S-methyl-5'-thioribose kinase mutants and their use in ribose-1-phosphate synthesis

Abstract

The application discloses an S-methyl-5'-thioribose kinase mutant and application thereof in ribose-1-phosphate synthesis, relates to the technical field of genetic engineering and enzyme engineering, and specifically discloses a high-activity S-methyl-5'-thioribose kinase mutant. The mutant is obtained through directional mutation, key mutations occur at the 128th position and the 240th position, and the catalytic efficiency on ribose-1-phosphate is significantly improved. The mutant is suitable for industrial production of ribose-1-phosphate and derivatives thereof.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, specifically to an S-methyl-5'-thioribokinase mutant and its application in the synthesis of ribose-1-phosphate. Background Technology

[0002] S-methyl-5'-thioribokinase (MTRK) is an ATP-dependent phosphotransferase belonging to the glycokinase family. Its natural substrate is S-methyl-5'-thioribose, which catalyzes the following reaction: S-methyl-5'-thioribose + ATP → ribose-1-phosphate + ADP + S-methylthiol.

[0003] MTRK participates in the sulfur metabolism pathway in microorganisms, converting S-methyl-5'-thioribose into ribose-1-phosphate, which can then enter the pentose phosphate pathway or nucleotide synthesis. In Escherichia coli, this enzyme is encoded by the gene mtnK and is regulated by sulfur sources (such as upregulated expression in methionine deficiency).

[0004] Ribo-1-phosphate is a key intermediate in the synthesis of nucleotides in living organisms. Its industrial applications mainly include: drug synthesis: nucleoside analogs: as precursors for antiviral drugs (such as ribavirin and acyclovir) and anticancer drugs (such as gemcitabine). mRNA vaccine excipients: used to synthesize modified nucleosides (such as pseudouridine ψ) to improve vaccine stability and translation efficiency.

[0005] Meanwhile, traditional chemical methods for synthesizing ribose-1-phosphate require multiple protection-deprotection reactions (such as ribose-5-phosphate isomerization), resulting in problems such as low yield (<40%), environmental pollution (using organophosphorus reagents and heavy metal catalysts), and low chiral purity (requiring additional resolution steps).

[0006] Phosphorylation reactions using biocatalysis have advantages such as high atom economy (theoretical yield of 100%), strict stereoselectivity (producing only β-ribose-1-phosphate), and mild conditions (room temperature and pressure, neutral pH). However, the activity defects of wild-type S-methyl-5'-thioribose enzymes limit their industrial application.

[0007] Therefore, it is of great significance to invent an S-methyl-5'-thioribose kinase mutant and apply it to catalyze the production of ribose-1-phosphate and its derivatives from S-methyl-5'-thioribose. Summary of the Invention

[0008] The purpose of this invention is to provide an S-methyl-5'-thioribokinase mutant and its application in ribose-1-phosphate synthesis, in order to solve the problems raised in the prior art.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] An S-methyl-5'-thioribokinase mutant was obtained by PCR amplification of the wild-type mtnK gene, followed by DNase ligation of the expression vector pET-28a(+) and the PCR amplification product into Escherichia coli BL21(DE3) and screening to obtain the S-methyl-5'-thioribokinase mutant.

[0011] Furthermore, the S-methyl-5'-thioribokinase DNA sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0012] Furthermore, the S-methyl-5'-thioribokinase mutants include single-point mutants and combined mutants.

[0013] Furthermore, the single-point mutant is obtained by replacing the aspartic acid at position 128 of the mtnK gene sequence with glycine or replacing the leucine at position 240 of the mtnK gene sequence with arginine.

[0014] Furthermore, the combined mutant is obtained by replacing the aspartic acid at position 128 and the leucine at position 240 of the mtnK gene sequence with glycine and arginine, respectively.

[0015] Furthermore, the mtnK gene of *E. coli* was obtained by extracting genomic DNA from *E. coli* BL21(DE3) using the CTAB method.

[0016] Furthermore, in the PCR amplification process, the amplification program includes pre-deformation, cycling, and final extension; the pre-deformation temperature is 98℃ and the time is 2 min; the cycling stage includes three steps: deformation, annealing, and extension; the temperatures are 98℃, 55℃, and 72℃, and the times are 10s, 15s, and 1.5 min, respectively; the cycling stage is repeated 30 times; the final extension temperature is 72℃ and the time is 5 min.

[0017] Furthermore, the forward and reverse primers are designed based on the corresponding genomic DNA sequences;

[0018] Furthermore, the mtnK gene of the *E. coli* is 1.2 kb in length.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] 1. Significantly improved catalytic efficiency

[0021] The specific activity was increased by 3.1 times: the specific activity of the mutant reached 47.3 U / mg (the wild type was only 15.2 U / mg), and the catalytic ability per unit amount of enzyme was greatly enhanced.

[0022] The kcat / Km value increased by 10.9 times: the catalytic efficiency of the mutant. Far exceeding wild type This indicates that its affinity for the substrate S-methyl-5'-thioribose and its conversion rate are both significantly optimized.

[0023] Substrate conversion rate >95%: Under the same reaction conditions (10mM substrate, 24h), the mutant almost completely converted the substrate, while the wild-type conversion rate was less than 60%.

[0024] 2. Significantly enhanced thermal stability

[0025] The half-life at 50°C is extended to 6.5 hours (compared to only 1.8 hours for the wild type), making it suitable for long-term use in high-temperature environments in industrial fermentation or in immobilization reactions.

[0026] pH stability broadened: The mutant maintains >80% activity in the pH range of 6.0-8.5 (the wild type is only stable in the pH range of 7.0-7.5), adapting to pH fluctuations in different fermentation or reaction systems.

[0027] 3. Advantages of industrial application

[0028] Reduced production costs: High catalytic efficiency reduces enzyme usage, resulting in a cost reduction of over 40% per unit output.

[0029] Simplified purification process: The yield of mutants in nickel column purification is >90% (wild type is prone to inclusion bodies, yield is only 60%).

[0030] Immobilized enzymes are reusable: after covalent coupling to epoxy resin, they retain 85% of their activity even after 10 consecutive batches of reaction, significantly reducing enzyme consumption.

[0031] 4. Environmental friendliness

[0032] Alternative chemical synthesis methods: avoid the use of toxic organophosphorus reagents and heavy metal catalysts, and reduce the discharge of waste.

[0033] The reaction is carried out under mild conditions: at room temperature and pressure, and at a neutral pH, which conforms to the principles of green chemistry.

[0034] 5. Expand application potential

[0035] Suitable for the synthesis of nucleoside analogues: such as the preparation of intermediates for the antiviral drug ribavirin, improving chiral purity (ee value > 99.9%).

[0036] Facilitating mRNA vaccine production: Efficiently synthesizing modified nucleosides such as pseudouridine (ψ) to improve vaccine stability and efficacy.

[0037] 6. The MTRK mutant obtained through rational design and directed evolution has achieved breakthroughs in catalytic efficiency, stability, and industrial applicability, providing a highly efficient tool enzyme for the green biomanufacturing of ribose-1-phosphate and its derivatives, and has significant industrial application value. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] In the examples, experimental methods without specific conditions are generally performed under conventional conditions.

[0040] In this embodiment, the genome extraction kit, plasmid extraction kit, and DNA purification and recovery kit were purchased from Nanjing Genscript Biotech Co., Ltd.; E. coli BL21(DE3) and plasmid pET-28a(+) were purchased from Qingke Biotechnology Co., Ltd.; DNA marker, low molecular weight standard protein, protein gel, etc. were purchased from Shanghai Sangon Biotech Co., Ltd.; primer synthesis and sequence sequencing were provided by Qingke Biotechnology Co., Ltd.

[0041] Example: Step 1: Mutant amplification

[0042] Genomic DNA of Escherichia coli BL21(DE3) was extracted using the CTAB method to obtain the wild-type mtnK gene; the wild-type mtnK gene was amplified by PCR using the wild-type mtnK gene as template DNA to obtain the PCR amplification product.

[0043] PCR amplification system:

[0044] 2×PhantaMax buffer: 25 μL;

[0045] dNTPs: 1 μL;

[0046] Forward primer: 1 μL;

[0047] Reverse primer: 1 μL;

[0048] Template DNA: 1 μL;

[0049] Phanta Super-Fidelity DNA polymerase: 0.5 μL;

[0050] ddH2O: 20.5 μL.

[0051] Design the corresponding PCR forward and reverse primers as shown above based on the corresponding genomic DNA sequence (SEQ ID NO.1);

[0052] The PCR amplification results were verified positive by DNA agarose gel electrophoresis. The results showed that the PCR amplification products were single bands, each approximately 1200 bp in size. The PCR products were then digested with DpnI enzyme, and the amplification products were purified and recovered using a DNA purification kit.

[0053] Step 2: Construction of expression vector and engineered bacteria

[0054] Expression vector pET-28a(+) and PCR amplification products were double-digested with their respective restriction endonucleases. After digestion, the digested products were purified using a DNA purification kit to remove restriction endonucleases and small nucleotide fragments. The double-digested PCR amplification products were ligated to expression vector pET-28a(+) with corresponding cut sites using T4 DNA ligase to construct expression vectors pET-28a(+)-R128G, pET-28a(+)-L240R, and pET-28a(+)-R128G-L240R. The constructed expression vectors were transformed into E. coli BL21(DE3) and plated on a substrate containing 50 mg / ml of urea. LB plates resistant to natriuretic peptide were incubated at 37°C for 8 hours. Clones were randomly selected, plasmids were extracted, and sequenced for identification. Recombinant E. coli containing the expression plasmids pET-28a(+)-R128G, pET-28a(+)-L240R, and pET-28a(+)-R128G-L240R were screened to obtain single-point mutants R128G, L240R, and combined mutants R128G / L240R.

[0055] Experiment: Enzyme activity detection:

[0056] The glycerol tubes of wild-type mtnK strain, single-point mutant R128G strain, single-point mutant L240R strain, and combined mutant R128G / L240R strain prepared in the above examples were inoculated at 1% in LB medium containing 50 mg / L kanamycin and cultured overnight at 37°C. The next day, they were inoculated at 5% in LB medium containing 50 mg / L kanamycin and cultured at 37°C with shaking at 200 rpm for 2 h. When the OD600 reached approximately 0.6, 0.2 mM IPTG was added and induced at 30°C with 200 rpm for 4 h. After induction, the bacterial culture was centrifuged at 12000 rpm for 2 minutes at 4°C, the supernatant was discarded, the culture was inverted to drain completely, and the cells were resuspended in a 1:4 (w / w) solution of lysis buffer (pH 7.4). The bacterial culture was then sonicated for 2 minutes. After centrifugation, the supernatant was collected as crude enzyme solution. Enzyme activity was detected after purification using a nickel column. The experimental results are shown in Tables 1 and 2 below. The product was ribose-1-phosphate.

[0057] Table 1 Enzyme Activity Data of Single-Point Mutants

[0058]

[0059] Conclusion: The single-point mutant R128G reduced Km and enhanced substrate affinity by eliminating the steric hindrance of Asp128; the single-point mutant L240R significantly increased kcat and improved catalytic rate by stabilizing ATP binding with arginine.

[0060] Table 2. Enzyme activity data of combined mutants

[0061]

[0062] Conclusion: The MTRK mutant obtained by rational design and directed evolution in this invention has achieved breakthroughs in catalytic efficiency, stability, and industrial applicability, providing a highly efficient tool enzyme for the green biomanufacturing of ribose-1-phosphate and its derivatives, and has significant industrial application value.

[0063] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. An S-methyl-5'-thioribokinase mutant, characterized in that: The S-methyl-5'-thioribokinase mutant is a mutant obtained by replacing arginine at position 128 of wild-type S-methyl-5'-thioribokinase with glycine and / or replacing leucine at position 240 with arginine. The wild-type S-methyl-5'-thioribokinase DNA sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. The application of the S-methyl-5'-thioribokinase mutant according to claim 1 in the synthesis of ribose-1-phosphate, characterized in that: Application of the S-methyl-5'-thioribokinase mutant in catalyzing the synthesis of ribose-1-phosphate from S-methyl-5'-thioribose.

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