A polyphosphate kinase mutant, a recombinant genetic engineering bacterium expressing the mutant and its application

Abstract

The present invention provides a polyphosphate kinase mutant, a recombinant genetic engineering bacterium expressing the mutant and an application thereof, belonging to the technical field of enzymes. The polyphosphate kinase mutant is one of F45S, V115D or A200D; the amino acid sequence of F45S is shown in SEQ ID NO.10 in the sequence list; the amino acid sequence of V115D is shown in SEQ ID NO.12 in the sequence list; the amino acid sequence of A200D is shown in SEQ ID NO.14 in the sequence list. Based on the wild-type PPGK enzyme, the present invention performs site-directed mutagenesis to change the molecular structure of the PPGK enzyme, obtains a PPGK enzyme mutant, improves the enzyme activity of the PPGK enzyme, and constructs a recombinant genetic engineering bacterium containing the PPGK enzyme mutant gene, which significantly improves the expression level of the PPGK enzyme.

Description

Technical Field

[0001] The invention relates to a polyphosphate kinase mutant, a recombinant genetic engineering bacterium expressing the mutant and application thereof, belonging to the technical field of enzymes. Background Art

[0002] Mannose is widely used in food, medicine, biotechnology and other fields. The traditional method of producing D-mannose generally adopts plant tissue hydrolysis method, or chemical synthesis using D-glucose as raw material. The chemical synthesis method requires the use of chemical catalysts and needs to be produced under strictly controlled temperature and acid concentration conditions.

[0003] However, the chemical synthesis of D-mannose has problems such as high energy consumption, high production cost, harsh reaction conditions, formation of by-products during the production process, and complex subsequent purification operations.

[0004] In recent years, many scholars have begun to study the use of biotransformation methods to achieve green production of mannose. The biotransformation method uses enzymes to catalyze reactions. In the prior art, mannose is prepared by multi-enzyme catalysis using glucose as a raw material. The specific process is that glucose generates glucose 6-phosphate under the catalysis of polyphosphokinase (referred to as PPGK enzyme), and then generates fructose 6-phosphate under the catalysis of glucose phosphoisomerase (referred to as PGI enzyme), and generates mannose 6-phosphate under the catalysis of mannose 6-phosphate isomerase (referred to as M6PI enzyme), and finally generates mannose under the catalysis of mannose 6-phosphate phosphatase (referred to as M6PP enzyme). PGI enzyme and M6PI enzyme can be replaced by bifunctional enzyme glucose phosphoisomerase / mannose 6-phosphate isomerase (referred to as PGI / M6PI enzyme).

[0005] The existing wild-type PPGK enzyme has low enzyme activity when used for the bioconversion production of mannose, which leads to low glucose conversion efficiency; the recombinant genetically engineered bacteria constructed with the gene of the wild-type PPGK enzyme also have a low expression level of the PPGK enzyme. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a polyphosphate kinase mutant, a recombinant genetically engineered bacterium expressing the mutant and applications thereof, to achieve the following invention objectives: to improve the enzymatic activity of the PPGK enzyme and to improve the expression level of the PPGK enzyme by the recombinant genetically engineered bacteria containing the PPGK enzyme mutant gene.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A polyphosphate kinase mutant, wherein the polyphosphate kinase mutant is one of F45S, V115D or A200D; the amino acid sequence of F45S is shown as SEQ ID NO.10 in the sequence list; the amino acid sequence of V115D is shown as SEQ ID NO.12 in the sequence list; the amino acid sequence of A200D is shown as SEQ ID NO.14 in the sequence list.

[0009] Compared with the wild-type PPGK enzyme, the phenylalanine at position 45 in the amino acid sequence of F45S mutates into serine; compared with the wild-type PPGK enzyme, the valine at position 115 in the amino acid sequence of V115D mutates into aspartic acid; compared with the wild-type PPGK enzyme, the alanine at position 200 in the amino acid sequence of A200D mutates into aspartic acid.

[0010] The nucleotide sequence of F45S is shown as SEQ ID NO.9 in the sequence listing; the nucleotide sequence of V115D is shown as SEQ ID NO.11 in the sequence listing; and the nucleotide sequence of A200D is shown as SEQ ID NO.13 in the sequence listing.

[0011] A recombinant genetic engineering bacterium expressing the polyphosphate kinase mutant.

[0012] The polyphosphate kinase mutant is used in synthesizing mannose.

[0013] Compared with the prior art, the present invention achieves the following beneficial effects:

[0014] 1. Based on the wild-type PPGK enzyme, the present invention performs site-directed mutagenesis to change the molecular structure of the PPGK enzyme, obtains a PPGK enzyme mutant, improves the enzyme activity of the PPGK enzyme, and constructs a recombinant genetically engineered bacterium containing the PPGK enzyme mutant gene, which significantly improves the expression level of the PPGK enzyme.

[0015] 2. The PPGK enzyme mutant of the present invention has a relative enzyme activity of 139.6-160.8% relative to the wild-type PPGK enzyme at 75°C.

[0016] 3. The recombinant genetically engineered bacteria containing the PPGK enzyme mutant V115D gene and the recombinant genetically engineered bacteria containing the PPGK enzyme mutant A200D gene constructed by the present invention have significantly improved expression levels of the PPGK enzyme compared to the recombinant genetically engineered bacteria containing the wild-type PPGK enzyme gene. DETAILED DESCRIPTION

[0017] The cloning host was E. coli DH5α, and the expression host was E. coli BL21 (DE3);

[0018] LB liquid medium: molecular grade yeast powder 5g / L, molecular grade peptone 10g / L, NaCl 10g / L;

[0019] LB solid medium: Add 2% (w / v) agar powder to LB liquid medium;

[0020] TB fermentation medium: peptone 12 g / L, glycerol 5 g / L, potassium dihydrogen phosphate 2.31 g / L, dipotassium hydrogen phosphate 16.43 g / L, yeast powder 12 g / L.

[0021] Example 1 Selection of mutation sites

[0022] The amino acid mutation sites of the PPGK enzyme mutant of the present invention are determined to be F45S, V115D, and A200D.

[0023] Design corresponding mutation primers based on the selected mutation sites.

[0024] The sequences of the mutagenesis primers are as follows:

[0025] F45S-F: as shown in SEQ ID NO.1 in the sequence listing;

[0026] F45S-R: as shown in SEQ ID NO.2 in the sequence listing;

[0027] V115D-F: as shown in SEQ ID NO.3 in the sequence listing;

[0028] V115D-R: as shown in SEQ ID NO.4 in the sequence listing;

[0029] A200D-F: as shown in SEQ ID NO.5 in the sequence listing;

[0030] A200D-R: as shown in SEQ ID NO.6 in the sequence listing.

[0031] Example 2 Construction of a recombinant plasmid containing a PPGK enzyme mutant

[0032] The nucleotide sequence of the wild-type PPGK enzyme was inserted into the pET24a plasmid to obtain the recombinant plasmid pET24a-PPGK; the whole plasmid PCR technology was used to perform site-directed mutagenesis with the recombinant plasmid pET24a-PPGK as a template to obtain the recombinant plasmids pET24a-PPGK-F45S, pET24a-PPGK-V115D, and pET24a-PPGK-A200D containing the PPGK enzyme mutants;

[0033] The wild-type PPGK enzyme has a nucleotide sequence as shown in SEQ ID NO.7 in the sequence list, and an amino acid sequence as shown in SEQ ID NO.8 in the sequence list.

[0034] The PPGK enzyme mutant contained in the recombinant plasmid pET24a-PPGK-F45S has a nucleotide sequence as shown in SEQ ID NO.9 in the sequence list, and an amino acid sequence as shown in SEQ ID NO.10 in the sequence list, wherein the phenylalanine (F) at position 45 is mutated into serine (S);

[0035] The PPGK enzyme mutant contained in the recombinant plasmid pET24a-PPGK-V115D has a nucleotide sequence as shown in SEQ ID NO.11 in the sequence list, and an amino acid sequence as shown in SEQ ID NO.12 in the sequence list, wherein the valine (V) at position 115 is mutated into aspartic acid (D);

[0036] The PPGK enzyme mutant contained in the recombinant plasmid pET24a-PPGK-A200D has a nucleotide sequence as shown in SEQ ID NO.13 in the sequence list, and an amino acid sequence as shown in SEQ ID NO.14 in the sequence list, wherein the alanine (A) at position 200 is mutated into aspartic acid (D).

[0037] Site-directed mutagenesis PCR system:

[0038] Forward primer: volume 1 μL (the concentration of the forward primer is 100 ng / μL);

[0039] Reverse primer: volume 1 μL (concentration of reverse primer is 100 ng / μL);

[0040] Template: volume 1 μL (template concentration 100 ng / μL);

[0041] DNA polymerase for high-fidelity PCR (TaKaRa R045A): volume 25 μL;

[0042] Add sterile water to make up to 50 μL.

[0043] PCR amplification conditions are:

[0044] Pre-denaturation at 95°C for 10 min; then enter the following cycle: denaturation at 98°C for 10 s, annealing at 62°C for 30 s, extension at 72°C for 2 min, 35 cycles; extension at 72°C for another 5 min; and insulation at 4°C.

[0045] After the PCR was successful, gel electrophoresis was performed using agarose gel electrophoresis. A clear bright band appeared in the target band, and the band was recovered using a gel recovery kit. The recovered mutant fragments were constructed into the vector pET24a, transferred into E. coli DH5α, and transformants were selected for sequencing. The sequencing verification results showed that no random mutations occurred except for the required mutation sites, indicating that the recombinant plasmids pET24a-PPGK-F45S, pET24a-PPGK-V115D, and pET24a-PPGK-A200D were successfully constructed. Each recombinant plasmid was extracted and the concentration was adjusted to 200 μg / mL.

[0046] Example 3 Construction of recombinant genetically engineered bacteria containing PPGK enzyme mutants

[0047] Add the E. coli BL21 (DE3) competent cells (concentration 1.5×10 8 / mL) were added with the obtained recombinant plasmid (2 μL of recombinant plasmid in 100 μL of competent cells), allowed to stand on ice for 30 min, heat-shocked at 42 °C for 90 s, quickly cooled on ice for 90 s, added with 890 μL of sterile SOC liquid culture medium, placed on a shaker at 37 °C and 100 r / min for 1 h, and 50 μL of the above bacterial solution was spread on an LB solid culture medium plate containing kanamycin at a final concentration of 50 mg / mL. After the bacterial solution was completely absorbed by the culture medium, the plate was inverted and cultured in a 37 °C constant temperature incubator for 15 h. Clones were randomly picked from the colonies grown on the plates and the plasmids were extracted for identification by agarose gel electrophoresis to obtain recombinant genetically engineered bacteria containing PPGK enzyme mutant genes: E. coli BL21 (DE3)-pET24a-PPGK-F45S, E. coli BL21 (DE3)-pET24a-PPGK-V115D, and E. coli BL21 (DE3)-pET24a-PPGK-A200D.

[0048] The recombinant plasmid pET24a-PPGK was constructed into Escherichia coli according to the above method to obtain the wild-type recombinant genetic engineering bacteria E. coli BL21 (DE3)-pET24a-PPGK.

[0049] Example 4 Fermentation culture of recombinant genetically engineered bacteria containing PPGK enzyme mutants

[0050] Pick one single colony from the obtained recombinant genetically engineered bacteria plate and put it into a test tube containing 4 mL of LB liquid, add kanamycin with a final concentration of 50 mg / mL, and culture at 37°C and 200 rpm for 12 h. Inoculate it into a sterilized LB liquid culture medium containing kanamycin with a final concentration of 0.1 mM according to an inoculum size of 1% (v / v), and culture at 37°C and 200 rpm until OD600 reaches 0.6. Then, add 0.1 mM IPTG inducer (final concentration), and continue to culture at 25°C and 160 rpm for 20 h to obtain a fermentation broth.

[0051] Take 3000mL of fermentation broth and centrifuge it at 8000rpm and 4℃ for 10min to obtain cell bodies. Add 150mL of PBS buffer (pH=7.1) to resuspend the cells. Use an ultrasonic disruptor to treat the resuspended cells in an ice bath. Then centrifuge the obtained disrupted liquid (8000G, 4℃) for 20min. The supernatant is the crude enzyme solution. After protein purification and desalting of the crude enzyme solution, pure enzyme solutions of E. coli BL21 (DE3)-pET24a-PPGK-F45S, E. coli BL21 (DE3)-pET24a-PPGK-V115D, E. coli BL21 (DE3)-pET24a-PPGK-A200D, and E. coli BL21 (DE3)-pET24a-PPGK were obtained, which are referred to as pure enzyme solution of PPGK enzyme mutant F45S, pure enzyme solution of PPGK enzyme mutant V115D, pure enzyme solution of PPGK enzyme mutant A200D, and pure enzyme solution of wild-type PPGK enzyme. Detection by microplate reader showed that the protein concentrations in the pure enzyme solutions were 3.5 g / L, 5.1 g / L, 4.5 g / L, and 3.7 g / L, respectively.

[0052] The results showed that the protein concentrations in the pure enzyme solutions of the PPGK enzyme mutants V115D and A200D were significantly higher than those in the pure enzyme solutions of the wild-type PPGK enzyme, indicating that the expression levels of the PPGK enzymes in the recombinant genetically engineered bacteria E. coli BL21 (DE3)-pET24a-PPGK-V115D and E. coli BL21 (DE3)-pET24a-PPGK-A200D were significantly higher than those in the wild-type recombinant genetically engineered bacteria.

[0053] The above pure enzyme solution was uniformly adjusted to a concentration of 3.5 g / L for the following experiment.

[0054] Example 5 Determination of enzyme activity of PPGK enzyme mutants

[0055] Enzyme activity detection method: prepare 100g / L glucose solution (using 50mM sodium hydrogen phosphate-citric acid buffer, pH7.1), divide it into 250mL triangular flasks, take 18mL glucose solution in each bottle, preheat it in a water bath at 75℃ for 10min, add 0.2mL pure enzyme solution of PPGK enzyme (pure enzyme solution of PPGK enzyme mutant or pure enzyme solution of wild-type PPGK enzyme) and 0.6mL PGI / M6PI enzyme solution, 0.6mL M6PP enzyme solution, 0.6mL M6PI enzyme solution, mix well, put it in a water bath at 75℃ for 30min, boil it for 10min to terminate the reaction. After diluting it 10 times with water, use HPLC to detect the mannose content after processing with a 0.22μm needle filter.

[0056] The concentration of the pure enzyme solution of the PPGK enzyme is 3.5 g / L;

[0057] The concentration of the PGI / M6PI enzyme solution is 1.25 g / L, the concentration of the M6PP enzyme solution is 1.03 g / L, and the concentration of the M6PI enzyme solution is 0.94 g / L.

[0058] Definition of enzyme activity: Under the conditions of 75°C and pH=7.1, one unit of enzyme activity is the amount of mannose produced per minute per milliliter of pure enzyme solution (3.5 g / L) of PPKG enzyme.

[0059] Under 75℃ conditions, the enzyme activities of the wild-type PPGK enzyme and the PPGK enzyme mutants F45S, PPGK enzyme mutants V115D and PPGK enzyme mutants A200D were 23.42U / mL, 32.7U / mL, 46.04U / mL and 37.66U / mL, respectively. The wild-type PPGK enzyme had a relative enzyme activity of 100%, and the PPGK enzyme mutants F45S, PPGK enzyme mutants V115D and PPGK enzyme mutants A200D had relative enzyme activities of 139.6%, 196.5% and 160.8%, respectively.

Claims

1. A polyphosphate kinase mutant, characterized in that: The polyphosphate kinase mutant is one of F45S, V115D or A200D; the amino acid sequence of F45S is shown in SEQ ID NO.10 in the sequence list; the amino acid sequence of V115D is shown in SEQ ID NO.12 in the sequence list; the amino acid sequence of A200D is shown in SEQ ID NO.14 in the sequence list.

2. A recombinant genetically engineered bacterium expressing the polyphosphate kinase mutant according to claim 1.

3. Use of the polyphosphate kinase mutant according to claim 1 in synthesizing mannose.