A polyphosphate kinase mutant and its application in glutamine synthesis

Abstract

The present invention discloses a polyphosphate kinase mutant and its application in glutamine synthesis, belonging to the field of biotechnology. The present invention has screened a polyphosphate kinase mutant with significantly improved enzyme activity. The catalytic activity of this mutant enzyme has increased by 182.2% compared to the original enzyme, enabling efficient regeneration of ATP and reducing the cost of biocatalysis. Using the polyphosphate kinase mutant constructed in the present invention for the production of glutamine significantly improves the sustainability of ATP regeneration. The glutamine yield can reach 94.4 mM after 8 h of reaction, and the conversion rate is 94.4%. The yield of glutamine produced by coupling with the original enzyme is increased by 41.5%.

Description

Technical Field

[0001] The present invention relates to a polyphosphate kinase mutant and its application in glutamine synthesis, belonging to the field of biotechnology. Background Art

[0002] Adenosine triphosphate (ATP) is a common high-energy phosphate compound in organisms, consisting of 1 molecule of adenine, 1 molecule of ribose, and 3 molecules of phosphate groups. It provides energy for processes such as synthesis, transportation, and information transfer in living cells. As an important cofactor, it participates in many energy-requiring biocatalytic reactions. Directly adding ATP to the system is costly and leads to the accumulation of by-products, complicating the downstream separation process. The literature has reported that multiple enzymes can be used for ATP regeneration, such as pyruvate kinase (PK), creatine kinase (CK), acetate kinase (AK), and polyphosphate kinase (PPK), etc. However, systems based on PEP, acetyl phosphate, and creatine have problems such as product inhibition, high cost, or low stability, which may limit the application of these technologies in large-scale production. So far, the vast majority of ATP regeneration systems start from ADP, such as the reduction of carboxylic acids to aldehydes, the formation of amide bonds, and cascade reactions in biocatalytic reactions. For S-adenosylmethionine-dependent alkylation reactions, ATP regeneration is required. Most AMP-to-ATP regeneration systems use two enzymes, such as adenylate kinase / acetate kinase, or more complex mixtures, such as Escherichia coli lysate supplemented with acetyl phosphate. Considering the cost limitation of biocatalysts to the process, the minimization of the number of enzymes and the catalysis in the most primitive form are preferred. Although the construction of such in vivo cascades may bring some disadvantages in terms of mass transfer limitations, product toxicity to the host organism, and complex downstream processes. PPKs are widely used in ATP regeneration due to the cheap and easily available substrate polyphosphate (polyP). Polyphosphate (polyP) is an inorganic linear polymer containing thousands of phosphate residues and multiple energy-rich phosphoanhydride bonds. It is an energy storage material and can be used as a stress and survival factor, energy source, for cell motility, biofilm formation, and metal ion chelation. In recent years, the reversible reaction of inorganic polyphosphate catalyzed by polyphosphate kinase (PPK) to generate ATP has attracted wide attention. In nature, polyP is synthesized and degraded by polyphosphate kinases (PPKs). PPKs can be divided into two major categories according to the phylogenetic tree and catalytic performance: PPK1 and PPK2. PPK1 tends to synthesize polyP, and PPK2 tends to synthesize ATP. In addition, according to different structures and biochemical characteristics, PPK2 can be further divided into three categories: PPK2Ⅰ, PPK2Ⅱ, and PPK2Ⅲ. Usually, the substrate preferences of PPK2Ⅰ and PPK2Ⅱ result in the generation of ATP from the corresponding ADP. PPK2Ⅲ is a bifunctional enzyme that can form ADP and ATP from AMP. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for efficiently producing ATP by screening and high-throughput mutating polyphosphate kinase, so as to overcome the deficiencies of the prior art.

[0004] Technical solution:

[0005] To solve the above technical problem, the present invention screens polyphosphate kinases from different sources, and through the determination of related properties, screens a polyphosphate kinase ChPPK with better catalytic ability.

[0006] The present invention provides a polyphosphate kinase mutant, in which the 103rd amino acid is mutated on the basis of the starting sequence shown in SEQ ID NO.8.

[0007] In one embodiment, the mutation is the mutation of the 103rd lysine to valine, glycine, aspartic acid or glutamic acid.

[0008] The present invention also provides a gene encoding the mutant.

[0009] The present invention also provides a recombinant plasmid carrying the gene.

[0010] In one embodiment, the plasmid includes but is not limited to pET28a(+) or pXMJ-19.

[0011] The present invention also provides a recombinant microorganism expressing the mutant.

[0012] In one embodiment, the microorganism includes but is not limited to Escherichia coli.

[0013] In one embodiment, the recombinant microorganism uses Escherichia coli E.coli BL21(DE3) as the host and pET28a(+) and pXMJ-19 plasmids as expression vectors.

[0014] In one embodiment, the recombinant microorganism also expresses glutamine synthetase.

[0015] In one embodiment, the gene encoding glutamine synthetase has the nucleotide sequence shown in SEQ ID NO.7.

[0016] The present invention also provides the application of the mutant or the recombinant Escherichia coli expressing the mutant in the production of glutamine.

[0017] In one embodiment, the recombinant Escherichia coli also expresses glutamine synthetase.

[0018] In one embodiment, the application is to add the cell culture solution of the mutant or the recombinant Escherichia coli into the reaction system; the reaction system contains glutamic acid, (NH4)2SO4, polyphosphate, ATP, and MgCl2.

[0019] In one embodiment, the polyphosphate includes, but is not limited to, polyphosphates with a degree of polymerization ≥ 3 or ≥ 6.

[0020] In one embodiment, the addition amount of the mutant in the reaction system is ≥ 4 U / mL.

[0021] In one embodiment, the reaction is carried out at 35 - 37 °C for at least 6 h.

[0022] The present invention also provides the application of the mutant, or the recombinant microorganism, or the recombinant Escherichia coli, or the method in the production of glutamine or glutamine-containing products.

[0023] Beneficial effects:

[0024] 1. By constructing a polyphosphate kinase PPK mutant library, the present invention screens for PPK enzymes with significantly improved enzyme activity through high-throughput screening. By constructing a PPK enzyme mutant library, the present invention fine-tunes the gene sequence to further change the tertiary structure of the enzyme, and screens for a mutant enzyme ChPPK with an expanded substrate spectrum range and improved enzyme activity. K103E The catalytic activity of this mutant enzyme is increased by 182.2% compared to the original enzyme, breaking through the drawback of the low efficiency of the original polyphosphate kinase in regenerating ATP.

[0025] 2. The present invention uses the screened polyphosphate kinase mutant for the production of glutamine, significantly improving the sustainability of ATP regeneration, enhancing the catalytic efficiency, enabling the glutamine production to reach 94.4 mM after 8 h of reaction, with a conversion rate of 94.4%, and the production of glutamine is increased by 41.5% compared to the production of glutamine by coupling with the original enzyme. Description of the drawings

[0026] Figure 1 : Catalytic activity of PPK enzyme towards phosphate chains of different lengths.

[0027] Figure 2 : Screening of the PPK mutant library.

[0028] Figure 3 : Determination of the enzyme activity of the saturation mutant at key sites.

[0029] Figure 4 : Mutant enzyme ChPPK K103E Enzyme activity properties.

[0030] Figure 5 : Mutant enzyme ChPPKK103E Verification of ATP production ability Detailed implementation manners

[0031] The present invention will be further described below in conjunction with specific embodiments.

[0032] The nucleotide sequence of polyphosphate kinase BlPPK derived from Escherichia coli is shown as SEQ ID NO.1; the nucleotide sequence of polyphosphate kinase CgPPK derived from Corynebacterium glutamicum is shown as SEQ ID NO.2; the nucleotide sequence of polyphosphate kinase PaPPK derived from Pseudomonas aeruginosa is shown as SEQ ID NO.3; the nucleotide sequence of polyphosphate kinase ChPPK derived from Callitriche stagnalis bacteria is shown as SEQ ID NO.4, and the amino acid sequence is shown as SEQ ID NO.8; the nucleotide sequence of green fluorescent protein gene gfp is shown as SEQ ID NO.5; the nucleotide sequence of promoter rrnB P1 is shown as SEQ ID NO.6; the nucleotide sequence of glutamine synthetase CgGS derived from Corynebacterium glutamicum is shown as SEQ ID NO.7.

[0033] The culture media involved in the following examples:

[0034] LB medium (1L): 10 g NaCl, 10 g tryptone, 5 g yeast extract.

[0035] The PPK enzyme activity detection method involved in the following examples is as follows:

[0036] The reaction mixture contains 100 mM Tris-HCl (pH 8.0), 10 mM glucose, 10 mM NADP, 25 mM MgCl2, 10 mM ADP, 10 mM polyP6, 5 U hexokinase (HK), 5 U glucose-6-phosphate dehydrogenase (G6PD) and PPK enzyme. React at 35 °C for 30 min and measure the absorbance value at 340 nm. The definition of one unit of PPK (1 U) is the amount of enzyme required to produce 1 μmol ATP per minute.

[0037] Example 1: Screening of polyphosphate kinase

[0038] The specific steps are as follows:

[0039] (1) Construction of overexpression plasmids pXMJ-19-BlPPK, pXMJ-19-CgPPK, pXMJ-19-PaPPK, pXMJ-19-ChPPK

[0040] According to the gene sequences of polyphosphate kinases published on the NCBI website, the polyphosphate kinase gene ppk sequences on the genomes of Escherichia coli BL21(DE3), Corynebacterium glutamicum ATCC 13032, Pseudomonas aeruginosa, and Calditrichaceae bacterium were selected. The ppk gene sequences were amplified from the genomes of Escherichia coli, Corynebacterium glutamicum, and Pseudomonas aeruginosa with primers P1 / P2, P3 / P4, and P5 / P6 respectively (SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 respectively). The polyphosphate kinase gene from Calditrichaceae bacterium (nucleotide sequence shown in SEQ ID NO.4) was synthesized by Genewiz Suzhou. The PCR program was: 95°C, 10 min; 95°C, 30 s; 58°C, 30 s; 72°C, 1 min; 72°C, 10 min, for 30 cycles. The obtained ppk gene fragments were purified and then ligated to the linearized plasmid pXMJ-19 (amplified by P7 / P8) through the homologous recombinase ClonExpress II One Step Cloning Kit (Novoprotein), and transformed into competent E. coli BL21(DE3) cells to obtain transformants. The transformants were spread on LB solid medium containing 20 μg / mL chloramphenicol and cultured at 37°C for 12 h. Positive colonies were picked, and colony PCR verification of the single colonies was performed with Taq DNA polymerase using P9 / P10 as primers. After the positive single colonies with the target band size were inoculated into small bottles containing LB liquid medium and cultured for 12 h, the plasmids were extracted and sent to Genewiz for correct sequencing. The successfully constructed plasmids were named pXMJ-19-BlPPK, pXMJ-19-CgPPK, pXMJ-19-PaPPK, and pXMJ-19-ChPPK respectively. The primer sequences involved are as follows:

[0041] P1: 5’-cgggtacaaaggaggacaaccatgggtcaggaaaagctatacatc-3’;

[0042] P2: 5’-agctcgttagtggtggtggtggtggtgttcaggttgttcgagtgatttgatg-3’;

[0043] P3: 5’-cgggtacaaaggaggacaaccatggctgaaaccaacgaaaatgat-3’;

[0044] P4: 5'-gaattcgagctcgctagtggtggtggtggtggtggtcaccgatctggtcgcgcc-3';

[0045] P5: 5'-ccccgggtacaaaggaggacaaccgtggccagcccggcgcag-3';

[0046] P6: 5'-tcgagctcgtcagtggtggtggtggtggtgggccgggatatccaggttcgcg-3';

[0047] P7: 5'-cgagctcgaattcagcttggct-3';

[0048] P8: 5'-actctagaggatccccgggtac-3';

[0049] P9: 5'-ctggcaaatattctgaaatgagctg-3';

[0050] P10: 5'-gcagttccctactctcgcat-3';

[0051] (2) Preparation of competent Escherichia coli BL21

[0052] Streak Escherichia coli E. coli BL21 on an antibiotic-free LB plate, incubate in an incubator at 37 °C, pick colonies after incubation and inoculate into a 10 mL LB vial for 12 h. Transfer with an inoculation amount of 1% to a 50 mL LB medium bottle. When the cell concentration reaches 0.4 - 0.6, prepare for the preparation of competent E. coli. Pre-cool relevant reagents and instruments in advance. Prepare solution A, solution B in the kit, 1.5 mL EP tubes and 50 mL centrifuge tubes and place them on ice. Set the centrifuge temperature to 4 °C. Aliquot 50 mL of the bacterial solution in a sterile workbench, centrifuge (8000 r·min -1 , 5 min), and discard the supernatant. Pipette 5 mL of solution A and blow-suspend, centrifuge (8000 r·min -1 , 5 min), and discard the supernatant. Pipette 5 mL of solution B and blow-suspend, aliquot into pre-cooled and pre-prepared EP tubes, with 100 μL in each tube.

[0053] (3) Construction of recombinant bacteria BL21 / pXMJ-19-BlPPK, BL21 / pXMJ-19-CgPPK, BL21 / pXMJ-19-PaPPK, BL21 / pXMJ-19-ChPPK

[0054] Transform the plasmids pXMJ-19-BlPPK, pXMJ-19-CgPPK, pXMJ-19-PaPPK, and pXMJ-19-ChPPK obtained in step (1) into competent Escherichia coli BL21 cells to obtain transformants. Then spread the transformants on LB solid medium containing 20 μg / mL chloramphenicol resistance, and culture at 37 °C for 12 h. Pick positive colonies and perform colony PCR verification on the single colonies using P9 / P10 as primers with Taq DNA polymerase. After culturing the positive single colonies with the target band size in a vial containing LB liquid medium for 12 h, extract the plasmids and send them to Genewiz for sequencing. Correct sequencing indicates successful construction of the recombinant strain.

[0055] Example 2: Expression and purification of polyphosphate kinase PPK

[0056] Inoculate a single colony of the successfully constructed recombinant strain in Example 1 into 10 ml of LB liquid medium and culture at 37 °C for 12 h. Then inoculate at an inoculation amount of 1% into 50 ml of LB liquid medium and culture until the OD 600 is approximately 0.8. Add IPTG and culture at 16 °C for 16 h. Wash the cells three times with PBS, resuspend the collected cells with PBS again, and break the cells with an ultrasonic disruptor, with the Escherichia coli broken for 1 s and paused for 3 s, for a total of 15 min. Centrifuge at 10,000 rpm for 20 min and take the supernatant to run a protein gel. Purify the crude enzyme solution through a protein purification nickel column and verify the pure enzyme by running a protein gel.

[0057] Add the pure enzyme to a reaction solution containing polyphosphate (polyP) of different lengths. The reaction system contains a final concentration of 100 mM Tris-HCl (pH 8.0), 10 mM glucose, 10 mM NADP, 25 mM MgCl2, 10 mM ADP, 10 mM polyP3 / polyP6 / polyP n (n > 6), 5 U / mL HK, 5 U / mL G6PD, and 4 U / mL PPK enzyme. React at 35 °C for 30 min and measure the absorbance value at 340 nm.

[0058] The results show that the substrate polyP6 has better catalytic ability than polyP3 and polyP n And ChPPK shows the best enzyme activity towards polyP6, with a specific activity of 605.2 ± 2.1 U / mg. Except for CgPPK, almost all PPKs did not show enzyme activity towards polyP3. The polyphosphate kinase of ChPPK has the highest catalytic activity towards the substrate polyP6, and this enzyme is selected for subsequent experiments.

[0059] Example 3: Construction of the ChPPK mutant library

[0060] (1) Construction of the pXMJ-19-rrnB P1-GFP plasmid

[0061] The activity of the ribosomal RNA promoter rrnB P1 has been shown to depend on the ATP concentration in Escherichia coli cells. According to the rrnB P1 sequence (nucleotide sequence shown in SEQ ID NO.6) in the complete genome nucleic acid sequence of Escherichia coli BL21(DE3) in NCBI, primers P12 / P13 were designed. The obtained gene fragment was fused with the gfp gene (shown in SEQ ID NO.5). The purified fragment was ligated to the linearized plasmid pXMJ-19 (amplified by P14 / P15) through the homologous recombinase ClonExpress II One Step Cloning Kit (Novoprotein), and then transformed into E. coli BL21(DE3) competent cells. The cells were spread on LB solid medium containing chloramphenicol at a concentration of 20 μg / mL and cultured at 37°C for 12 h. Positive colonies were picked and verified by colony PCR of single colonies using Taq DNA polymerase with P10 / P11 as primers. After culturing positive single colonies with the target band size in a vial containing LB liquid medium for 12 h, the plasmid was extracted and sent to Genewiz for sequencing. If the sequencing was correct, the pXMJ-19-rrnB P1-GFP plasmid was successfully constructed. The primer sequences involved are as follows:

[0062] P12: 5’-atggcaatgacgccaggagctgaa-3’;

[0063] P13: 5’-actcatggttgtcctcctttgtttgccgttgttccgtgtcagtg-3’;

[0064] P14: 5’-gctcctggcgtcattgccatgttgagatccagttcgatgtaacc-3’;

[0065] P15: 5’-gctcctggcgtcattgccatgttgagatccagttcgatgtaacc-3’;

[0066] (2) Construction of the ChPPK mutant library

[0067] Design primers P17 / P18, and use an error-prone PCR kit to randomly mutate the ChPPK gene fragment (shown in SEQ ID NO.4). The amplification conditions are as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 60 s, for 30 cycles; final extension at 72°C for 5 min. Design primers P19 / P20, use the pXMJ-19-rrnB P1-GFP plasmid as a template to amplify the linearized plasmid. The amplification conditions are as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 120 s, for 30 cycles; final extension at 72°C for 5 min. Use a gel recovery kit to purify and recover the PCR products, and detect the concentration of the recovered products. The obtained mutant gene fragments are respectively ligated with the linearized plasmid pXMJ-19 through the homologous recombinase ClonExpress II One Step Cloning Kit (Vazyme), transformed into E. coli BL21(DE3) competent cells, and spread on LB solid medium containing chloramphenicol at a concentration of 20 μg / mL, and cultured at 37°C for 12 h. The grown transformants are used for subsequent high-throughput screening. The primer sequences involved are as follows:

[0068] P17: 5’-ctgcaggtcgactctagaggatcc-3’;

[0069] P18: 5’-ttcagcttggctgttttggcggat-3’;

[0070] P19: 5’-taaggtactgtgcgtacccggggatcctctagag-3’;

[0071] P20: 5’-atcaccaccaccaccaccacatgagtaaaggagaagaacttttcactg-3’.

[0072] Example 4: High-throughput screening of ChPPK mutant strains

[0073] (1) Construction of the ChPPK mutant strain library

[0074] The transformants were inoculated into 24-well plates, ADP and polyP6 were added to the medium, and cultured at 37 °C for 12 h; the collected cells were reacted at 40 °C for 30 min. Through the color reaction based on ATP-TMB of 1008 strains of bacteria, 188 mutants with higher absorbance values and potentially better catalytic activity compared with the wild type were screened. The 188 mutant strains were inoculated onto 24-well plates containing LB medium, cultured at 37 °C for 2 h, then 0.5 mM IPTG, ADP and polyP6 were added, induced at 16 °C for 15 h, the strains were collected, and their fluorescence values and OD 600 values were detected using a microplate reader, with the wild type as a control.

[0075] Select mutant strains with fluorescence value / OD 600 values higher than those of the wild type to extract plasmids and sequence. The sequencing results showed that most of the mutation sites were located at the 103 residue sites. Therefore, site-directed saturation mutagenesis was carried out at this site, and the 103 residue lysine of ChPPK was mutated into the other 19 amino acids. Primers (P21-P58) were designed, using the ChPPK gene fragment as a template, and the mutant fragments were obtained by PCR amplification. The amplification conditions were: pre-denaturation at 95 °C for 5 min: denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, 30 cycles; final extension at 72 °C for 5 min. Primers P59 / P60 were designed, using the pXMJ-19 plasmid as a template, and the linearized plasmid was obtained by amplification. The amplification conditions were: pre-denaturation at 95 °C for 5 min: denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 120 s, 30 cycles; final extension at 72 °C for 5 min. The PCR products were purified and recovered using a gel recovery kit, and the concentration of the recovered products was detected. The purified mutant fragments were respectively ligated with the linearized plasmid pXMJ-19 through the homologous recombinase ClonExpress II One Step Cloning Kit (Vazyme), transformed into competent cells of Escherichia coli E. coli BL21(DE3), and spread on LB solid medium containing chloramphenicol at a concentration of 20 μg / mL, and cultured at 37 °C for 12 h. Positive colonies were picked, and colony PCR verification of the single colonies was carried out using Taq DNA polymerase with P10 / P11 as primers; after the positive single colonies with the target band size were inoculated into small bottles containing LB liquid medium and cultured for 12 h, plasmids were extracted. If the sequencing by Genewiz was correct, the library of 103 site-saturated mutant strains of ChPPK was successfully constructed, that is, the strains BL21 / pXMJ-19-ChPPK K103A 、BL21 / pXMJ-19-ChPPK K103R 、BL21 / pXMJ-19-ChPPK K103N 、BL21 / pXMJ-19-ChPPK K103D, BL21 / pXMJ-19-ChPPK K103C , BL21 / pXMJ-19-ChPPK K103Q , BL21 / pXMJ-19-ChPPK K103E , BL21 / pXMJ-19-ChPPK K103G , BL21 / pXMJ-19-ChPPK K103H , BL21 / pXMJ-19-ChPPK K103I , BL21 / pXMJ-19-ChPPK K103L , BL21 / pXMJ-19-ChPPK K103M , BL21 / pXMJ-19-ChPPK K103F , BL21 / pXMJ-19-ChPPK K103P , BL21 / pXMJ-19-ChPPK K103S , BL21 / pXMJ-19-ChPPK K103T , BL21 / pXMJ-19-ChPPK K103W , BL21 / pXMJ-19-ChPPK K103Y , BL21pXMJ-19-ChPPK K103V Construction successful.

[0076] P21: 5’-tgacttccttcgaagtgccatccaagatcgaactgtccca-3’;

[0077] P22: 5’-tggcacttcgaaggaagtcaccttcacgccttgtg-3’;

[0078] P23: 5’-tgacttccttcgcagtgccatccaagatcgaactgtccca-3’;

[0079] P24: 5’-tggcactgcgaaggaagtcaccttcacgccttgtg-3’;

[0080] P25: 5’-tgacttccttcgatgtgccatccaagatcgaactgtccca-3’;

[0081] P26: 5’-tggcacatcgaaggaagtcaccttcacgccttgtg-3’;

[0082] P27: 5’-tgacttccttcttcgtgccatccaagatcgaactgtccca-3’;

[0083] P28: 5’-tggcacgaagaaggaagtcaccttcacgccttgtg-3’;

[0084] P29: 5’-tgacttccttcggcgtgccatccaagatcgaactgtccca-3’;

[0085] P30: 5’-tggcacgccgaaggaagtcaccttcacgccttgtg-3’;

[0086] P31: 5’-tgacttccttcatcgtgccatccaagatcgaactgtccca-3’;

[0087] P32: 5’-tggcacgatgaaggaagtcaccttcacgccttgtg-3’;

[0088] P33: 5’-tgacttccttcctggtgccatccaagatcgaactgtccca-3’;

[0089] P34: 5’-tggcaccaggaaggaagtcaccttcacgccttgtg-3’;

[0090] P35: 5’-tgacttccttcaacgtgccatccaagatcgaactgtccca-3’;

[0091] P36: 5’-tggcacgttgaaggaagtcaccttcacgccttgtg-3’;

[0092] P37: 5’-tgacttccttcgtggtgccatccaagatcgaactgtccca-3’;

[0093] P38: 5’-tggcaccacgaaggaagtcaccttcacgccttgtg-3’;

[0094] P39: 5’-tgacttccttctgggtgccatccaagatcgaactgtccca-3’;

[0095] P40: 5’-tggcacccagaaggaagtcaccttcacgccttgtg-3’;

[0096] P41: 5’-tgacttccttctacgtgccatccaagatcgaactgtccca-3’;

[0097] P42: 5’-tggcacgtagaaggaagtcaccttcacgccttgtg-3’;

[0098] P43: 5’-tgacttccttccaagtgccatccaagatcgaactgtccca-3’;

[0099] P44: 5’-tggcacttggaaggaagtcaccttcacgccttgtg-3’;

[0100] P45: 5’-tgacttccttcatggtgccatccaagatcgaactgtccca-3’;

[0101] P46: 5’-tggcaccatgaaggaagtcaccttcacgccttgtg-3’;

[0102] P47: 5’-tgacttccttctccgtgccatccaagatcgaactgtccca-3’;

[0103] P48: 5’-tggcacggagaaggaagtcaccttcacgccttgtg-3’;

[0104] P49: 5’-tgacttccttcaccgtgccatccaagatcgaactgtccca-3’;

[0105] P50: 5’-tggcacggtgaaggaagtcaccttcacgccttgtg-3’;

[0106] P51: 5’-tgacttccttcgcagtgccatccaagatcgaactgtccca-3’;

[0107] P52: 5’-tggcactgcgaaggaagtcaccttcacgccttgtg-3’;

[0108] P53: 5’-tgacttccttcccagtgccatccaagatcgaactgtccca-3’;

[0109] P54: 5’-tggcactgggaaggaagtcaccttcacgccttgtg-3’;

[0110] P55: 5’-tgacttccttccacgtgccatccaagatcgaactgtccca-3’;

[0111] P56: 5’-tggcacgtggaaggaagtcaccttcacgccttgtg-3’;

[0112] P57: 5’-tgacttccttccgcgtgccatccaagatcgaactgtccca-3’;

[0113] P58: 5’-tggcacgcggaaggaagtcaccttcacgccttgtg-3’;

[0114] (1) Determination of the enzyme activity of the ChPPK mutant strain

[0115] Inoculate a single colony of the ChPPK saturated mutant strain constructed in Example 4 (1) into 10 ml of LB liquid culture and culture at 37 °C for 12 hours. Then inoculate it into 50 ml of LB liquid medium at an inoculation amount of 1%, and culture until the OD 600 is approximately 0.8. Add IPTG and culture at 16 °C for 16 h. Wash the cells three times with PBS, resuspend the collected bacteria with PBS, break the cells with an ultrasonic cell disruptor, and purify the crude enzyme solution through a protein purification nickel column to obtain the mutant pure enzyme of ChPPK. The final concentrations of the reaction mixture for measuring the enzyme activity are 100 mM Tris-HCl (pH 8.0), 10 mM glucose, 10 mM NADP, 25 mM MgCl2, 10 mM ADP, 10 mM polyP6, 5 U HK, 5 U G6PD, and the enzyme (4 U / mL). React at 35 °C for 30 min and measure the absorbance value at 340 nm. The definition of one unit of PPK (1 U) is the amount of enzyme required to produce 1 μmol of ATP per minute. The results are expressed as relative enzyme activity (%) (taking the specific enzyme activity of the original ChPPK enzyme as 100%).

[0116] The results showed that compared with the wild type, the relative enzyme activities of K103V, K103G and K103D were 146.0%, 120.0% and 143.0%, respectively, while the relative enzyme activity of K103E was 182.2% (the specific enzyme activity of the original ChPPK was 605.2±2.1 U / mg, taken as 100%). K103E The catalytic activity of BL21 / pXMJ-19-ChPPK was improved most significantly, and the specific enzyme activity could reach 1102.7±2.08U / mg. K103E Used in subsequent experiments.

[0117] Example 5: Mutant enzyme ChPPK K103E The enzyme activity

[0118] BL21 / pXMJ-19-ChPPK constructed in Example 4 K103E A single colony was inoculated into 10 ml of LB liquid medium and cultured at 37 °C for 12 h. A 1% inoculum was inoculated into 50 ml of LB liquid medium and cultured until OD 600 The concentration of the enzyme was about 0.8, IPTG was added, and the cells were cultured at 16°C for 16 h. The cells were washed three times with PBS, the collected bacteria were resuspended in PBS, the cells were broken by ultrasonic disruptor, and the crude enzyme solution was purified by protein purification nickel column to obtain ChPPK K103E Pure enzyme. The catalytic activity of the mutant enzyme on phosphate chains of different lengths was determined. The reaction system included a final concentration of 100 mM Tris-HCl (pH 8.0), 10 mM glucose, 10 mM NADP, 25 mM MgCl2, 10 mM ADP, 10 mM polyP3 / polyP6 / polyP n (n>6), 5U HK, 5U U6PD and 4U / mL mutant enzyme ChPPK K103E The reaction was carried out at 35°C for 30 min.

[0119] The results showed that the original enzyme ChPPK was effective for polyP3, polyP6, and polyP n The specific enzyme activities of the mutant enzymes were 11.37±0.79U / mg, 605.2±2.1U / mg, and 166.76±2.8U / mg, respectively. n The enzyme activities of the two enzymes were significantly increased. Compared with the original enzyme, the enzyme activities increased by 707.1%, 182.1% and 136.7%, respectively. This shows that the mutation of lysine at position 103 of ChPPK to glutamate effectively improved the substrate spectrum and catalytic activity of the enzyme.

[0120] Example 6: Coupling mutant enzyme ChPPK K103E Production of glutamine

[0121] (1) Construction of overexpression plasmid pET28a-CgGS

[0122] According to the gene sequence of glutamine synthetase GS from Corynebacterium glutamicum published on the NCBI website, using the genome of Corynebacterium glutamicum ATCC13032 as a template, primers P59 / P60 were designed, and the CgGS gene (the nucleotide sequence of CgGS is SEQ ID NO.7) was amplified by PCR. The amplification conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, for 30 cycles; final extension at 72°C for 5 min. Using the pET28a plasmid as a template, primers P61 / P62 were designed, and the linearized plasmid was amplified by PCR. The amplification conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 2.5 min, for 30 cycles; final extension at 72°C for 5 min. The CgGS gene was ligated to the linearized plasmid pET28a through the homologous recombinase ClonExpress II One Step Cloning Kit (Novoprotein), transformed into E. coli BL21(DE3) competent cells, and spread on LB solid medium containing kanamycin at a concentration of 50 μg / mL. After culturing at 37°C for 12 h, positive colonies were picked, and colony PCR verification of the single colonies was performed using Taq DNA polymerase with P63 / P64 as primers; positive single colonies with the target band size were inoculated into small bottles containing LB liquid medium and cultured for 12 h, then the plasmids were extracted and sent to Suzhou Genewiz for sequencing. If the sequencing was correct, the pET28a-CgGS plasmid was successfully constructed. The primer sequences involved are as follows:

[0123] P59: 5’-tgggtcgcggatccgaattcgtggcgtttgaaaccccggaagaa-3’;

[0124] P60: 5’-caagcttgtcgacggagctcttagcagtcgaagtacaattcgaattc-3’;

[0125] P61: 5’-cttcgactgctaagagctccgtcgacaagcttgcg-3’;

[0126] P62: 5’-ggtttcaaacgccacgaattcggatccgcgacccatttg-3’;

[0127] P63: 5’-agaggatcgagatctcgatcccgc-3’;

[0128] P64: 5’-atccggatatagttcctcctttca-3’;

[0129] (2) Construction of overexpression plasmids pET28a-CgGS-ChPPK and pET28a-CgGS-ChPPK K103E Construction

[0130] Using the pET28a-CgGS plasmid as a template, primers P65 / P66 were designed, and a linearized plasmid was obtained by PCR amplification. The ChPPK gene and the ChPPK K103E gene were respectively ligated to the linearized plasmid pET28a-CgGS through the homologous recombinase ClonExpress II OneStep Cloning Kit (Novoprotein), transformed into E. coli BL21(DE3) competent cells, and spread on LB solid medium containing kanamycin at a concentration of 50 μg / mL. After culturing at 37 °C for 12 h, positive colonies were picked, and colony PCR verification of the single colonies was performed using P63 / P64 as primers by Taq DNA polymerase; after culturing the positive single colonies with the target band size in a vial containing LB liquid medium for 12 h, the plasmids were extracted and sent to Suzhou Genewiz for sequencing. If the sequencing was correct, the plasmids pET28a-CgGS-ChPPK and pET28a-CgGS-ChPPK K103E were successfully constructed, and the BL21 / pET28a-CgGS-ChPPK and BL21 / pET28a-CgGS-ChPPK K103E strains with correct sequencing were stored in glycerol tubes. The primer sequences involved are as follows:

[0131] P65: 5’-tggctgttttggccgcactcgagcaccaccaccaccaccactgaga-3’;

[0132] P66: 5’-acagctcatttcagaatattgccgcaagcttgtcgacggagctc-3’;

[0133] (3) Verification of the production of glutamine by the coupled mutant enzyme ChPPK K103E Verification

[0134] The BL21 / pET28a-CgGS-ChPPK and BL21 / pET28a-CgGS-ChPPK K103E strains were induced and purified. Induced expression of CgGS-ChPPK and CgGS-ChPPK was carried out according to step (1) of Example 2 K103E, the cells were collected and disrupted by an ultrasonic crusher for 15 min to obtain crude enzyme solutions of CgGS-ChPPK (7.8 U / mg) and CgGS-ChPPK K103E (8.5 U / mg). BL21 / pET28a-CgGS, BL21 / pET28a-CgGS-ChPPK and BL21 / pET28a-CgGS-ChPPK K103E were named EB01, EB02 and EB03 respectively.

[0135] The glutamine production system contained, by final concentration: 100 mM Tris-HCl (pH 7), 100 mM glutamic acid, 100 mM (NH4)2SO4, 10 mM polyP6, 5 mM ATP, 20 mM MgCl2 and 6 U / mL enzyme solution, and the reaction temperature was 37 °C. 10 mM of polyP6 was added continuously after 2 h and 4 h of reaction respectively.

[0136] At 8 h of reaction, 94.4 mM of glutamine could be obtained in the reaction system participated by the enzyme expressed by EB03, and the conversion rate was 94.4%, which was 95.3% and 41.5% higher than that of EB01 and EB02 respectively. In the ATP regeneration model of glutamine, 1 mM of glutamine requires 1 mM of ATP molecules. Therefore, EB03 needs 94.4 mM of ATP molecules to produce 94.4 mM of glutamine, while only 5 mM of ATP was added to the system to initiate the ATP cycle. EB03 produced 89.4 mM of ATP. In the glutamine model of EB03, one ATP cycle produced 1 mM of ATP. Therefore, with only 5 mM of ATP added, the number of ATP regeneration cycles in the glutamine regeneration model was about 89 times, greatly improving the energy supply efficiency. Therefore, the addition of the mutant enzyme ChPPK K103E could significantly improve the sustainability of ATP regeneration, thereby improving the catalytic efficiency and saving the cost of high-energy-consuming catalytic reactions.

[0137] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A polyphosphate kinase mutant, characterized in that, The sequence of the mutant is based on the starting sequence shown in SEQ ID NO.8, with the 103rd amino acid mutated to valine, glycine, aspartic acid or glutamic acid.

2. A gene encoding the mutant according to claim 1.

3. A recombinant plasmid carrying the gene according to claim 2.

4. The recombinant plasmid according to claim 3, wherein The plasmid includes, but is not limited to, pET28a(+) or pXMJ-19.

5. A recombinant microorganism expressing the mutant according to claim 1.

6. A recombinant Escherichia coli, characterized in that, Using Escherichia coli E.coli BL21(DE3) as the host and pET28a(+) or pXMJ-19 plasmid as the expression vector to express the polyphosphate kinase mutant according to claim 1.

7. The recombinant Escherichia coli according to claim 6, wherein It also expresses glutamine synthetase.

8. Use of the mutant according to claim 1, or the recombinant Escherichia coli according to any one of claims 6 to 7 in the production of glutamine.

9. The application according to claim 8, characterized in that, Adding the cell lysate of the mutant according to claim 1 or the recombinant Escherichia coli according to any one of claims 6 to 7 to the reaction system; the reaction system contains glutamate, (NH4)2SO4, polyphosphate, ATP and MgCl2.

10. The application according to claim 9, wherein The polyphosphate includes polyphosphates with a degree of polymerization ≥3.

11. The application according to claim 9, wherein The polyphosphate includes polyphosphates with a degree of polymerization ≥6.

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