Polyphosphokinase mutant and application thereof

Abstract

The invention belongs to the field of gene engineering, and particularly relates to a polyphosphate kinase mutant and application thereof. According to the invention, modification and screening are carried out on the basis of amino acids of wild type polyphosphate kinase PPK (polyP-polyphosphate kinase [EC: 2.7.4. 1]) from Mycobacterium tuberculosis, and the polyphosphate kinase with high enzyme activity, high catalytic efficiency and high ATP regeneration efficiency is obtained. The amino acid sequence of the polyphosphate kinase mutant is as shown in SEQ ID NO.11, and the polyphosphate kinase mutant is obtained by mutating valine at the 267th site of wild type polyphosphate kinase into glutamic acid and simultaneously mutating threonine at the 369th site into proline. Uridine monophosphate and cytidine monophosphate are produced through catalysis of the polyphosphokinase, the conversion yield is larger than 95%, meanwhile, cyclic regeneration of ATP is efficiently achieved, the ATP adding amount is reduced by 97%-98%, the product concentration is increased, the production cost is reduced, the catalysis application potential is greatly improved, and the important industrial application prospect is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to a polyphosphate kinase mutant and an application thereof. Background Art

[0002] Adenosine triphosphate (ATP) is a high-energy phosphate compound that serves as an energy donor to fuel catalytic reactions and is commonly used in industry to synthesize high-energy compounds in a variety of biocatalytic reactions. However, ATP's high cost limits its industrial use. Patent CN114606213A discloses a polyphosphate kinase mutant, but it only uses 75% less ATP. Therefore, a more efficient ATP recycling system is needed.

[0003] Polyphosphate kinase (PPK) utilizes inexpensive polyphosphate as a phosphate donor to regenerate ATP and catalyze the synthesis of products. This method is cost-effective and simple to operate. However, PPK currently suffers from low catalytic efficiency and product conversion yields. Therefore, the discovery of more active PPKs is of great application value. Summary of the Invention

[0004] In the first aspect, the technical problem to be solved by the present invention is to provide a polyphosphate kinase mutant in view of the shortcomings of the prior art such as low polyphosphate kinase activity, low polyphosphate kinase catalytic efficiency, and poor ATP regeneration efficiency.

[0005] In a second aspect, the technical problem to be solved by the present invention is to provide a nucleotide sequence encoding the polyphosphate kinase mutant.

[0006] In a third aspect, the technical problem to be solved by the present invention is to provide a recombinant expression vector.

[0007] In a fourth aspect, the technical problem to be solved by the present invention is to provide a recombinant genetically engineered bacterium.

[0008] In a fifth aspect, the technical problem to be solved by the present invention is to provide the application of the polyphosphate kinase mutant in constructing an ATP recycling regeneration system.

[0009] In a sixth aspect, the technical problem to be solved by the present invention is to provide the use of the polyphosphate kinase mutant in catalyzing the synthesis of pyrimidine nucleotides.

[0010] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0011] A polyphosphate kinase mutant, wherein the polyphosphate kinase mutant is obtained by mutating the valine at position 267 of the wild-type polyphosphate kinase to glutamic acid and the threonine at position 369 to proline;

[0012] The wild-type polyphosphate kinase is derived from Mycobacterium tuberculosis, and its amino acid sequence is shown in SEQ ID NO.7. The corresponding nucleotide sequence encoding the wild-type polyphosphate kinase gene is shown in SEQ ID NO.8.

[0013] Specifically, the activity of the polyphosphate kinase mutant is significantly improved after the amino acid site modification, and the enzyme activity is about 2.0 times that of the wild-type polyphosphate kinase.

[0014] Specifically, the enzyme activity is defined as the amount of enzyme required to produce 1 μmol ATP per minute, and the enzyme activity unit is calculated per gram of wet bacteria.

[0015] Wherein, the amino acid sequence of the polyphosphate kinase mutant is shown in SEQ ID NO.11.

[0016] The nucleotide sequence encoding the polyphosphate kinase mutant is also within the scope of protection of the present invention.

[0017] Specifically, the nucleotide sequence is shown in SEQ ID NO.12.

[0018] A recombinant expression vector containing the nucleotide sequence is also within the scope of protection of the present invention.

[0019] The recombinant expression vector can be constructed by conventional methods in the art by ligating the nucleotide sequence of the polyphosphate kinase mutant gene of the present invention to various commercially available empty vectors. The commercially available empty vector can be any conventional plasmid vector in the art, as long as the recombinant expression vector can replicate normally in the corresponding expression host and express the polyphosphate kinase mutant.

[0020] In some embodiments of the present invention, the commercially available empty vector is a pET28a vector for the host Escherichia coli BL21 (DE3).

[0021] A recombinant genetically engineered bacterium containing the nucleotide sequence or the recombinant expression vector is also within the scope of protection of the present invention.

[0022] The use of the polyphosphate kinase mutant in constructing an ATP recycling system is also within the scope of protection of the present invention.

[0023] Specifically, by using the polyphosphate kinase mutant of the present invention, 97-98% of the amount of ATP added can be saved in the ATP recycling system.

[0024] The use of the polyphosphate kinase mutant in catalyzing the synthesis of pyrimidine nucleotides is also within the scope of protection of the present invention.

[0025] The catalysis uses pyrimidine nucleoside, sodium hexametaphosphate, and ATP as substrates and magnesium ions as auxiliary substrates, and catalyzes the synthesis of pyrimidine nucleotides by adding enzyme solutions of pyrimidine nucleoside kinase and polyphosphate kinase mutants.

[0026] Specifically, when the pyrimidine nucleoside is uridine, the pyrimidine nucleotide is uridine monophosphate; when the pyrimidine nucleoside is cytidine, the pyrimidine nucleotide is cytidine monophosphate.

[0027] Specifically, the pyrimidine nucleoside kinase is uridine cytidine kinase UDK, and the uridine cytidine kinase UDK is the substrate-tolerant uridine kinase M2 disclosed in Chinese patent CN118256466A (which can catalyze both uridine and cytidine).

[0028] The catalytic reaction comprises a reaction system comprising: 50-150 mM pyrimidine nucleoside (uridine / cytidine), 1-5 mM ATP, 5-20 mM magnesium chloride, 25-75 mM sodium hexametaphosphate, 1-10 g / L uridine cytidine kinase (UDK) enzyme solution, and 0.1-2 g / L polyphosphate kinase enzyme solution; and reaction conditions comprising: 35-40° C., pH 7.0-8.0, and reaction time of 1-10 h.

[0029] Beneficial effects: The present invention obtains a polyphosphate kinase with high enzyme activity, high catalytic efficiency and high ATP regeneration efficiency by site-directed modification and screening of wild-type polyphosphate kinase derived from Mycobacterium tuberculosis. The amino acid sequence of the polyphosphate kinase mutant is shown in SEQ ID NO.11, and its enzyme activity is more than 2.0 times that of the wild-type polyphosphate kinase. In the present invention, the highly active polyphosphate kinase is used to catalyze the production of uridine and cytidylic acid, and the conversion yield is greater than 95%. At the same time, the regeneration of the ATP energy cycle is efficiently achieved, the amount of ATP added is saved by 97-98%, the product concentration is increased, and the production cost is reduced, which greatly improves the potential of catalytic applications and has important industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0031] Figure 1 It is a recombinant expression vector of the gene encoding the polyphosphate kinase mutant. DETAILED DESCRIPTION

[0032] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0033] Example 1: Construction, expression and screening of polyphosphate kinases from different sources

[0034] Based on the enzyme activity and gene sequences of polyphosphate kinases reported in the literature, the Brenda database, and the NCBI database, the gene sequences of polyphosphate kinases from Corynebacterium glutamicum, Escherichia coli, the thermophilic bacterium Meiothermus cerbereus, Mycobacterium tuberculosis, and Pseudomonas aeruginos were selected and fully synthesized by General Biotechnology (Anhui) Co., Ltd. The genes were ligated into the pET28a vector to obtain plasmids pET28a-CgPPK, pET28a-EcPPK, pET28a-McPPK, pET28a-MtPPK, and pET28a-PaPPK, respectively. Each plasmid was transformed into Escherichia coli BL21(DE3) competent cells to obtain recombinant E. coli expressing different polyphosphate kinases.

[0035] Among them, the amino acid sequence of the polyphosphate kinase from Corynebacterium glutamicum is shown in SEQ ID NO.1, and the corresponding nucleotide sequence is shown in SEQ ID NO.2; the amino acid sequence of the polyphosphate kinase from Escherichia coli is shown in SEQ ID NO.3, and the corresponding nucleotide sequence is shown in SEQ ID NO.4; the amino acid sequence of the polyphosphate kinase from the thermophilic bacterium Meiothermus cerbereus is shown in SEQ ID NO.5, and the corresponding nucleotide sequence is shown in SEQ ID NO.6; the amino acid sequence of the polyphosphate kinase from Mycobacterium tuberculosis is shown in SEQ ID NO.7, and the corresponding nucleotide sequence is shown in SEQ ID NO.8. The amino acid sequence of the polyphosphate kinase from Pseudomonas aeruginos is shown in SEQ ID NO.9, and the corresponding nucleotide sequence is shown in SEQ ID NO.10.

[0036] Recombinant E. coli expressing different polyphosphate kinases were cultured in LB medium (containing kanamycin at a final concentration of 50 mg / L) at 37°C and 200 rpm for 12 hours. A 1% v / v inoculum was transferred to 1 L TB medium (containing kanamycin at a final concentration of 50 mg / L) and cultured at 37°C and 200 rpm until the OD 600 When the pH value was 0.6-0.8, IPTG was added to a final concentration of 0.2 mM for induction. Culture was continued at 28°C and 200 rpm for 12-16 h. The cells were collected by centrifugation and stored in a -80°C refrigerator.

[0037] Weigh 2g of each polyphosphate kinase bacterial cell into a 50mL centrifuge tube, wash twice with 0.1mol / L PBS (pH 7.5), collect the cells by centrifugation, and resuspend in 20mL of 0.1mol / L PBS (pH 7.5) to a 100g / L bacterial suspension. Disrupt the cells using an ultrasonic disruptor in an ice-water bath at 200W power, sonicating for 3s on, 7s off, for approximately 40 minutes until the suspension becomes clear. Centrifuge at 8000rpm at 4°C for 10 minutes, and collect the supernatant, which is the crude polyphosphate kinase enzyme solution.

[0038] The enzyme activity assay system in a 50 mL volume consists of 10 mM ADP, 10 mM magnesium chloride, and 5 mM sodium hexametaphosphate. After adjusting the pH to 7.5, 0.5 g / L crude polyphosphate kinase enzyme solution was added. The reaction was shaken at 200 rpm and 37°C. Samples were taken every 10 minutes and quenched in a boiling water bath. Enzyme activity was defined as the amount of enzyme required to produce 1 μmol ATP per minute and was calculated per gram of wet bacterial cells.

[0039] The enzyme activities of the above five polyphosphate kinases were measured. The results are shown in Table 1. The polyphosphate kinase MtPPK from Mycobacterium tuberculosis had the highest enzyme activity of 194.0 U / g. Subsequently, the polyphosphate kinase MtPPK was mutated.

[0040] Table 1 Comparison of polyphosphate kinase activity from different sources

[0041]

[0042]

[0043] Example 2: Construction and screening of polyphosphate kinase single mutants

[0044] The construction of polyphosphate kinase single mutants was carried out by whole-plasmid PCR. Single-point mutations were made at positions 146, 217, 267, 326, 368, 369, 482, 533, 587, and 720, respectively. The designed mutagenesis primers are shown in Table 2. PCR amplification was performed using the plasmid pET28a-MtPPK constructed in Example 1 as a template and the corresponding primers. The PCR reaction system included: 5 μL 10× PCR Buffer, 5 μL 2 mM dNTPs, 3 μL 25 mM magnesium sulfate, 1.5 μL each of 10 pmol / μL primer F and primer R, 1 μL plasmid template, 1 μL KOD-Plus-Neo polymerase (purchased from Toyobo Co.), and finally, the volume was adjusted to 50 μL with sterile distilled water; the PCR reaction parameters were: melting at 94°C for 2 minutes, denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, extension at 68°C for 4 minutes, 30 cycles, fill at 68°C for 5 minutes, and then storage at 4°C.

[0045] Table 2 Primer sequences corresponding to each single point mutation site

[0046]

[0047] The amplified PCR product was digested with DpnI (purchased from TaKaRa) using the following system: 8 μL of PCR product, 1 μL of 10× QuickCut Buffer, and 1 μL of DpnI at 37°C for 0.5 h. The cells were then transformed into competent E. coli BL21 (DE3) cells and cultured on LB solid plates (containing kanamycin at a final concentration of 50 mg / L) at 37°C for 12 hours to obtain clones. Three clones were selected and cultured in LB medium (containing kanamycin at a final concentration of 50 mg / L) at 37°C and 200 rpm for 12 hours before sequencing. After correct sequencing, the polyphosphate kinase single mutant was induced for expression using the same process as described for the polyphosphate kinase in Example 1.

[0048] Following the cell disruption and enzyme activity assay procedures described in Example 1, enzyme activity assays were performed on the polyphosphate kinase single mutants. The results are shown in Table 3. The wild-type enzyme activity was 194.0 U / g. After the single-point mutations, the enzyme activity of the single mutants increased and decreased. When the mutation sites were V267E, T369P, and A533D, the enzyme activity of the single mutants was significantly improved, with the corresponding relative enzyme activities all exceeding 130%. Combinations of any two or three of these sites were subsequently selected for combined mutations.

[0049] Table 3 Comparison of enzyme activities of wild-type polyphosphate kinase and single mutants

[0050] Single mutant Enzyme activity (U / g) Relative enzyme activity (%) WT (control) 194.0 100.0 S146D 160.4 82.7 Q217E 181.3 93.5 V267E 262.6 135.4 S326P 192.1 99.0 R368P 170.2 87.7 T369P 283.2 146.0 A482V 120.8 62.3 A533D 256.3 132.1 H587Q 149.4 77.0 A720P 182.7 94.2

[0051] Example 3: Construction and screening of polyphosphate kinase combination mutants

[0052] (1) Construction and expression of the double mutant V267E-T369P

[0053] The plasmid of the single mutant V267E was used as a template and primers 369-F and 369-R were used for PCR amplification. The PCR amplification process, PCR product digestion and transformation, and induced expression of the double mutant V267E-T369P were the same as in Example 2.

[0054] (2) Construction and expression of the double mutant V267E-A533D

[0055] The plasmid of the single mutant V267E was used as a template and primers 533-F and 533-R were used for PCR amplification. The PCR amplification process, PCR product digestion and transformation, and induced expression of the double mutant V267E-A533D were the same as in Example 2.

[0056] (3) Construction and expression of the double mutant T369P-A533D

[0057] The plasmid of the single mutant T369P was used as a template and primers 533-F and 533-R were used for PCR amplification. The PCR amplification process, PCR product digestion and transformation, and induced expression of the double mutant T369P-A533D were the same as in Example 2.

[0058] (4) Construction and expression of the three variants V267E-T369P-A533D

[0059] The double mutant V267E-T369P plasmid was used as a template and PCR amplification was performed using primers 533-F and 533-R. The PCR amplification process, PCR product digestion and transformation, and induction expression of the triple mutant V267E-T369P-A533D were the same as in Example 2.

[0060] The enzyme activity of the combined mutants was tested in the same manner as in Example 1. The results are shown in Table 4.

[0061] Table 4 Comparison of enzyme activities of wild-type polyphosphate kinase and combined mutants

[0062] mutant Enzyme activity (U / g) Relative enzyme activity (%) WT 194.0 100.0 V267E-T369P 393.2 203.7 V267E-A533D 200.3 103.2 T369P-A533D 233.6 120.4 V267E-T369P-A533D 284.1 146.4

[0063] The results showed that after combined mutations, the double mutant V267E-T369P had the highest enzyme activity, 393.2 U / g, and its relative activity was more than 2.0 times that of the wild-type. The amino acid sequence of this polyphosphate kinase mutant is shown in SEQ ID NO. 11, and the corresponding nucleotide sequence is shown in SEQ ID NO. 12. Figure 1 The diagram of the construction of the recombinant expression vector of the polyphosphate kinase mutant encoding gene is shown.

[0064] Example 4: Application of polyphosphate kinase mutant (double mutant V267E-T369P) in enzyme-catalyzed production of uridine monophosphate

[0065] To a 3 L reaction system, add 100 mM uridine, 50 mM sodium hexametaphosphate, 2 mM ATP, and 10 mM magnesium chloride, respectively. After adjusting the pH to 7.5, add 1 g / L crude enzyme solution of a polyphosphate kinase mutant and 5 g / L crude enzyme solution of uridine cytidine kinase (UDK) at 37°C. React for 5 hours, terminate the reaction in a boiling water bath for 3 minutes, and measure the uridine monophosphate concentration.

[0066] Comparison of the catalytic performance of wild-type polyphosphate kinase and the polyphosphate kinase mutant V267E-T369P is shown in Table 5. The mutant polyphosphate kinase generated a uridine monophosphate concentration of 31.0 g / L with a conversion yield of 95.6%, while the wild-type polyphosphate kinase generated a uridine monophosphate concentration of 20.6 g / L with a conversion yield of 63.5%. Compared to the wild-type polyphosphate kinase, the mutant generated a significantly higher uridine monophosphate concentration and a significantly improved conversion efficiency.

[0067] In a 3L reaction system, 100mM uridine substrate was added with 100mM ATP and 10mM magnesium chloride, the pH was adjusted to 7.5, and then 5g / L crude uridine cytidine kinase enzyme solution was added at 37°C and reacted for 5 hours. As a control group, the control group achieved a conversion rate of over 95% only with the addition of 100mM ATP. The reaction system of this example only required the addition of 2mM ATP to achieve this effect, saving 98% of the ATP addition and achieving efficient ATP recycling.

[0068] Table 5 Comparison of catalytic effects of wild-type and mutant polyphosphate kinases

[0069] mutant Uridylate production (g / L) Conversion rate (%) WT 20.6 63.5 V267E-T369P 31.0 95.6

[0070] Example 5: Application of polyphosphate kinase mutants in the enzymatic production of cytidylic acid

[0071] In a 3 L reaction system, 70 mM cytidine, 35 mM sodium hexametaphosphate, 2 mM ATP, and 10 mM magnesium chloride were added respectively. After adjusting the pH to 7.5, 1 g / L crude polyphosphate kinase enzyme solution and 5 g / L crude uridine cytidine kinase UDK enzyme solution were added at 37°C. The reaction was allowed to react for 5 hours. The reaction was terminated by boiling in a water bath for 3 minutes, and the concentration of cytidylic acid was measured.

[0072] Comparing the catalytic performance of wild-type polyphosphate kinase and the mutant V267E-T369P, as shown in Table 6, the mutant generated 21.9 g / L of cytidylic acid (CMP) with a conversion yield of 96.8%. Wild-type polyphosphate kinase generated 13.1 g / L of CMP with a conversion yield of 57.9%. Compared to the wild-type, the mutant also significantly increased the CMP concentration and conversion efficiency.

[0073] In a 3 L reaction system, 70 mM cytidine substrate was added with 70 mM ATP and 10 mM magnesium chloride, the pH was adjusted to 7.5, and then 5 g / L crude uridine cytidine kinase enzyme solution was added at 37°C and reacted for 5 hours. This control group achieved a conversion rate of over 95% only with the addition of 70 mM ATP. The reaction system of this example only required the addition of 2 mM ATP, saving 97.14% of the added ATP.

[0074] At the same time, the application of the polyphosphate kinase mutant to catalyze the preparation of uridine and cytidylic acid shows that the enzyme can achieve the cyclic regeneration of ATP in different catalytic systems and has universal applicability.

[0075] Table 6 Comparison of catalytic effects of wild-type and mutant polyphosphate kinases

[0076] mutant Cytidine production (g / L) Conversion rate (%) WT 13.1 57.9 V267E-T369P 21.9 96.8

[0077] The present invention provides a polyphosphate kinase and its application concept and method. There are numerous methods and approaches for implementing this technical solution. The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A polyphosphate kinase mutant, characterized in that The polyphosphate kinase mutant is obtained by mutating the 267th valine of the wild-type polyphosphate kinase to glutamic acid and the 369th threonine to proline. Wherein, the amino acid sequence of the wild-type polyphosphate kinase is shown in SEQ ID NO.

7.

2. The polyphosphate kinase mutant according to claim 1, characterized in that The amino acid sequence of the polyphosphate kinase mutant is shown in SEQ ID NO.

11.

3. A nucleotide sequence, characterized in that Encoding the polyphosphate kinase mutant according to claim 1 or claim 2.

4. A recombinant expression vector, characterized in that: Containing the nucleotide sequence according to claim 3.

5. A recombinant genetically engineered bacterium, characterized in that: Containing the nucleotide sequence of claim 3 or the recombinant expression vector of claim 4.

6. Use of the polyphosphate kinase mutant according to any one of claims 1 to 3 in constructing an ATP recycling system.

7. Use of the polyphosphate kinase mutant according to any one of claims 1 to 3 in catalyzing the synthesis of pyrimidine nucleotides.

8. The use according to claim 7, characterized in that The catalysis uses pyrimidine nucleoside, sodium hexametaphosphate, and ATP as substrates and magnesium ions as co-substrates, and catalyzes the synthesis of pyrimidine nucleotides by adding an enzyme solution of pyrimidine nucleoside kinase and an enzyme solution of the polyphosphate kinase mutant according to any one of claims 1 to 3.

9. The use according to claim 8, characterized in that When the pyrimidine nucleoside is uridine, the pyrimidine nucleotide is uridine monophosphate; when the pyrimidine nucleoside is cytidine, the pyrimidine nucleotide is cytidine monophosphate.

10. The use according to claim 8, characterized in that The pyrimidine nucleoside kinase is uridine cytidine kinase UDK; the catalytic synthesis has the following reaction conditions: 35-40° C., pH=7.0-8.0, and reaction time of 1-10 hours.

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