Fusion protein and application thereof in preparation of L-cysteine

Abstract

The invention relates to the technical field of biology, in particular to a fusion protein and application thereof in preparation of L-cysteine. The invention provides a fusion protein, the fusion protein contains an alpha subunit of tryptophan synthetase TrpS and a beta subunit of tryptophan synthetase TrpS, and has tryptophan synthetase catalytic activity, through a genetic engineering method, on the premise that respective spatial conformations of the alpha subunit and the beta subunit of the tryptophan synthetase TrpS are retained, the fusion protein can be used for preparing the tryptophan synthetase. TrpB and TrpA are connected into a single fusion protein TrpSL (TrpB-Linker-TrpA) through Linker, dimer dissociation is avoided, the immobilized fusion enzyme can be repeatedly used only through simple cleaning after reaction, the industrial production efficiency is remarkably improved, and the economic cost is reduced.

Description

TECHNICAL FIELD

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

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

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

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

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

[0006] To this end, the first aspect of the present application provides a fusion protein. According to an embodiment of the present application, the fusion protein comprises an alpha subunit of tryptophan synthase TrpS, a beta subunit of tryptophan synthase TrpS, and a connecting peptide. The amino acid sequence of the connecting peptide is shown in SEQ ID NO: 20-22.

[0007] SEQ ID NO: 20 ​AEAAAKEAAAKAAVLEYLTAEILELAAAVLEYLTAEILELAGGAAVLEYLTAEILELAAAVLEYLTAEILELA SEQ ID NO: 21 ASAKMLHEMQRKNEQGGGGAAVLEYLTAEILELAAAVLEYLTAEILELAGGGASAKMLHEMQRKNEQ SEQ ID NO: 22 ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK The present application discloses a fusion protein, an expression vector, a host cell and an application thereof. Enterobacteriaceae The present application discloses a fusion protein, an expression vector, a host cell and an application thereof.

[0008] According to an embodiment of the present application, the C-terminal of the beta subunit of the tryptophan synthetase TrpS is connected to the alpha subunit of the tryptophan synthetase TrpS through the connecting peptide.

[0009] According to an embodiment of the present application, the amino acid sequence of the alpha subunit of the tryptophan synthetase TrpS is shown as SEQ ID NO: 1. The amino acid sequence of the beta subunit of the tryptophan synthetase TrpS is shown as SEQ ID NO: 2.

[0010] The present application discloses a fusion protein, an expression vector, a host cell and an application thereof.

[0011] The present application discloses a fusion protein, an expression vector, a host cell and an application thereof.

[0012] The present application discloses a fusion protein, an expression vector, a host cell and an application thereof.

[0013] The fifth aspect of the present invention provides use of the fusion protein of the first aspect, the isolated nucleic acid of the second aspect, the expression vector of the third aspect, and the host cell of the fourth aspect in preparing L-cysteine ​​or L-cystine.

[0014] A sixth aspect of the present invention provides a method for immobilizing tryptophan synthase. According to an embodiment of the present invention, the method comprises: (1) Performing ion exchange equilibrium on the resin to obtain a balanced resin; (2) contacting the equilibrated resin with the fusion protein described in the first aspect, and / or the fusion protein expressed by at least one of the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, and the host cell described in the fourth aspect, so that the fusion protein is fixed on the resin.

[0015] According to an embodiment of the present invention, in step (2), when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 20, the resin is ES-103B resin.

[0016] According to an embodiment of the present invention, in step (2), when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 21, the resin is LX-109S resin.

[0017] According to an embodiment of the present invention, in step (2), when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 22, the resin is ES-103B resin.

[0018] A seventh aspect of the present invention provides a tryptophan synthase immobilized resin column. According to an embodiment of the present invention, the tryptophan synthase immobilized resin column comprises: Resin column and fusion protein immobilized thereon, The fusion protein is selected from the fusion protein described in the first aspect, and / or a fusion protein expressed by at least one of the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, and the host cell described in the fourth aspect.

[0019] According to an embodiment of the present invention, when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 20, the resin is ES-103B resin.

[0020] According to an embodiment of the present invention, when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 21, the resin is LX-109S resin.

[0021] According to an embodiment of the present invention, when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 22, the resin is ES-103B resin.

[0022] An eighth aspect of the present invention provides a method for preparing L-cysteine. According to an embodiment of the present invention, the method comprises: The immobilized enzyme is obtained by the method for immobilizing tryptophan synthase described in the sixth aspect and / or the tryptophan synthase immobilized resin column described in the seventh aspect is used to catalyze the synthesis of L-cysteine ​​using L-serine and a sulfur donor as substrates.

[0023] A ninth aspect of the present invention provides a method for preparing L-cystine. According to an embodiment of the present invention, the method comprises: S1: obtaining an immobilized tryptophan synthase by the method for immobilizing tryptophan synthase described in the sixth aspect and / or utilizing the tryptophan synthase immobilized resin column described in the seventh aspect, catalyzing the synthesis of L-cysteine ​​using L-serine and a sulfur donor as substrates; S2: Using the L-cysteine ​​synthesized in step S1 as a substrate, an oxidation reaction is performed to synthesize L-cystine.

[0024] The present invention successfully constructed a linker-connected tryptophan synthase fusion that can be used to synthesize L-cysteine. It cleverly solved the problem that TrpB and TrpA in tryptophan synthase could not complete multiple rounds of reactions after resin immobilization, opened up new ideas for the immobilization reaction of dual enzymes or even multi-enzyme complexes, and significantly improved the efficiency and economy of industrial production.

[0025] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which: Figure 1 The plasmid map of TrpA constructed into the E. coli dual expression vector pETDuet-1 with two promoters is shown; Figure 2 The plasmid map of TrpB constructed into the E. coli dual expression vector pETDuet-1 with two promoters is shown; Figure 3 The three-dimensional structure of the protein was obtained by protein modeling of the TrpS sequence using the new artificial intelligence software RoseTTA Fold2 online software. Figure 4The results of SDS-PAGE gel electrophoresis of the supernatant and precipitate obtained after culturing the genetically engineered mutant LK1-LK10 bacteria are shown. DETAILED DESCRIPTION

[0027] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0028] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0029] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0030] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present invention belongs.

[0031] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0032] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0033] According to a specific embodiment of the present invention, the present invention provides a fusion protein, which comprises the α subunit of tryptophan synthase TrpS, the β subunit of tryptophan synthase TrpS and a connecting peptide. The amino acid sequence of the connecting peptide is shown in SEQ ID NO: 20-22.

[0034] It should be noted that in the fusion protein provided by the present invention, the α subunit of tryptophan synthase TrpS and the β subunit of tryptophan synthase TrpS are connected by a connecting peptide shown in SEQ ID NO: 20-22. There is no particular limitation on the connection mode. For example, the N-terminus or C-terminus of the α subunit of tryptophan synthase TrpS can be connected to the C-terminus or N-terminus of the β subunit of tryptophan synthase TrpS through a connecting peptide, or the N-terminus or C-terminus of the β subunit of tryptophan synthase TrpS can be connected to the C-terminus or N-terminus of the α subunit of tryptophan synthase TrpS through a connecting peptide. According to a preferred embodiment of the present invention, the C-terminus of the β subunit of tryptophan synthase TrpS is connected to the α subunit of tryptophan synthase TrpS through the connecting peptide.

[0035] It should be noted that the amino acid sequences of the α and β subunits of tryptophan synthase TrpS are not specifically shown, including but not limited to those derived from Enterobacteriaceae The tryptophan synthase TrpS is composed of an α subunit and a β subunit, wherein the amino acid sequence of the α subunit TrpA (GenBank: WP_000443067.1) is shown in the sequence list as SEQ ID NO: 1, and the amino acid sequence of the β subunit TrpB (GenBank: WP_000209520.1) is shown in the sequence list as SEQ ID NO: 2. Alternatively, the amino acid sequences of the α subunit and the β subunit of the tryptophan synthase TrpS can also be the same as those derived from Enterobacteriaceae The amino acid sequence of the tryptophan synthase TrpS of the present invention has a tryptophan synthase derivative with more than 80% or more than 90% homology. These tryptophan synthase derivatives maintain the catalytic ability and enzyme activity of the tryptophan synthase itself, but the amino acid sequence is slightly different from the amino acid sequence of the wild-type tryptophan synthase TrpS. Enterobacteriaceae The tryptophan synthase TrpS and its derivatives are all included in the scope of the wild-type tryptophan synthase described in the present invention.

[0036] The present invention is based on Enterobacteriaceae The tryptophan synthase TrpS (the amino acid sequences of the α subunit TrpA and the β subunit TrpB are shown in SEQ ID NOs: 1 and 2, respectively) are connected through a specific linker, which avoids dimer dissociation while ensuring enzyme activity. The immobilized fusion enzyme only needs simple cleaning after the reaction and can be reused for multiple rounds, significantly improving the efficiency of industrial production and reducing economic costs.

[0037] According to a specific embodiment of the present invention, the amino acid sequence of the α subunit of tryptophan synthase TrpS is shown in SEQ ID NO: 1; The amino acid sequence of the β subunit of the tryptophan synthase TrpS is shown in SEQ ID NO: 2.

[0038] According to a specific embodiment of the present invention, the present invention provides a nucleic acid sequence encoding the fusion protein.

[0039] According to a specific embodiment of the present invention, the present invention provides a recombinant vector containing a nucleic acid sequence encoding the fusion protein, and a genetically engineered bacterium containing a nucleic acid sequence encoding the fusion protein. Specifically, the vector can be any expression vector, including but not limited to any one of the pET expression vector, the pCW expression vector, the pUC expression vector, or the pPIC9k expression vector. The host cell of the genetically engineered bacterium can be any suitable host cell, including but not limited to Escherichia coli, Bacillus subtilis, Streptomyces, or Pichia pastoris.

[0040] According to a specific embodiment of the present invention, the present invention also relates to the use of the fusion protein, the isolated nucleic acid, the expression vector, and the host cell in the preparation of L-cysteine ​​or L-cystine.

[0041] According to a specific embodiment of the present invention, the present invention provides a method for immobilizing tryptophan synthase, comprising: (1) Performing ion exchange equilibrium on the resin to obtain a balanced resin; (2) contacting the equilibrated resin with the aforementioned fusion protein, and / or the fusion protein expressed by at least one of the aforementioned isolated nucleic acid, the aforementioned expression vector, and the aforementioned host cell, so that the fusion protein is fixed on the resin.

[0042] There is no particular limitation on the cleaning solution used in the ion exchange equilibration process, and it may be, for example, PBK solution, PBS solution, etc. All types of cleaning solutions that can perform ion exchange equilibration and are available in the art are within the scope of protection of the present invention.

[0043] According to a specific embodiment of the present invention, in step (2), when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 20, the resin is ES-103B resin.

[0044] According to a specific embodiment of the present invention, in step (2), when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 21, the resin is LX-109S resin.

[0045] According to a specific embodiment of the present invention, in step (2), when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 22, the resin is ES-103B resin.

[0046] According to a specific embodiment of the present invention, the present invention provides a tryptophan synthase immobilized resin column, comprising: Resin column and fusion protein immobilized thereon, The fusion protein is selected from the aforementioned fusion proteins, and / or fusion proteins expressed by at least one of the aforementioned isolated nucleic acid, the aforementioned expression vector, and the aforementioned host cell.

[0047] According to a specific embodiment of the present invention, when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQID NO: 20, the resin is ES-103B resin; when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQID NO: 21, the resin is LX-109S resin; when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQID NO: 22, the resin is ES-103B resin.

[0048] According to a specific embodiment of the present invention, the present invention provides a method for preparing L-cysteine, comprising: The immobilized enzyme is obtained by the aforementioned method for immobilizing tryptophan synthase and / or the aforementioned tryptophan synthase immobilized resin column is used to catalyze the synthesis of L-cysteine ​​using L-serine and a sulfur donor (such as sodium bisulfide) as substrates.

[0049] It should be noted that there are no particular limitations on the conditions for preparing L-cysteine ​​using tryptophan synthase, and these conditions can be those commonly used in the art for enzymatic catalysis. Regardless of whether optimized or conventional reaction conditions are employed, the fusion protein of the present invention enables multiple rounds of catalytic reactions compared to unmodified wild-type tryptophan synthase. This cleverly addresses the issue of TrpB and TrpA in tryptophan synthase being unable to complete multiple rounds of reactions after resin immobilization, opens new avenues for immobilized reactions involving dual or even multi-enzyme complexes, and significantly improves the efficiency and cost-effectiveness of industrial production.

[0050] According to a specific embodiment of the present invention, the present invention provides a method for preparing L-cystine, comprising: S1: obtaining an immobilized tryptophan synthase by the aforementioned method for immobilizing tryptophan synthase and / or utilizing the aforementioned tryptophan synthase immobilized resin column to catalyze the synthesis of L-cysteine ​​using L-serine and a sulfur donor (e.g., sodium bisulfide) as substrates; S2: Using the L-cysteine ​​synthesized in step S1 as a substrate, an oxidation reaction is performed to synthesize L-cystine.

[0051] It should be noted that there are no particular limitations on the conditions for preparing L-cystine using the tryptophan synthase mutant, and these conditions can be any known condition for oxidizing L-cysteine ​​to L-cystine. Regardless of whether optimized conditions or conventional reaction conditions are employed, the fusion protein of the present invention enables multiple rounds of catalytic reactions compared to unmodified wild-type tryptophan synthase. This cleverly addresses the issue of TrpB and TrpA in tryptophan synthase being unable to complete multiple rounds of reactions after resin immobilization, and opens new avenues for the immobilization of dual-enzyme or even multi-enzyme complexes.

[0052] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0053] Materials and methods 1) Materials: Plasmid pET28a was purchased from Changsha Abiwei Biotechnology Co., Ltd.; restriction enzymes such as BamHI and HindIII, high-fidelity enzyme premix, and one-step rapid cloning kit were purchased from Shanghai Yisheng Biotechnology Co., Ltd.; synthetic primers, E. coli BL21 (DE3) competent cells, DNA marker, plasmid extraction kit, DNA gel recovery and purification kit, kanamycin sulfate, ampicillin, isopropyl-β-d-thiogalactopyranoside, L-cysteine, and L-cystine standard were purchased from Shanghai Sangon Biotechnology Co., Ltd.; HisSep Ni-NTA Agarose Resin (His-tagged protein agarose purification resin) was purchased from Shanghai Yisheng Biotechnology Co., Ltd., centrifugal ultrafiltration tubes (10 kDa) were purchased from Millipore, and immobilized enzyme carrier resins such as LX-107S were purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.; all chemical reagents were of analytical grade from Chinese medicine. For plasmid extraction procedures, refer to the instructions for the plasmid extraction kit; for DNA gel recovery procedures, refer to the instructions for the DNA gel recovery and purification kit; for DNA fragment ligation procedures, refer to the instructions for the one-step rapid cloning kit; LB medium (g / L): 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, sterilized at 121°C for 20 min. 3) Tryptophan synthase TrpS substrate reaction solution: 200 mM L-serine, 240 mM sodium bisulfide, 0.1 mM pyridoxal phosphate (PLP), pH 8.2; 4) Enzyme purification and immobilization buffer: Binding Buffer: 50 mM PBK buffer (pH 7.8); Wash Buffer: 50 mM PBK buffer (pH 7.8), 30 mM imidazole.

[0054] Elution Buffer: 50 mM PBK buffer (pH 7.8), 300 mM imidazole.

[0055] 5) Determination of enzyme activity: Accurately measure the supernatant of the sonicated bacterial suspension and add it to the substrate reaction solution preheated to 35°C to a final bacterial concentration of 20 mg / mL. Stir the reaction in a 35°C water bath for 30 min. Terminate the reaction by adding an equal volume of 20% trichloroacetic acid solution, shake well, and centrifuge. The supernatant is analyzed by HPLC using L-cysteine ​​and L-cystine standards as controls. 6) HPLC analysis: Thermo Fisher ODS-2 Hypersil™ (4.6 × 250 mm, 5 µm); flow rate: 1.0 mL / min; detection wavelength: 210 nm; mobile phase for the TrpS enzyme reaction: methanol: 0.05 mol / L potassium dihydrogen phosphate + 0.005 mol / L sodium decane sulfonate (pH 2.30) = 23:77; 7) Definition of enzyme activity unit: 1 U is the amount of enzyme required to produce 1 micromole (μmol) of L-cysteine ​​per unit time at pH 8.2 and temperature 35°C.

[0056] Example 1: Construction of wild-type tryptophan synthase TrpS genetically engineered bacteria (1) Wild type trpS gene synthesis According to the information published in the NCBI database, EnterobacteriaceaeThe tryptophan synthase TrpS is composed of an α-subunit and a β-subunit. The amino acid sequence of the α-subunit TrpA (GenBank: WP_000443067.1) is shown in SEQ ID NO. 1 in the sequence listing, and the amino acid sequence of the β-subunit TrpB (GenBank: WP_000209520.1) is shown in SEQ ID NO. 2 in the sequence listing. (To facilitate subsequent purification, 6 x His tags were added to the C-termini of TrpA and TrpB, respectively.) The gene coding sequence was codon-optimized according to the codon preference of Escherichia coli, and BamHI and HindIII restriction sites were designed and added at both ends of the coding sequence. The resulting proteins were then sent to a biotechnology company for artificial synthesis, yielding TrpA (SEQ ID NO: 3) and TrpB (SEQ ID NO: 4), respectively.

[0057] The amino acid sequence of tryptophan synthase α subunit TrpA is shown in SEQ ID NO: 1: MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIIDTLIEAGADALELGIPFSPLADGPTIQNATLRAFAAGVTPAQCFEMLALIRQKHPTIPIGLLMYANLVFNKGIDEFYAQCEKVGVDSVLVADVPVEESA PFRQAALRHNVAPIFICPPNADDDLLRQIASYGRGYTYLLSRAGVTGAENRAALPLNHLVAKLKEYNAAPPLQGFGISAPDQVKAAIDAGAAGAISGSAIVKIIEQHINEPEKMLAALKVFVQPMKAATRSHHHHHH The amino acid sequence of tryptophan synthase β subunit TrpB is shown in SEQ ID NO: 2: MTTLLNPYFGEFGGMYVPQILMPALRQLEEAFVSAQKDPEFQAQFNDLLKNYAGRPTALTKCQNITAGTNTTLYLKREDLLHGGAHKTNQVLGQALLAKRMGKTEIIAETGAGQHGVASALASALLGLKCRIYMGAKDVERQSPNVFRMRLMGAEVIPVHSGSATLKDACNEALRDWSGSYETAHYMLGTAAGPHPYPTIV REFQRMIGEETKAQILEREGRLPDAVIACVGGGSNAIGMFADFINETNVGLIGVEPGGHGIETGEHGAPLKHGRVGIYFGMKAPMMQTEDGQIEESYSISAGLDFPSVGPQHAYLNSTGRADYVSITDDEALEAFKTLCLHEGIIPALESSHALAHALKMMRENPDKEQLLVVNLSGRGDKDIFTVHDILKARGEIHHHHHH The TrpA gene coding sequence after codon optimization is shown in SEQ ID NO: 3: ggatccatggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcgtcacgctcggtgatccgggcattgagcagtcattgaaaattatcgatacgctaattgaagccggtgctgacgcgctggagttaggtatccccttctccgacccactggcggatggcccgacgattcaaaacgccactctgcgcgcctttgcggcaggtgtgactccggcacaatgttttgaaatgctggcactgattcgccagaaacacccgaccattcccattggcctgttgatgtatgccaatctggtgtttaacaaaggcattgatgagttttatgcccagtgcgaaaaagtcggcgtcgattcggtgctggttgccgatgtgccagttgaagagtccgcgcccttccgccaggccgcgttgcgtcataatgtcgcacctatcttcatctgcccgccaaatgccgatgacgacctgctgcgccagatagcctcttacggtcgtggttacacctatttgctgtcacgagcaggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcgaagctgaaagagtacaacgctgcacctccattgcagggatttggtatttccgccccggatcaggtaaaagcagcgattgatgcaggagctgcgggcgcgatttctggttcggccattgttaaaatcatcgagcaacatattaatgagccagagaaaatgctggcggcactgaaagtttttgtacaaccgatgaaagcggcgacgcgcagttaa aagctt The sequence of the codon-optimized TrpB gene coding sequence is shown in SEQ ID NO: 4: ggatcc aagctt (2) Construction of wild-type TrpS-pETDuet-1 vector TrpA was constructed into the multiple cloning site 1 of the E. coli dual expression vector pETDuet-1 with two promoters (plasmid map as shown in Figure 1 Primers for vector construction were designed, and 1-F (5'-ggcagcagccatcaccatcatcaccac-3', as shown in SEQ ID NO: 5) / 1-R (5'- ggtatatctccttcttaaagttaaacaaaatta -3', as shown in SEQ ID NO: 6) were used as primers, and the purchased vector pETDuet-1 was used as template. The linearized vector was amplified by PCR using 2×Hieff Canace® Plus PCR Master Mix (With Dye) high-fidelity enzyme premix; 2-F (5'- ttaagaaggagatataccatggaacgctacgaatct-3', as shown in SEQ ID NO: 7) / 2-R (5'- atggtgatggctgctgccttagtggtggtggtggtgatg -3', as shown in SEQ ID NO: 8) were used as primers, and the synthetic TrpA The gene DNA fragment (SEQ ID NO: 3) was used as a template, and the high-fidelity enzyme premix PCR amplified the 5' and 3' ends with sequences completely consistent with the two ends of the linearized vector. TrpA Gene DNA fragments were detected by 1% agarose gel electrophoresis, and the amplified linearized vector (5.4 kb) and TrpA The linearized vector recovered from the gel was cloned into a DNA fragment of the gene (0.8 kb). The Hieff Clone®Plus One Step Cloning Kit was used to clone the linearized vector and TrpA The gene fragments were homologously recombined. The recombination system was: 2.5 μL of pETDuet-1 linearized vector, TrpA 2.5 μL of gene fragment and 5 μL of 2×Hieff Clone® Enzyme Premix were added. The reaction was carried out at 50℃ for 30 min. The wild type was then obtained by (3). TrpA The plasmid vector TrpA-pETDuet-1 carrying the gene.

[0058] Similarly, TrpB was constructed into the multiple cloning site 2 of the E. coli dual expression vector pETDuet-1 with two promoters (plasmid map as shown in Figure 2The linearized vector was amplified by PCR using 3-F (5'-gcagatctcaattggatatcggc-3', as shown in SEQ ID NO: 9) / 3-R (5'- atgtatatctccttcttatacttaactaatatactaag -3', as shown in SEQ ID NO: 10) as primers and the successfully constructed TrpA-pETDuet-1 as template and 2×Hieff Canace® Plus PCR Master Mix (With Dye) high-fidelity enzyme premix; the linearized vector was amplified by PCR using 4-F (5'- gaaggagatatacatatgacaacattacttaacccctat-3', as shown in SEQ ID NO: 11) / 4-R (5'- tatccaattgagatctgctcagtggtggtggtggtg -3', as shown in SEQ ID NO: 12) as primers and the artificially synthesized TrpB The gene DNA fragment (SEQ ID NO: 4) was used as a template, and the high-fidelity enzyme premix PCR amplified the 5' and 3' ends with sequences completely consistent with the two ends of the linearized vector. TrpB Gene DNA fragments were detected by 1% agarose gel electrophoresis, and the amplified linearized vector (6.2 kb) and TrpB The linearized vector recovered from the gel was cloned into a 1.2 kb DNA fragment of the gene. The Hieff Clone® Plus OneStep Cloning Kit was used to clone the linearized vector and TrpB The gene fragments were homologously recombined. The recombination system was: TrpA-pETDuet-1 linearized vector 2.5 μL, TrpB 2.5 μL of gene fragment and 5 μL of 2× HieffClone® Enzyme Premix were added. The reaction was carried out at 50°C for 30 min.

[0059] (3) Construction of wild-type TrpS genetically engineered bacteria The homologous recombination product obtained in (2) was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method. The transformation product was spread on LB plate containing 50 μg / mL ampicillin. After overnight culture at 37°C, positive transformants were selected and sent to sequencing company for sequencing. Transformants with correct sequencing contained wild type TrpA Gene and wild type TrpB The plasmid vector TrpS-pETDuet-1 of the gene, the transformant is also a wild-type TrpS genetically engineered bacterium.

[0060] Example 2: Preparation and expression of tryptophan synthase TrpSLX genetically engineered bacteria (1) LinkerX gene synthesis In order to connect the C-terminus of TrpB to the N-terminus of TrpA with a linker, the protein modeling of the TrpS sequence was performed using the new artificial intelligence software tool RoseTTA Fold2 online software (http: / / robetta.bakerlab.org / ) to obtain its three-dimensional spatial structure as shown in the figure. Figure 3 The model was visualized and processed using PyMol. Based on literature research and protein design, 10 linkers were selected. Codon optimization of the gene coding sequence was performed based on the codon preference of E. coli. BamHI and HindIII restriction sites were added at both ends of the coding sequence. The resulting proteins, L1-L10, were synthesized at a biotechnology company, as shown in Table 1.

[0061] Table 1:

[0062] (2) Construction of fusion protein TrpSLX-pET28a vector The primer sequences used to design and obtain the fusion protein are shown in Table 2: Table 2:

[0063] The plasmid TrpB-pET28a synthesized artificially from a biological company was used as a template to obtain the recombinant plasmid TrpS-X-pETDuet-1 carrying the mutant of tryptophan synthetase. The linearized vector large fragment (B) carrying TrpB was amplified by PCR using 5-F (5'-gcggcgacgcgcagttaaaagcttgcggccgcactc-3', as SEQ ID NO: 53) 5-R (5'-gatttcccctcgtgcttt-3', as SEQ ID NO: 54) primers, 2xHieff Canace® Plus PCR Master Mix (With Dye) high-fidelity enzyme premix. The small fragment gene (A) carrying TrpA was amplified by PCR using 6-F (5'-atggaacgctacgaatct-3', as SEQ ID NO: 55) / 6-R (5'-gcggcgacgcgcagttaa-3', as SEQ ID NO: 56) primers, TrpA-pET28a plasmid as a template, 2xHieff Canace® Plus PCR Master Mix (With Dye) high-fidelity enzyme premix. The small fragment gene 1 carrying L1 was amplified by PCR using L1-F / L2-R primers, L1-pET28a plasmid as a template, 2xHieff Canace® Plus PCR Master Mix (With Dye) high-fidelity enzyme premix. The 5' end of the small fragment gene 1 carried a sequence identical to the 3' end sequence of the large fragment, and the 3' end carried a sequence identical to the 5' end sequence of the small fragment gene 2. The 3' end of the small fragment gene 2 carried a sequence identical to the 5' end sequence of the large fragment. The amplified DNA of the large and small fragments was detected by 1% agarose gel electrophoresis and purified using a DNA gel recovery purification kit. The DNA of the large and small fragments was subjected to homologous recombination using Hieff Clone® Plus One Step Cloning Kit one-step cloning kit. The recombination system was: 1 μL of DNA of the large fragment (B), 2 μL of DNA of the small fragment (A), 2 μL of DNA of the small fragment 1, 5 μL of 2xHieff Clone® Enzyme Premix. 50°C for 30 min.

[0064] The homologous recombination products were transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method. The transformation products were spread on LB plates containing 50 μg / mL kanamycin sulfate and cultured overnight at 37°C. Positive transformants were selected and sent to Shanghai Shenggong Bioengineering Technology Service Co., Ltd. for sequencing. The transformants with correct sequencing were the plasmid vector TrpSL1-pET28a containing the TrpB-L1-TrpA gene. This transformant was also a TrpB-L1-TrpA genetically engineered bacterium and was numbered LK1. In the same way, the engineered bacteria LK2 with TrpB-L2-TrpA, LK3 with TrpB-L3-TrpA, LK4 with TrpB-L4-TrpA, LK5 with TrpB-L5-TrpA, LK6 with TrpB-L6-TrpA, LK7 with TrpB-L7-TrpA, LK8 with TrpB-L8-TrpA, LK9 with TrpB-L9-TrpA, and LK10 with TrpB-L10-TrpA were constructed.

[0065] The correctly sequenced mutants LK1-LK10 genetically engineered bacteria were inoculated into test tubes containing 4 mL LB medium containing 50 μg / mL kanamycin sulfate. After overnight culture at 37°C and 200 rpm, they were transferred to 50 mL LB medium and cultured at 37°C and 200 rpm until the OD 600 The pH value was 0.6-0.8, IPTG was added to a final concentration of 0.2 mM, and the culture was continued at 20°C and 200 rpm for 18 h. The fermentation broth was centrifuged at 9000 rpm for 5 min, and the supernatant was removed. The bacterial pellet was retained and resuspended in water to a bacterial concentration of 100 mg / mL. Ultrasonic disruption was performed for 10 min, and the pellet was removed by centrifugation. The supernatant was retained for SDS-PAGE gel electrophoresis analysis. The results are shown in Figure 2. Figure 4 As shown, these proteins were expressed normally.

[0066] Example 3: Determination of enzyme activity of fusion protein TrpSLX Add 100 μL of enzyme solution to a centrifuge tube containing 900 μL of substrate solution (200 mM L-serine, 240 mM sodium bisulfide, and 0.1 mM pyridoxal phosphate (PLP) in water) and incubate at 35°C for 30 min. After the reaction is complete, terminate it with an equal volume of 20% trichloroacetic acid solution. Shake well and centrifuge. The supernatant is analyzed by HPLC using cysteine ​​and cystine standards as controls.

[0067] The relative enzyme activity results of each single mutant are shown in Table 3: Table 3:

[0068] The results in Table 3 show that after the three linkers L8-L10 connected TrpB and TrpA into fusion proteins, the enzyme activity did not show significant loss compared with the wild-type enzyme activity. Among them, the fusion protein LK9 containing L9 retained 99.6% of the wild-type enzyme activity.

[0069] Example 4: Purification of TrpSLX fusion protein The fusion protein LK8-10, which has no obvious loss of enzyme activity compared with the wild-type enzyme activity, was selected. The wild-type TrpS and the LK8-10 fusion protein TrpSL8-TrpSL10 were purified using the pre-constructed 6xHis tag using HisSep Ni-NTA Agarose Resin (His-tagged protein agarose purification resin) to obtain high-purity proteins. After removing imidazole through a centrifugal ultrafiltration tube (10KDa), they were used for immobilization.

[0070] The specific steps of enzyme purification are as follows: (1) Filling of HisSep Ni-NTA Agarose Resin gravity column: Take a 50mL gravity chromatography column, install the lower gasket, add an appropriate amount of pure water to rinse the column tube and gasket, and close the lower outlet. Mix the HisSep Ni-NTA Agarose Resin evenly, and use a pipette to draw an appropriate amount of slurry into the gravity column (the actual volume of the filler should account for half of the suspension). Open the lower outlet to drain the protective solution. Add an appropriate amount of pure water to rinse the filler. Once the liquid in the column tube has drained by gravity, close the lower outlet.

[0071] (2) Equilibration: Equilibrate the gravity column with 5 column volumes (CV) of Binding Buffer at a flow rate of 1-2 mL / min, repeat 2-3 times; (3) Sample loading: According to the method in Example 2, the fusion protein was induced to express and ultrasonically disrupted to obtain the supernatant crude enzyme solution which was slowly loaded onto the column bed and the binding time was 1-2 hours at 4°C; (4) Washing: Use 10-15 CV Wash Buffer to wash away non-specifically bound proteins; (5) Elution: Elute the target protein in sections using 5-10 CV Elution Buffer, collecting 1 CV in each section at a flow rate of 1 mL / min. Detect the UV absorbance (A280) of each section using a UV spectrophotometer until elution is complete.

[0072] (6) Column regeneration: Wash the column with 5 CV of 0.5 M NaOH (to remove residual proteins), rinse with 5 CV of 20% ethanol, and store at 4°C to prevent microbial contamination.

[0073] (7) Concentration and replacement of liquid: Add the collected eluate to the concentrator tube and centrifuge at 4°C to concentrate it to 1 / 10 of the original volume. Add Binding Buffer to the concentrator tube to the original volume, centrifuge again and concentrate to the target volume. Repeat the dilution-concentration step 3 times to obtain pure enzyme and detect the enzyme concentration.

[0074] Example 5: Immobilized resin screening of TrpS wild-type protein After the wild-type TrpS protein was purified according to the method in Example 4, 9 different enzyme immobilization carrier resins were selected for immobilization to screen out the resin with the best immobilization effect.

[0075] The specific steps of enzyme immobilization are as follows: (1) Resin equilibration: Weigh 10 g of resin into a beaker, wash the resin with 3 CV of 2 M PBK solution and filter, repeat 2-4 times; (2) Immobilization: Transfer the pure enzyme solution obtained in Example 4 into a beaker containing resin and gently stir at room temperature for 20 h; (3) Filtration and washing: Filter the resin and collect the filtrate. Wash the resin with 4CV of Binding Buffer and filter, repeat 2-4 times. Collect the washing solution and measure the concentration of unimmobilized enzyme in the filtrate and washing solution to evaluate the resin immobilization efficiency. The immobilization efficiencies of the nine enzyme immobilization support resins for the wild-type TrpS pure enzyme are shown in Table 4. Table 4 shows that among the nine enzyme immobilization support resins, LX-107S has the highest immobilization efficiency of 76.4% for the wild-type TrpS pure enzyme, with 30.54 mg of enzyme immobilized per gram of resin.

[0076] Table 4:

[0077] Example 6: Immobilized resin screening of TrpSL8 fusion protein The TrpSL8 fusion protein was purified according to the methods in Examples 4 and 5, and the immobilization resin with the best immobilization effect was screened.

[0078] The immobilization efficiencies of the nine enzyme immobilization support resins for the pure TrpSL8 fusion protein are shown in Table 5. Table 5 shows that among the nine enzyme immobilization support resins, ES-103B has the highest immobilization efficiency of 72.5% for the pure TrpSL8 fusion protein, with 28.98 mg of enzyme immobilized per gram of resin.

[0079] Table 5:

[0080] Example 7: Immobilized resin screening of TrpSL9 fusion protein The TrpSL9 fusion protein was purified according to the methods in Examples 4 and 5, and the immobilization resin with the best immobilization effect was screened.

[0081] The immobilization efficiency of the nine enzyme immobilization support resins for the pure TrpSL9 fusion protein enzyme is shown in Table 6. As shown in Table 6, among the nine enzyme immobilization support resins, LX-109S has the highest immobilization efficiency of 69.2% for the pure TrpSL9 fusion protein enzyme, with 27.69 mg of enzyme immobilized per gram of resin.

[0082] Table 6:

[0083] Example 8: Immobilized resin screening of TrpSL10 fusion protein The TrpSL10 fusion protein was purified according to the methods in Examples 4 and 5, and the immobilization resin with the best immobilization effect was screened.

[0084] The immobilization efficiencies of the nine enzyme immobilization support resins for pure TrpSL10 fusion protein enzyme are shown in Table 7. Table 7 shows that among the nine enzyme immobilization support resins, ES-103B has the highest immobilization efficiency of 73.9% for pure TrpSL10 fusion protein enzyme, with 29.57 mg of enzyme immobilized per gram of resin.

[0085] Table 7:

[0086] Example 9: Round reaction of TrpSL8-10 fusion protein Prepare 100 mL of a reaction solution (containing 380 mM L-serine, 438 mM sodium bisulfide, and 0.1 mM pyridoxal phosphate (PLP)). Based on Examples 5-8, select the resin with the highest immobilization efficiency for wild-type TrpS and the fusion protein TrpSL8-10. Weigh 200 mg of the resin to immobilize the pure enzyme and add it to the reaction solution. Incubate at 35°C for 6 hours, adjusting the pH to 8.2-8.6 with HCl. Monitor the reaction progress by HPLC, using L-serine, L-cysteine, and cystine as standards. Calculate the cysteine ​​conversion at the end of the reaction.

[0087] After the reaction, the resin was filtered, washed with 3CV of Binding Buffer, and then re-added to a new reaction solution. The reaction-washing process was repeated until the reaction rate was significantly reduced. The cysteine ​​conversion rate and reaction round number at the end of each reaction were recorded (see Table 8).

[0088] As can be seen from the results in Table 8, in the first round of reaction, both the WT group and the LK8-10 group completed the reaction in 6h and completed the conversion of serine. However, after washing with the cleaning solution, the second round of reaction showed the effect of Linker on the immobilization of TrpS. After the physical shearing of the wild-type TrpS during the washing process, the weak interaction between TrpB and TrpA was opened, resulting in the washing away of TrpB or TrpA that was not cross-linked with the resin, and the inability to form a complete dimer structure, which greatly reduced the reaction rate of the WT group. The connection of TrpB and TrpA through Linker, on the one hand, enhanced the interaction between TrpB and TrpA, making the dimer structure more compact. On the other hand, TrpB and TrpA were connected by Linker to form a fusion protein. No matter which part of TrpB and TrpA was cross-linked by the resin, it was difficult to be washed away, so that the reaction rate of the LK8-10 group in the second round remained the same. After the third round of reaction, wild-type TrpS was no longer able to generate cysteine. In the LK8-10 group, LK8 and LK10 maintained this performance until the ninth and eighth rounds, respectively, but achieved only 5.9% and 0.9% cysteine ​​conversion rates at 6 hours, respectively, with no cysteine ​​production in subsequent rounds. LK9, on the other hand, performed the best, achieving 87.4-92.4% cysteine ​​conversion rates at 6 hours after 10 rounds of reaction.

[0089] The above results indicate that the present invention has successfully constructed a linker-connected tryptophan synthase fusion that can be used to synthesize L-cysteine, cleverly solving the problem that TrpB and TrpA in tryptophan synthase cannot complete multiple rounds of reactions after resin immobilization. This opens up new ideas for the immobilization reaction of dual-enzymes or even multi-enzyme complexes, significantly improving the efficiency and economy of industrial production.

[0090] Table 8:

[0091] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0092] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein includes the α subunit of tryptophan synthase TrpS, the β subunit of tryptophan synthase TrpS and a connecting peptide. The amino acid sequence of the connecting peptide is shown in SEQ ID NO: 20-22.

2. The fusion protein according to claim 1, characterized in that The C-terminus of the β-subunit of the tryptophan synthase TrpS is connected to the α-subunit of the tryptophan synthase TrpS through the connecting peptide.

3. The fusion protein according to claim 1, wherein The amino acid sequence of the α subunit of the tryptophan synthase TrpS is shown in SEQ ID NO: 1; The amino acid sequence of the β subunit of the tryptophan synthase TrpS is shown in SEQ ID NO:

2.

4. An isolated nucleic acid, characterized in that The nucleic acid encodes the fusion protein according to any one of claims 1 to 3.

5. An expression vector, characterized in that The expression vector comprises the isolated nucleic acid of claim 4.

6. A host cell, characterized in that The host cell comprises the expression vector according to claim 5.

7. Use of the fusion protein according to any one of claims 1 to 3, the isolated nucleic acid according to claim 4, the expression vector according to claim 5, and the host cell according to claim 6 in the preparation of L-cysteine ​​or L-cystine.

8. A method for immobilizing tryptophan synthase, characterized in that: include: (1) Performing ion exchange equilibrium on the resin to obtain a balanced resin; (2) contacting the equilibrated resin with the fusion protein of any one of claims 1 to 3, and / or the fusion protein expressed by at least one of the isolated nucleic acid of claim 4, the expression vector of claim 5, and the host cell of claim 6, so that the fusion protein is immobilized on the resin.

9. The method according to claim 8, characterized in that In step (2), when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 20, the resin is ES-103B resin; Or, when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 21, the resin is LX-109S resin; Alternatively, when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 22, the resin is ES-103B resin.

10. A tryptophan synthase immobilized resin column, characterized in that: include: Resin column and fusion protein fixed thereon, The fusion protein is selected from the fusion protein of any one of claims 1 to 3 and / or a fusion protein expressed by at least one of the isolated nucleic acid of claim 4, the expression vector of claim 5, and the host cell of claim 6.

11. The tryptophan synthase immobilized resin column according to claim 10, characterized in that: When the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 20, the resin is ES-103B resin; Or, when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 21, the resin is LX-109S resin; Alternatively, when the amino acid sequence of the connecting peptide in the fusion protein is as shown in SEQ ID NO: 22, the resin is ES-103B resin.

12. A method for preparing L-cysteine, characterized in that: include: The immobilized enzyme is obtained by the method for immobilizing tryptophan synthase according to claim 8 or 9 and / or the tryptophan synthase immobilized resin column according to claim 10 or 11 is used to catalyze the synthesis of L-cysteine ​​using L-serine and a sulfur donor as substrates.

13. A method for preparing L-cystine, characterized in that: include: S1: obtaining an immobilized tryptophan synthase by the method for immobilizing tryptophan synthase according to claim 8 or 9 and / or utilizing the tryptophan synthase immobilized resin column according to claim 10 or 11 to catalyze the synthesis of L-cysteine ​​using L-serine and a sulfur donor as substrates; S2: Using the L-cysteine ​​synthesized in step S1 as a substrate, an oxidation reaction is performed to synthesize L-cystine.

Images