Construction method of high-activity mutant of chondroitin-6-O-sulfotransferase

Abstract

The invention discloses a construction method of a high-activity mutant of chondroitin 6-O sulfotransferase, and belongs to the field of biological enzyme engineering and synthetic biology. Interaction sites of MusC6 protein and a substrate PAPS are accurately analyzed through a computer-aided molecular docking technology, and key active sites are directionally modified by adopting a strategy of combining rational design and saturated mutation. Experimental results show that the catalytic performance of the obtained mutant S139T / S140L / Y364M / K412V is remarkably improved, and the enzyme activity of the mutant reaches 1182U / L and is improved by 4.4 times compared with the enzyme activity of a wild type 270U / L. According to the invention, the technical bottleneck of low catalytic efficiency of a wild type enzyme on a natural substrate is solved through a multi-site synergistic mutation strategy, and a biocatalyst with industrial application value is provided for efficiently synthesizing chondroitin sulfate C by a microbiological method.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for constructing a high-activity mutant of chondroitin-6-O sulfotransferase, in particular to a method for rationally designing a high-activity mutant of chondroitin-6-O-sulfotransferase (C6ST) based on computer-aided molecular docking and multi-site synergistic mutation, and the application of the mutant in the efficient synthesis of chondroitin sulfate C (CSC), belonging to the technical field of biological enzyme engineering and synthetic biology. BACKGROUND

[0002] 6-O-sulfotransferase (C6ST) is a key modification enzyme in the biosynthesis of chondroitin sulfate (CS), responsible for catalyzing the transfer of sulfate groups from the active co-substrate 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the C6 position of the galactosamine hydroxyl group of chondroitin, forming chondroitin sulfate C (CSC). CSC is an important member of the chondroitin sulfate family, and its unique 6-O-sulfation modification endows it with biological activities such as anticoagulation, anti-inflammatory, and promotion of nerve regeneration, and it is widely used in the treatment of osteoarthritis, tissue engineering, and drug delivery systems.

[0003] Currently, CSC is mainly produced by animal tissue extraction method, but there are problems such as limited raw materials and large batch differences. Microbial synthesis method has the advantage of sustainability, but the core challenge is the low catalytic efficiency and insufficient substrate affinity of natural C6ST, which leads to the yield of product cannot meet the industrial demand. Studies have shown that the binding ability of wild-type C6ST (such as MusC6) to PAPS is weak, and the conformational flexibility of the active center is insufficient, which seriously limits the synthesis efficiency of CSC.

[0004] Traditional enzyme engineering methods such as directed evolution rely on high-throughput screening, which is costly and random; single rational design is limited by insufficient understanding of enzyme-substrate interaction mechanisms, making it difficult to achieve multi-site synergistic optimization. In recent years, although there have been studies to improve the activity of C6ST through point mutations, most of the modifications are focused on a single active site, and there is a lack of systematic study on the synergistic effect of co-substrate binding region and conformational regulation region, resulting in limited enzyme activity improvement (usually less than 2 times). SUMMARY

[0005] In the prior art, the low catalytic efficiency and substrate binding defects of natural C6ST, combined with the randomness and high cost of traditional enzyme engineering methods and the limitations of single-site modification, have hindered the industrialization process of microbial synthesis of CSC. The present application aims to break through the above technical bottlenecks by precisely designing a multi-site synergistic mutation strategy, and to simultaneously improve the catalytic efficiency and stability of C6ST.

[0006] The first technical solution provided by the application is a chondroitin-6-O-sulfotransferase MusC6ST mutant, wherein 38 amino acids are truncated from the N terminus of chondroitin-6-O-sulfotransferase MusC6 (the amino acid sequence is shown as SEQ ID NO. 1) to obtain the amino acid sequence of the recombinant chondroitin-6-O-sulfotransferase MusC6ST shown as SEQ ID NO. 2 and the nucleotide sequence shown as SEQ ID NO. 3.

[0007] In some embodiments, the mutant is the chondroitin-6-O-sulfotransferase parent shown as SEQ ID NO. 1, wherein 38 amino acids are truncated from the N terminus, and any one of the amino acids at positions 134, 135, 136, 137, 138, 139, 140, 141, 157, 267, 295, 303, 364, 397, 398, 409, 410, 411, 412, 413, 414, and 417 is mutated to alanine, and the amino acids and nucleotides before and after mutation are shown in Table 1. It is particularly pointed out that the counting order of all mutation sites in the application is counted in the order of the amino acid sequence of the wild-type chondroitin-6-O-sulfotransferase.

[0008] In some embodiments, the mutant is the chondroitin-6-O-sulfotransferase parent shown as SEQ ID NO. 1, wherein 38 amino acids are truncated from the N terminus, and the amino acids at positions 139, 140, 364, 398, 412, and / or 417 are mutated. The 18 amino acids and nucleotides after mutation are shown in Table 2.

[0009] In some embodiments, the mutant is the chondroitin-6-O-sulfotransferase parent shown as SEQ ID NO. 1, wherein 38 amino acids are truncated from the N terminus, and the amino acids at positions 139, 140, 364, 398, 412, and / or 417 are mutated to alanine.

[0010] In some embodiments, the mutant is the chondroitin-6-O-sulfotransferase parent shown as SEQ ID NO. 1, wherein 38 amino acids are truncated from the N terminus, and the amino acids at positions 139, 140, 364, 398, 412, and / or 417 are mutated to alanine.

[0011] In some embodiments, the mutant is the chondroitin-6-O-sulfotransferase parent shown as SEQ ID NO. 1, wherein 38 amino acids are truncated from the N terminus, and the amino acids at positions 139, 140, 364, 398, 412, and / or 417 are mutated to alanine.

[0012] The second technical solution provided by the present application is a gene encoding the chondroitin-6-O-sulfotransferase MusC6ST mutant of the first technical solution.

[0013] The third technical solution provided by the present application is a recombinant vector carrying the gene of the second technical solution.

[0014] In some embodiments, the recombinant vector uses Ppic9k as an expression vector.

[0015] The fourth technical solution provided by the present application is a recombinant cell expressing the chondroitin-6-O-sulfotransferase MusC6ST mutant of the first technical solution, or containing the gene of the second technical solution, or transformed with the recombinant vector of the third technical solution.

[0016] In some embodiments, the host of the recombinant cell includes Escherichia coli or Pichia pastoris.

[0017] In some embodiments, the Escherichia coli is one of Escherichia coli JM109, DH5α or Top10.

[0018] In some embodiments, the Pichia pastoris is K.phaffii GS115 strain.

[0019] The fifth technical solution provided by the present application is a yeast engineering bacterium, which uses K.phaffii GS115 strain as a host and integrates expression of the chondroitin-6-O-sulfotransferase MusC6ST mutant of the first technical solution.

[0020] In some embodiments, the chondroitin-6-O-sulfotransferase mutant is integrated into the HIS mutant site of the K.phaffii GS115 strain genome.

[0021] The present application also provides a method for obtaining the above-mentioned recombinant chondroitin-6-O-sulfotransferase MusC6 mutant, which comprises the following steps:

[0022] (1) Based on the wild-type MusC6 encoding gene (GenBank accession number: BAA29054.1), the target mutation site is determined by molecular docking;

[0023] (2) Using the wild-type MusC6 gene as a template, the mutant gene fragment is amplified by overlap extension PCR site-directed mutagenesis technology; the methylated template DNA is digested by DpnI enzyme, and the mutant MusC6 gene is obtained by purification. Design specific PCR primers to introduce base substitution to the mutation site;

[0024] (3) The mutant gene was cloned into the eukaryotic expression system pPIC9K; the recombinant plasmid pPIC9K-MusC6-Mut was constructed by ligation with T4 ligase.

[0025] (4) The recombinant plasmid was digested with restriction endonuclease salI and transformed into Pichia pastoris GS115; it was cultured in BMGY medium at 30℃ for 24h, and then centrifuged to transfer the cells to BMMY containing 1% methanol for 72h.

[0026] (5) The bacterial cells were disrupted by sonication and the intracellular supernatant was collected by centrifugation at 12,000 rpm for 30 min. The His-tagged fusion protein was purified by affinity chromatography with a nickel column and eluted with gradient imidazole (20-500 mM).

[0027] The sixth technical solution provided by the present invention is a method for preparing chondroitin sulfate C (CSC), wherein the method involves adding the chondroitin-6-O sulfotransferase MusC6ST mutant described in the first technical solution to a reaction system containing chondroitin and PAPS to prepare CSC.

[0028] In some embodiments, the 1 mL reaction system contains 100 mL of chondroitin (2-20 g / L chondroitin), 0.5 mM PAPS, 500 μL of crude enzyme, and 20-100 mM Tris-HCl, and is brought to a final volume of 1 mL.

[0029] In some embodiments, the reaction temperature is 30-42°C and the reaction time is 2-24 hours.

[0030] The seventh technical solution provided by the present invention is the application of the chondroitin-6-O sulfotransferase MusC6ST mutant described in the first technical solution, or the gene described in the second technical solution, or the recombinant vector described in the third technical solution, or the recombinant cell described in the fourth technical solution, or the yeast engineered strain described in the fifth technical solution, or the method described in the sixth technical solution in the preparation of CSC or CSC-containing products.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] (1) Through molecular dynamics simulation and binding energy calculation, the interaction network between MusC6 and PAPS was accurately analyzed, and key sites such as S139, Y364 and Q417 were identified.

[0033] (2) By combining rational design with saturation mutation, the limitations of single-site modification are overcome, and the synergistic optimization of the active site and the co-substrate binding domain is achieved.

[0034] (3) The mutant MusC6 S139T / Y364M / Q417W obtained has an enzyme activity of 943 U / L, which is 3.5 times that of the wild type (270 U / L), and the stability is significantly enhanced. This technology provides the first high-performance biological catalyst with multiple site engineering for the efficient microbial synthesis of CSC, which has clear industrial application potential.

[0035] (4) The C6ST enzyme activities of the superposition mutants S139T / S140L / Y364M / K412V and S139T / S140L / K412V / Q417C are at a higher level in the figure, which is significantly higher than that of the single mutant, and the enzyme activities reach 1203 and 1138 U / L, respectively. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is the molecular docking site of MusC6 wild type and PAPS.

[0037] Figure 2 is the alanine scanning result of the molecular docking site.

[0038] Figure 3 is the single site mutation result.

[0039] Figure 4 is the superposition mutation result.

[0040] Figure 5 is the western blot detection of MusC6. DETAILED DESCRIPTION

[0041] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are used to better explain the present application and are not used to limit the present application.

[0042] Method in the example:

[0043] 1. Enzyme activity detection method:

[0044] Chondroitin-6-O-sulfotransferase MusC6 enzyme activity detection method: C6ST activity is evaluated by monitoring the change in absorbance of p-nitrophenol (PNP) at 400 nm wavelength. The increase in absorbance corresponds to the amount of PNP formed, thereby quantitatively measuring C6ST activity. The total volume of the reaction system is 1 mL, which includes 100 μL of chondroitin, 500 μL of C6ST crude enzyme solution or pure enzyme solution prepared from cell lysate, 50 mM p-nitrophenyl sulfate (PNPS), 1 mM PAPS and 2 mg / mL aryl sulfotransferase IV (AST IV). The reaction mixture is incubated at 37°C for 2 h.

[0045] 2. Preparation of CSC using chondroitin-6-O-sulfotransferase:

[0046] The total volume of the reaction system was 1 mL, including 100 μL of 20 g / L chondroitin, 500 μL of C6ST crude enzyme solution prepared from cell lysate or pure enzyme solution, 10 mM PAPS, and 20-100 mM Tris-HCl, and the volume was made up to 1 mL. The reaction temperature was 30-42°C, and the reaction time was 2-24 h.

[0047] Materials used in the examples:

[0048] 1. Escherichia coli Top 10, JM109, DH5α and Pichia pastoris GS115 were commercial strains; pPIC9K was a commercial plasmid; DpnI and SalI enzymes were from Takara Co.

[0049] 2. All culture media were from the National Pharmaceutical Group.

[0050] 3. Culture medium:

[0051] LB medium: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast powder.

[0052] BMGY medium: 10 g / L yeast extract: 20 g / L tryptone, 100 mL / L potassium phosphate buffer (1 M, pH 6.0), 13.4 g / L YNB (Yeast Nitrogen Base): 0.4 mg / L biotin, 10 g / L glycerol, pH 6.0.

[0053] BMMY medium: 10 g / L yeast extract: 20 g / L tryptone, 100 mL / L potassium phosphate buffer (1 M, pH 6.0), 13.4 g / L YNB (Yeast Nitrogen Base): 0.4 mg / L biotin, 0.5% methanol, pH 6.0.

[0054] Table 1 Changes in amino acids and nucleotides before and after mutation

[0055]

[0056] Table 2 6 kinds of amino acids are respectively mutated into another 18 kinds of amino acids and nucleotide sequences

[0057]

[0058]

[0059] Example 1 Expression and purification of chondroitin-6-O-sulfotransferase in Pichia pastoris GS115 strain

[0060] A 38-amino acid truncated fragment of the MusC6ST gene (PEO ID NO.2) was inserted into the pPIC9K vector using T4 ligase. AOX1 Downstream of the promoter, a recombinant plasmid pPIC9K-MusC6ST was constructed. The ligation product was transformed into *E. coli* TOP10 competent cells and plated on LB agar plates containing 100 μg / mL kanamycin, and incubated at 37°C for 12 hours. Single colonies were picked for colony PCR and sequencing verification to confirm pPIC9K-MusC6ST. AOX1 Correct ligation of the promoter to the MusC6ST gene and sequence integrity.

[0061] The validated recombinant plasmid was linearized with SalI, purified, and then transformed into Pichia pastoris GS115 competent cells via electroporation (parameters: 2.0 kV, 25 μF, 200 Ω). After transformation, cells were plated on MD plates (1.34% YNB, ×10⁻⁶). -5 Screening for His (1% biotin, 1% glucose) by culturing at 30℃ for 3-5 days. + Transformants. Genomic PCR was performed using primers 5'-AOX1 (5'-GACTGGTTCCAATTGACAAGC-3') and 3'-MusC6ST (5'-AATCCAATCTTCAACTTGTGGAGTCAA-3') to verify the specific integration of the target gene at the HIS4 site.

[0062] Positive clones were inoculated into BMGY medium (1% yeast extract, 2% peptone, 1.34% YNB, 1% glycerol) and incubated at 30°C with shaking at 250 rpm for 24 hours until OD reached. 600 ≈6. Centrifuge to collect bacterial cells, transfer to BMMY induction medium (containing 0.5% methanol), add methanol every 24 hours to a final concentration of 0.5%, induce at 30°C for 72 hours, and centrifuge to collect bacterial cells.

[0063] After high-pressure homogenization (1000 bar, 5 min), the supernatant was collected by centrifugation at 12,000 × g for 30 min. Use The protein purification system, using a HisTrap HP 5mL nickel column (Cytiva), was used for purification. Figure 1 Solution A (binding / washing buffer): 20mM KH2PO4, 500mM NaCl, 20mM imidazole (pH 7.4); Solution B (elution buffer): 20mM KH2PO4, 500mM NaCl, 500mM imidazole (pH 7.4).

[0064] Example 2 Construction of alanine scanning mutant

[0065] Based on the substrate binding domain and catalytic pocket prediction of MusC6ST parent (SEQ ID NO. 1), 22 key residues (134-141, 157, 267, 295, 303, 364, 397-398, 409-414, 417) were selected for alanine scanning. Complementary overlapping primers were designed for each site, with the mutation primer center containing an alanine codon (GCT). The amino acid sequences before and after mutation are shown in Table 1.

[0066] PCR was performed using pPIC9K-MusC6ST as a template: the gene was amplified using the mutant primer pair (94°C for 30 s, 55°C for 30 s, 72°C for 15 s / kb, 42 cycles); phosphorylation and ligation, transformation of E. coli TOP10, and kanamycin plate screening for clones. The correct plasmid was digested with Sal I, the fragment was purified and then transformed into GS115 competent cells, and strains that could grow on MD plates and had large colonies on G418 resistance plates were screened.

[0067] The results are shown in Table 2. Figure 2 、 3 Among them, after the mutation of S139, T140, Y364, K398, R412, Q417 sites, the enzyme activity was significantly higher than that of the parent (WT), and the enzyme activity of the six mutants was 346.1, 353.7, 371.3, 363.6, 464.6 and 432.9 U / L, respectively, compared with 265.6 U / L of the parent.

[0068] Example 3 Saturation mutation of enzyme activity improvement sites

[0069] Saturation mutation was performed on the target residues (S139, T140, Y364, K398, R412, Q417) obtained in Example 2, and the six sites were mutated to other 19 kinds of amino acids, respectively. The mutation results are shown in Table 2, Figure 4 Through the construction of saturation mutation library and high-throughput screening of the six key sites (S139, T140, Y364, K398, R412, Q417) of MusC6ST, five high-activity mutants (S139C, S140L, Y364M, R412V, Q417C, activity >150%) were obtained, among which S139C had the highest activity of 2.16 times that of wild type (578 U / mg), and the mutation of the significant sites Y364 and Q417 had the greatest impact on enzyme activity (activity fluctuation >200%). About 12% of the mutants in the library had increased activity, 68% remained neutral, and 20% were significantly inactivated, indicating that Y364 is a key catalytic residue and Q417 is involved in substrate binding. The high-activity mutants need to be sequenced and the effects of combined mutations need to be verified, and the specific analysis is shown in Table 3.

[0070] Table 3 Mutation result analysis

[0071]

[0072] Example 4 Combined mutations on high enzyme activity sites

[0073] Based on the single mutations constructed in Example 3, further mutations were introduced using the same strategy to construct double, triple, quadruple, and quintuple mutants. The PCR amplification products were digested with Dpn I restriction enzyme and purified by column method. The obtained linearized fragments were assembled by Gibson assembly method, then transformed into E. coli Top 10 cells, and the cells were plated on culture medium containing kanamycin. Positive clones were selected for further culture, and plasmids were extracted. After Sal I enzyme digestion, the purified plasmids were transformed into GS115 strain for expression, and the mutants were obtained by the method in Example 2. Figure 5The middle strain number: 1-14 are double mutations, respectively S139T / S140L, S139T / Y364M, S139T / K412V, S140L / Y364M, S140L / K412V, Y364M / K412V, S139T / Q417C, S139T / Q417W, S140L / Q417C, S140L / Q417W, Y364M / Q417C, Y364M / Q417W, K412V / Q417C, K412V / Q417W; 15-30 are triple mutations, respectively S139T / S140L / Y364M, S139T / S140L / K412V, S139T / Y364M / K412V, S140L / Y364M / K412V, S139T / S140L / Q417C, S139T / S140L / Q417W, S139T / Y364M / Q417C, S139T / Y364M / Q417W, S139T / K412V / Q417C, S139T / K412V / Q417W, S140L / Y364M / Q417C, S140L / Y364M / Q417W, S140L / K412V / Q417C, S140L / K412V / Q417W, Y364M / K412V / Q417C, Y364M / K412V / Q417W; 31-39 are four mutations, respectively S139T / S140L / Y364M / K412V, S139T / S140L / Y364M / Q417C, S139T / S140L / Y364M / Q417W, S139T / S140L / K412V / Q417C, S139T / S140L / K412V / Q417W, S139T / Y364M / K412V / Q417C, S139T / Y364M / K412V / Q417W, S140L / Y364M / K412V / Q417C, S140L / Y364M / K412V / Q417W, 40, 41 are five mutations, respectively S139T / S140L / Y364M / K412V / Q417C, S139T / S140L / Y364M / K412V / Q417W.

[0074] The C6ST enzyme activity of the superimposed mutants S139T / S140L / Y364M / K412V and S139T / S140L / K412V / Q417C in the figure is at a higher level in the figure, which is significantly higher than that of single mutant (S139T, S140L, Y364M, K412V, Q417C, Q417W) and double mutant (S139T / S140L, S139T / Y364M, S139T / K412V, S140L / Y364M, S140L / K412V, Y364M / K412V). Figure 5), and the enzyme activities reached 1203, 1138 U / L, respectively. This means that these mutation sites have a promoting effect on C6ST enzyme activity after superposition, and there may be a synergistic effect between different site mutations, which enhances enzyme activity by changing the spatial structure of the enzyme or the ability to bind to the substrate. This shows that the multi-site combined mutation of C6ST is an effective strategy to improve its enzyme activity, and provides a direction for optimizing the function of C6ST using genetic engineering, such as improving the synthesis efficiency of target products in related biosynthetic pathways.

[0075] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application. Therefore, the protection scope of the present application should be defined by the claims.

Claims

1. A chondroitin-6-O-sulfotransferase MusC6ST mutant, characterized in that, The mutant is a chondroitin-6-O sulfotransferase parent with N-terminal truncation of 38 amino acids of the amino acid sequence shown in SEQ ID NO. 1, and mutation of amino acids at positions 139, 140, 364, 398, 412 and / or 417.

2. The Chondroitin-6-O-sulfotransferase MusC6ST mutant according to claim 1, characterized in that, The mutant is a chondroitin-6-O sulfotransferase parent with N-terminal truncation of 38 amino acids of the amino acid sequence shown in SEQ ID NO. 1, and mutation of amino acids at positions 139, 140, 364, 398, 412 or 417 to alanine.

3. The Chondroitin-6-O-sulfotransferase MusC6ST mutant according to claim 1, characterized in that, The mutant is a chondroitin-6-O sulfotransferase parent with N-terminal truncation of 38 amino acids of the amino acid sequence shown in SEQ ID NO. 1, and at least one of the following mutations: (1) mutation of serine at position 139 to threonine; (2) mutation of serine at position 140 to leucine; (3) mutation of tyrosine at position 364 to methionine; (4) mutation of arginine at position 412 to valine; (5) mutation of glutamine at position 417 to tryptophan or cysteine.

4. A gene encoding the chondroitin-6-O sulfotransferase mutant MusC6ST according to any one of claims 1 to 3.

5. A recombinant vector carrying the gene according to claim 4.

6. A recombinant cell expressing the chondroitin-6-O sulfotransferase mutant MusC6ST according to any one of claims 1 to 3, or containing the gene according to claim 4, or transformed with the recombinant vector according to claim 5.

7. A yeast engineering bacterium, characterized in that, The yeast engineering takes Pichia pastoris GS115 strain as host, and integrates expression of the chondroitin-6-O sulfotransferase mutant MusC6ST according to claims 1 to 3.

8. A method for preparing chondroitin sulfate C, characterized by, The method is to add the chondroitin-6-O sulfotransferase mutant MusC6ST according to any one of claims 1 to 3 to a reaction system containing chondroitin and PAPS, and to perform reaction to prepare chondroitin sulfate C.

9. The method of claim 8, wherein, The reaction temperature is 30-42℃, and the reaction time is 2-24h.

10. Use of the chondroitin-6-O sulfotransferase mutant MusC6ST according to any one of claims 1 to 3, or the gene according to claim 4, or the recombinant vector according to claim 5, or the recombinant cell according to claim 6, or the yeast engineering bacteria according to claim 7, or the method according to claim 8 or 9 in preparation of chondroitin sulfate C or a product containing chondroitin sulfate C.

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