Polyphosphorylase mutants, methods of making and using the same

Abstract

The application discloses a polyphosphate kinase mutant, a preparation method and application thereof, and wild-type polyphosphate kinase from Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1, The active site is mutated by using a directed evolution method, and a polyphosphate kinase mutant with high activity in catalyzing synthesis of ATP is obtained, the relative activity of the mutant to a substrate ADP or AMP is much higher than that of a wild type, and the mutation is at least one of D307M, W408A and Y431P, or at least one of E304V, P331S and Y431P. The application realizes efficient synthesis from a cheap substrate to a high-value product, is helpful to industrial production of ATP, and has a good application prospect in the fields of medicine and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to an enzyme mutant and its preparation method and application, and belongs to the technical field of enzyme engineering and genetic engineering. BACKGROUND

[0002] Polyphosphate Kinase (PPK) can catalyze the mutual transformation of inorganic polyphosphate (polyP) and adenosine diphosphate (ADP), and synthesize important energy molecules-adenosine triphosphate (ATP) in vivo. In industrial production, PPK provides important support for the fields of 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 cycle system with other enzymes, such as combining with adenosine kinase (AK) to regenerate ATP, and promoting the conversion of adenosine monophosphate (AMP) or ADP into target nucleotides to improve production efficiency.

[0003] However, the existing PPK has low catalytic activity and other problems, which limits its industrial application. Chinese patent CN105624238A discloses a method for regenerating ATP using a rationally designed enzyme. The polyphosphate kinase from Medicago sativa Chinese root nodule is rationally modified and heterologously expressed by gene mutation, which can use ADP as substrate and low-cost tetraphosphoric acid as phosphate donor to economically and efficiently produce ATP. Chinese patent application CN115806957A discloses a polyphosphate kinase mutant and its application in glutamine synthesis. The mutant enzyme ChPPKK103E is derived from a bacterial of the family Hydrocotylaceae, which has the advantages of expanded substrate spectrum and improved enzyme activity, and its catalytic activity is increased by 182.2% compared with the original enzyme. Due to the differences in sequence, structure and function of PPK from different sources, it is necessary to develop more polyphosphate kinases with high catalytic activity to meet the needs of industrial production. SUMMARY

[0004] The purpose of the present application 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 application 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 the amino acid sequences thereof are SEQ ID NO. 2-8, respectively.

[0007] The polyphosphate kinase (PaPPK, PDB:3CZP) of Pseudomonas aeruginosa PAO1 is used as the original enzyme (wild type) in the present application, and the amino acid sequence thereof is SEQ ID NO. 1, and the gene sequence encoding the enzyme is SEQ ID NO. 9. The PaPPK gene is selected according to the principles of protein structure similarity, conserved site analysis and host source diversity by performing non-redundant search in databases such as PDB using the sequence and structure information of the polyphosphate kinase reported in the prior art, and the pure enzyme is obtained through functional expression and purification in an Escherichia coli expression system. The polyphosphate kinase mutants with high synthetic ATP activity are finally obtained by amplifying the PaPPK gene and using semi-rational design for directed evolution modification.

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

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

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

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

[0012] The present application also provides a preparation method of the polyphosphate kinase mutant, comprising the following steps:

[0013] (1) Designing a point mutation primer, using a plasmid with a wild-type polyphosphate kinase gene as a template, and performing PCR reaction using the point mutation primer, and obtaining a mutant gene fragment and a linearized plasmid after purification;

[0014] (2) connecting the mutant gene fragment with the linearized plasmid to construct an expression vector, and transforming the expression vector into a host cell to induce expression of the polyphosphate kinase mutant.

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

[0016] Mutant 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] Component Volume 10 x Buffer for KOD-Plus- 2.5 μL 2 mM dNTP 2.5 μL 25 mM MgSO4 1.5 μL DMSO 1 μL 10 pmol / μL Forward Primer 0.75 μL 10 pmol / μL Reverse Primer 0.75 μL DNA template <100 ng KOD-Plus- 1 μL ddH2O up to 25 μL .

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

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

[0021] The present application also provides use of the product in synthesis of ATP.

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

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

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

[0025] Figure 2 Schematic diagram of relative activity of original enzyme WT and mutants in catalyzing synthesis of ATP with AMP as substrate. DETAILED DESCRIPTION

[0026] The technical solutions of the present application are further described below with reference to the accompanying drawings.

[0027] The example takes the polyphosphate kinase (PaPPK. Pdb: 3CZP) of Pseudomonas aeruginosa PAOl as the original enzyme (wild type). According to the coding sequence of the enzyme in the Pdb database, the gene fragment is synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd. and constructed on the pET22b vector, named pET22b-PaPPK gene. The 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 the N-terminal 408th position of the wild type enzyme amino acid sequence 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 is transformed into E. coli Top10 strain, screened with ampicillin-resistant LB plates, and a clone is obtained. One clone is selected and inoculated in an ampicillin-resistant LB medium test tube with a liquid volume of 20 mL and cultured at 37°C, 220 rpm for 12 h. After the culture is completed, the cells are collected by centrifugation at 12,000 rpm for 1 min, and the plasmid is extracted from E. coli Top10 / pET22b-PaPPK using a high-purity plasmid extraction kit as a template for iterative mutation. The plasmid pET22b-PaPPK mutant is constructed, sequenced by Suzhou Jinyuzhi Biotechnology Co., Ltd., and a clone with correct insertion of the vector and no mutation is selected to obtain a polyphosphate kinase gene expression vector, named pET22b-PaPPK.

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

[0033] The engineered bacteria pET22b-PaPPK / BL21(DE3) is inoculated into the culture medium at a inoculation amount of 0.1%, and the liquid volume of the shake flask is 100 mL / 250 mL (containing 100 mg / L of ampicillin). When the OD 600 of the bacterial solution reaches 0.6-0.8, isopropyl thiogalactoside (IPTG) is added at a final concentration of 0.5 mM for induction, and the temperature is reduced to 18°C for continuous culture for about 20 h. Centrifugation is performed at 4000 rpm for 10 min, the cells are resuspended with 100 mM pH 7.5 KPi buffer, and the cells containing polyphosphate kinase are obtained.

[0034] 2. Preparation of polyphosphatase mutant W408A

[0035] The target mutant gene was obtained by whole plasmid PCR, and the 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, the amplification product was detected by 0.9% agarose gel electrophoresis, and the results showed that the amplification product was a single band with a size of about 7000 bp. The amplification product was purified and recovered by using a DNA recovery purification kit.

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

[0040] The recombinant expression plasmid pET22b successfully sequenced was transformed into E. coli BL21 (DE3) as an expression host, and the recombinant mutant expression strain E. coli BL21 (DE3) / pET22b-PaPPK was constructed.

[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 single colonies were picked and inoculated in 0.5 mL of LB medium containing resistance at 37°C and 200 rpm overnight. A 1% inoculum was transferred to 2 mL of TB medium containing resistance, and the OD 600When the OD600 reaches about 0.6, IPTG is added to a final concentration of 0.5 mM, and the induction is carried out at 18°C for about 20 h. After induction, the deep-well plates are centrifuged, and the cells containing the mutant polyphosphokinase are resuspended in 1.5 mL of KPi buffer (pH 7.5) to a final concentration of 100 mM.

[0042] Example 2: A polyphosphokinase 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 mutation primers are as follows:

[0044] Y431P upstream primer: CCCC GTATAAGCGT CCG AAAATCACCGAAGAAG

[0045] Y431P downstream primer: GCCAGTCTTCTTCGGTGATTTT CGG ACGCTT

[0046] Example 3: A polyphosphokinase mutant D307M / Y431P

[0047] In this example, the tyrosine at position 431 of the N-terminal amino acid sequence of the wild-type enzyme 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 primers are as follows:

[0048] D307M upstream primer: GGTGGCCGTTTTTGAAGGTAAT ATG GCAGCCGGC

[0049] D307M downstream primer: GGCACCGCCTTTGCCGGCTGC CAT ATTACCTTC

[0050] Y431P upstream primer: CCCC GTATAAGCGT CCG AAAATCACCGAAGAAG

[0051] Y431P downstream primer: GCCAGTCTTCTTCGGTGATTTT CGG ACGCTT

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

[0053] In this example, the tyrosine at position 431 of the N-terminal of the wild-type enzyme amino acid sequence is mutated to proline, and 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 primers are as follows:

[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: CCCC GTATAAGCGT 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 of the wild-type enzyme amino acid sequence 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 mutation primers are as follows:

[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 and the tyrosine at position 431 of the wild-type enzyme amino acid sequence are mutated. 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 substantially the same as in Example 1, except that the mutation primers are as follows:

[0066] E304V upstream primer: GTCTGGTGGCCGTTTTTG T AGGTAATGATGCAG

[0067] E304V downstream primer: GCCGGCTGCATCATTACCT A CAAAAACGGCCAC

[0068] Y431P upstream primer: CCCC GTATAAGCGT 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, the proline at position 331 and the tyrosine at position 431 of the wild-type enzyme amino acid sequence are mutated.

[0072] E304V upstream primer: GTCTGGTGGCCGTTTTTG T AGGTAATGATGCAG

[0073] E304V downstream primer: GCCGGCTGCATCATTACCT A CAAAAACGGCCAC

[0074] Y431P upstream primer: CCCC GTATAAGCGT CGG 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: detection of polyphosphate kinase catalyzed synthesis of ATP activity

[0079] Because ATP is very polar, it is difficult to detect by liquid chromatography, so a three-enzyme cascade reaction of polyphosphate kinase, hexokinase, and glucose-6-phosphate dehydrogenase is established to detect the absorbance change of NADPH generated in the reaction at 340 nm, and to indirectly detect the enzyme activity of polyphosphate kinase. The cascade reaction process is as follows:

[0080]

[0081] Detection method: add the resuspended bacterial liquid to the enzyme-labeled plate, add 50 mM glucose, 50 mM MgCl2, 50 mM sodium hexametaphosphate, 10 mM NAD + , 10 mM ADP or AMP to each well, immediately detect with an enzyme-labeled instrument, and record the absorbance change at 340 nm from 0 to 4 min, once per minute. Calculate the enzyme activity according to the detected absorbance change of the sample.

[0082] The detection results are shown in Tables 1, 2, Figure 1 , Figure 2 The polyphosphate kinase mutant with the best ADP reaction effect is W408A / D307M / Y431P, and its relative activity is 221% of the wild type; the mutant with the best AMP reaction effect is E304V / P331S / Y431P, and its relative activity is 198% of the wild type.

[0083] Table 1 Original enzyme WT, mutant D307M / Y431P / W408A absorbance at 340 nm

[0084]

[0085] Table 2 Original enzyme WT, mutant E304V / P331S / W408A absorbance at 340 nm

[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 D307M / W408A / Y431P, starting from the third position of the sequence.

2. A polynucleotide, characterized in that, Encodes the polyphosphate kinase mutant of claim 1.

3. A recombinant vector, characterized in that, It comprises the polynucleotide of claim 2.

4. A recombinant cell, characterized in that, It includes the recombinant vector as described in claim 3.

5. A method for preparing the polyphosphokinase mutant according to claim 1, characterized in that, Includes the following steps: (1) Design point mutation primers, use plasmids containing wild-type polyphosphate kinase genes as templates, perform PCR reactions using point mutation primers, and obtain 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 as follows: 。 8. A product for producing ATP, characterized in that, The product comprises the polyphosphokinase mutant of claim 1, or the polynucleotide of claim 2, or the recombinant vector of claim 3, or the recombinant cell of claim 4.

9. The use of the product of claim 8 in the synthesis of ATP.

10. The application according to claim 9, characterized in that, The substrate for the synthesis of ATP is ADP.

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