Method for synthesizing S-sulfo-L-cysteine through multi-enzyme cascade catalysis

Abstract

The invention relates to the technical field of biology, in particular to a method for synthesizing S-sulfo-L-cysteine through multi-enzyme cascade catalysis. The invention provides a new route for biosynthesis of S-sulfo-L-cysteine, and a test result shows that serine, AMP, sodium hexametaphosphate and sodium thiosulfate are used as substrates, and the S-sulfo-L-cysteine is synthesized by a one-step method. A crude enzyme liquid which contains serine kinase, polyphosphoric acid-AMP phosphotransferase and phosphoserine dependent type S-sulfo-L-cysteine synthetase in which the background of tryptophanase and glutaredoxin cells is knocked out is used for synthesizing the S-sulfo-L-cysteine, the steps are simple, the product yield is high, the cost is low, and the method has a relatively strong industrial application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for synthesizing S-sulfo-L-cysteine ​​by multi-enzyme cascade catalysis. Background Art

[0002] S-sulfo-L-cysteine ​​(SSC) is a new modified amino acid launched by Merck that can replace cysteine, which is essential for cell culture medium, while increasing the level of glutathione in cells, thereby improving the production of recombinant proteins (CN107847549B). In bacteria and plants, SSC is synthesized in two steps using serine, acetyl-CoA and thiosulfate as substrates, catalyzed by 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).

[0003] Biosynthesis is currently a popular synthesis method, but because acetyl-CoA is only available in milligram-level reagents at high prices, the above biosynthesis route is difficult to apply industrially. Therefore, it is particularly necessary to develop a biosynthesis method with low material cost, high conversion efficiency and good product purity. Summary of the invention

[0004] In view of this, 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.

[0005] The present invention provides a combined enzyme, which comprises: phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase SSC03 (S256P ∆CT), serine kinase PkSerK derived from Pyrococcus furiosus and polyphosphate-AMP phosphotransferase PAP derived from Pseudomonas syringae pv. tomato or Pseudomonas aeruginosa.

[0006] Furthermore, in the combined enzyme of the present invention,

[0007] The amino acid sequence of the phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase SSC03 (S256P ∆CT) is shown in SEQ ID NO: 5;

[0008] The amino acid sequence of the serine kinase PkSerK from Pyrococcus furiosus is shown in SEQ ID NO: 1;

[0009] The amino acid sequence of the polyphosphate-AMP phosphotransferase PsPAP derived from Pseudomonas syringae pv. tomato is shown in SEQ ID NO: 3;

[0010] The amino acid sequence of the polyphosphate-AMP phosphotransferase PaPAP derived from Pseudomonas aeruginosa is shown in SEQ ID NO:9.

[0011] The present invention provides a biomaterial comprising at least one of the following A) to C):

[0012] A), a nucleic acid encoding the combined enzyme of the present invention;

[0013] B), a recombinant vector containing the nucleic acid as described in A);

[0014] C), transfecting or transforming host cells with the recombinant vector described in B).

[0015] Furthermore, in the biomaterial of the present invention,

[0016] The nucleotide sequence of the nucleic acid encoding the phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase SSC03 (S256P ∆CT) is shown in SEQ ID NO: 6;

[0017] The nucleotide sequence of the nucleic acid encoding the serine kinase PkSerK derived from Pyrococcus furiosus is shown in SEQ ID NO: 2;

[0018] The nucleotide sequence of the nucleic acid encoding the polyphosphate-AMP phosphotransferase PsPAP derived from Pseudomonas syringae pv. tomato is shown in SEQ ID NO: 4;

[0019] The nucleotide sequence of the nucleic acid encoding the polyphosphate-AMP phosphotransferase PsPAP derived from Pseudomonas aeruginosa is shown in SEQ ID NO:10.

[0020] The nucleic acid described in the present invention can be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, the nucleic acid is in the form of DNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. Nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acids can be linear or circular in topology. Nucleic acids can be part of a vector (such as an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from a natural source, or can be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0021] In the present invention, the codons of the nucleic acid are optimized, and these optimizations include but are not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repeat sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.

[0022] The present invention provides a recombinant vector comprising the nucleic acid of the present invention.

[0023] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule, which contains the desired coding sequence and the appropriate nucleic acid sequence or element that is essential for the expression of the operably connected coding gene in a specific host organism. The nucleic acid sequence or element necessary for the expression in the model animal or mammalian cell includes a promoter, a ribosome binding site and possible other sequences. It is known that prokaryotes use promoters, enhancers and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" can sometimes be interchanged and used, because plasmid is the most commonly used vector form at present. However, the present invention is intended to include such other forms of expression vectors, which play an equivalent role, which are known or will become known in the art, including but not limited to: plasmid, phage particle, viral vector and / or only potential genomic insert. In a specific embodiment, the nucleic acid encoding the fusion protein provided by the present invention can be constructed in various prokaryotic expression vectors. For example, its backbone vector can be a pET series vector, specifically pET28a and / or pETDuet.

[0024] Further, in the present invention, the combined enzyme can be integrated into the recombinant vector in a single or combined manner, and the backbone of the recombinant vector can be pET28a or pETDuet; in a specific embodiment of the present invention, the recombinant vector includes the following forms:

[0025] pET28a-PsPAP;

[0026] pET28a-SSC03 (S256P ΔCT);

[0027] pET28a-PkSerK;

[0028] pETDuet- PsPAP-SSC03 (S256P ΔCT);

[0029] The present invention provides a host cell, the source of which includes plants, animals, bacteria, fungi, bacteriophages or viruses, which are not limited by the present invention. In a specific embodiment of the present invention, the host cell is derived from bacteria, specifically Escherichia coli, and the bacterial cell is Escherichia coli BL21 DE3 or Escherichia coli with a modified genetic background.

[0030] The Escherichia coli with modified gene background is a genetically engineered bacterium in the present invention; the genetically engineered bacterium lacks the tryptophanase EcTnaA and glutaredoxin EcNrdH functions (BSSC-d02), and is transfected or transformed with the recombinant vector in the biomaterial of the present invention.

[0031] In a specific embodiment of the present invention, the recombinant vector can be transformed or transfected into BL21 DE3, or can be transformed or transfected into BSSC-d02 to obtain the genetically engineered bacteria; in the present invention, specific strain forms include:

[0032] pET28a-PsPAP (BL21 DE3);

[0033] pET28a-SSC03(S256P ΔCT)(BL21 DE3);

[0034] pET28a-PkSerK(BL21 DE3);

[0035] pET28a-PsPAP-SSC03 (S256P ΔCT) (BSSC-d02);

[0036] pET28a-PkSerK(BSSC-d02);

[0037] The present invention provides the use of at least one of the following I) to III) in the synthesis of S-sulfo-L-cysteine:

[0038] 1), the combined enzyme of the present invention;

[0039] II), the biomaterial of the present invention;

[0040] III), the genetically engineered bacteria of the present invention;

[0041] IV), culturing the host cells in the biological material of the present invention or the culture obtained by the genetically engineered bacteria of the present invention.

[0042] The present invention provides a method for biosynthesizing S-sulfo-L-cysteine ​​for the first time. The above-mentioned specific strain forms can be used in the synthesis of S-sulfo-L-cysteine, and the present invention does not limit this. In addition, the substrates used for the synthesis of the intermediate product of S-sulfo-L-cysteine ​​shown in I) to III) above can be serine, ADP, or serine, AMP, sodium hexametaphosphate, and the present invention does not limit this; experimental results show that the introduction of the ADP regeneration system (with serine, AMP, and sodium hexametaphosphate as substrates) reduces the amount of ADP / AMP used, and at the same time replaces the expensive ADP with the relatively cheap AMP, and the unit price of AMP is about one tenth of that of ADP, which greatly reduces the production cost.

[0043] The present invention provides a method for preparing S-sulfo-L-cysteine, which comprises synthesizing S-sulfo-L-cysteine ​​using at least one of the following i) to iii):

[0044] i), the combined enzyme of the present invention;

[0045] ii), the biomaterial of the present invention;

[0046] iii) the genetically engineered bacteria of the present invention;

[0047] iv) culturing the host cells in the biological material of the present invention or the culture obtained by the genetically engineered bacteria of the present invention.

[0048] Furthermore, the preparation method of the present invention comprises the following steps: serine kinase PkSerK, polyphosphate-AMP phosphotransferase PsPAP and phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase SSC03 (S256P ∆CT) catalyze the substrate to generate S-sulfo-L-cysteine ​​in the presence of magnesium ions;

[0049] The substrates include serine, AMP, sodium hexametaphosphate and sodium thiosulfate.

[0050] Further, the thiosulfate is selected from at least one of sodium thiosulfate, potassium thiosulfate and / or ammonium thiosulfate; in a specific embodiment of the present invention, the thiosulfate is sodium thiosulfate or ammonium thiosulfate;

[0051] In the preparation method of the present invention, the order of adding the substances participating in the reaction is not limited, and the substances participating in the afterimage can be added in two steps; or in one step; in a specific embodiment of the present invention, the specific steps when the two-step addition is adopted are:

[0052] Step 1, serine kinase and polyphosphate-AMP phosphotransferase catalyze substrate 1 to generate OPS in the presence of magnesium ions;

[0053] Step 2: Phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase uses sodium thiosulfate as a substrate to convert OPS into SSC.

[0054] Furthermore,

[0055] The catalytic conditions are pH 7.0 and temperature 38°C.

[0056] The concentration of serine is 100 mM to 200 mM, and specifically 200 mM is optimal;

[0057] The concentration of the salt containing AMP is 1 mM to 10 mM, and specifically 5 mM is optimal;

[0058] The concentration of the sodium hexametaphosphate is 25 mM to 65 mM, and 65 mM is optimal;

[0059] The concentration of the thiosulfate is 100-300 mM, specifically 200-260 mM;

[0060] The concentration of magnesium ions is 20mM~60mM, and the optimal concentration is 40mM;

[0061] The amount of bacteria containing serine kinase PkSerK added is 1g / L~5g / L;

[0062] The amount of bacteria containing polyphosphate-AMP phosphotransferase PsPAP added is 10g / L~50g / L;

[0063] The amount of bacteria containing phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase SSC03 (S256P ∆CT) added is 10g / L~50g / L;

[0064] Specifically, as in Example 5 of the present invention, the amount of PkSerK added is 2.5 g / L; the amount of bacteria containing polyphosphate-AMP phosphotransferase and phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase SSC03 (S256P ∆CT) added is 25 g / L or 30 g / L;

[0065] In the present invention, the bacterial cell may contain both polyphosphate-AMP phosphotransferase PsPAP and phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase SSC03 (S256P ∆CT), or may contain the above two enzymes alone; in a specific embodiment of the present invention, the host cell containing the above two enzymes alone is BL21 DE3, and the host cell containing the above two enzymes at the same time is BSSC-d02 lacking the tryptophanase EcTnaA and glutaredoxin EcNrdH functions; in the preparation method used in the specific embodiment of the present invention, the bacterial cell containing the enzymes participating in the reaction includes at least one of the following two combinations:

[0066] Combination a: pET28a-PsPAP (BL21 DE3); pET28a-SSC03 (S256P ΔCT) (BL21 DE3); pET28a-PkSerK (BL21 DE3);

[0067] Combination b: pET28a-PsPAP-SSC03 (S256P ∆CT) (BSSC-d02); pET28a-PkSerK (BSSC-d02);

[0068] The test results show that, compared with adding the reactants in two steps in combination a, adding the reactants in one step in combination b has simpler steps, higher product yields and faster reactions.

[0069] Furthermore, even though pET28a-PsPAP-SSC03 (S256P ∆CT) (BSSC-d02) contained two enzymes, the activities of the corresponding enzymes did not decrease compared with the expression of the two enzymes alone in BSSC-d02, and the growth rate of the bacteria did not change.

[0070] The present invention designs a new S-sulfo-L-cysteine ​​biosynthetic route, using serine (L-serine), adenosine diphosphate (ADP), and thiosulfate (thiosulfate) as substrates, and synthesizing SSC under the catalytic action of serine kinase (SerK) and phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase (CysK2). On the opened route, an ADP regeneration system composed of polyphosphate-AMP phosphotransferase (PAP), adenosine monophosphate (AMP) and sodium hexametaphosphate is further introduced to avoid the use of expensive ADP, thereby reducing the material cost of biosynthesizing SSC. On the basis of optimizing the reaction substrate concentration and conditions, a special Escherichia coli competent cell is constructed in which tryptophanase and glutaredoxin that degrade S-sulfo-L-cysteine ​​are knocked out, and the above-mentioned catalytic enzyme is expressed in the competent cell, which greatly improves the reaction conversion rate and the concentration of the product S-sulfo-L-cysteine.

[0071] The invention provides a new route for the biosynthesis of S-sulfo-L-cysteine. Experimental results show that, by using serine, AMP, sodium hexametaphosphate and sodium thiosulfate as substrates and using a crude enzyme solution containing serine kinase, polyphosphate-AMP phosphotransferase and phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase in a cell background in which tryptophanase and glutaredoxin have been knocked out, the S-sulfo-L-cysteine ​​synthesis process is simple, the product yield is high, the cost is low, and the S-sulfo-L-cysteine ​​has strong industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The biosynthetic route of S-sulfo-L-cysteine ​​(SSC) used in the present invention is shown;

[0073] Figure 2 Shows protein electrophoresis of serine kinases PkSerK and SaSbnI;

[0074] Figure 3 It shows the pH optimization of PkSerK enzyme reaction;

[0075] Figure 4 Shows the thermal stability of PkSerK enzyme;

[0076] Figure 5 Shows the temperature optimization of PkSerK enzyme reaction;

[0077] Figure 6 Shows the protein electrophoresis of polyphosphate-AMP phosphotransferase PaPAP and PsPAP;

[0078] Figure 7 The thermal stability of PaPAP enzyme and PsPAP enzyme is shown;

[0079] Figure 8It shows that PaPAP enzyme-PkSerK enzyme and PsPAP enzyme-PkSerK enzyme synthesize OPS;

[0080] Fig. 9 The figure shows the optimization of AMP concentration in the PsPAP enzyme-PkSerK enzyme reaction;

[0081] Fig.10 The figure shows the optimization of enzyme amount in the PsPAP enzyme-PkSerK enzyme reaction;

[0082] Fig.11 Shows pH optimization of SSC03 (S256P ∆CT) enzyme reaction;

[0083] Fig.12 Shows the temperature optimization of SSC03 (S256P ∆CT) enzyme reaction;

[0084] Fig.13 Shows the effect of BSSC-d01 / d02 host on OPS production;

[0085] Fig.14 Shows the effect of BSSC-d01 / d02 host on SSC generation. DETAILED DESCRIPTION

[0086] The present invention provides a method for synthesizing S-sulfo-L-cysteine ​​by multi-enzyme cascade catalysis. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the method. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention.

[0087] The amino acid sequence of the serine kinase PkSerK from Pyrococcus kodakaraensis is: MGVEKVPKYDIPTKKVDYVFIELDKMKPHEQLVQKELEAFIESVTGSGIFWKPMLLAKVPGEDMYLIVDGHHRWAGLQKLGAKRAPSVILDYFSDDVKVYTWYPAFKGDLNEVVERLKKEGLEVIEDPEAEEKAERGEIAFALVGEKSFAIPGGLEEQKKVSKVLDEMSVEGKIELIYYGLKEDAREDMAKGEIDYVFIRKAPTKEEVMELVKRGEVYSPKTTRHVLPFNPDKIDVKLEELF (SEQ ID NO: 1);

[0088] Nucleotide sequence of serine kinase PkSerK (codon-optimized): atgggagttgaaaaagtacccaagtatgatatcccaacgaaaaaggtggattacgttttcatcgaattggacaaaatgaaaccgcacgaacaactggtgcagaaggagctggaggctttcatcgaaagcgttaccggtagcggtattttttggaaaccgatgctgctggcgaaggtgccgggcgaggacatgtatctgattgttgatggtcatcatcgttgggcaggcttacagaagcttggtgcgaagcgcgcaccgtccgtgattttggactacttctctgatgatgtaaaggtctacacttggtatccggcattcaaaggcgatctcaatgaagttgttgaacgtctgaaaaaagagggcctggaggtcatcgaagatccggaagctgaagaaaaggcggagcgcggcgagatcgccttcgccctggttggtgagaaatcctttgcgattccgggcggtttggaggagcaaaagaaggtgagcaaagtgttggacgaaatgagcgttgaaggcaaaatcgagctgatttattatggtctgaaagaagacgcgcgtgaagacatggctaagggtgaaattgactacgtgtttatccgtaaggcgccaaccaaagaggaggtcatggaactggttaagcgcggtgaggtgtactcgccgaagaccacccgtcacgttctgccgtttaacccggataagatcgacgtgaaactggaggagttgttttaa (SEQ ID NO:2);

[0089] The amino acid sequence of the polyphosphate-AMP phosphotransferase PsPAP from Pseudomonas syringae pv. tomato is: MFESAEIGHAIDDDTYEAALPSLREALLEAQIDLHEQAKRQIIVLINGIEGAGKGETVKLLSEWMDPRLIEVRTFDQQTDEELAHPPVWRYWRQLPAKGRMGIFFGNWYSQMLQGRVHGQYKDAVLDQAISGAERLEKMLCDEGALIFKFWFHLSKKQMKLRLKTLKDDPLHSWRISPLDWQQSKTYDKFVRFGERVLRRTSRDYAPWHVIEGVDANYRSLTVGRLLLEGMQAAL NKVEPESSALTIGPLAIHNNERTLLDSLDLSLHLSKEDYQHELIAEQARLSGNLRDKRMKSHALVAVFEGNDAAGKGGAIRRVAAALDPRQYAIVPIAAPTQDERAQPYLWRFWRQIPARGKFTIFDRSWYGR VLVERVEGFCSESDWKRAYAEINDFEEQLTEAGVVVVKFWLAIDEQTQLERFQEREKIPFKRYKITEDDWRNRKKWPDYRQAVGDMVDRTSTEIAPWTLIEANDKRWARVKVLRTINEALEKAFARDKKK (SEQ ID NO:3);

[0090]

[0091] The modified phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase SSC03 (S256P ΔCT) amino acid sequence: MTHVLSIDTPRISRDHHTSCRYPVGAERFRPAPQQCRHAPDRYRRVGAMVGNTPVLWVGEPFGRGERGFWAKLEGTNPGGMKDRPAMHMVERAGLRGELLPGARIVESTSGTLGLGLALAGQVYRHPVTLVTDPGMEPIIRHMLAAYGARVDLVSQPHPVGGWQQARKDRVAELLAAEPDAWCPDQYSNPD NIDAYRPLALELLDQLGDIDVLVCSVGTGGHSAGVARVLRQHNPELELIGVDTIGSTIFGQPAGPRLMRGLGSSIYPRNVDYSAFTEVHWVAPAEAVWAARTLAATYYTSGGWSVGAVALVAGWAARTYPRGTRIAAVFPDGPQRYFDTIYNDDYCRDHHLLDTNPPSDPEEISTPTQAVVNRWTRCTTVVDPTATTT (SEQ ID NO:5);

[0092]

[0093] Amino acid sequence of serine kinase SaSbnI (1 - 240): MNHIHEHLKLVPVDKIDLHETFEPLRLEKTKSSIEADDFIRHPILVTAMQHGRYMVIDGVHRYTSLKALGCKKVPVQEIHETQYSISTWQHKVPFGVWWETLQQEHRLPWTTETRQEAPFITMCHGDTEQYLYTKDLGEAHFQVWEKVVASYSGCCSVERIAQGTYPCLSQQDVLMKYQPLSYKEIEAVVHKGETVPAGVTRFNISGRCLNLQVPLALLKQDDDVEQLRNWKQFLADKFA (SEQ ID NO:7);

[0094] Nucleotide sequence (codon-optimized) of serine kinase SaSbnI (1 - 240): atgaaccacatccatgaacacctgaaactggtccctgtagataaaattgacctgcatgaaactttcgaaccgctgcgcctggaaaagaccaaatcctccatcgaagcggatgatttcatccgtcatccgattctggttaccgcaatgcagcatggtcgttacatggtgatcgatggcgttcaccgctacacttctctgaaagctctgggctgcaaaaaagtgccggtgcaggaaatccacgagacccagtattccatcagcacctggcagcacaaagtgccgttcggcgtttggtgggagactctgcaacaggagcaccgtctgccgtggacgactgaaacgcgtcaggaagcaccgttcattacgatgtgccacggcgacactgaacagtacctgtacaccaaggacctgggtgaagcccactttcaggtttgggaaaaagtggtagcgtcctactctggctgctgctctgttgaacgcatcgcacagggtacctatccgtgtctgtctcagcaagacgtgctgatgaagtaccagccactgagctataaagaaatcgaggcggttgttcacaaaggtgaaaccgtgccggccggtgtaacccgtttcaacatctctggtcgttgcctgaatctgcaagttccactggctctgctgaagcaggacgacgatgtggaacagctgcgtaactggaaacagttcctggcggacaaattcgcgtaa (SEQ ID NO:8);

[0095] The amino acid sequence of the polyphosphate-AMP phosphotransferase PaPAP enzyme from Pseudomonas aeruginosa is: MFESAEVGHSIDKDTYEKAVIELREALLEAQFELKQQARFPVIILINGIEGAGKGETVKLLNEWMDPRLIEVQSFLRPSDEELERPPQWRFWRRLPPKGRTGIFFGNWYSQMLYARVEGHIKEAKLDQAIDAAERFERMLCDEGALLFKFWFHLSKKQLKERLKALEKDPQHSWKLSPLDWKQSEVYDRFVHYGERVLRRTSRDYAPWYVVEGADERYRALTVGRILLEGLQ AALATKERAKRQPHAAPLVSSLDNRGLLDSLDLGQYLDKDAYKEQLAAEQARLAGLIRDKRFRQHSLVAVFEGNDAAGKGGAIRRVTDALDPRQYHIVPIAAPTEEERAQPYLWRFWRHIPARRQFTIFDRSWY GRVLVERIEGFCAPADWLRAYGEINDFEEQLSEYGIIVVKFWLAIDKQTQMERFKEREKTPYKRYKITEDWRNRDKWDQYVDAVGDMVDRTSTEIAPWTLVEANDKRFARVKVLRTINDAIEAAYKKDK (SEQ ID NO:9);

[0096]

[0097] tnaA-gRNA sequence: ctggctcaataacacgaatg (SEQ ID NO: 11);

[0098] nrdh-gRNA sequence: aaacgggcgatggaaaaccg (SEQ ID NO: 12);

[0099] The overall scheme of the present invention is as follows:

[0100] Through experiments, the present invention screened out serine kinase PkSerK from Pyrococcus kodakaraensis, polyphosphate-AMP phosphotransferase PsPAP from Pseudomonas syringae pv. tomato and modified phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase SSC03 (S256P ∆CT) for the synthesis of S-sulfo-L-cysteine.

[0101] The nucleotides encoding the above enzymes were codon-optimized and then fully synthesized. The synthesized genes were then constructed into pET28a vectors, transformed into BL21 (DE3) expression hosts, and then punctured or preserved in glycerol tubes (Beijing Qingke Biotechnology). The punctured or glycerol tube-preserved bacteria were cultured in LB medium (Kan) at two levels at 16°C with 0.1 mM IPTG to induce expression, and the cells were collected by centrifugation. 1 g of wet cells were added with a disruption buffer (20 mM Na2HPO4 / NaH2PO4, 100 mM NaCl, pH 7.6), resuspended, and then disrupted to obtain a crude enzyme solution.

[0102] First, PkSerK enzyme was used to synthesize the intermediate product phosphoserine (O-phospho-L-serine, OPS) with serine and ADP as substrates. Single factor optimization obtained the suitable reaction pH 5.0~8.0 (optimum pH 6.0) and suitable temperature 38~52℃ (optimum temperature 45℃). After introducing the ADP regeneration system composed of PsPAP enzyme, sodium hexametaphosphate, and adenosine monophosphate (AMP), the appropriate enzyme amount of PkSerK enzyme was optimized to be 0.5%~10% (optimum enzyme amount 1%, v / v the same below), the appropriate enzyme amount of PsPAP enzyme was 5%~20% (optimum enzyme amount 15%), the appropriate concentration of serine was 100~200mM (optimum 200mM), the appropriate concentration of AMP was 1~10mM (optimum 5mM), the appropriate concentration of magnesium chloride hexahydrate was 20~60mM (optimum 40mM), and the appropriate concentration of sodium hexametaphosphate was 35~65mM (optimum 65mM).

[0103] Then, the OPS-containing reaction solution obtained by the aforementioned PkSerK enzyme and PsPAP enzyme dual enzyme system and thiosulfate (one of sodium thiosulfate, potassium thiosulfate, and ammonium thiosulfate) were used as substrates, and SSC03 (S256P ∆CT) enzyme was added to synthesize the final product SSC, and the appropriate pH was optimized to 6.0~8.0 (optimum pH 7.0) and the appropriate temperature was 30~45℃ (optimum 38℃).

[0104] Then, the nucleotide sequences of PsPAP enzyme and SSC03-S256P ∆CT enzyme were inserted into the co-expression vector pETDuet-1, and then the two plasmids pETDuet-PsPAP-SSC03 (S256P ∆CT) and pET28a-PkSerK were respectively transferred into Escherichia coli competent cells BSSC-d02 in which tryptophanase EcTnaA and glutaredoxin EcNrdH were knocked out for culture and induced expression.

[0105] Finally, prepare a reaction solution with 100-200 mM serine (optimum 200 mM), 20-60 mM magnesium chloride hexahydrate (optimum 60 mM), 35-65 mM sodium hexametaphosphate (optimum 65 mM), 1-10 mM AMP (optimum 5 mM), and 1.1 eq-1.3 eq thiosulfate equivalent (optimum 1.3 eq), adjust the pH to 6.0-8.0 (optimum pH 7.0), make up the volume, preheat to 25-45 °C (optimum 38 °C), add 5%-20% (optimum 12%) PsPAP+SSC03 (S256P ∆CT) enzyme and 0.5%-10% (optimum 1%) PkSerK enzyme under low-speed stirring, and start the reaction timer.

[0106] In the present invention, vt% is the volume fraction, which refers to the ratio of the volume of the solute to the total volume of the solution, and is calculated as follows: volume fraction (vt%) = (volume of the solute / total volume of the solution) × 100%; in the present invention, vt% is equivalent to %v / v or v / v%.

[0107] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0108] Example 1 Obtaining crude enzyme solution

[0109] Through experiments, the present invention screened out serine kinase PkSerK from Pyrococcus kodakaraensis, polyphosphate-AMP phosphotransferase PsPAP from Pseudomonas syringae pv. tomato and modified phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase SSC03 (S256P ∆CT) for the synthesis of S-sulfo-L-cysteine.

[0110] The nucleotides encoding the above enzymes were codon-optimized and then fully synthesized. Then, the synthesized genes were constructed into pET28a vectors, transformed into BL21 (DE3) expression hosts, and then punctured or stored in glycerol tubes (Beijing Qingke Biotechnology). The punctured or glycerol tube-stored strains were cultured in LB medium (Kan) at two levels at 16°C with 0.1 mM IPTG to induce expression, and the cells were collected by centrifugation. A disruption buffer (20 mM Na2HPO4 / NaH2PO4, 100 mM NaCl, pH 7.6) was added according to 1 g of wet cells, resuspended, and then disrupted to obtain a crude enzyme solution for subsequent experiments (the crude enzyme solution of other strains of enzymes not mentioned here was obtained according to the operation here. Unless the source of the above enzyme is specifically stated, the source of the enzyme mentioned in the abbreviated form later corresponds to that here).

[0111] Example 2 Synthesis of OPS (O-phospho-L-serine) using serine and ADP as substrates

[0112] Weigh 0.53 g of serine (100 mM), 3.53 g of adenosine diphosphate disodium salt (ADP-Na2) (150 mM), and 0.3 g of magnesium chloride hexahydrate (30 mM) and dissolve them in 40 mL of pure water. Adjust the pH to 7.5 and make the volume to 45 mL. After preheating at 38 °C, add 5 mL of PkSerKBL21 (DE3) crude enzyme solution (see protein expression). Figure 2 ).

[0113] A sample was taken from the reaction solution and diluted 10 times with 60% acetonitrile. The concentration of OPS was detected by high performance liquid chromatography (HPLC). After reacting for 15 minutes, 81.63 mM OPS was generated.

[0114] 2. Optimization of PkSerK enzyme reaction conditions in the process of synthesizing OPS using serine and ADP as substrates

[0115] 1. Optimization of pH of PkSerK enzyme reaction

[0116] Weigh 1.58g of serine (100mM), 2.83g of adenosine diphosphate disodium salt (ADP-Na2) (40mM), and 0.61g of magnesium chloride hexahydrate (20mM) and dissolve them in 120mL of pure water. Divide into 5 portions, adjust the pH to 5, 6, 7, 8, and 9 respectively, and make the volume to 29.85mL. After preheating at 38℃, add 0.15mL of PkSerK BL21 (DE3) crude enzyme solution. Take a sample and detect the generation of OPS by HPLC. Figure 3 As shown, the product OPS concentration in the pH 6 group was the highest, and 9.59 mM OPS could be generated after 5 minutes of reaction with 0.5% PkSerK enzyme.

[0117] 2. Thermal stability test of PkSerK enzyme

[0118] The crude enzyme solution of PkSerK BL21 (DE3) was placed in a water bath at 38°C, 50°C, and 65°C for 10 min, and then centrifuged and the supernatant was subjected to protein electrophoresis. The target bands were semi-quantitatively analyzed using ImageJ software. Figure 4 As shown, the PkSerK enzyme has good thermal stability, with only a 15% to 20% loss at 65°C.

[0119] 3. Optimization of PkSerK enzyme reaction temperature

[0120] Weigh 1.26g of serine (100mM), 2.26g of adenosine diphosphate disodium salt (ADP-Na2) (40mM), and 0.49g of magnesium chloride hexahydrate (20mM) and dissolve them in 100mL of pure water. Adjust the pH to 6.0 and make the volume to 119.4mL. Divide the mixture into 4 portions, preheat at 30℃, 38℃, 45℃, and 52℃ respectively, and add 0.15mL of PkSerK BL21 (DE3) crude enzyme solution. Take a sample and test the generation of OPS by HPLC. Figure 5 As shown, the product OPS concentration in the 45°C group was the highest, and 10.62 mM OPS could be generated after 5 minutes of reaction.

[0121] Example 2 Synthesis of OPS using serine, AMP and sodium hexametaphosphate as substrates

[0122] 1. Synthesis of OPS using serine, AMP and sodium hexametaphosphate as substrates

[0123] Weigh 1.05 g of serine (100 mM), 0.20 g of adenosine monophosphate disodium salt (AMP-Na2) (5 mM), 0.81 g of magnesium chloride hexahydrate (40 mM), and 2.14 g of sodium hexametaphosphate (35 mM) and dissolve them in 80 mL of pure water. Adjust the pH to 6.0 and make up to 94 mL. Divide into two portions and preheat at 38 °C before adding 2.5 mL of crude enzyme solution of PsPAP enzyme BL21 (DE3) to each portion (see protein expression). Figure 6) and PkSerKBL21 (DE3) crude enzyme solution 0.5 mL, polyphosphate-AMP phosphotransferase PaPAP enzyme BL21 (DE3) crude enzyme solution 2.5 mL from Pseudomonas aeruginosa (protein expression see Figure 6 ) and PkSerK BL21 (DE3) crude enzyme solution 0.5 mL. Because both PsPAP enzyme and PaPAP enzyme cannot tolerate 45℃ heat treatment ( Figure 7 ), the reaction temperature was adjusted to 38° C. Samples were taken and HPLC was used to detect the generation of OPS.

[0124] like Figure 8 As shown, the combination of 5vt% PsPAP enzyme and 1vt% PkSerK enzyme produced OPS faster, with 72.63mM OPS produced after 1h of reaction.

[0125] 2. Optimization of reaction conditions in the synthesis of OPS using serine, AMP and sodium hexametaphosphate as substrates

[0126] 1. Optimizing the AMP concentration of the PsPAP enzyme-PkSerK enzyme reaction

[0127] Weigh 1.26g of serine (100mM), 0.98g of magnesium chloride hexahydrate (40mM), and 2.57g of sodium hexametaphosphate (35mM) and dissolve them in 100mL of pure water. Divide them into 4 portions, add 0.01g (1mM), 0.02g (2mM), 0.06g (5mM), and 0.12g (10mM) of adenosine monophosphate disodium salt (AMP-Na2), respectively, adjust the pH to 6.0 and make the volume to 26.7mL. After preheating at 38℃, add 3mL of PsPAPBL21 (DE3) crude enzyme solution and 0.3mL of PkSerK BL21 (DE3) crude enzyme solution. Take samples and detect the generation of OPS by HPLC.

[0128] like Fig. 9 As shown, under the combination of 10vt% PsPAP enzyme and 1vt% PkSerK enzyme, the OPS production rate of the 1mM AMP group was significantly lower than that of the 2~10mM group.

[0129] 2. Optimizing the amount of PsPAP and PkSerK enzymes at 200 mM serine

[0130] Prepare the reaction solution according to Table 1, weigh serine, adenosine monophosphate disodium salt (AMP-Na2), magnesium chloride hexahydrate (MgCl2·6H2O), and sodium hexametaphosphate and dissolve them in pure water, adjust the pH to 6.0 and make up to volume, preheat at 38°C, add different volumes of PsPAP BL21 (DE3) crude enzyme solution and PkSerK BL21 (DE3) crude enzyme solution, and add water to make up the reaction volume to 30 mL. Take samples and detect the generation of OPS by HPLC.

[0131] Table 1

[0132]

[0133] like Fig.10 As shown, the combination of 15vt% PsPAP enzyme and 1vt% PkSerK enzyme is optimal, and the conversion rate is close to 100% after 3h of reaction.

[0134] Example 3 Synthesis of SSC using serine, AMP, sodium hexametaphosphate and sodium thiosulfate as substrates

[0135] 1. Step-by-step synthesis of SSC using serine, AMP, sodium hexametaphosphate and sodium thiosulfate as substrates

[0136] Weigh 4.20g of serine (200mM), 0.39g of adenosine monophosphate disodium salt (AMP-Na2) (5mM), 1.63g of magnesium chloride hexahydrate (40mM), and 7.95g of sodium hexametaphosphate (65mM) and dissolve them in 160mL of pure water, adjust the pH to 6.0 and make the volume to 168mL, preheat at 38℃, add 30mL of PsPAP BL21 (DE3) crude enzyme solution and 2mL of PkSerK BL21 (DE3) crude enzyme solution. After 3h of reaction, the OPS concentration no longer increases and 190.75mM is generated.

[0137] Weigh 7.80 g (210 mM, 1.3 eq) of sodium thiosulfate and add it to the above reaction solution containing OPS to dissolve, adjust the pH to 7.0, add 35 mL of SSC03 (S256P ∆CT) BL21 (DE3) crude enzyme solution, and react at 25°C.

[0138] A sample was taken from the reaction solution and diluted 10 times with 60% acetonitrile. The concentration of SSC was detected by high performance liquid chromatography (HPLC). After 8 hours of reaction, 76.30 mM was generated, and the conversion rate was 47%.

[0139] 2. Optimization of the step-by-step synthesis of SSC using serine, AMP, sodium hexametaphosphate, and sodium thiosulfate as substrates

[0140] 2.1 Optimizing the pH of the SSC03 (S256P ∆CT) enzyme reaction using OPS reaction solution as substrate

[0141] Dilute the OPS reaction solution to 167mM, take 108mL and add 3.13g sodium thiosulfate (165mM, 1.1eq) to dissolve, divide into 4 portions, adjust pH to 5, 6, 7, 8 respectively, preheat at 25℃ and then add 3mL of SSC03 (S256P ∆CT) BL21 (DE3) crude enzyme solution respectively. Take samples and detect the generation of SSC by HPLC. Fig.11 As shown, the product SSC concentration in the pH 7.0 group was the highest, and the intermediate product OPS150mM. When the enzyme amount of SSC03 (S256P ∆CT) BL21 (DE3) was 10vt%, SSC 50.0mM was generated after 4h of reaction.

[0142] 2.2 Optimizing the temperature of SSC03 (S256P ∆CT) enzyme reaction using OPS reaction solution as substrate

[0143] Dilute the OPS reaction solution to 167mM, take 108mL and add 3.13g sodium thiosulfate (165mM, 1.1eq) to dissolve, divide into 4 portions, adjust the pH to 7.0, preheat at 25℃, 30℃, 38℃, and 45℃ respectively, and add 3mL of SSC03 (S256P ∆CT) BL21 (DE3) crude enzyme solution. Take a sample and detect the generation of SSC by HPLC. Fig.12 As shown, the SSC concentration of the product in the 38°C group was the highest, and 76.6 mM SSC could be generated after 2 h of reaction.

[0144] Example 4 Further Optimization - Construction of Optimized Expression Strains for PsPAP, SSC03 (S256P ∆CT) and PkSerK

[0145] 1. Construction of optimized expression strains for PsPAP enzyme, SSC03-S256P ∆CT enzyme and PkSerK enzyme

[0146] PsPAP and SSC03 (S256P ∆CT) were expressed as fusion proteins, and PkSerK was expressed alone; their expressions were carried out in Escherichia coli knocked out for tryptophanase EcTnaA and glutaredoxin EcNrdH.

[0147] The steps for constructing E. coli competent cells BSSC-d02 with knockout of tryptophanase EcTnaA and glutaredoxin EcNrdH are as follows:

[0148] First, construct the pGRB-tnaA-gRNA plasmid containing the tnaA-gRNA sequence, and electroporate it together with the ∆tnaA donar DNA into the BL21 (DE3) competent cells containing the pEcCas plasmid. Then, plate and screen the E. coli cells with successful knockout of the tryptophan enzyme EcTnaA. Then, obtain the E. coli cells BSSC-d01-pEcCas with the pGRB-tnaA-gRNA plasmid eliminated, and then obtain the E. coli cells BSSC-d01 with the pEcCas plasmid eliminated. Make them into competent cells for later use.

[0149] The pGRB-nrdh-gRNA plasmid containing the nrdh-gRNA sequence was then constructed and electroporated into the E. coli competent cells BSSC-d01-pEcCas together with the ∆nrdh donar DNA. The E. coli cells in which the tryptophanase EcTnaA and the glutaredoxin EcNrdH were successfully knocked out were screened by plating. Then, the E. coli cells BSSC-d02 (whose tryptophanase EcTnaA and glutaredoxin EcNrdH functions were lost) that had eliminated the pGRB-nrdh-gRNA plasmid and the pEcCas plasmid were obtained and made into competent cells for use.

[0150] Then, the nucleotide sequences of PsPAP enzyme and SSC03 (S256P ∆CT) enzyme were co-inserted into the vector pETDuet-1 to obtain the plasmid pETDuet-PsPAP-SSC03 (S256P ∆CT); the plasmids pET28a-PkSerK and pETDuet-PsPAP-SSC03 (S256P ∆CT) in Example 1 and the plasmid were respectively transferred into BSSC-d02 for culture and induction of expression, and then the bacteria were collected by centrifugation. A crushing buffer (20 mM Na2HPO4 / NaH2PO4, 100 mM NaCl, pH7.6) was added according to 1 g of wet bacteria, and the cells were resuspended and crushed to obtain a crude enzyme solution.

[0151] 2. Verify the influence of BSSC-d01 / d02 host on OPS generation

[0152] Weigh 3.15 g of serine (200 mM), 0.29 g of adenosine monophosphate disodium salt (AMP-Na2) (5 mM), 1.22 g of magnesium chloride hexahydrate (40 mM), and 5.96 g of sodium hexametaphosphate (65 mM) and dissolve them in 120 mL of pure water. Adjust the pH to 6.0 and make the volume to 126 mL. Divide into 3 portions, preheat at 38 °C, add 7.5 mL of PsPAP BL21 (DE3) crude enzyme solution and 0.5 mL of PkSerK BL21 (DE3) crude enzyme solution, 7.5 mL of PsPAP BSSC-d01 crude enzyme solution and 0.5 mL of PkSerK BSSC-d01 crude enzyme solution, 7.5 mL of PsPAP BSSC-d02 crude enzyme solution and 0.5 mL of PkSerK BSSC-d02 crude enzyme solution, respectively. Take samples and detect the generation of OPS by HPLC.

[0153] like Fig.13 As shown, knockout of the tryptophanase EcTnaA in the host BSSC-d01 inhibited the production of OPS, while knockout of the tryptophanase EcTnaA and glutaredoxin EcNrdH in the host BSSC-d02 accelerated the production of OPS.

[0154] 3. Verify the influence of BSSC-d01 / d02 host on SSC generation

[0155] Weigh 2.78 g of phosphoserine (OPS) (100 mM) and 3.56 g of sodium thiosulfate (150 mM) and dissolve them in 120 mL of pure water. Adjust the pH to 7.0 and make up to 135 mL. Divide into three portions and preheat at 25°C. Add 5 mL of SSC03 (S256P ∆CT) BL21 (DE3) crude enzyme solution, 5 mL of SSC03 (S256P ∆CT) BSSC-d01 crude enzyme solution, and 5 mL of SSC03 (S256P ∆CT) BSSC-d02 crude enzyme solution. Take samples and detect the generation of SSC by HPLC.

[0156] like Fig.14 As shown, the crude enzyme solution of SSC03 (S256P ∆CT) expressed in hosts BSSC-d01 and BSSC-d02 can significantly increase the production of SSC compared to the original host BL21 (DE3).

[0157] Example 5 One-step synthesis of SSC using serine, AMP, sodium hexametaphosphate and sodium thiosulfate as substrates

[0158] 1. Small system test

[0159] Weigh 2.10g of serine (200mM), 3.48g of sodium thiosulfate (220mM, 1.1eq), 0.20g of adenosine monophosphate disodium salt (AMP-Na2) (5mM), 0.81g of magnesium chloride hexahydrate (40mM), and 3.98g of sodium hexametaphosphate (65mM) and dissolve them in 80mL of pure water. Adjust the pH to 7.0 and make the volume to 89mL. After preheating at 38℃, add 10mL of PsPAP+SSC03 (S256P ∆CT) BSSC-d02 crude enzyme solution and 1mL of PkSerK BSSC-d02 crude enzyme solution. Sample HPLC was used to detect the generation of SSC. The results showed that 154.77mM SSC was generated after 2h of reaction.

[0160] 2. System scale-up test

[0161] Weigh 21.02g of serine (200mM), 41.11g of sodium thiosulfate (260mM, 1.3eq), 1.96g of adenosine monophosphate disodium salt (AMP-Na2) (5mM), 12.20g of magnesium chloride hexahydrate (60mM), and 39.77g of sodium hexametaphosphate (65mM) and dissolve them in 800mL of pure water, adjust the pH to 7.0 and make the volume to 870mL, preheat at 38℃ and add 120mL of PsPAP+SSC03 (S256P ∆CT) BSSC-d02 crude enzyme solution and 10mL of PkSerK BSSC-d02 crude enzyme solution. Reaction for 6h to generate 174.92mM SSC, with a conversion rate of 87.5%.

[0162] Comparative Example 1 OPS was synthesized using serine kinase SaSbnI (1-240) from Staphylococcus aureus with serine and adenosine triphosphate (ATP) as substrates

[0163] Weigh 0.53 g of serine (100 mM), 2.76 g of adenosine triphosphate disodium salt (ATP-Na2) (100 mM), and 0.3 g of magnesium chloride hexahydrate (30 mM) and dissolve them in 40 mL of pure water. Adjust the pH to 7.5 and make the volume to 45 mL. After preheating at 38 °C, add 5 mL of SaSbnI (1-240) BL21 (DE3) crude enzyme solution (see protein expression). Figure 2 ).

[0164] The reaction solution was sampled and diluted 10 times, and then the OPS concentration was detected by HPLC. No OPS was detected after 6 hours of reaction, which may be due to protein expression in the precipitate.

[0165] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A combined enzyme, characterized in that include: Phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase, a serine kinase from Pyrococcus furiosus, and a polyphosphate-AMP phosphotransferase 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 Including 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: The method comprises synthesizing S-sulfo-L-cysteine ​​by 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: The method comprises the following steps: serine kinase, polyphosphate-AMP phosphotransferase and phosphoserine-dependent S-sulfo-L-cysteine ​​synthetase catalyze a 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.

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