Polyphosphokinase mutant as well as preparation method and application thereof

Abstract

The invention discloses a polyphosphate kinase mutant and a preparation method and application thereof, wild type polyphosphate kinase comes from Pseudomonas aeruginosa PAO1, active sites of the wild type polyphosphate kinase are mutated by means of directed evolution to obtain the polyphosphate kinase mutant with high catalytic synthesis ATP (adenosine triphosphate) activity, the relative activity of the polyphosphate kinase mutant to a substrate ADP (adenosine diphosphate) or AMP (adenosine monophosphate) is far higher than that of the wild type, and the polyphosphate kinase mutant can be used for preparing an ATP (adenosine monophosphate). The mutation is at least one of D307M, W408A and Y431P, or at least one of E304V, P331S and Y431P. According to the method, efficient synthesis from a cheap substrate to a high-value product is realized, industrial production of ATP is facilitated, and the method has a good application prospect in the fields of medicines and the like.

Description

Technical Field

[0001] The invention relates to an enzyme mutant and a preparation method and application thereof, belonging to the technical field of enzyme engineering and gene engineering. Background Art

[0002] Polyphosphate Kinase (PPK) can catalyze the conversion of inorganic polyphosphate (polyP) and adenosine diphosphate (ADP) to synthesize an important energy molecule in the body - adenosine triphosphate (ATP). In industrial production, PPK provides important support for biocatalysis, biosynthesis and environmental governance: using PPK to catalyze polyP and ADP to generate ATP is an efficient, environmentally friendly and economical method for synthesizing ATP; PPK can also form an energy circulation system with other enzymes, such as combining with adenylate kinase (AK) to regenerate ATP, promote the conversion of adenosine monophosphate (AMP) or ADP into target nucleotides, and improve production efficiency.

[0003] However, the existing PPK has problems such as low catalytic activity, which limits its industrial application. Chinese patent CN105624238A discloses a method for regenerating ATP by rationally designing an enzyme. The modified polyphosphate kinase from Sinorhizobium alfalfa is rationally transformed and heterologously expressed by gene mutation. It can use ADP as a substrate and use cheap tetrapolyphosphate as a phosphate donor to produce ATP economically and efficiently. Chinese patent application CN115806957A discloses a polyphosphate kinase mutant and its application in glutamine synthesis. The mutant enzyme ChPPKK103E is derived from the bacteria of the water horse tooth family. It has the advantages of expanding the substrate spectrum and improving enzyme activity. Its catalytic activity is increased by 182.2% compared with the original enzyme. Since PPKs from different sources are different in sequence, structure, function, etc., it is necessary to develop more polyphosphate kinases with high catalytic activity to meet the needs of industrial production. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a polyphosphate kinase mutant with high ATP synthesis activity, and to provide related nucleotide sequences, recombinant vectors, recombinant cells and preparation methods.

[0005] Technical solution: The polyphosphate kinase mutant of the present invention is obtained by amino acid mutation of the sequence shown in SEQ ID NO.1, wherein the mutation is at least one of D307M, W408A, and Y431P; or, the mutation is at least one of E304V, P331S, and Y431P.

[0006] The polyphosphate kinase mutants are preferably W408A, Y431P, D307M / Y431P, D307M / W408A / Y431P, E304V, E304V / Y431P, E304V / P331S / Y431P, and their amino acid sequences are SEQ ID NOs. 2 to 8, respectively.

[0007] The present invention uses the polyphosphate kinase (PaPPK. PDB: 3CZP) of Pseudomonas aeruginosa PAO1 as the original enzyme (wild type), whose amino acid sequence is SEQ ID NO.1, and the gene sequence encoding the enzyme is SEQID NO.9. The present invention uses the sequence and structural information of the publicly reported polyphosphate kinase, performs non-redundant retrieval in databases such as PDB, selects the PaPPK gene based on the principles of protein structure similarity, conservative site analysis and host source diversity, and obtains a pure enzyme through functional expression and purification in an Escherichia coli expression system. By amplifying the PaPPK gene and using semi-rational design to perform directed evolution transformation on it, a polyphosphate kinase mutant with high ATP synthesis activity is finally obtained.

[0008] The present invention uses the standard single-letter code for amino acids and the standard substitution notation, for example: W408A means that the tryptophan (W) at position 408 at the N-terminus is mutated to alanine (A); D307M / Y431P means that the aspartic acid (D) at position 307 at the N-terminus is mutated to methionine (M), and the tyrosine (Y) at position 316 at the N-terminus is mutated to proline (P).

[0009] The present invention also provides a nucleotide sequence encoding the polyphosphate kinase mutant. The nucleotide sequence can be obtained by base mutation of the sequence shown in SEQ ID NO.9, such as SEQ ID NO.10-16.

[0010] The present invention also provides a recombinant vector comprising the nucleotide sequence, including a cloning vector or an expression vector, which may be a plasmid or a virus, and can maintain replication ability in a host cell, and amplify or express the nucleotide sequence.

[0011] The present invention also provides a recombinant cell, comprising the recombinant vector.

[0012] The present invention also provides a method for preparing the polyphosphate kinase mutant, comprising the following steps:

[0013] (1) Designing point mutation primers, using a plasmid carrying a wild-type polyphosphate kinase gene as a template, and using the point mutation primers to perform PCR reactions, respectively, and obtaining mutant gene fragments and linearized plasmids after purification;

[0014] (2) The mutant gene fragment is connected to the linearized plasmid to construct an expression vector, and the expression vector is transferred into the host bacteria to induce the expression of the polyphosphate kinase mutant.

[0015] Preferably, in step (1), the point mutation primer is:

[0016] Mutation primer name Sequence (5'-3') W408A_F GGCATTATTGTGGTTAAGTTCGCGCTGGCCATTG W408A_R CTGGGTCTGTTTATCAATGGCCAGCGCGAACTTAACC Y431P_F CCCCGTATAAGCGTCCGAAAATCACCGAAGAAG Y431P_R GCCAGTCTTCTTCGGTGATTTTCGGACGCTT D307M_F GGTGGCCGTTTTTGAAGGTAATATGGCAGCCGGC D307M_R GGCACCGCCTTTGCCGGCTGCCATATTACCTTC E304V_F GTCTGGTGGCCGTTTTTGTAGGTAATGATGCAG E304V_R GCCGGCTGCATCATTACCTACAAAAACGGCCAC P331S_F CGTCAGTATCATATTGTGTCGATTGCCGCACCG P331S_R CTTCTTCGGTCGGTGCGGCAATCGACACAATATG .

[0017] Preferably, in step (1), the PCR reaction system is:

[0018] Composition volume 10×BufferforKOD-Plus- 2.5μL 2mM dNTP 2.5μL <![CDATA[25mMMgSO4]]> 1.5μL DMSO 1μL 10pmol / μLForwardPrimer 0.75μL 10pmol / μLReversePrimer 0.75μL DNA template <100ng KOD-Plus- 1μL <![CDATA[ddH2O]]> up to 25 μL .

[0019] Preferably, in step (2), after expression is completed, the cells are collected; or the cells are disrupted and the crude enzyme solution is collected; or the cells are disrupted and the separated and purified polyphosphate kinase mutant is collected.

[0020] The present invention also provides a product, which comprises the polyphosphate kinase mutant, or the nucleotide sequence, or the recombinant vector, or the recombinant cell.

[0021] The invention also provides application of the product in synthesizing ATP.

[0022] Preferably, the substrate for synthesizing ATP is AMP or ADP.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the present invention provides a polyphosphate kinase mutant with high ATP synthesis activity, the relative activity of the mutant D307M / W408A / Y431P to the substrate ADP is 221% of that of the wild type, and the relative activity of the mutant E304V / P331S / Y431P to the substrate AMP is 198% of that of the wild type. The present invention realizes the efficient synthesis of high-value ATP from cheap polyphosphate, is conducive to the industrial production of ATP, and has good application prospects in the fields of medicine and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the relative activities of the original enzyme WT and mutants in catalyzing the synthesis of ATP using ADP as substrate;

[0025] Figure 2 Schematic diagram of the relative activities of the original enzyme WT and the mutant in catalyzing the synthesis of ATP using AMP as substrate. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0027] The polyphosphate kinase (PaPPK. Pdb: 3CZP) of Pseudomonas aeruginosa PAO1 was used as the original enzyme (wild type). According to the coding sequence of the enzyme in the Pdb database, Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the gene fragment and construct it on the pET22b vector, named pET22b-PaPPK gene. Its amino acid sequence is shown in SEQ ID NO.1, and the gene sequence is shown in SEQ ID NO.9.

[0028] Example 1: A polyphosphate kinase mutant W408A

[0029] In this example, the tryptophan at position 408 of the wild-type enzyme amino acid sequence N-terminal is mutated to alanine. The preparation method is as follows:

[0030] 1. Preparation of wild-type polyphosphate kinase PaPPK

[0031] The pET22b-PaPPK gene vector was transformed into the E.coli Top10 strain and screened with an ampicillin-resistant LB plate to obtain clones. One clone was selected and inoculated into a 20 mL ampicillin-resistant LB medium test tube and cultured at 37°C and 220 rpm for 12 hours. After the culture, the bacteria were centrifuged at 12,000 rpm for 1 minute and the cells were collected. The plasmid was extracted from E.coli Top10 / pET22b-PaPPK using a high-purity plasmid extraction kit as a template for iterative mutation, and the plasmid pET22b-PaPPK mutant was constructed. It was sequenced by Suzhou Jinweizhi Biotechnology Co., Ltd., and clones with correct insertion into the vector and no mutation were selected to obtain the polyphosphate kinase gene expression vector, named pET22b-PaPPK.

[0032] The expression vector pET22b-PaPPK is transformed into E. coli BL21 (DE3) to obtain the E. coli genetic engineering bacteria pET22b-PaPPK / BL21 (DE3) capable of expressing polyphosphate kinase.

[0033] The engineered bacteria pET22b-PaPPK / BL21 (DE3) was inoculated into the culture medium at a 0.1% inoculum volume, and the volume of the shake flask was 100 mL / 250 ml (containing 100 mg / L ampicillin). When the bacterial solution OD 600 When it reaches 0.6-0.8, add isopropylthiogalactoside (IPTG) with a final concentration of 0.5mM for induction, cool to 18°C ​​and continue culturing for about 20h, centrifuge at 4000rpm for 10min, collect the bacteria, and resuspend with 100mM KPi buffer at pH7.5 to obtain bacteria containing polyphosphate kinase.

[0034] 2. Preparation of polyphosphate kinase mutant W408A

[0035] The target mutant gene was obtained by whole plasmid PCR and primers were designed. The amino acid sequence of W408A is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.10.

[0036] W408A upstream primer: GGCATTATTGTGGTTAAGTTC GC GCTGGCCATTG

[0037] W408A downstream primer: CTGGGTCTGTTTATCAATGGCCAGC GC GAACTTAACC

[0038] After the PCR amplification was completed, the amplified product was detected by 0.9% agarose gel electrophoresis, and the result showed that the amplified product was a single band with a size of about 7000 bp. The amplified product was purified and recovered using a DNA recovery and purification kit.

[0039] The purified gene fragment was digested with DpnI to remove the template and then recombined with recombinase. The recombinant product was transformed into E.coli DH5α competent cells, spread on the surface of LB solid culture medium containing 100μg / mL ampicillin, cultured at 37℃ for 12h, picked a single colony to LB liquid culture, identified the successfully constructed positive transformant by PCR, and verified the correctness of the mutation site by sequencing. After verification, a part of it was added with sterile glycerol at a final concentration of 25%, numbered, and stored at -80℃ for use, and a part of the bacteria was extracted with a plasmid extraction kit, and the recombinant plasmid was stored in a -20℃ refrigerator.

[0040] The recombinant expression plasmid pET22b that was successfully sequenced was transferred into E. coli BL21 (DE3) as the expression host to construct the recombinant mutant expression strain E. coli BL21 (DE3) / pET22b-PaPPK.

[0041] The successfully constructed recombinant mutant expression strain E. coli BL21 (DE3) / pET22b-PaPPK was spread on a plate containing ampicillin at a final concentration of 100 μg / mL, and a single colony was picked and inoculated into 0.5 mL of LB medium containing resistance and cultured at 37 ° C and 200 rpm overnight. 1% of the inoculum was transferred to 2 mL of TB medium containing resistance and cultured at OD 600When the p-value reached about 0.6, IPTG was added to a final concentration of 0.5 mM, and the mixture was induced for about 20 h at 18° C. After induction, the deep-well plate was centrifuged and resuspended to 1.5 mL with 100 mM KPi buffer (pH 7.5) to obtain cells containing polyphosphate kinase mutants.

[0042] Example 2: A polyphosphate kinase mutant Y431P

[0043] In this example, the tyrosine at position 431 of the N-terminal amino acid sequence of the wild-type enzyme is mutated to proline. The amino acid sequence of the mutant is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.11. The preparation method is basically the same as that of Example 1, except that the mutant primer is:

[0044] Y431P upstream primer: CCCCGTATAAGCGT CCG AAAATCACCGAAGAAG

[0045] Y431P downstream primer: GCCAGTCTTCTTCGGTGATTTT CGG ACGCTT

[0046] Example 3: A polyphosphate kinase mutant D307M / Y431P

[0047] In this example, the tyrosine at position 431 of the wild-type enzyme amino acid sequence is mutated to proline, and the aspartic acid at position 307 is mutated to methionine. The amino acid sequence of the mutant is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.12. The preparation method is basically the same as that of Example 1, except that the mutation primer is:

[0048] D307M upstream primer: GGTGGCCGTTTTTGAAGGTAAT ATG GCAGCCGGC

[0049] D307M downstream primer: GGCACCGCCTTTGCCGGCTGC CAT ATTACCTTC

[0050] Y431P upstream primer: CCCCGTATAAGCGT CCG AAAATCACCGAAGAAG

[0051] Y431P downstream primer: GCCAGTCTTCTTCGGTGATTTT CGG ACGCTT

[0052] Example 4: A polyphosphate kinase mutant W408A / D307M / Y431P

[0053] In this example, the tyrosine at position 431 of the wild-type enzyme amino acid sequence is mutated to proline, the aspartic acid at position 307 is mutated to methionine, and the tryptophan at position 408 is mutated to alanine. The amino acid sequence of the mutant is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.13. The preparation method is basically the same as that of Example 1, except that the mutation primer is:

[0054] D307M upstream primer: GGTGGCCGTTTTTGAAGGTAAT ATG GCAGCCGGC

[0055] D307M downstream primer: GGCACCGCCTTTGCCGGCTGC CAT ATTACCTTC

[0056] W408A upstream primer: GGCATTATTGTGGTTAAGTTC GC GCTGGCCATTG

[0057] W408A downstream primer: CTGGGTCTGTTTATCAATGGCCAGC GC GAACTTAACC

[0058] Y431P upstream primer: CCCCGTATAAGCGT CCG AAAATCACCGAAGAAG

[0059] Y431P downstream primer: GCCAGTCTTCTTCGGTGATTTT CGG ACGCTT

[0060] Example 5: A polyphosphate kinase mutant E304V

[0061] In this example, the glutamic acid at position 304 of the N-terminal amino acid sequence of the wild-type enzyme is mutated to valine. The amino acid sequence of the mutant is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO.14. The preparation method is basically the same as that of Example 1, except that the mutant primer is:

[0062] E304V upstream primer: GTCTGGTGGCCGTTTTTG T AGGTAATGATGCAG

[0063] E304V downstream primer: GCCGGCTGCATCATTACCT A CAAAAACGGCCAC

[0064] Example 6: A polyphosphate kinase mutant E304V / Y431P

[0065] In this example, the glutamic acid at position 304 of the wild-type enzyme amino acid sequence N-terminal is mutated to valine, and the tyrosine at position 431 is mutated to proline. The amino acid sequence of the mutant is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.15. The preparation method is basically the same as that of Example 1, except that the mutation primer is:

[0066] E304V upstream primer: GTCTGGTGGCCGTTTTTG T AGGTAATGATGCAG

[0067] E304V downstream primer: GCCGGCTGCATCATTACCT A CAAAAACGGCCAC

[0068] Y431P upstream primer: CCCCGTATAAGCGT CCG AAAATCACCGAAGAAG

[0069] Y431P downstream primer: GCCAGTCTTCTTCGGTGATTTT CGG ACGCTT

[0070] Example 7: A polyphosphate kinase mutant E304V / P331S / Y431P

[0071] In this example, the glutamic acid at position 304 of the N-terminal amino acid sequence of the wild-type enzyme is mutated to valine, the proline at position 331 is mutated to serine, and the tyrosine at position 431 is mutated to proline.

[0072] E304V upstream primer: GTCTGGTGGCCGTTTTTG T AGGTAATGATGCAG

[0073] E304V downstream primer: GCCGGCTGCATCATTACCT A CAAAAACGGCCAC

[0074] Y431P upstream primer: CCCCGTATAAGCGT CCG AAAATCACCGAAGAAG

[0075] Y431P downstream primer: GCCAGTCTTCTTCGGTGATTTT CGG ACGCTT

[0076] P331S upstream primer: CGTCAGTATCATATTGTG T CGATTGCCGCACCG

[0077] P331S downstream primer: CTTCTTCGGTCGGTGCGGCAATCG A CACAATATG

[0078] Performance test: Detect the activity of polyphosphate kinase in catalyzing the synthesis of ATP

[0079] Since ATP has a high polarity and is difficult to detect using liquid chromatography, a three-enzyme cascade reaction of polyphosphate kinase, hexokinase, and glucose-6-phosphate dehydrogenase was established to detect the absorbance change of NADPH generated by the reaction at 340nm to indirectly detect the enzyme activity of polyphosphate kinase. The cascade reaction process is as follows:

[0080]

[0081] Detection method: Add the resuspended bacterial solution to the ELISA plate, add 50mM glucose, 50mM MgCl2, 50mM sodium hexametaphosphate, 10mM NAD + , 10mM ADP or AMP, and immediately detect with a microplate reader, record the change of absorbance at 340nm from 0 to 4min, and record once every minute. Calculate the enzyme activity based on the change of sample absorbance detected.

[0082] The test results are shown in Table 1, Table 2, Figure 1 , Figure 2 The best polyphosphate kinase mutant for ADP response was W408A / D307M / Y431P, whose relative activity was 221% of the wild type; the best mutant for AMP response was E304V / P331S / Y431P, whose relative activity was 198% of the wild type.

[0083] Table 1 Absorbance of original enzyme WT and mutant D307M / Y431P / W408A at 340nm

[0084]

[0085] Table 2 Absorbance of original enzyme WT and mutant E304V / P331S / W408A at 340nm

[0086]

Claims

1. A polyphosphate kinase mutant obtained by amino acid mutation of the sequence shown in SEQ ID NO.1, characterized in that: The mutation is at least one of D307M, W408A, and Y431P; or, the mutation is at least one of E304V, P331S, and Y431P.

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

3. A recombinant vector, characterized in that: Comprising the nucleotide sequence of claim 2.

4. A recombinant cell, characterized in that Comprising the recombinant vector according to claim 3.

5. A method for preparing the polyphosphate kinase mutant according to claim 1, characterized in that: The steps include: (1) Designing point mutation primers, using a plasmid carrying a wild-type polyphosphate kinase gene as a template, and using the point mutation primers to perform PCR reactions, respectively, and obtaining mutant gene fragments and linearized plasmids after purification; (2) The mutant gene fragment is connected to the linearized plasmid to construct an expression vector, and the expression vector is transferred into the host bacteria to induce the expression of the polyphosphate kinase mutant.

6. The preparation method according to claim 5, characterized in that: In step (1), the point mutation primer is: 。 7. The preparation method according to claim 5, characterized in that: In step (1), the PCR reaction system is:

8. A product, characterized in that The product comprises the polyphosphate kinase mutant according to claim 1, or the nucleotide sequence according to claim 2, or the recombinant vector according to claim 3, or the recombinant cell according to claim 4.

9. Use of the product according to claim 8 in synthesizing ATP.

10. The use according to claim 9, characterized in that: The substrate for synthesizing ATP is AMP or ADP.

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