A mutant with improved S-adenosylmethionine synthetase activity and its application

Abstract

The invention discloses a mutant with improved activity of S-adenosylmethionine synthetase and its application, belonging to the field of bioenzyme engineering technology. Site-directed combined mutations were performed on positions 189, 200, 234, 266 and 371 of S-adenosylmethionine synthetase MAT to obtain a series of mutants with improved enzyme activity, among which I189V / N371R had the most obvious improvement, with a specific enzyme activity of 1.092±0.0497U·mg ‑1 , which is 342.82% higher than the parent WT. The enzyme mutant provided in this application has broad application prospects in producing S-adenosylmethionine and constructing genetically engineered microorganisms of S-adenosylmethionine.

Description

Technical Field

[0001] The invention relates to a mutant with improved S-adenosylmethionine synthetase activity and application thereof, belonging to the technical field of bioenzyme engineering. Background Art

[0002] S-adenosylmethionine (SAM) is an important metabolite in organisms. It participates in a variety of biochemical reactions in the body and is involved in almost all cellular metabolic processes. It mainly acts as a methyl donor and participates in various biochemical reactions such as transsulfuration, transamination and transribosylation in the body. It can also act as a signal molecule to regulate the metabolism of microorganisms. In addition, SAM is related to the activity of a variety of enzymes. The lack of SAM in the body will affect the normal metabolic function of the entire organism. It has outstanding clinical application value and is currently mainly used for the treatment of liver, nervous system diseases, osteoarthritis, anti-tumor and other aspects. Due to the biological functions and clinical application value of SAN as mentioned above, its preparation method has attracted more and more research attention.

[0003] At present, the microbial fermentation method has become the mainstream method for industrial production of SAM due to its advantages such as easy scale-up and lower raw material cost, but this method does not have an advantage in terms of fermentation cycle and late product extraction and purification. The whole-cell catalytic method not only has the advantage of easy scale-up of the microbial fermentation method, but also does not require cell disruption, reducing the cost of downstream separation and purification. However, the currently known S-adenosylmethionine synthetase (MAT) has the problems of low enzyme activity and poor stability, resulting in a low substrate conversion level, and the product yield is much lower than that of the microbial fermentation method, and the common MAT has serious substrate and product inhibition. The whole-cell catalytic method for synthesizing SAM has the advantages of easy scale-up, short production cycle, simple preparation, and elimination of the cumbersome enzyme purification process, but the current whole-cell synthesis of SAM has low yields and cannot reach industrial levels. Summary of the invention

[0004] In view of the problems of low catalytic efficiency and poor stability of synthetase in the current industrial production of S-adenosylmethionine, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a mutant with improved activity of S-adenosylmethionine synthetase, and to increase the yield of S-adenosylmethionine synthesized by the whole-cell catalytic method, which has a universal significance for promoting the synthesis of S-adenosylmethionine.

[0005] The technical solution adopted by the present invention is as follows:

[0006] The S-adenosylmethionine synthetase MAT encoded by the sam2 gene from Saccharomyces cerevisiae was cloned, and a protein model was established using the Swiss-Model. The protein structure was analyzed, the protein was molecularly docked with the substrate, and sites were selected for site-directed mutagenesis. The activity of the mutated enzyme was measured to determine the sites that had an important influence on the enzyme activity. Then, combined mutations were performed and the mutation advantages were superimposed to obtain mutants with improved activity and stability compared to the wild type.

[0007] The present invention provides an S-adenosylmethionine synthetase mutant, which is subjected to any of the following mutations based on the amino acid sequence shown in SEQ ID NO.1:

[0008] (1) Mutation of isoleucine at position 189 to valine (I189V);

[0009] (2) mutating isoleucine at position 189 to valine and asparagine at position 371 to arginine (I189V / N371R);

[0010] (3) mutating isoleucine at position 189 to valine and aspartic acid at position 200 to glutamic acid (I189V / D200E);

[0011] (4) mutating isoleucine at position 189 to valine and glutamine at position 234 to asparagine (I189V / Q234N);

[0012] (5) The isoleucine at position 189 was mutated to valine, the valine at position 266 was mutated to histidine, and the asparagine at position 371 was mutated to arginine (I189V / V266H / N371R).

[0013] The present invention also provides a gene encoding the mutant.

[0014] The invention also provides an expression vector carrying the gene.

[0015] The present invention also provides a microbial cell expressing the mutant or carrying the expression vector.

[0016] The present invention also provides a method for producing S-adenosylmethionine, wherein the mutant or the microbial cell is used as a catalyst for the reaction.

[0017] In one embodiment of the present invention, the microbial cells are cultured in a culture system until OD 600 The pH value was 0.6-0.8, the inducer was added and induced at 25°C for 24 hours, and the bacteria were collected; the bacteria were added to the reaction system containing the substrate and reacted for 2 hours.

[0018] In one embodiment of the present invention, the recombinant monoclonal clone is inoculated into 10 mL LB liquid medium containing kanamycin, cultured at 37°C and 220 rpm for 10-12 h, and then 2% of the culture solution is transferred to a new 50 mL LB liquid medium containing kanamycin and cultured for about 2-3 h until the OD 600 =0.6-0.8, add filter-sterilized inducer IPTG to a final concentration of 0.1 mM, then culture at 25°C 220 rpm for 12-24 h, and collect the bacteria by centrifugation.

[0019] In one embodiment of the present invention, the substrate comprises L-methionine, adenosine-5'-triphosphate disodium salt, potassium chloride, and magnesium chloride. The substrate concentrations in the reaction system are 20 to 60 mM, and the strain OD 600 =(3.2)±0.2, reacted at pH 8±1, 37°C.

[0020] The present invention also provides a method for improving the catalytic activity of S-adenosylmethionine synthetase, comprising performing any of the following mutations on the basis of the amino acid sequence shown in SEQ ID NO.1:

[0021] (1) Mutation of isoleucine at position 189 to valine;

[0022] (2) mutating isoleucine at position 189 to valine and asparagine at position 371 to arginine;

[0023] (3) mutating the isoleucine at position 189 to valine and the aspartic acid at position 200 to glutamic acid;

[0024] (4) mutating isoleucine at position 189 to valine and glutamine at position 234 to asparagine;

[0025] (5) The isoleucine at position 189 was mutated to valine, the valine at position 266 was mutated to histidine, and the asparagine at position 371 was mutated to arginine.

[0026] The present invention also provides the use of the mutant or the microbial cell in preparing S-adenosylmethionine or a product containing S-adenosylmethionine.

[0027] Beneficial effects:

[0028] (1) A series of mutants with improved enzyme activity were obtained by mutating one or more sites at positions 189, 200, 234, 266, and 371 of S-adenosylmethionine synthetase MAT. The isoleucine at position 189 was mutated to valine (I189V), and the specific enzyme activity was increased by 60.67% compared with that of the parent. The isoleucine at position 189 was mutated to valine, and the asparagine at position 371 was mutated to arginine (I189V / N371R), and the specific enzyme activity was increased by 342.82% compared with that of the parent. The isoleucine at position 189 was mutated to valine, and the asparagine at position 200 was mutated to glutamic acid (I189V / N371R). The specific enzyme activity of the mutant was increased by 159.00% compared with the parent by mutating the isoleucine at position 189 to valine and the glutamine at position 234 to asparagine (I189V / Q234N), and the specific enzyme activity was increased by 147.45% compared with the parent; the isoleucine at position 189 was mutated to valine, the valine at position 266 was mutated to histidine, and the asparagine at position 371 was mutated to arginine (I189V / V266H / N371R), and the specific enzyme activity was increased by 98.18% compared with the parent, and the crude enzyme activity of the corresponding combined mutations was also significantly increased compared with the parent. The specific enzyme activity of the single point mutations at positions 189, 200, 234, 266, and 371 was increased to varying degrees compared with the parent, indicating that the enzyme activity of the mutant was greatly improved.

[0029] (2) The catalytic performance was compared by whole-cell catalysis. After 2 h of catalysis, the concentration of S-adenosylmethionine synthesized by the parent (WT) was 276.32 mg / L, the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V was 479.91 mg / L, the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V / N371R was 1119.34 mg / L, the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V / D200E was 695.81 mg / L, the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V / Q234N was 581.65 mg / L, and the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V / N371R was 1119.34 mg / L. The concentration of S-adenosylmethionine synthesized by BL21-pET28a-sam2-I189V / V266H / N371R was 1552.81 mg / L, which was significantly higher than that of the parent. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The wild enzyme was used as the control and the enzyme activity was set as 100%, and the relative enzyme activity of the mutants obtained by site-directed mutagenesis;

[0031] Figure 2 Comparison of whole-cell catalysis between the wild-type enzyme and the mutant. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0033] In the following examples, the enzyme activity unit (U) is defined as the amount of MAT enzyme required to produce 1 μmol SAM per minute, and the specific enzyme activity (U·mg -1 ) to measure. SAM enzyme activity assay system (based on final concentration): 100mM Tris-HCl (pH8.0), 20mM L-Met, 8mM reduced glutathione, 50mM KCl, 20mM MgCl2, 20mM ATP, add 400μL crude enzyme solution to each 1mL reaction system, react in a water bath at 37℃ for 30min, take out 500μL and mix with an equal volume of 20% perchloric acid solution to terminate the reaction, after ice bathing on ice for 30min, 12000r·min -1 After centrifugation for 10 min, the supernatant was collected and the SAM concentration was determined by HPLC.

[0034] Detection method of S-adenosylmethionine content: SAM concentration was determined by high performance liquid chromatography. The fermentation supernatant was filtered with a 0.22 μm water filter membrane, HPLC analysis column: chromatographic column Diamonsil C18 (250x 4.6mm, 5 μm), mobile phase V (2mM sodium heptane sulfonate and 40mM diammonium phosphate solution): V (methanol) = 82:18, pH was adjusted to 3.0 with phosphoric acid, flow rate was 0.8 mL / min, column temperature was 30°C, UV detection wavelength was 254 nm, injection volume was 10 μL.

[0035] Definition of enzyme activity: Under these conditions, the amount of enzyme required to catalyze the production of 1 μmol S-adenosylmethionine (SAM) per minute is one enzyme activity unit (1U). In the following examples, crude enzyme activity refers to the enzyme activity per unit volume of crude enzyme solution, with the unit of U·mL -1 .

[0036] Definition of specific enzyme activity: the number of enzyme activity units per unit weight (mg) of protein. The calculation formulas for enzyme activity and specific enzyme activity are as follows:

[0037]

[0038]

[0039] In the formula, A SAMis the molar concentration of the product S-adenosylmethionine in the reaction solution, V is the volume of the reaction solution, t is the conversion time (min), and the unit of enzyme activity is U = μmol·min -1 .

[0040] Example 1 Construction of mutants

[0041] 1. Construction of E. coli BL21 / pET-28a(+)-sam2 wild-type strain

[0042] The S-adenosylmethionine synthetase gene sam2 derived from Saccharomyces cerevisiae and codon-optimized for Escherichia coli was artificially synthesized by GENEWIZ (Suzhou GENEWIZ Biotechnology Co., Ltd.). The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.1.

[0043] The target fragment sam2 was amplified according to the primers in Table 1, and then connected with the plasmid pET-28a double-enzymed with restriction endonucleases (EcoR I, Hind III) using the homologous recombinase ClonExpress IIOne Step Cloning Kit in a 37°C metal bath for 30 minutes to obtain the recombinant plasmid pET28a-sam2, and the recombinant plasmid pET28a-sam2 was transformed into Escherichia coli BL21 (DE3), spread on an LB plate containing Kan, and when a single colony grew, the transformant was picked for colony PCR verification. If the bands were consistent, they were sent to Tianlin Biotechnology (Shanghai) Co., Ltd. for sequencing to obtain the recombinant Escherichia coli BL21-pET28a-sam2 expressing wild-type S-adenosylmethionine synthetase.

[0044] Table 1 Primers used for construction of recombinant strains

[0045]

[0046] Note: The lowercase sequence is the recombinant sequence added to the 5' end

[0047] 2. Construction of mutant strains

[0048] (1) Screening of mutation sites

[0049] AlphaFold 2 and SWISS MODEL (https: / / swissmodel.expasy.org / ) were used to predict the three-dimensional model of MAT protein and evaluate the model. The substrate and protein were docked by AutoDock Vina, and the best docking position was selected based on the docking analysis. The protein was visualized and analyzed using the software Pymol.

[0050] Single site mutant construction:

[0051] The site-directed mutation site was introduced by the inverse PCR method, and the recombinant plasmid pET28a-sam2 obtained in Example 1 was used as a template for single-point mutation, and the primers were as shown in Table 2. Then, the methylation site was recognized by Dpn I enzyme and the template was digested by enzyme digestion, and transformed into Escherichia coli BL21 (DE3), and spread on the LB plate containing Kan, and a single colony was grown, and finally the bacteria were picked and sequenced to obtain the recombinant Escherichia coli expressing the mutant. Taking the construction of recombinant Escherichia coli expressing mutant I189V as an example, the recombinant plasmid pET28a-sam2 was used as a template, and I189V-F and I189V-R were used as primers to perform reverse PCR to obtain the recombinant plasmid pET28a-sam2-I189V. The methylation site was then recognized by Dpn I enzyme and the template was digested by enzyme digestion and transformed into Escherichia coli BL21 (DE3). The template was spread on an LB plate containing Kan and a single colony was grown. Finally, the colony was picked and sequenced to obtain the recombinant E. coli BL21-pET28a-sam2-I189V expressing mutant I189V.

[0052] Table 2 Primers used for site-directed mutagenesis

[0053]

[0054] Construction of multi-site mutants:

[0055] Taking the strain E. coli BL21-pET28a-sam2-I189V / V266H / N371R as an example, the plasmid pET28a-sam2-I189V was used as a template, and the primers V266H-F and V266H-R were selected for inverse PCR to construct the recombinant plasmid pET28a-sam2-I189V / V266H. Then, the plasmid pET28a-sam2-I189V / V266H was used as a template, and N371R-F and N371R-R were used as primers to introduce the site-directed mutation site by inverse PCR to construct the recombinant plasmid pET28a-sam2-I189V / V266H / N371R. I enzyme recognized the methylation site and digested the template, transformed into Escherichia coli BL21 (DE3), spread on the LB plate containing Kan, waited for single colonies to grow, and finally performed bacterial picking and sequencing verification to obtain recombinant E. coli BL21-pET28a-sam2-I189V / V266H / N371R expressing the mutant I189V / V266H / N371R.

[0056] Mutant screening:

[0057] In order to screen mutants with improved enzyme activity, the enzyme activity of the unmutated strain E. coli BL21 / pET-28a(+)-sam2(WT) was used as a blank control, defined as 100%. The 15 constructed mutants and WT were induced at 25°C for 28 hours, and their enzyme activities were measured. The culture conditions were as follows: the seed solution cultured at 37°C and 220 rpm for 12 hours was transferred to the fermentation medium at an inoculum of 2%, and cultured for about 1-2 hours until the OD 600 =0.6-0.8, add IPTG with a final concentration of 0.1mM, and culture at 25°C and 220rpm for 24h. At the end of the culture, the bacterial concentration in the fermentation liquid is controlled to be OD 600 About 3.3±0.3.

[0058] Take 2 mL of fermentation liquid and heat it at 4 °C and 8000 r / min -1 Centrifuge for 5 minutes, resuspend and wash the cells with 50mM Tris-HCl (pH 8.0) buffer, centrifuge for 5 minutes, and repeat the washing twice. Take 1mL of buffer to suspend the cells, perform ultrasonic disruption, and then spin at 4℃ and 12000r·min. -1 Centrifuge for 10 minutes, and the supernatant is the crude enzyme solution, and its crude enzyme activity is determined. In addition, the mutant crude enzyme is purified by nickel column affinity chromatography, and its specific enzyme activity is determined under the optimal conditions.

[0059] Under the same conditions, the relative enzyme activity of the mutant and the wild type was calculated with the wild type relative enzyme activity as 100%. Figure 1 and Table 3.

[0060] Table 3 Enzyme activities of mutants

[0061]

[0062] The results are as follows Figure 1 As shown in Table 3, the specific enzyme activity was 0.2466±0.0022 U·mg compared with the original -1 The crude enzyme activity and specific enzyme activity of WT, mutants I189V, I189V / N371R, I189V / Q234N, I189V / D200E, and I189V / V266H / N371R were improved to varying degrees, among which I189V / N371R was the most significantly improved, with a crude enzyme activity of 0.1012 U·ml -1 , which was increased by 303.19% compared with the parent WT; the specific enzyme activity was 1.092±0.0497U·mg -1 , an increase of 342.82% compared with the parent WT.

[0063] Example 2: Whole-cell catalytic synthesis of SAM

[0064] The wild-type E. coli BL21-pET28a-sam2 and mutant strains E. coli BL21-pET28a-sam2-I189V, E. coli BL21-pET28a-sam2-I189V / N371R, E. coli BL21-pET28a-sam2-I189V / Q234N, E. coli BL21-pET28a-sam2-I189V / D200E, and E. coli BL21-pET28a-sam2-I189V / V266H / N371R were streaked and activated, and single colonies were picked and transferred to 10 mL LB liquid culture medium containing Kan, cultured at 37°C, 220 rpm for 12 h, and transferred to 50 mL LB liquid culture medium containing Kan with a 2% inoculum. Incubate in TB medium at 37°C and 220 rpm for about 1.5-2.5 h until the biomass OD 600 =0.6-0.8, add IPTG with a final concentration of 0.1 mM, and culture at 25°C and 220 rpm for 24 h. Collect the cells, wash them twice with 50 mM Tris-HCl (pH 8.0) buffer, and perform whole-cell catalysis.

[0065] Whole cell catalytic system (based on final concentration): biomass OD 600 =5±0.4, 100mM Tris-HCl Buffer (pH 8.0), 20mM L-Met, 50mM KCl, 20mM MgCl2, 20mM ATP, 220rpm, 37℃ for 2h, then add an equal volume of 20% perchloric acid to terminate the reaction, centrifuge and take the supernatant, and use HPLC to detect the production of SAM. The specific results are shown in Figure 2 . Figure 2 The results showed that after 2h of whole-cell reaction, the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V was 479.91mg / L, the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V / N371R was 1119.34mg / L, the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V / D200E was 695.81mg / L, the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V / Q234N was 581.65mg / L, and the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V / N371R was 1119.34mg / L. The highest concentration of SAM synthesized by BL21-pET28a-sam2-I189V / V266H / N371R was 1552.81 mg / L, which was about 461.96% higher than that of the wild type WT (276.32 mg / L).

[0066] In summary, the present invention heterologously expresses S-adenosylmethionine synthetase from Saccharomyces cerevisiae, constructs recombinant Escherichia coli, transforms S-adenosylmethionine synthetase protein, performs site-directed mutagenesis on it, and improves enzyme activity. Through whole-cell catalysis, the output of S-adenosylmethionine synthesized by mutant I189V / V266H / N371R reaches 1440.41 mg / L. The present invention successfully constructs an exogenous expression pathway for S-adenosylmethionine, further improves enzyme activity, saves raw materials and costs, improves economic benefits, and lays a foundation for industrial production.

[0067] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A mutant of S-adenosylmethionine synthetase, characterized in that Based on the amino acid sequence shown in SEQ ID NO.1, any of the following mutations are performed: (1) Mutation of isoleucine at position 189 to valine; (2) mutating isoleucine at position 189 to valine and asparagine at position 371 to arginine; (3) mutating the isoleucine at position 189 to valine and the aspartic acid at position 200 to glutamic acid; (4) mutating isoleucine at position 189 to valine and glutamine at position 234 to asparagine; (5) The isoleucine at position 189 was mutated to valine, the valine at position 266 was mutated to histidine, and the asparagine at position 371 was mutated to arginine.

2. A gene encoding the mutant according to claim 1.

3. An expression vector carrying the gene according to claim 2.

4. A microbial cell expressing the mutant according to claim 1 or carrying the expression vector according to claim 3.

5. A recombinant Escherichia coli, characterized in that The mutant according to claim 1 is expressed using Escherichia coli BL21 (DE3) as a host and pET28a as an expression vector.

6. A method for producing S-adenosylmethionine, characterized in that: The mutant according to claim 1, or the lysate of the microbial cells according to claim 4, or the lysate of the recombinant Escherichia coli according to claim 5 is used as a catalyst, and L-methionine, adenosine-5'-triphosphate disodium salt, sodium chloride, and magnesium chloride are used as substrates to carry out the reaction.

7. The method according to claim 6, characterized in that The reaction was carried out at pH 7-9 and 37-60°C for at least 2 h.

8. A method for improving the catalytic activity of S-adenosylmethionine synthetase, characterized in that: Based on the amino acid sequence shown in SEQ ID NO.1, any of the following mutations are performed: (1) Mutation of isoleucine at position 189 to valine; (2) mutating isoleucine at position 189 to valine and asparagine at position 371 to arginine; (3) mutating the isoleucine at position 189 to valine and the aspartic acid at position 200 to glutamic acid; (4) mutating isoleucine at position 189 to valine and glutamine at position 234 to asparagine; (5) The isoleucine at position 189 was mutated to valine, the valine at position 266 was mutated to histidine, and the asparagine at position 371 was mutated to arginine.

9. Use of the mutant according to claim 1, or the microbial cell according to claim 4, or the recombinant Escherichia coli according to claim 5 in the preparation of S-adenosylmethionine or a product containing S-adenosylmethionine.

10. The use according to claim 9, characterized in that: The products include food, medicine or daily chemical products.

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