subjected to HPLC analysis, with cysteine and cystine standards as controls.

[0122] The results of the determination of relative enzyme activity of each single mutant are shown in Table 3:

[0123] Table 3:

[0124]

[0125] From the results of Table 3, it can be seen that the three Linkers L8-L10 connecting TrpB and TrpA into fusion proteins have no significant loss of enzyme activity compared with the wild-type enzyme activity, and the fusion protein LK9 containing L9 retains 99.6% of the wild-type enzyme activity.

[0126] Example 4: Purification of TrpSLX fusion proteins

[0127] The fusion proteins LK8-10 with no significant loss of enzyme activity compared with the wild-type enzyme activity were selected, and the wild-type TrpS and LK8-10 fusion proteins TrpSL8-TrpSL10 were fused with 6xHis tags to purify the high-purity proteins using HisSep Ni-NTA Agarose Resin, and then the imidazole was removed by centrifugal ultrafiltration tube (10KDa) to obtain the immobilized proteins.

[0128] The specific steps of enzyme purification are as follows:

[0129] (1) Packing of HisSep Ni-NTA Agarose Resin gravity column:

[0130] Take 50 mL of gravity chromatography column, install the lower gasket, add appropriate amount of pure water to rinse the column and gasket, and close the lower outlet. Mix the HisSep Ni-NTA Agarose Resin evenly, use the gun head to suck the appropriate slurry, and add it to the gravity column (the actual volume of the filler accounts for half of the suspension), open the lower outlet to dry the protective solution. Add appropriate amount of pure water to rinse the filler, and close the lower outlet after the liquid in the column tube flows dry by gravity.

[0131] (2) Equilibrium: equilibrate the gravity column with 5 times the column volume (CV) of Binding Buffer at a flow rate of 1-2 mL / min, repeat 2-3 times;

[0132] (3) Loading: load the supernatant crude enzyme solution obtained after induction expression and ultrasonic disruption of the fusion protein according to the method in Example 2 into the column bed slowly, and combine at 4°C for 1-2 hours;

[0133] (4) Washing: wash away the non-specifically bound impurities with 10-15 CV of Wash Buffer;

[0134] (5) Elution: elute the target protein in sections with 5-10 CV of Elution Buffer, collect 1 CV per section, and the flow rate is 1 mL / min. Use ultraviolet spectrophotometer to detect the ultraviolet absorption (A280) of each stage until the elution is complete.

[0135] (6) Column regeneration: wash the column with 5 CV of 0.5 M NaOH (to remove residual protein) and 5 CV of 20% ethanol, and store at 4°C to prevent microbial contamination.

[0136] (7) Concentration and buffer exchange: add the collected eluate to a concentration tube and centrifuge at 4°C to concentrate to 1 / 10 of the original volume. Add Binding Buffer to the original volume to the concentration tube, and centrifuge again to concentrate to the target volume. Repeat the dilution and concentration steps 3 times to obtain pure enzyme, and detect the enzyme concentration.

[0137] Example 5: Screening of immobilization resins for wild-type TrpS protein

[0138] After purifying the wild-type TrpS protein according to the method in Example 4, 9 different enzyme immobilization carrier resins were selected for immobilization to screen the best resin for immobilization.

[0139] The specific steps for enzyme immobilization are as follows:

[0140] (1) Resin equilibration: weigh 10 g of resin into a beaker, wash the resin with 3 CV of 2 M PBK solution and filter, repeat 2-4 times;

[0141] (2) Immobilization: transfer the pure enzyme solution obtained in Example 4 to the beaker containing the resin, and gently stir at room temperature for 20 h;

[0142] (3) Filtration and washing: filter the resin and collect the filtrate. Wash the resin with 4 CV of Binding Buffer and filter, repeat 2-4 times. Collect the washing solution and detect the concentration of unimmobilized enzyme in the filtrate and washing solution to evaluate the immobilization efficiency of the resin;

[0143] The immobilization efficiency of 9 enzyme immobilization carrier resins for wild-type TrpS pure enzyme is shown in Table 4. According to Table 4, among the 9 enzyme immobilization carrier resins, LX-107S has the highest immobilization efficiency for wild-type TrpS pure enzyme, which is 76.4%, and the amount of immobilized enzyme per gram of resin is 30.54 mg.

[0144] Table 4:

[0145]

[0146] Example 6: Screening of immobilization resins for TrpSL8 fusion protein

[0147] Purify the TrpSL8 fusion protein according to the methods in Examples 4 and 5, and screen the best immobilization resin for immobilization.

[0148] The enzyme immobilization carrier resin efficiency of TrpSL8 fusion protein pure enzyme is shown in Table 5. According to Table 5, among the 9 enzyme immobilization carrier resins, ES-103B has the highest immobilization efficiency of 72.5% for the fusion protein TrpSL8 pure enzyme, and the amount of immobilized enzyme per gram of resin is 28.98 mg.

[0149] Table 5:

[0150]

[0151] Example 7: Immobilization resin screening of TrpSL9 fusion protein

[0152] The TrpSL9 fusion protein was purified according to the methods in Examples 4 and 5, and the best immobilization resin for immobilization was screened.

[0153] The enzyme immobilization carrier resin efficiency of TrpSL9 fusion protein pure enzyme is shown in Table 6. According to Table 6, among the 9 enzyme immobilization carrier resins, LX-109S has the highest immobilization efficiency of 69.2% for the fusion protein TrpSL9 pure enzyme, and the amount of immobilized enzyme per gram of resin is 27.69 mg.

[0154] Table 6:

[0155]

[0156] Example 8: Immobilization resin screening of TrpSL10 fusion protein

[0157] The TrpSL10 fusion protein was purified according to the methods in Examples 4 and 5, and the best immobilization resin for immobilization was screened.

[0158] The enzyme immobilization carrier resin efficiency of TrpSL10 fusion protein pure enzyme is shown in Table 7. According to Table 7, among the 9 enzyme immobilization carrier resins, ES-103B has the highest immobilization efficiency of 73.9% for the fusion protein TrpSL10 pure enzyme, and the amount of immobilized enzyme per gram of resin is 29.57 mg.

[0159] Table 7:

[0160]

[0161] Example 9: TrpSL8-10 fusion protein round reaction

[0162] The reaction solution 100 mL (containing 380 mM L-serine, 438 mM sodium hydrosulfide, 0.1 mM pyridoxal phosphate (PLP)) was prepared according to the embodiments 5-8, and the resins with the highest immobilization efficiency for wild-type TrpS and the fusion protein TrpSL8-10 were selected. The corresponding resin was weighed according to the amount of 200 mg of the immobilized pure enzyme and added to the reaction solution, which was placed at 35°C for 6 h. During the reaction, the pH was adjusted to 8.2-8.6 with HCl. The reaction progress was monitored by HPLC, and L-serine, L-cysteine and cystine were used as standard samples to calculate the cysteine conversion rate at the end of the reaction.

[0163] After the reaction was completed, the resin was filtered, washed with 3 CV of Binding Buffer, and then put into a new reaction solution for repeated reaction-washing until the reaction rate was significantly reduced. The cysteine conversion rate at the end of each reaction round and the reaction round were recorded (see Table 8).

[0164] As can be seen from the results in Table 8, in the first reaction round, both the WT group and the LK8-10 group completed the conversion of serine in 6 h. However, after washing with the washing solution, the second reaction round showed the effect of the linker on the immobilization of TrpS. After the physical shearing during the washing process, the weak interaction between TrpB and TrpA of the wild-type TrpS was opened, resulting in the removal of TrpB or TrpA not crosslinked to the resin, which could not form a complete dimer structure, so that the reaction rate of the WT group was greatly reduced. The TrpB and TrpA were connected by the linker, which on the one hand enhanced the interaction between TrpB and TrpA to make the dimer structure more compact, and on the other hand, TrpB and TrpA were connected by the linker to form a fusion protein, so that any part of TrpB and TrpA crosslinked to the resin could not be washed away, so that the reaction rate of the LK8-10 group was still maintained in the second reaction round. After the third reaction round, the wild-type TrpS could not generate cysteine, and in the LK8-10 group, LK8 and LK10 maintained the ninth and eighth rounds, respectively, but their cysteine conversion rates were only 5.9% and 0.9% at 6 h, respectively, and then no cysteine was generated in the subsequent rounds. LK9 performed the best, and after 10 rounds of reaction, it still achieved a cysteine conversion rate of 87.4-92.4% at 6 h.

[0165] The above results show that the present application successfully constructs a tryptophan synthetase fusion based on linker connection, which can be used for the synthesis of L-cysteine, and ingeniously solves the problem that TrpB and TrpA in tryptophan synthetase cannot complete multiple rounds of reaction after resin immobilization, which opens up ideas for the immobilization reaction of double-enzyme or even multi-enzyme complexes, and significantly improves the efficiency and economy of industrial production.

[0166] Table 8:

[0167]

[0168] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", "some implementations" or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0169] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A fusion protein, characterized in that, the fusion protein comprises an alpha subunit of tryptophan synthetase TrpS, a beta subunit of tryptophan synthetase TrpS and a linker peptide, an amino acid sequence of the linker peptide is shown as SEQ ID NO: 20-22, a C-terminal of the beta subunit of tryptophan synthetase TrpS is connected to the alpha subunit of tryptophan synthetase TrpS through the linker peptide, an amino acid sequence of the alpha subunit of tryptophan synthetase TrpS is encoded by a nucleic acid sequence shown as SEQ ID NO: 3; an amino acid sequence of the beta subunit of tryptophan synthetase TrpS is encoded by a nucleic acid sequence shown as SEQ ID NO:

4.

2. An isolated nucleic acid, comprising, the nucleic acid encodes the fusion protein of claim 1.

3. An expression vector, characterized by, the expression vector comprises the isolated nucleic acid of claim 2.

4. A host cell, characterized in that, the host cell comprises the expression vector of claim 3.

5. Use of the fusion protein of claim 1, the isolated nucleic acid of claim 2, the expression vector of claim 3, the host cell of claim 4 in the preparation of L-cysteine or L-cystine.

6. A method of immobilizing tryptophan synthetase, characterized by, comprising: (1) ion exchange equilibration of a resin to obtain an equilibrated resin; (2) contacting the equilibrated resin with the fusion protein of claim 1, and / or a fusion protein expressed by at least one of the isolated nucleic acid of claim 2, the expression vector of claim 3, the host cell of claim 4, so as to immobilize the fusion protein on the resin.

7. The method of claim 6, wherein, In step (2), when the amino acid sequence of the linker peptide in the fusion protein is shown as SEQ ID NO: 20, the resin is ES-103B resin; or, when the amino acid sequence of the linker peptide in the fusion protein is shown as SEQ ID NO: 21, the resin is LX-109S resin; or, when the amino acid sequence of the linker peptide in the fusion protein is shown as SEQ ID NO: 22, the resin is ES-103B resin.

8. A tryptophan synthetase immobilized resin column, characterized in that, comprising: a resin column and a fusion protein immobilized thereon, the fusion protein is selected from the fusion protein of claim 1, and / or a fusion protein expressed by at least one of the isolated nucleic acid of claim 2, the expression vector of claim 3, the host cell of claim 4.

9. The tryptophan synthetase immobilized resin column of claim 8, wherein, when the amino acid sequence of the linker peptide in the fusion protein is shown as SEQ ID NO: 20, the resin is ES-103B resin; or, when the amino acid sequence of the linker peptide in the fusion protein is shown as SEQ ID NO: 21, the resin is LX-109S resin; or, when the amino acid sequence of the linker peptide in the fusion protein is shown as SEQ ID NO: 22, the resin is ES-103B resin.

10. A method of preparing L-cysteine, characterized by, comprising: the immobilized enzyme obtained by the method for immobilizing tryptophan synthetase of claim 6 or 7, and / or the tryptophan synthetase immobilized resin column of claim 8 or 9, catalyzing the synthesis of L-cysteine using L-serine and a sulfur donor as substrates.

11. A method for preparing L-cystine, characterized by, comprising: S1: obtaining the immobilized enzyme by the method for immobilizing tryptophan synthetase according to claim 6 or 7 and / or using the tryptophan synthetase immobilized resin column according to claim 8 or 9, to catalyze the synthesis of L-cysteine with L-serine and a sulfur donor as substrates; S2: taking the L-cysteine synthesized in step S1 as a substrate, and through an oxidation reaction, so as to synthesize L-cystine.

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