CN119351371A - Method for synthesizing S-sulfo-L-cysteine by multi-enzyme cascade catalysis - Google Patents

Abstract

The present invention relates to the field of biotechnology, and in particular to a method for synthesizing S-sulfo-L-cysteine by multi-enzyme cascade catalysis. The present invention provides a new route for the biosynthesis of S-sulfo-L-cysteine, and test results show that using serine, AMP, sodium hexametaphosphate and sodium thiosulfate as substrates, using a crude enzyme solution containing serine kinase, polyphosphate-AMP phosphotransferase and phosphoserine-dependent S-sulfo-L-cysteine synthetase in a cell background with tryptophanase and glutaredoxin knocked out, the steps for synthesizing S-sulfo-L-cysteine are simple, the product yield is high, the cost is low, and it has strong industrial application prospects.

Description

Method for synthesizing S-sulfo-L-cysteine by multienzyme cascade catalysis

Technical Field

The invention relates to the technical field of biology, in particular to a method for synthesizing S-sulfo-L-cysteine by multienzyme cascade catalysis.

Background

S-sulfo-L-cysteine (SSC) is a novel modified amino acid which is pushed out by merck, can replace cysteine which is necessary for a cell culture medium, and can increase the level of glutathione in cells, thereby improving the yield of recombinant protein (CN 107847549B). In bacteria and plants, SSC is synthesized in two steps by catalytic synthesis of serine, acetyl-CoA and thiosulfate as substrates, via serine acetyltransferase (SAT, EC 2.3.1.30) and acetylserine-dependent S-sulfo-L-cysteine synthetase (CysM or CS26, EC 2.5.1.144) （Olsen et al. 2004, Kumar et al. 2011, Yi et al. 2013, Dharavath et al. 2022, Nakamura et al. 1983, Nakamura, Iwahashi, and Eguchi 1984, Claus et al. 2005, Chattopadhyay et al. 2007, Bermúdez et al. 2010）.

Biosynthesis is a currently hot synthetic method, but the above biosynthesis route is difficult to be industrially applied because acetyl-CoA is only sold with a high-cost milligram-grade reagent. Therefore, the development of a biosynthesis method with low material cost, high conversion efficiency and good product purity is particularly necessary.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for synthesizing S-sulfo-L-cysteine by multi-enzyme cascade catalysis.

The present invention provides a combination enzyme comprising phosphoserine-dependent S-sulfo-L-cysteine synthetase SSC03 (S256P. Mu.CT), serine kinase PkSerK from Pyrococcus furiosus and polyphosphate-AMP phosphotransferase PAP from Pseudomonas syringae tomato pathogenic variety or Pseudomonas aeruginosa.

Furthermore, in the combination enzyme of the present invention,

The amino acid sequence of the phosphoserine dependent S-sulfo-L-cysteine synthetase SSC03 (S256P fatlutea CT) is shown as SEQ ID NO. 5;

the amino acid sequence of serine kinase PkSerK from the flamingo is shown as SEQ ID NO. 1;

The amino acid sequence of the polyphosphoric acid-AMP phosphotransferase PsPAP from the pathogenic variety of the pseudomonas syringae tomato is shown as SEQ ID NO. 3;

The amino acid sequence of the polyphosphoric-acid-AMP phosphotransferase PaPAP derived from Pseudomonas aeruginosa is shown in SEQ ID NO. 9.

The invention provides a biological material, which comprises at least one of the following components A) to C):

a) Nucleic acid encoding a combination enzyme according to the invention;

b) A recombinant vector comprising the nucleic acid of A);

c) Transfecting or transforming a host cell with the recombinant vector of B).

Further, in the biomaterial of the present invention,

The nucleotide sequence of the nucleic acid for encoding the phosphoserine-dependent S-sulfo-L-cysteine synthetase SSC03 (S256P fatlutea CT) is shown as SEQ ID NO. 6;

the nucleotide sequence of the nucleic acid for encoding the serine kinase PkSerK from the streptococcus ragmitis is shown as SEQ ID NO. 2;

the nucleotide sequence of the nucleic acid for encoding the polyphosphoric-acid-AMP phosphotransferase PsPAP from the pathogenic variety of Pseudomonas syringae tomato is shown as SEQ ID NO. 4;

The nucleotide sequence of the nucleic acid encoding the Pseudomonas aeruginosa-derived polyphosphate-AMP phosphotransferase PsPAP is shown in SEQ ID NO. 10.

The nucleic acid according to the invention may be DNA, RNA, cDNA or PNA. In an embodiment of the invention, the nucleic acid is in the form of DNA. The DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. Nucleic acids may include nucleotide sequences having different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acids may be topologically linear or circular. The nucleic acid may be part of a vector (e.g., an expression or cloning vector), or a fragment. The nucleic acids may be obtained directly from natural sources or may be prepared by recombinant, enzymatic or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

In the present invention, the codons of the nucleic acids are optimized, including, but not limited to, codon usage bias, elimination of secondary structures that are detrimental to expression (e.g., hairpin structures), changes in GC content, cpG dinucleotide content, secondary structures of mRNA, cryptic splice sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA instability regions, repeat sequences (direct repeats, inverted repeats, etc.), and restriction sites that may affect cloning.

The present invention provides recombinant vectors comprising the nucleic acids of the present invention.

The recombinant vector of the present invention, referred to as a recombinant nucleic acid vector, is a recombinant DNA molecule comprising the desired coding sequence and appropriate nucleic acid sequences or elements necessary for expression of the operably linked coding gene in a particular host organism. Nucleic acid sequences or elements necessary for expression in model animal or mammalian cells include promoters, ribosome binding sites and possibly other sequences. Prokaryotic cells are known to utilize promoters, enhancers and terminators. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes interchangeable, as the plasmid is the most commonly used form of vector at present. However, the present invention is intended to include such other forms of expression vectors that perform equivalent functions, which are or will become known in the art, including but not limited to plasmids, phage particles, viral vectors and/or just potential genomic inserts. In particular embodiments, nucleic acids encoding fusion proteins provided herein can be constructed in a variety of prokaryotic expression vectors. For example, the backbone vector may be a pET series vector, specifically pET28a and/or petdeet.

Furthermore, in the invention, the combined enzyme can be integrated into the recombinant vector in a single or combined mode, the skeleton of the recombinant vector can be pET28a or pETDuet, and in the specific embodiment of the invention, the recombinant vector comprises the following forms:

pET28a-PsPAP;

pET28a-SSC03(S256P ∆CT);

pET28a-PkSerK;

pETDuet- PsPAP-SSC03(S256P ∆CT);

The present invention provides host cells of which sources include plants, animals, bacteria, fungi, phages or viruses, as the invention is not limited in this regard. In the specific embodiment of the invention, the bacteria are derived from, specifically, escherichia coli, and more specifically, escherichia coli BL21 DE3 or escherichia coli modified by genetic background.

The escherichia coli modified by the genetic background is a genetically engineered bacterium, and the genetically engineered bacterium has functions of tryptophan enzyme EcTnaA and glutaredoxin EcNrdH (BSSC-d 02) which are deleted, and is transfected or transformed into a recombinant vector in the biological material.

In the specific embodiment of the invention, the recombinant vector can be transformed or transfected into BL21 DE3 or into BSSC-d02 to obtain the genetically engineered bacterium, and the specific strain form comprises:

pET28a-PsPAP(BL21 DE3);

pET28a-SSC03(S256P ∆CT)(BL21 DE3);

pET28a-PkSerK(BL21 DE3);

pET28a-PsPAP-SSC03(S256P ∆CT)(BSSC-d02);

pET28a-PkSerK(BSSC-d02);

The invention provides an application of at least one of the following I) to III) in S-sulfo-L-cysteine synthesis:

i) The combination enzyme of the invention;

II) the biomaterial according to the invention;

III), the genetically engineered bacterium disclosed by the invention;

IV), a host cell in the biological material of the present invention or a culture obtained by the genetically engineered bacterium of the present invention.

The present invention provides for the first time a method for biosynthesis of S-sulfo-L-cysteine, and the specific strain forms mentioned above can be used in the synthesis of S-sulfo-L-cysteine, and the invention is not limited thereto. The invention is not limited to serine, ADP, AMP and sodium hexametaphosphate, and experimental results show that the introduction of an ADP regeneration system (taking serine, AMP and sodium hexametaphosphate as substrates) reduces the ADP/AMP consumption, and simultaneously replaces expensive ADP with relatively cheap AMP, wherein the unit price of the AMP is about one tenth of that of the ADP, thereby greatly reducing the production cost.

The invention provides a preparation method of S-sulfo-L-cysteine, which comprises the step of synthesizing the S-sulfo-L-cysteine by utilizing at least one of the following steps of i) to iii):

i) The combination enzyme of the invention;

ii) the biomaterial according to the invention;

iii) The genetically engineered bacterium disclosed by the invention;

iv) culturing host cells in the biological material of the invention or a culture obtained from the genetically engineered bacterium of the invention.

Further, the preparation method comprises the steps of catalyzing a substrate to generate S-sulfo-L-cysteine by serine kinase PkSerK, polyphosphate-AMP phosphotransferase PsPAP and phosphoserine dependent S-sulfo-L-cysteine synthetase SSC03 (S256P (CT)) in the presence of magnesium ions;

the substrate includes serine, AMP, sodium hexametaphosphate, and sodium thiosulfate.

Further, the thiosulfate is at least one selected from sodium thiosulfate, potassium thiosulfate and/or ammonium thiosulfate; in a specific embodiment of the present invention, the thiosulfate is sodium thiosulfate or ammonium thiosulfate;

In the preparation method of the invention, the adding sequence of the substances participating in the reaction is not limited, the substances participating in the ghost can be added in two steps or in one step, and in the specific embodiment of the invention, the specific steps when two steps are adopted are as follows:

step 1, catalyzing a substrate 1 to generate OPS by serine kinase and polyphosphate-AMP phosphotransferase in the presence of magnesium ions;

Step 2, phosphoserine-dependent S-sulfo-L-cysteine synthetase converts OPS into SSC using sodium thiosulfate as a substrate.

Further, the method comprises the steps of,

The catalytic conditions were pH 7.0 and temperature 38 ℃.

The concentration of serine is 100 mM-200 mM, and is particularly 200mM optimal;

The concentration of the salt containing the AMP is 1 mM-10 mM, and particularly 5mM is optimal;

the concentration of the sodium hexametaphosphate is 25 mM-65 mM, and particularly 65mM is optimal;

The concentration of the thiosulfate is 100-300 mM, specifically 200-260 mM;

the concentration of magnesium ions is 20 mM-60 mM, and is particularly 40mM optimal;

The addition amount of the bacterial cells containing serine kinase PkSerK is 1g/L to 5g/L;

The addition amount of the thallus containing the polyphosphoric acid-AMP phosphotransferase PsPAP is 10 g/L-50 g/L;

The addition amount of the bacterial cells containing phosphoserine dependent S-sulfo-L-cysteine synthetase SSC03 (S256P fatin CT) is 10g/L to 50g/L;

Specifically, as in example 5 of the present invention, pkSerK was added in an amount of 2.5g/L, and the amount of cells containing the polyphosphate-AMP phosphotransferase and the phosphoserine-dependent S-sulfo-L-cysteine synthetase SSC03 (S256P. Sub.CT) was added in an amount of 25g/L or 30g/L;

In the present invention, the bacterial cells may contain both the polyphosphoric-AMP phosphotransferase PsPAP and the phosphoserine-dependent S-sulfo-L-cysteine synthetase SSC03 (S256P. Mu.CT) or may contain both the above-mentioned enzymes alone, in a specific embodiment of the present invention, the host cell containing both the above-mentioned enzymes alone is BL21 DE3, and the host cell containing both the above-mentioned enzymes is BSSC-d02 having a functional deficiency of tryptophan enzyme EcTnaA and glutaredoxin EcNrdH, and in the preparation method used in the specific embodiment of the present invention, the bacterial cells containing the enzymes involved in the reaction include at least one of the following two combinations:

combination a, pET28a-PsPAP (BL 21 DE 3), pET28a-SSC03 (S256 P+CT) (BL 21 DE 3), pET28a-PkSerK (BL 21 DE 3);

Combination b, pET28a-PsPAP-SSC03 (S256 father CT) (BSSC-d 02), pET28a-PkSerK (BSSC-d 02);

the test results show that the addition of the participating reaction substances in one step in combination b is simpler in steps, higher in product yield and quicker in reaction compared with the addition of the participating reaction substances in two steps in combination a.

Further, even though pET28a-PsPAP-SSC03 (S256 fatlutect) (BSSC-d 02) contains two enzymes, the activity of the corresponding enzyme is not decreased and the growth rate of the cell is not changed as compared with the expression of the two enzymes in BSSC-d02 alone.

The invention designs a new S-sulfo-L-cysteine biosynthesis route, which takes serine (L-serine), adenosine Diphosphate (ADP) and thiosulfate (thiosulfate) as substrates to synthesize SSC under the catalysis of serine kinase (SerK) and phosphoserine dependent S-sulfo-L-cysteine synthetase (CysK 2). On the open route, an ADP regeneration system consisting of polyphosphoric acid-AMP phosphotransferase (PAP), adenosine Monophosphate (AMP) and sodium hexametaphosphate is further introduced, so that expensive ADP is avoided, and the material cost of biosynthesis of SSC is further reduced. On the basis of optimizing the concentration and condition of the reaction substrate, a special E.coli competent cell for knocking out tryptophan enzyme and glutaredoxin degrading S-sulfo-L-cysteine is constructed, and the catalytic enzyme is expressed in the competent cell, so that the reaction conversion rate and the concentration of the product S-sulfo-L-cysteine are greatly improved.

The invention provides a new route for S-sulfo-L-cysteine biosynthesis, and test results show that the steps of S-sulfo-L-cysteine synthesis are simple, the product yield is high, the cost is low, and the industrial application prospect is stronger by using serine, AMP, sodium hexametaphosphate and sodium thiosulfate as substrates and using crude enzyme liquid containing serine kinase, polyphosphate-AMP phosphotransferase and phosphoserine dependent S-sulfo-L-cysteine synthetase which are used for knocking out tryptophan enzyme and glutaredoxin cell background.

Drawings

FIG. 1 shows the S-sulfo-L-cysteine (SSC) biosynthetic pathway employed in the present invention;

FIG. 2 shows protein electrophoresis of serine kinases PkSerK and SaSbnI;

FIG. 3 shows the pH optimization of PkSerK enzyme reactions;

FIG. 4 shows the thermostability of PkSerK enzymes;

FIG. 5 shows the temperature optimization of PkSerK enzyme reactions;

FIG. 6 shows protein electrophoresis of polyphosphate-AMP phosphotransferase PaPAP and PsPAP;

FIG. 7 shows the thermostability of PaPAP and PsPAP enzymes;

FIG. 8 shows the synthesis of OPS by PaPAP enzyme-PkSerK enzyme, psPAP enzyme-PkSerK enzyme;

FIG. 9 shows the AMP concentration optimization of PsPAP enzyme-PkSerK enzyme reaction;

FIG. 10 shows the enzyme amount optimization of PsPAP enzyme-PkSerK enzyme reaction;

FIG. 11 shows the pH optimization of SSC03 (S256 fatting CT) enzyme reaction;

FIG. 12 shows the temperature optimization of SSC03 (S256 fatting CT) enzyme reaction;

FIG. 13 shows the effect of BSSC-d01/d02 host on OPS production;

FIG. 14 shows the effect of BSSC-d01/d02 host on SSC production.

Detailed Description

The invention provides a method for synthesizing S-sulfo-L-cysteine by multienzyme cascade catalysis, and a person skilled in the art can properly improve the technological parameters by referring to the content of the description. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.

Amino acid sequence of serine kinase PkSerK from pyrococcus furiosus Pyrococcus kodakaraensis ：MGVEKVPKYDIPTKKVDYVFIELDKMKPHEQLVQKELEAFIESVTGSGIFWKPMLLAKVPGEDMYLIVDGHHRWAGLQKLGAKRAPSVILDYFSDDVKVYTWYPAFKGDLNEVVERLKKEGLEVIEDPEAEEKAERGEIAFALVGEKSFAIPGGLEEQKKVSKVLDEMSVEGKIELIYYGLKEDAREDMAKGEIDYVFIRKAPTKEEVMELVKRGEVYSPKTTRHVLPFNPDKIDVKLEELF（SEQ ID NO:1）;

Nucleotide sequence of serine kinase PkSerK (codon optimized) ）：atgggagttgaaaaagtacccaagtatgatatcccaacgaaaaaggtggattacgttttcatcgaattggacaaaatgaaaccgcacgaacaactggtgcagaaggagctggaggctttcatcgaaagcgttaccggtagcggtattttttggaaaccgatgctgctggcgaaggtgccgggcgaggacatgtatctgattgttgatggtcatcatcgttgggcaggcttacagaagcttggtgcgaagcgcgcaccgtccgtgattttggactacttctctgatgatgtaaaggtctacacttggtatccggcattcaaaggcgatctcaatgaagttgttgaacgtctgaaaaaagagggcctggaggtcatcgaagatccggaagctgaagaaaaggcggagcgcggcgagatcgccttcgccctggttggtgagaaatcctttgcgattccgggcggtttggaggagcaaaagaaggtgagcaaagtgttggacgaaatgagcgttgaaggcaaaatcgagctgatttattatggtctgaaagaagacgcgcgtgaagacatggctaagggtgaaattgactacgtgtttatccgtaaggcgccaaccaaagaggaggtcatggaactggttaagcgcggtgaggtgtactcgccgaagaccacccgtcacgttctgccgtttaacccggataagatcgacgtgaaactggaggagttgttttaa（SEQ ID NO:2）;

Amino acid sequence of polyphosphate-AMP phosphotransferase PsPAP from Pseudomonas syringae tomato pathogenic variety Pseudomonas syringae pv. chemical ：MFESAEIGHAIDDDTYEAALPSLREALLEAQIDLHEQAKRQIIVLINGIEGAGKGETVKLLSEWMDPRLIEVRTFDQQTDEELAHPPVWRYWRQLPAKGRMGIFFGNWYSQMLQGRVHGQYKDAVLDQAISGAERLEKMLCDEGALIFKFWFHLSKKQMKLRLKTLKDDPLHSWRISPLDWQQSKTYDKFVRFGERVLRRTSRDYAPWHVIEGVDANYRSLTVGRLLLEGMQAALNKVEPESSALTIGPLAIHNNERTLLDSLDLSLHLSKEDYQHELIAEQARLSGNLRDKRMKSHALVAVFEGNDAAGKGGAIRRVAAALDPRQYAIVPIAAPTQDERAQPYLWRFWRQIPARGKFTIFDRSWYGRVLVERVEGFCSESDWKRAYAEINDFEEQLTEAGVVVVKFWLAIDEQTQLERFQEREKIPFKRYKITEDDWRNRKKWPDYRQAVGDMVDRTSTEIAPWTLIEANDKRWARVKVLRTINEALEKAFARDKKK（SEQ ID NO:3）;

Nucleotide sequence of polyphosphoric-acid-AMP phosphotransferase PsPAP (codon optimized) ）：atgttcgaaagtgccgaaattggtcatgccattgatgatgatacctatgaagcagcactgccgagcctgcgtgaagccctgctggaagcccagattgatctgcatgaacaggccaaacgccagattattgtgctgattaatggtattgaaggtgcaggcaaaggcgaaaccgttaaactgctgagtgaatggatggacccgcgtctgattgaagtgcgcacctttgatcagcagaccgatgaagaactggcccatccgccggtgtggcgctattggcgccagctgccggccaaaggccgtatgggtattttctttggtaattggtatagtcagatgctgcagggccgcgtgcatggccagtataaagatgcagttctggatcaggccattagtggcgcagaacgcctggaaaaaatgctgtgtgatgaaggtgcactgatttttaaattttggtttcatctgagcaagaagcagatgaaactgcgtctgaaaaccctgaaagatgatccgctgcatagttggcgcattagcccgctggattggcagcagagcaaaacctatgataaatttgtgcgttttggtgaacgtgttctgcgtcgtaccagtcgtgattatgccccgtggcatgttattgaaggtgtggatgccaattatcgcagcctgaccgttggtcgtctgctgctggaaggcatgcaggccgcactgaataaggtggaaccggaaagtagtgcactgaccattggcccgctggcaattcataataatgaacgcaccctgctggatagcctggatctgagtctgcatctgagcaaagaagattatcagcatgaactgattgcagaacaggcccgcctgagcggcaatctgcgtgataaacgtatgaaaagccatgccctggtggcagtttttgaaggcaatgatgccgcaggcaaaggtggcgcaattcgccgtgtggcagccgcactggacccgagacagtatgcaattgttccgattgccgccccgacccaggatgaacgcgcccaaccgtatctgtggcgcttttggcgccagattccggcacgtggtaaattcaccatttttgatcgcagctggtatggtcgcgtgctggtggaacgcgttgaaggcttttgtagcgaaagcgattggaaacgtgcctatgccgaaattaatgattttgaagaacagctgaccgaagcaggtgttgtggttgtgaaattttggctggcaattgatgaacagacccagctggaacgctttcaggaacgtgaaaaaattccgtttaaacgttataagatcaccgaagatgattggcgcaatcgtaaaaaatggccggattatcgccaggcagtgggtgacatggtggatcgcaccagcaccgaaattgcaccgtggaccctgattgaagccaatgataaacgctgggcacgcgtgaaagttctgcgtaccattaatgaagccctggaaaaagcatttgcacgtgataaaaagaaataa（SEQ ID NO:4）;

The amino acid sequence of the modified phosphoserine-dependent S-sulfo-L-cysteine synthetase SSC03 (S256P fatT) ：MTHVLSIDTPRISRDHHTSCRYPVGAERFRPAPQQCRHAPDRYRRVGAMVGNTPVLWVGEPFGRGERGFWAKLEGTNPGGMKDRPAMHMVERAGLRGELLPGARIVESTSGTLGLGLALAGQVYRHPVTLVTDPGMEPIIRHMLAAYGARVDLVSQPHPVGGWQQARKDRVAELLAAEPDAWCPDQYSNPDNIDAYRPLALELLDQLGDIDVLVCSVGTGGHSAGVARVLRQHNPELELIGVDTIGSTIFGQPAGPRLMRGLGSSIYPRNVDYSAFTEVHWVAPAEAVWAARTLAATYYTSGGWSVGAVALVAGWAARTYPRGTRIAAVFPDGPQRYFDTIYNDDYCRDHHLLDTNPPSDPEEISTPTQAVVNRWTRCTTVVDPTATTT（SEQ ID NO:5）;

The nucleotide sequence of the modified phosphoserine-dependent S-sulfo-L-cysteine synthetase SSC03 (S256P fatCT) was modified (codon optimized) ）：atgactcacgttctgtctatcgataccccgcgcatttctcgtgaccatcacacctcctgtcgctaccctgtgggtgcagaacgcttccgtccggctccgcaacagtgccgtcacgcgcctgaccgttaccgtcgtgttggcgcgatggttggtaacaccccagtactgtgggtaggcgagccttttggccgcggcgaacgcggcttttgggcaaaactggagggtaccaacccgggcggcatgaaagatcgcccggcaatgcacatggttgaacgtgccggcctgcgtggtgaactgctgcctggtgctcgtatcgttgaaagcacctctggcaccctgggcctgggtctggcactggcgggccaagtgtaccgccacccggtgaccctggtgactgatccgggcatggaaccgatcatccgccacatgctggccgcatacggtgctcgtgtggatctggtgtcccagccgcacccggtaggcggttggcagcaagcccgtaaagatcgcgtggcagaactgctggctgccgaaccggatgcatggtgcccggaccagtattccaacccagacaacatcgacgcctatcgtccgctggccctggaactgctggaccagctgggtgacatcgacgttctggtttgttctgtcggcactggcggccactctgcaggtgtggcacgtgtactgcgtcagcacaacccggagctggaactgatcggtgtggacactatcggttctaccatctttggtcagccggccggccctcgcctgatgcgcggcctgggttcctccatctatccacgcaacgtagactactccgcattcactgaagttcactgggttgccccagcagaagcggtttgggcagcccgtactctggcggccacttactacacctccggtggttggtccgtgggtgctgttgccctggtagctggttgggcagcccgtacctacccacgtggtacccgtattgcagcggtgttcccagacggtccacagcgttacttcgacactatctacaacgatgattactgccgcgatcaccacctgctggacacgaaccctccgtccgatccggaagaaatctccactccaacccaggcggtggtaaaccgttggactcgttgtaccaccgtggttgatccgaccgcgactaccacctaa（SEQ ID NO:6）;

Amino acid sequence of serine kinase SaSbnI (1-240) ：MNHIHEHLKLVPVDKIDLHETFEPLRLEKTKSSIEADDFIRHPILVTAMQHGRYMVIDGVHRYTSLKALGCKKVPVQEIHETQYSISTWQHKVPFGVWWETLQQEHRLPWTTETRQEAPFITMCHGDTEQYLYTKDLGEAHFQVWEKVVASYSGCCSVERIAQGTYPCLSQQDVLMKYQPLSYKEIEAVVHKGETVPAGVTRFNISGRCLNLQVPLALLKQDDDVEQLRNWKQFLADKFA（SEQ ID NO:7）;

Nucleotide sequence (codon optimized) of serine kinase SaSbnI (1-240) ）：atgaaccacatccatgaacacctgaaactggtccctgtagataaaattgacctgcatgaaactttcgaaccgctgcgcctggaaaagaccaaatcctccatcgaagcggatgatttcatccgtcatccgattctggttaccgcaatgcagcatggtcgttacatggtgatcgatggcgttcaccgctacacttctctgaaagctctgggctgcaaaaaagtgccggtgcaggaaatccacgagacccagtattccatcagcacctggcagcacaaagtgccgttcggcgtttggtgggagactctgcaacaggagcaccgtctgccgtggacgactgaaacgcgtcaggaagcaccgttcattacgatgtgccacggcgacactgaacagtacctgtacaccaaggacctgggtgaagcccactttcaggtttgggaaaaagtggtagcgtcctactctggctgctgctctgttgaacgcatcgcacagggtacctatccgtgtctgtctcagcaagacgtgctgatgaagtaccagccactgagctataaagaaatcgaggcggttgttcacaaaggtgaaaccgtgccggccggtgtaacccgtttcaacatctctggtcgttgcctgaatctgcaagttccactggctctgctgaagcaggacgacgatgtggaacagctgcgtaactggaaacagttcctggcggacaaattcgcgtaa（SEQ ID NO:8）;

Amino acid sequence of polyphosphate-AMP phosphotransferase PaPAP enzyme derived from Pseudomonas aeruginosa Pseudomonas aeruginosa ：MFESAEVGHSIDKDTYEKAVIELREALLEAQFELKQQARFPVIILINGIEGAGKGETVKLLNEWMDPRLIEVQSFLRPSDEELERPPQWRFWRRLPPKGRTGIFFGNWYSQMLYARVEGHIKEAKLDQAIDAAERFERMLCDEGALLFKFWFHLSKKQLKERLKALEKDPQHSWKLSPLDWKQSEVYDRFVHYGERVLRRTSRDYAPWYVVEGADERYRALTVGRILLEGLQAALATKERAKRQPHAAPLVSSLDNRGLLDSLDLGQYLDKDAYKEQLAAEQARLAGLIRDKRFRQHSLVAVFEGNDAAGKGGAIRRVTDALDPRQYHIVPIAAPTEEERAQPYLWRFWRHIPARRQFTIFDRSWYGRVLVERIEGFCAPADWLRAYGEINDFEEQLSEYGIIVVKFWLAIDKQTQMERFKEREKTPYKRYKITEEDWRNRDKWDQYVDAVGDMVDRTSTEIAPWTLVEANDKRFARVKVLRTINDAIEAAYKKDK（SEQ ID NO:9）;

Nucleotide sequence of polyphosphate-AMP phosphotransferase PaPAP enzyme derived from Pseudomonas aeruginosa Pseudomonas aeruginosa (codon optimized) ）：atgtttgaaagcgcagaagtaggtcacagcatcgacaaggacacctatgaaaaagctgtgattgaactgcgtgaagcgctgctggaagctcagttcgagctgaaacagcaggcacgtttcccagtaatcatcctgatcaacggcatcgaaggtgcaggtaaaggtgagaccgtaaagctgctgaacgaatggatggacccgcgtctgattgaagtacagtctttcctgcgcccttctgatgaggaactggaacgcccgccgcaatggcgcttctggcgtcgtctgccaccgaaaggtcgtactggcatcttctttggcaactggtactctcagatgctgtatgctcgtgtggaaggccacatcaaggaagcgaagctggaccaggctatcgacgcggcggagcgctttgaacgtatgctgtgcgatgaaggcgctctgctgttcaaattttggttccacctgtccaaaaaacagctgaaagaacgtctgaaagccctggaaaaggacccgcagcactcctggaaactgagcccgctggattggaaacagtctgaagtgtacgatcgcttcgtccattatggcgaacgcgtgctgcgccgcacctcccgtgattacgccccgtggtacgtcgttgagggtgctgatgaacgttaccgcgctctgacggttggtcgcatcctgctggaaggtctgcaagcagcactggccaccaaagagcgtgccaaacgtcaaccgcatgccgcaccgctggtatcttctctggataaccgcggtctgctggatagcctggacctgggccagtatctggataaggatgcatacaaggaacagctggcagcagaacaagcgcgcctggctggcctgatccgtgataaacgtttccgccagcatagcctggttgctgtgttcgaaggcaacgacgcagcgggtaaaggtggcgcaatccgtcgcgttaccgatgccctggatccgcgtcagtatcacatcgtaccgatcgctgcgccgaccgaagaggaacgtgcccagccgtacctgtggcgtttctggcgtcatattccggcacgtcgtcagttcaccatcttcgatcgcagctggtacggccgtgtactggttgaacgcatcgagggcttctgtgcaccggctgactggctgcgtgcgtacggtgaaattaacgactttgaggaacagctgtccgaatacggcatcattgtggttaagttctggctggctatcgacaaacagacccagatggaacgtttcaaagaacgcgagaaaaccccgtataaacgctacaagattaccgaagaggattggcgtaaccgcgataagtgggaccagtatgtcgacgcggtgggtgacatggttgatcgcacttccactgagatcgcgccgtggaccctggtggaagctaatgacaaacgcttcgcccgtgtgaaagttctgcgtaccatcaacgacgctatcgaggccgcttacaaaaaagataaataa（SEQ ID NO:10）;

The tnaA-gRNA sequence CTGGCTCAATAACACGAATG (SEQ ID NO: 11);

nrdh-gRNA sequence AAACGGGCGATGGAAAACCG (SEQ ID NO: 12);

The whole scheme of the invention is as follows:

the invention provides a method for synthesizing S-sulfo-L-cysteine by screening serine kinase PkSerK derived from Pyrococcus furiosus Pyrococcus kodakaraensis, polyphosphate-AMP phosphotransferase PsPAP derived from Pseudomonas syringae tomato pathogenic variety Pseudomonas syringae pv. to and modified phosphoserine dependent S-sulfo-L-cysteine synthetase SSC03 (S256P (CT)).

The nucleotide coding the enzyme is subjected to complete gene synthesis after codon optimization, then the synthesized genes are respectively constructed in pET28a vectors and converted into BL21 (DE 3) expression hosts, then puncture bacteria or glycerol tube preservation (Beijing engine family organisms) is carried out, the puncture bacteria or glycerol tube preservation strains are subjected to two-stage expansion culture in LB culture medium (Kan) at the temperature of 16 ℃ for 0.1 mM IPTG induction expression, then thalli are centrifugally collected, and then a crushing buffer solution (20 mM Na ₂HPO₄/NaH₂PO₄, 100mM NaCl,pH 7.6) is added according to the ratio of 1g to 4mL of wet thalli for re-suspension and then crushing is carried out, so that crude enzyme liquid is obtained.

Firstly, using PkSerK enzyme to synthesize an intermediate product phosphoserine (O-phospho-L-serine, OPS) by taking serine and ADP as substrates, and obtaining the reaction with proper pH value of 5.0-8.0 (optimal pH value of 6.0) and proper temperature of 38-52 ℃ by single factor optimization (optimal temperature of 45 ℃). After introducing an ADP regeneration system consisting of PsPAP enzyme, sodium hexametaphosphate and Adenosine Monophosphate (AMP), optimizing to obtain the proper enzyme amount of PkSerK enzyme by 0.5% -10% (the optimal enzyme amount is 1%, v/v is the same), the proper enzyme amount of PsPAP enzyme by 5% -20% (the optimal enzyme amount is 15%), the proper concentration of serine by 100% -200 mM (the optimal 200 mM), the proper concentration of AMP by 1% -10 mM (the optimal 5 mM), the proper concentration of magnesium chloride hexahydrate by 20% -60 mM (the optimal 40 mM) and the proper concentration of sodium hexametaphosphate by 35% -65 mM (the optimal 65 mM).

And adding SSC03 (S256P (CT)) enzyme into the OPS-containing reaction solution obtained by the PkSerK enzyme and PsPAP enzyme double enzyme system serving as a substrate to synthesize a final product SSC, and optimizing to obtain a proper pH value of 6.0-8.0 (the proper pH value of 7.0) and a proper temperature of 30-45 ℃ (the proper temperature of 38 ℃).

Then, the nucleotide sequences of PsPAP enzyme and SSC03-S256P fatlutet enzyme are inserted into a coexpression vector pETDuet-1, and then two plasmids pETDuet-PsPAP-SSC03 (S256P fatCT) and pET28a-PkSerK are respectively transferred into an escherichia coli competent cell BSSC-d02 from which tryptophan enzyme EcTnaA and glutaredoxin EcNrdH are knocked out for culture induction expression.

Finally, preparing a reaction solution of serine 100-200 mM (most suitable 200 mM), magnesium chloride hexahydrate 20-60 mM (most suitable 60 mM), sodium hexametaphosphate 35-65 mM (most suitable 65 mM), AMP 1-10 mM (most suitable 5 mM), thiosulfate equivalent 1.1 eq-1.3 eq (most suitable 1.3 eq), regulating pH to 6.0-8.0 (most suitable pH 7.0), constant volume, preheating to 25-45 ℃ (most suitable 38 ℃), adding PsPAP +SSC03 (S256P-20% (most suitable 12%) enzyme amount, pkSerK enzyme amount 0.5% -10% (most suitable enzyme amount 1%) under low-speed stirring, and starting reaction timing.

In the invention, vt% is the volume fraction, which refers to the ratio of the volume of solute to the total volume of solution, and the calculation formula is as follows, volume fraction (vt%) = (volume of solute/total volume of solution) ×100%, wherein vt% is equal to% v/v or v/v%.

The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:

EXAMPLE 1 obtaining crude enzyme solution

The invention provides a method for synthesizing S-sulfo-L-cysteine by screening serine kinase PkSerK derived from Pyrococcus furiosus Pyrococcus kodakaraensis, polyphosphate-AMP phosphotransferase PsPAP derived from Pseudomonas syringae tomato pathogenic variety Pseudomonas syringae pv. to and modified phosphoserine dependent S-sulfo-L-cysteine synthetase SSC03 (S256P (CT)).

The nucleotide coding the enzyme is subjected to complete gene synthesis after codon optimization, then the synthesized genes are respectively constructed in a pET28a vector and transformed into a BL21 (DE 3) expression host, then puncture bacteria or glycerol tube preservation (Beijing engine family organism) is carried out, after the puncture bacteria or glycerol tube preservation bacteria are subjected to two-stage expansion culture in LB culture medium (Kan) at 16 ℃ for 0.1 mM IPTG induction expression, thalli are centrifugally collected, a crushing buffer solution (20 mM Na ₂HPO₄/NaH₂PO₄, 100mM NaCl,pH 7.6) is added according to the ratio of 1g to 4mL of the thalli to be crushed, a crude enzyme solution is obtained, and subsequent experiments are carried out (other strains not mentioned herein are all operated according to the crude enzyme solution, unless the sources of the enzymes are specifically indicated, the sources of the enzymes are mentioned herein in a short way, and correspond to the enzymes).

EXAMPLE 2 Synthesis of OPS (O-phospho-L-serine, OPS) Using serine and ADP as substrates

0.53G (100 mM) of serine, 3.53g (150 mM) of adenosine disodium diphosphate (ADP-Na 2) and 0.3g (30 mM) of magnesium chloride hexahydrate were weighed and dissolved in 40mL of pure water, pH was adjusted to 7.5 and the volume was set to 45mL, and after preheating at 38℃was added to 5mL of PkSerK BL (DE 3) crude enzyme solution (protein expression, see FIG. 2).

The reaction mixture was sampled and diluted 10 times with 60% acetonitrile, and the concentration of OPS was detected by High Performance Liquid Chromatography (HPLC) and reacted for 15 minutes to give OPS 81.63mM.

2. Optimization of PkSerK enzyme reaction conditions in OPS synthesis process using serine and ADP as substrates

1. Optimization of pH for PkSerK enzymatic reactions

1.58G (100 mM) of serine, 2.83g (40 mM) of adenosine disodium diphosphate (ADP-Na 2) and 0.61g (20 mM) of magnesium chloride hexahydrate were weighed and dissolved in 120mL of pure water, and 5 parts were dispensed, pH 5, 6, 7, 8 and 9 were adjusted to a constant volume of 29.85mL, and after preheating at 38 ℃, pkSerK BL (DE 3) of crude enzyme solution was added to 0.15mL. Sampling HPLC detects OPS formation. As shown in FIG. 3, the highest concentration of OPS was obtained in the pH 6 group, and the amount of PkSerK enzyme was 0.5vt% and reacted for 5min to yield OPS 9.59mM.

2. Thermal stability test of PkSerK enzyme

PkSerK BL21 (DE 3) crude enzyme solution was taken and placed in water baths at 38 ℃, 50 ℃ and 65 ℃ for 10min, and the supernatant was centrifuged for protein electrophoresis detection, and the target bands were semi-quantitatively analyzed by using imageJ software. As shown in FIG. 4, pkSerK enzyme has better heat stability, and only has 15% -20% loss at 65 ℃.

3. Optimization of PkSerK enzyme reaction temperature

1.26G (100 mM) of serine, 2.26g (40 mM) of adenosine disodium diphosphate (ADP-Na 2) and 0.49g (20 mM) of magnesium chloride hexahydrate are weighed and dissolved in 100mL of pure water, the pH is adjusted to 6.0 to a constant volume of 119.4mL, 4 parts are split, and after preheating at 30 ℃, 38 ℃,45 ℃ and 52 ℃ respectively, pkSerK BL (DE 3) crude enzyme solution is added to 0.15mL. Sampling HPLC detects OPS formation. As shown in FIG. 5, the highest concentration of OPS was obtained for the 45℃group, and OPS 10.62mM was obtained by the reaction for 5 min.

EXAMPLE 2 Synthesis of OPS Using serine, AMP, sodium hexametaphosphate as substrates

1. Synthesis of OPS with serine, AMP, sodium hexametaphosphate as substrates

1.05G (100 mM) of serine, 0.20g (5 mM) of adenosine disodium monophosphate (AMP-Na 2), 0.81g (40 mM) of magnesium chloride hexahydrate, 2.14g (35 mM) of sodium hexametaphosphate were weighed and dissolved in 80mL of pure water, pH was adjusted to 6.0 and the volume was fixed to 94mL, 2 portions were dispensed, and after preheating at 38℃, 2.5mL (protein expression see FIG. 6) of PsPAP enzyme BL21 (DE 3) crude enzyme solution and 0.5mL (DE 3) crude enzyme solution of PkSerK BL (protein expression see FIG. 6) were added, respectively, and 2.5mL (protein expression see FIG. 6) of the crude enzyme liquid of polyphosphoric acid-AMP phosphotransferase PaPAP enzyme BL21 (DE 3) derived from Pseudomonas aeruginosa Pseudomonas aeruginosa and 0.5mL (protein expression see FIG. 6) of PkSerK BL (DE 3) crude enzyme solution were added. Since PsPAP enzyme and PaPAP enzyme did not withstand 45 ℃ heat treatment (fig. 7), the reaction temperature was adjusted to 38 ℃. Sampling HPLC detects OPS formation.

As shown in FIG. 8, the combination of PsPAP vt% and PkSerK enzyme 1vt% produced OPS at a faster rate, and reacted for 1h to produce OPS 72.63mM.

2. Optimization of reaction conditions in synthesis of OPS using serine, AMP, sodium hexametaphosphate as substrates

1. Optimization of AMP concentration in PsPAP enzyme-PkSerK enzyme reaction

1.26G (100 mM) of serine, 0.98g (40 mM) of magnesium chloride hexahydrate and 2.57g (35 mM) of sodium hexametaphosphate were weighed and dissolved in 100mL of pure water, and 4 portions of the solution were dispensed, and 0.01g (1 mM), 0.02g (2 mM), 0.06g (5 mM) and 0.12g (10 mM) of adenosine disodium monophosphate (AMP-Na 2), 0.02g (2 mM) and 0.7 mL of pH6.0 were each added, and after preheating at 38 ℃, 3mL of PsPAP BL (DE 3) crude enzyme solution and 0.3mL of PkSerK BL (DE 3) crude enzyme solution were each added. Sampling HPLC detects OPS formation.

As shown in FIG. 9, the OPS production rate of the 1mM AMP group was significantly lower than that of the 2-10 mM group in the combination of PsPAP enzyme 10vt% and PkSerK enzyme 1 vt%.

2. Optimizing the enzyme amounts of PsPAP and PkSerK enzymes at 200mM serine

The reaction solution was prepared in accordance with Table 1, serine, adenosine disodium monophosphate (AMP-Na 2), magnesium chloride hexahydrate (MgCl ₂·6H₂ O) and sodium hexametaphosphate were weighed and dissolved in pure water, pH was adjusted to 6.0 and the volume was fixed, and after preheating at 38℃different volumes of PsPAP BL (DE 3) crude enzyme solution and PkSerK BL (DE 3) crude enzyme solution were added respectively and water was added to make up to 30mL of reaction volume. Sampling HPLC detects OPS formation.

TABLE 1

As shown in FIG. 10, the PsPAP enzyme 15vt% and PkSerK enzyme 1vt% combination are optimal, and the reaction 3h conversion rate is close to 100%.

EXAMPLE 3 stepwise Synthesis of SSC Using serine, AMP, sodium hexametaphosphate, sodium thiosulfate as substrates

1. SSC is stepwise synthesized by using serine, AMP, sodium hexametaphosphate and sodium thiosulfate as substrates

4.20G (200 mM) of serine, 0.39g (5 mM) of adenosine disodium monophosphate (AMP-Na 2), 1.63g (40 mM) of magnesium chloride hexahydrate and 7.95g (65 mM) of sodium hexametaphosphate were weighed and dissolved in 160mL of pure water, pH 6.0 was adjusted and the volume was adjusted to 168mL, and 30mL of PsPAP BL (DE 3) crude enzyme solution and 2mL of PkSerK BL (DE 3) crude enzyme solution were added after preheating at 38 ℃. The OPS concentration did not increase again in reaction 3h, yielding 190.75mM.

7.80G (210 mM,1.3 eq) of sodium thiosulfate was weighed and added to the OPS-containing reaction solution to dissolve, the pH was adjusted to 7.0, 35mL of SSC03 (S256P. Sub.CT) BL21 (DE 3) crude enzyme solution was added and reacted at 25 ℃.

The reaction mixture was sampled and diluted 10 times with 60% acetonitrile, and the concentration of SSC was measured by High Performance Liquid Chromatography (HPLC) and reacted for 8 hours to give 76.30mM, with a conversion of 47%.

2. Optimization in stepwise synthesis of SSC using serine, AMP, sodium hexametaphosphate, sodium thiosulfate as substrates

2.1 Optimizing the pH of SSC03 (S256 fatly CT) enzyme reaction by using OPS reaction liquid as a substrate

The OPS reaction solution was diluted to 167mM, and 108mL of the solution was dissolved in sodium thiosulfate (165 mM,1.1 eq), and 4 parts of the solution were dispensed, and after preheating at pH 5, 6, 7, 8, and 25℃respectively, 3mL of the crude enzyme solution of SSC03 (S256P. Sub.CT) BL21 (DE 3) was added. Sample HPLC detects SSC formation. As shown in FIG. 11, the highest concentration of SSC was obtained in the pH 7.0 group, and 150mM of OPS as an intermediate was reacted for 4 hours to yield 50.0mM of SSC when the enzyme amount of SSC03 (S256 fatting CT) BL21 (DE 3) was 10 vt.

2.2 Using OPS reaction liquid as substrate, optimizing SSC03 (S256 fatT) enzyme reaction temperature

The OPS reaction solution was diluted to 167mM, and 108mL of the solution was dissolved in 3.13g (165 mM,1.1 eq) of sodium thiosulfate, and 4 parts of the solution were dispensed, and the pH was adjusted to 7.0, and the solution was preheated to 25℃30℃38℃45℃and then 3mL of the crude enzyme solution of SSC03 (S256P-L CT) BL21 (DE 3) was added. Sample HPLC detects SSC formation. As shown in FIG. 12, the concentration of SSC in the 38℃group product was the highest, and SSC 76.6mM was produced in 2 hours of reaction.

Example 4 construction of optimized expression strains for further optimizing the-PsPAP enzyme, SSC03 (S256 fatin CT) enzyme and PkSerK enzyme

1. Construction of strains optimized for expression of PsPAP enzyme, SSC03-S256 fatter CT enzyme and PkSerK enzyme

PsPAP enzyme, SSC03 (S256 fatin CT) enzyme are expressed in the form of fusion protein, pkSerK enzyme is expressed separately, and the expression of the PsPAP enzyme and SSC03 (S256 fatin CT) enzyme are performed in escherichia coli from which tryptophan enzyme EcTnaA and glutaredoxin EcNrdH are knocked out;

The construction of E.coli competent cell BSSC-d02, from which tryptophan enzyme EcTnaA and glutaredoxin EcNrdH were knocked out, was as follows:

First, a pGRB-tnaA-gRNA plasmid containing a tnaA-gRNA sequence is constructed, and is electrically transferred to BL21 (DE 3) competent cells containing a pEcCas plasmid together with fatliquor tnaA donar DNA, and E.coli cells which successfully knock out tryptophan enzyme EcTnaA are screened by plating, E.coli cells BSSC-d01-pEcCas which eliminate pGRB-tnaA-gRNA plasmid are obtained, and E.coli cells BSSC-d01 which eliminate pEcCas plasmid are obtained and are respectively manufactured into competent cells for standby.

Then constructing pGRB-nrdh-gRNA plasmid containing nrdh-gRNA sequence, and electrotransferring to E.coli competent cell BSSC-d01-pEcCas together with father nrdh donar DNA, plating and screening to obtain E.coli cell BSSC-d02 (whose tryptophan enzyme EcTnaA and glutaredoxin EcNrdH functions are deleted) which is eliminated pGRB-nrdh-gRNA plasmid and pEcCas plasmid, and making into competent cell for standby.

Then, the nucleotide sequences of PsPAP enzyme and SSC03 (S256P fatlute CT) enzyme were co-inserted into the vector pETDuet-1 to obtain the plasmid pETDuet-PsPAP-SSC03 (S256P fatct), the plasmids pET28a-PkSerK and pETDuet-PsPAP-SSC03 (S256P fatct) in example 1 and plasmid were transferred into BSSC-d02 respectively for culture induction expression, and then the cells were collected by centrifugation, and then the cells were resuspended in a disruption buffer (20 mM Na ₂HPO₄/NaH₂PO₄, 100mM NaCl,pH 7.6) according to a wet cell ratio of 1 g/4 mL and disrupted to obtain a crude enzyme solution.

2. Verification of the Effect of BSSC-d01/d02 host on OPS production

3.15G (200 mM) of serine, 0.29g (5 mM) of adenosine disodium monophosphate (AMP-Na 2), 1.22g (40 mM) of magnesium chloride hexahydrate and 5.96g (65 mM) of sodium hexametaphosphate are weighed and dissolved in 120mL of pure water, the pH is adjusted to 6.0 and the volume is fixed to 126mL, 3 parts are packed, and after preheating at 38 ℃, psPAP BL (DE 3) crude enzyme solution 7.5mL and PkSerK BL (DE 3) crude enzyme solution 0.5mL, psPAP BSSC-d01 crude enzyme solution 7.5mL and PkSerK BSSC-d01 crude enzyme solution 0.5mL, psPAP BSSC-d02 crude enzyme solution 7.5mL and PkSerK BSSC-d02 crude enzyme solution 0.5mL are respectively added. Sampling HPLC detects OPS formation.

As shown in FIG. 13, the host BSSC-d01 knocked out of tryptophan enzyme EcTnaA inhibited OPS production, whereas the host BSSC-d02 knocked out of tryptophan enzyme EcTnaA and glutaredoxin EcNrdH accelerated OPS production.

3. Verification of the Effect of BSSC-d01/d02 host on SSC production

2.78G (100 mM) of phosphoserine (OPS) and 3.56g (150 mM) of sodium thiosulfate are weighed and dissolved in 120mL of pure water, the pH is regulated to 7.0, the volume is fixed to 135mL, 3 parts are packed, 5mL of SSC03 (S256P (CT)) BL21 (DE 3) crude enzyme solution, 5mL of SSC03 (S256P (CT)) BSSC-d01 crude enzyme solution and 5mL of SSC03 (S256P (CT)) BSSC-d02 crude enzyme solution are respectively added after 25 ℃ pre-heating. Sample HPLC detects SSC formation.

As shown in FIG. 14, the SSC03 (S256 fatly CT) crude enzyme solutions expressed in the hosts BSSC-d01 and BSSC-d02 can greatly increase the SSC production compared with the original host BL21 (DE 3).

EXAMPLE 5 one-step Synthesis of SSC Using serine, AMP, sodium hexametaphosphate, sodium thiosulfate as substrates

1. Small system test

2.10G (200 mM) of serine, 3.48g (220 mM,1.1 eq) of sodium thiosulfate, 0.20g (5 mM) of adenosine monophosphate disodium salt (AMP-Na 2), 0.81g (40 mM) of magnesium chloride hexahydrate and 3.98g (65 mM) of sodium hexametaphosphate were dissolved in 80mL of pure water, the pH was adjusted to 7.0 and the volume was set to 89mL, and PsPAP +SSC03 (S256P. Sub.CT) BSSC-d02 crude enzyme solution 10mL and PkSerK BSSC-d02 crude enzyme solution 1mL were added after preheating at 38 ℃. Sample HPLC detects SSC formation. The results showed that SSC 154.77mM was produced after 2h of reaction.

2. Systematic magnification test

21.02G (200 mM) of serine, 41.11g (260 mM, 1.3 eq) of sodium thiosulfate, 1.96g (5 mM) of adenosine monophosphate disodium salt (AMP-Na 2), 12.20g (60 mM) of magnesium chloride hexahydrate and 39.77g (65 mM) of sodium hexametaphosphate were dissolved in 800mL of pure water, pH7.0 was adjusted and the volume was set to 870mL, and PsPAP +SSC03 (S256 P.sub.CT) BSSC-d02 crude enzyme solution 120mL and PkSerK BSSC-d02 crude enzyme solution 10mL were added after preheating at 38 ℃. The reaction time was 6 hours to give SSC 174.92mM with a conversion of 87.5%.

Comparative example 1 OPS was synthesized using serine kinase SaSbnI (1-240) derived from Staphylococcus aureus Staphylococcus aureus with serine, adenosine Triphosphate (ATP) as substrate

0.53G (100 mM) of serine, 2.76g (100 mM) of adenosine disodium triphosphate (ATP-Na 2) and 0.3g (30 mM) of magnesium chloride hexahydrate were weighed and dissolved in 40mL of pure water, pH7.5 was adjusted and the volume was set to 45mL, and SaSbnI (1-240) BL21 (DE 3) crude enzyme solution (protein expression, see FIG. 2) was added after preheating at 38 ℃.

The concentration of OPS was detected by high performance liquid chromatography after 10-fold dilution of the reaction solution, and OPS formation was not detected yet after 6 hours of reaction, possibly due to protein expression in the precipitate.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A combination enzyme, characterized in that it includes: phosphoserine-dependent S-sulfo-L-cysteine synthetase, serine kinase derived from Pyrococcus furiosus, and polyphosphate-AMP phosphotransferase derived from Pseudomonas syringae pv. tomato or Pseudomonas aeruginosa. 2. The combined enzyme according to claim 1, characterized in that The amino acid sequence of the phosphoserine-dependent S-sulfo-L-cysteine synthetase is shown in SEQ ID NO: 5; The amino acid sequence of the serine kinase derived from Pyrococcus furiosus is shown in SEQ ID NO: 1; The amino acid sequence of the polyphosphate-AMP phosphotransferase derived from Pseudomonas syringae pv. tomato is shown in SEQ ID NO: 3; The amino acid sequence of the polyphosphate-AMP phosphotransferase derived from Pseudomonas aeruginosa is shown in SEQ ID NO:9. 3. Biomaterial, characterized in that it comprises at least one of the following A) to C): A), a nucleic acid encoding the combined enzyme according to claim 1 or 2; B), a recombinant vector containing the nucleic acid as described in A); C), transfecting or transforming host cells with the recombinant vector described in B). 4. The biomaterial according to claim 3, characterized in that The nucleotide sequence of the nucleic acid encoding the phosphoserine-dependent S-sulfo-L-cysteine synthetase is shown in SEQ ID NO: 6; The nucleotide sequence of the nucleic acid encoding the serine kinase derived from Pyrococcus furiosus is shown in SEQ ID NO: 2; The nucleotide sequence of the nucleic acid encoding the polyphosphate-AMP phosphotransferase derived from Pseudomonas syringae pv. tomato is shown in SEQ ID NO: 4; The nucleotide sequence of the nucleic acid encoding the polyphosphate-AMP phosphotransferase derived from Pseudomonas aeruginosa is shown as SEQ ID NO:10. 5. A genetically engineered bacterium characterized in that the tryptophanase EcTnaA and glutaredoxin EcNrdH are functionally deficient and the recombinant vector in the biological material according to claim 3 is transfected or transformed. 6. Application of at least one of the following I) to III) in the synthesis of S-sulfo-L-cysteine: 1), the combined enzyme according to claim 1 or 2; II), the biological material according to claim 3 or 4; III), the genetically engineered bacteria according to claim 5; IV), culturing the host cells in the biological material according to claim 3 or 4 or the culture obtained by the genetically engineered bacteria according to claim 5. 7. A method for preparing S-sulfo-L-cysteine, characterized in that it comprises synthesizing S-sulfo-L-cysteine using at least one of the following i) to iv): i), the combined enzyme according to claim 1 or 2; ii), the biological material according to claim 3 or 4; iii) The genetically engineered bacteria according to claim 5; iv) culturing the host cells in the biological material according to claim 3 or 4 or the culture obtained by the genetically engineered bacteria according to claim 5. 8. The preparation method according to claim 7, characterized in that it comprises the following steps: serine kinase, polyphosphate-AMP phosphotransferase and phosphoserine-dependent S-sulfo-L-cysteine synthetase catalyze the substrate to generate S-sulfo-L-cysteine in the presence of magnesium ions; The substrates include serine, AMP, sodium hexametaphosphate and thiosulfate. 9. The preparation method according to claim 8, characterized in that: The concentration of serine is 100mM~200mM; The concentration of the salt containing AMP is 1-10 mM; The concentration of the sodium hexametaphosphate is 35mM~65mM; The concentration of the thiosulfate is 100-300 mM; The concentration of magnesium ions is 20mM~60mM; The amount of bacteria containing serine kinase added is 1g/L~5g/L; The amount of bacteria containing polyphosphate-AMP phosphotransferase added is 10g/L~50g/L; The amount of bacteria containing phosphoserine-dependent S-sulfo-L-cysteine synthetase added is 10 g/L to 50 g/L. 10. The preparation method according to claim 9, characterized in that the catalytic conditions are pH 6.0-8.0 and temperature 25-45°C.