WO2023142253A1 - 一种多聚磷酸激酶突变体、工程菌及其应用 - Google Patents

一种多聚磷酸激酶突变体、工程菌及其应用 Download PDF

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WO2023142253A1
WO2023142253A1 PCT/CN2022/082772 CN2022082772W WO2023142253A1 WO 2023142253 A1 WO2023142253 A1 WO 2023142253A1 CN 2022082772 W CN2022082772 W CN 2022082772W WO 2023142253 A1 WO2023142253 A1 WO 2023142253A1
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mutated
mutant
buffer
polyphosphate kinase
chppk
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薛亚平
薛语真
张诗嘉
沈其
郑裕国
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浙江工业大学
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Definitions

  • the invention belongs to the technical field of bioengineering, in particular to a polyphosphokinase (PPK) mutant, engineering bacteria and applications thereof, and to develop an efficient and cheap ATP regeneration system.
  • PPK polyphosphokinase
  • Adenosine triphosphate is a key molecule in the regulation of various biological processes such as energy metabolism, RNA and DNA synthesis, and signal transduction in organisms.
  • the activity of a large number of potential biocatalysts, including ligases, kinases and synthetases, is also dependent on ATP. Introducing an ATP regeneration system during these biotransformations can significantly reduce ATP consumption.
  • Most ATP regeneration systems include a phosphate donor and a phosphotransferase that catalyzes the connection between ADP and the phosphate donor.
  • polyphosphokinase 2-class III (PPK2-III) enzymes have been intensively studied because they convert AMP to ATP.
  • PPK2-III polyphosphokinase 2-class III
  • the availability and stability of phosphate donors are crucial for the application of ATP regeneration systems.
  • Inorganic polyphosphate (polyP) is a stable phosphate donor for the ATP regeneration system composed of PPK2-III enzyme.
  • long-chain polyP was used as a phosphate donor.
  • short-chain polyP as a phosphate donor can make the ATP generation system more attractive because short-chain polyP is less expensive than long-chain polyP.
  • polyphosphoric acid which is a mixture of linear polyphosphoric acid with different lengths, and is widely used in the manufacture of pigments, petroleum catalysts, spices and fire retardants.
  • PPA polyphosphoric acid
  • ChPPK molecularly engineered polyphosphatase
  • the purpose of the present invention is to solve the problem that the existing polyphosphate kinase activity is not high, to provide a polyphosphate kinase mutant and to use the polyphosphate kinase mutant gene recombinant bacteria or its crude enzyme solution as a biocatalyst for ATP
  • the regeneration system is constructed to solve the problem of low efficiency of the existing ATP regeneration system.
  • the present invention provides a polyphosphate kinase mutant, said polyphosphate kinase mutant is the 79th, 106th, 108th, 111th or 285th amino acid sequence shown in SEQ ID NO: 2 Obtained by performing single or multiple mutations; the nucleotide sequence of the gene encoding the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 1.
  • the polyphosphate kinase mutant is to mutate the amino acid sequence shown in SEQ ID NO: 2 into one of the following: (1) the 79th alanine is mutated into glycine (A79G); (2) the 106th Serine is mutated to cysteine (S106C); (3) Isoleucine at position 108 is mutated to phenylalanine, asparagine or tyrosine (I108F, I108N, I108Y); (4) Serine at position 111 Mutation to glutamic acid or lysine or alanine (S111E, S111K, S111A); (5) Leucine at position 285 is mutated to proline (L285P); (6) Alanine at position 79 is mutated to Glycine, 108th isoleucine is mutated to phenylalanine (A79G/I108F); (7) 79th alanine is mutated to glycine, 106th serine is
  • the present invention also provides a recombinant genetically engineered bacterium prepared by the coding gene of the polyphosphate kinase mutant, the recombinant vector constructed by the coding gene, and the recombinant vector transformed host bacterium;
  • the vector is a pET expression vector, a pCW expression vector or pPIC Expression vector, preferably plasmid pET-28a (+); described host cell is Escherichia coli, Bacillus subtilis, streptomyces, Saccharomyces cerevisiae, Pichia pastoris or mammalian cell, preferably Escherichia coli Escherichia coli (E.coli) BL21 (DE3).
  • the present invention also provides an application of the polyphosphate kinase mutant in constructing an ATP regeneration system, wherein the application is to replace part of ATP with the polyphosphate kinase mutant and polyphosphoric acid (PPA), and the polyphosphate kinase
  • PPA polyphosphoric acid
  • the mutant acts in the form of crude enzyme liquid or pure enzyme extracted from the wet thalline obtained by fermenting and culturing the polyphosphokinase mutant genetically engineered bacteria.
  • the polyphosphate kinase mutant of the present invention can replace part of ATP in all ATP-dependent biotransformation reactions.
  • the present invention also provides an application of the polyphosphate kinase mutant in the synthesis of nicotinamide mononucleotide (NMN), the method of which is: using the polyphosphate kinase mutant genetically engineered bacteria and tobacco
  • the wet cells obtained by the amide ribokinase (NRK) genetically engineered bacteria were resuspended in buffer, and the supernatant after sonication was used as a catalyst, and adenine nucleosine triphosphate (ATP) and nicotinamide ribose ( NR) as a substrate, magnesium chloride and polyphosphoric acid (PPA) were added, and a buffer solution with pH 6.5 was used as a reaction medium, and the reaction was carried out at 37°C (preferably for 6h), to obtain nicotinamide mononucleotide (NMN).
  • NRK amide ribokinase
  • ATP adenine nucleosine triphosphate
  • the adenosine triphosphate (ATP) addition is 10-100mM in buffer volume, preferably 25mM; the NR addition is 50-200mM in buffer volume, preferably 100mM; the magnesium chloride addition is The buffer volume is 5-20mM, preferably 10mM; the PPA addition is 1-10g/L by buffer volume, preferably 4.8g/L; the polyphosphokinase mutant supernatant is added in an amount to break
  • the pre-wet bacteria volume is 2-30 mg/mL buffer, preferably 4 mg/mL; the added amount of the nicotinamide ribokinase supernatant is 5-30 mg/mL buffer based on the wet bacteria volume before breaking, preferably 8 mg /mL.
  • the buffer is pH 6.5, 50mM potassium phosphate buffer.
  • the catalyst is prepared as follows: Inoculate polyphosphate kinase mutant genetically engineered bacteria into LB liquid medium containing 50 ⁇ g/mL kanamycin resistance, cultivate at 37°C for 12 hours at 200 rpm, and then inoculate with 1 % (v/v) inoculum was inoculated into fresh LB liquid medium containing 50 ⁇ g/mL kanamycin resistance, cultivated at 37°C and 150 rpm until the OD600 of the bacteria reached 0.6, and the final concentration of 0.1 mM was added.
  • IPTG after induction culture at 28°C for 12h, centrifuge at 4°C and 8000rpm for 10min, discard the supernatant, and collect the wet cell precipitate; the collected wet cell (preferably in the amount of 40g/L) is washed with 50mM phosphoric acid with a pH of 7.2 Resuspend in potassium buffer solution (PBS) and crush using an ultrasonic cell pulverizer with a crushing power of 50W, each working for 1s, intermittently for 2s, and crushing for a total of 20min; collect the cell lysate, centrifuge at 12000g for 1min, and take the supernatant, namely For the crude enzyme solution.
  • PBS potassium buffer solution
  • the supernatant of said nicotinamide ribokinase (NRK) genetically engineered bacteria is prepared in the same way as the supernatant of polyphosphate kinase mutant genetically engineered bacteria.
  • the NRK amino acid sequence is shown in SEQ ID NO: 4
  • the nucleotide sequence is shown in SEQ ID NO: 3
  • the vector for constructing engineering bacteria is pET-28a(+)
  • the host bacteria is E.coli BL21(DE3) .
  • the present invention also provides an application of the polyphosphokinase mutant in the synthesis of glucose 6 phosphate (G6P), the method of which is: using the polyphosphate kinase mutant genetically engineered bacteria and hexokinase (HK ) Genetically engineered bacteria were resuspended with buffer and the supernatant after ultrasonic crushing was used as a catalyst, and adenosine triphosphate (ATP) and glucose were used as substrates, and magnesium chloride and poly Phosphoric acid (PPA), using 50mM potassium phosphate buffer at pH 7.2 as the reaction medium, reacted at 37°C (preferably 8h) to obtain G6P.
  • G6P glucose 6 phosphate
  • the adenosine triphosphate (ATP) addition is 10-100mM in buffer volume, preferably 25mM; the glucose addition is 20-150mM in buffer volume, preferably 100mM; the magnesium chloride addition is The buffer volume is 5-20mM, preferably 10mM; the PPA addition is 1-10g/L by buffer volume, preferably 4.8g/L; the polyphosphokinase mutant supernatant is added in an amount to break
  • the pre-wet bacterial volume is 2-30 mg/mL buffer, preferably 4 mg/mL; the added amount of the HK supernatant is 5-30 mg/mL buffer, preferably 12 mg/mL, based on the wet bacterial volume before crushing.
  • the buffer is pH 7.2, 50mM potassium phosphate buffer.
  • the preparation of the supernatant of the hexokinase (HK) genetically engineered bacteria is the same as the supernatant of the polyphosphokinase mutant genetically engineered bacteria.
  • the HK amino acid sequence is shown in SEQ ID NO: 6, the nucleotide sequence is shown in SEQ ID NO: 5, the vector for constructing engineering bacteria is pET-28a(+), and the host bacteria is E.coli BL21(DE3) .
  • the present invention provides a variety of polyphosphate kinase mutants derived from Cytophaga hutchinsonii.
  • the specific enzyme activity of these mutants is 2.7-17.9 times higher than that of the parent polyphosphate kinase.
  • the ATP regeneration system formed by these mutants can synthesize NMN and In the reaction of G6P, the amount of ATP used can be reduced by more than 70% without affecting the final yield. Therefore, the present invention has wide industrial application prospect.
  • Fig. 1 is the relative enzyme activity of the crude enzyme solution containing the mutant prepared by the method in Example 4.
  • Fig. 2 is the relative enzyme activity of the mutant prepared by the method in Example 5.
  • Figure 3 is the relative enzyme activity of the mutants prepared by the method in Example 6.
  • Fig. 4 is the effect of temperature on the enzyme activity of mutant ChPPK/A79G/S106C/I108F/L285P and wild type in Example 7.
  • Fig. 5 is the effect of pH on the enzyme activity of mutant ChPPK/A79G/S106C/I108F/L285P and wild type in Example 7.
  • Figure 6 shows the effect of AMP concentration on the enzyme activity of mutant ChPPK/A79G/S106C/I108F/L285P and wild type in Example 7.
  • Figure 7 shows the effect of PPA concentration on the enzyme activity of mutant ChPPK/A79G/S106C/I108F/L285P and wild type in Example 7.
  • Fig. 8 is a graph showing NMN production in different reaction systems in Example 9.
  • Fig. 9 is a histogram of G6P yields in different reaction systems in Example 10.
  • the experimental methods without specific experimental conditions are usually carried out according to conventional conditions, such as the conditions described in the Molecular Cloning Experiment Guide (Third Edition, J. Sambrook et al.).
  • LB plate composition 10g/L tryptone, 10g/L sodium chloride, 5g/L yeast extract, 15g/L agar, the solvent is water, and the pH is natural.
  • composition of LB liquid medium 10g/L tryptone, 10g/L sodium chloride, 5g/L yeast extract, the solvent is water, and the pH is natural.
  • the recombinant gene ChPPK was inserted under the T7 promoter of pET-28a(+) to obtain the expression plasmid pET28-ChPPK.
  • Example 2 Induced expression of wild-type Ecoli.BL21(DE3)-ChPPK and extraction of wild-type polyphosphate kinase
  • the collected wet bacteria were resuspended with 50mM potassium phosphate buffer (PBS) with a pH of 7.2 in an amount of 40g/L to form a bacterial suspension, and crushed using an ultrasonic cell pulverizer with a crushing power of 50W, each working for 1s, and intermittently for 2s , a total of 20 minutes of crushing. Collect the cell disruption solution, centrifuge it at 12000rpm for 1min, and take the supernatant, which is the crude enzyme solution.
  • PBS potassium phosphate buffer
  • elution buffer 500mM sodium chloride + 250mM imidazole pH 7.2, 20mM potassium phosphate buffer
  • elution buffer 500mM sodium chloride + 250mM imidazole pH 7.2, 20mM potassium phosphate buffer
  • collect the eluate containing the target protein collect the eluate containing the target protein
  • 20mM pH 7.2 Potassium phosphate buffer was dialyzed in a dialysis bag (molecular weight cut-off 14KDa) for 48 hours, and the retentate was taken as pure enzyme.
  • concentration of pure enzyme was determined by Biyuntian BCA protein concentration kit (P0012). content meter.
  • Embodiment 3 the mensuration of enzyme activity
  • PPA polyphosphoric acid
  • the instrument used for HPLC is Agilent 1260 Infinity II (Agilent Technologies Co., Ltd., USA), equipped with Agilent 2414 UV detector, Agilent 1525 pump, and Agilent 717 injector.
  • the chromatographic column is XBridge C18 column (C18, 5 ⁇ m, 4.6 ⁇ 250mm, Waters, California, USA).
  • the mobile phase rate was 1 mL/min
  • the UV detection wavelength was 254 nm
  • the mobile phase was potassium phosphate buffer (50 mM, pH 7.0)
  • the injection volume was 10 ⁇ L
  • the detection time was 9 min.
  • the peak area data obtained by injecting samples with different concentrations of ATP (0.125mM, 0.25mM, 0.5mM, 1.0mM, 2.0mM and 4.0mM) was used to obtain the standard curve of ATP concentration and peak area.
  • Enzyme activity definition An enzyme activity is defined as the amount of enzyme required to produce 1 ⁇ mol ATP per minute under the above conditions (the first 5 minutes).
  • the enzyme activities of 0.4mg crude enzyme solution and 0.05mg pure enzyme solution in Example 2 are 0.0029U and 0.0932U respectively.
  • the substrate PPA is represented by polyP containing 5 phosphates. Docking of ChPPK to substrate simulation (AMP with polyP of 5 phosphates) was performed using Autodock vina 1.1.2. According to the results of the combination of the enzyme and the substrate, the distance between most of the enzyme and the substrate is selected (D77, G80, K81, D82, F102, K103, V104, P105, R117, R133, E137, N138, V141 and R208) and the distance between the part and the substrate in (S106, I108, S111) amino acids.
  • Example 1 Using the pET28-ChPPK plasmid in Example 1 as a template, using the Quick-change mutation method, using the primers listed in Table 1, the amino acids at each site were mutated into alanine.
  • the mutant PCR system contains 25 ⁇ l 2 ⁇ Phanta Max mixed solution, 0.4 ⁇ M upstream and downstream primers, about 10 ng template, and finally add pure water to 50 ⁇ l.
  • the PCR conditions are: 95° C. for 1 min. Then 20 amplification cycles, each amplification cycle including 95°C for 10s, 55°C for 30s, and 72°C for 5min. Finally, 72°C for 5min.
  • A79 and L285 were also selected for saturation mutation for the following reasons: in the ChPPK-substrate binding model, the distance between the A79 site and polyP is within range. Since the sequence itself is alanine, no alanine mutation was performed in Example 4. In Example 5, saturation mutation was directly performed on A79. In addition, L285 is a key site determining the lid structure of ChPPK, so saturation mutation was also performed in Example 5.
  • the pET28-ChPPK plasmid constructed by the method in Example 1 was used as a template, and the primers in Table 2 were used to select sites A79, G80, K81, F102, K103, P105, S106, I108, S111, R117, Saturation mutations were performed at R208 and L285.
  • Example 6 The combination of beneficial mutants improves the activity of ChPPK
  • the enzyme activity-enhancing sites of the single-residue mutation are located at positions 79, 106, 108, 111 and 285 of the ChPPK amino acid sequence. According to the results of enzyme-substrate docking, 79, 106, 108 and 111 are located in the substrate binding pocket, while 285 is far from these substrate binding sites. Therefore, it is less likely that the 285 site will interact with other beneficial mutations. Therefore, combinatorial mutations were first performed at positions 79, 106, 108, and 111. In order to combine beneficial mutations at positions 79, 106, 108 and 111, we used PCR to obtain a fragment containing all possible beneficial mutations at positions 79, 106, 108 and 111.
  • the PCR uses the pET28-ChPPK plasmid as a template, and PPK-M-b primer F, PPK-M-b primer R1, and PPK-M-b primer R2 as primers.
  • PPK-M-b primer F is the upstream primer
  • the mixture of PPK-M-b primer R1 and PPK-M-b primer R2 (the molar ratio of 1:1 added in the PCR reaction) is the downstream primer.
  • the upstream and downstream primers contain degenerative bases, which can generate all possible beneficial mutations.
  • PPK-M-b primer F TCAGGCAATGGATGCAGSAGGTAAAGATGGTA;
  • PPK-M-b primer R1 ACAGATAATCATGCTYCAGTTCAWWTTTASACGGAAC.
  • PPK-M-b primer R2 ACAGATAATCATGTGMCAGTTCAWWTTTASACGGAAC
  • pET-PPK primer F CATGATTATCTGTGGCGTCATTATGTG;
  • pET-PPK primer R TGCATCCATTGCCTGAAAAACAATCAG.
  • the pET-ChPPK with three mutations of A79G/S106C/I108F was further mutated to introduce beneficial mutation S111A mutation.
  • the pET28-ChPPK plasmid containing the three-site mutation of A79G/S106C/I108F was used as a template, and the 111-site alanine mutation amplification primer in Table 1 was used for mutation.
  • the above mutated plasmid was transformed into the host strain E.coli BL21(DE3), screening, expression and purification were carried out as in Examples 1, 2 and 3.
  • Reverse primer TAATCATGTTCCAGTTCAATTTTGGACG.
  • the pET-ChPPK with the three-site mutation of A79G/S106C/I108F was further mutated to introduce the L285P mutation.
  • the pET28-ChPPK plasmid containing the three-site mutation of A79G/S106C/I108F was used as a template, and the following primers were used for mutation.
  • screening, expression and purification were carried out as in Examples 1, 2 and 3. Results As shown in Figure 3, the four-position combined mutant (ChPPK/A79G/S106C/I108F/L285P) with A79G/S106C/I108F/L285P had the highest activity.
  • Reverse primer TTCTGTTCTGGGCTCACGGTCGGAAA.
  • the engineering bacteria E.coli BL21(DE3)-ChPPK and E.coli BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P constructed by the methods of Examples 1 and 6 were used to prepare pure enzymes by the method of Example 2.
  • the enzyme activity was measured by the method in Example 3, and the temperature for measuring the enzyme activity was changed to 25° C., 30° C., 35° C., 37° C., 40° C., 45° C., 50° C., 55° C. or 60° C. respectively.
  • the engineering bacteria E.coli BL21(DE3)-ChPPK and E.coli BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P constructed by the methods of Examples 1 and 6 were used to prepare pure enzymes by the method of Example 2.
  • the engineering bacteria E.coli BL21(DE3)-ChPPK and E.coli BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P constructed by the methods of Examples 1 and 6 were used to prepare pure enzymes by the method of Example 2.
  • the engineering bacteria E.coli BL21(DE3)-ChPPK and E.coli BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P constructed by the methods of Examples 1 and 6 were used to prepare pure enzymes by the method of Example 2.
  • the optimal PPA concentration of wild-type ChPPK is 1.6g/L
  • the mutant ChPPK/A79G/S106C/I108F/L285P exhibits the highest relative activity in the presence of 2.24g/L PPA, indicating that the mutant Type ChPPK/A79G/S106C/I108F/L285P was more resistant to PPA.
  • Example 8 Determination of Kinetic Parameters of Wild-type ChPPK and Mutant A79G/S106C/I108F/L285P Using Pure Enzyme
  • the wild-type Ecoli.BL21(DE3)-ChPPK constructed in Example 1 and the engineered bacteria Ecoli.BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P constructed by the method in Example 6 were used to prepare wild Pure enzymes of type ChPPK and mutant ChPPK/A79G/S106C/I108F/L285P.
  • Km and Kcat values were calculated using pseudo-one-substrate kinetic model.
  • the Km values of wild-type ChPPK for AMP and PPA were similar to those of mutant ChPPK/A79G/S106C/I108F/L285P (Table 3 and Table 4).
  • the increased enzymatic activity of mutant ChPPK/A79G/S106C/I108F/L285P could be explained by its increased turnover (kcat value) towards AMP and PPA.
  • the catalytic efficiencies (kcat/Km) of the mutant ChPPK/A79G/S106C/I108F/L285P towards AMP and PPA were 16-fold and 18-fold higher than those of the wild-type ChPPK, respectively.
  • nicotinamide ribokinase can catalyze the biosynthesis of nicotinamide mononucleotide (NMN) from nicotinamide riboside (NR), the nicotinamide ribokinase (NRK, GenBank number: XP_035204248.1) from brown hardtail duck (Oxyura jamaicensis) ) amino acid sequence as a template, artificially synthesized nicotinamide ribokinase gene optimized for Escherichia coli codons, the nucleotide sequence is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:4.
  • the recombinant gene NRK (shown in SEQ ID NO: 3) optimized for E. coli codons was inserted under the T7 promoter of pET-28a(+) to obtain the expression plasmid pET28-NRK.
  • the crude enzyme liquid was prepared under the same conditions as in Example 2, and the usage amount of NRK was calculated according to the amount of bacteria before crushing.
  • Reaction 1 25mM ATP: In 1mL of potassium phosphate buffer (50mM, pH 6.5), add ATP at a final concentration of 25mM, NR at a final concentration of 100mM, magnesium chloride at a final concentration of 10mM and NRK crude enzyme solution at a concentration of 8mg/mL, at 37°C After reacting for 6h, samples were taken at 0.5h, 1.5h, 3h and 6h, and detected by the HPLC method in Example 3 (NMN retention time is 2.9 minutes).
  • the concentration change of NMN generated in reaction 1 is shown in Figure 8, and the yield of NMN after 6 hours was 34 mM.
  • Reaction 2 (100mM ATP): Change the ATP concentration in Reaction 1 to 100mM, and the other operations are the same. The results are shown in Figure 8. When the input ATP reaches 100mM, the output of NMN increases to 85mM after 6 hours.
  • Reaction 3 25mM ATP+wild-type ChPPK: add 4mg/mL wild-type ChPPK crude enzyme solution prepared by the method of Example 2 and 4.8g/L PPA to reaction 1, other operations are the same, and the concentration of NMN changes as shown in Figure 8 It was shown that when the ATP regeneration system containing wild-type ChPPK was introduced into NMN synthesis, the production of NMN increased to 55mM after 6 hours.
  • Reaction 4 25mM ATP+mutant ChPPK/A79G/S106C/I108F/L285P: add 4mg/mL of mutant ChPPK/A79G/S106C/I108F/L285P crude enzyme solution prepared by the method in Example 6 to reaction 1 and 4.8g /L of PPA, other operations are the same, the results are shown in Figure 8, when the ATP regeneration system containing mutant ChPPK/A79G/S106C/I108F/L285P was introduced into NMN synthesis, the output of NMN was raised to 86mM after 6 hours. It shows that the ATP regeneration system composed of the introduced mutant can save 75% of ATP input without affecting the final NMN output.
  • hexokinase can catalyze the biosynthesis of glucose 6 phosphate (G6P) from glucose
  • G6P glucose 6 phosphate
  • the amino acid sequence of hexokinase (HK, GenBank number: NP_013551.1) from Saccharomyces cerevisiae was used as a template to artificially synthesize
  • the optimized hexokinase gene has a nucleotide sequence as shown in SEQ ID NO:5, and an amino acid sequence as shown in SEQ ID NO:6.
  • the recombinant gene HK (nucleotide sequence shown in SEQ ID NO: 5) optimized for codons in Escherichia coli was inserted under the T7 promoter of pET-28a(+) to obtain the expression plasmid pET28-HK.
  • the crude enzyme solution was prepared under the same conditions as in Example 2, wherein the usage amount of the HK crude enzyme solution was calculated according to the amount of bacteria before crushing.
  • Reaction 1 25mM ATP: Add final concentration of 25mM ATP, final concentration of 100mM glucose, final concentration of 10mM magnesium chloride and 12mg/mL HK crude enzyme solution in 5mL potassium phosphate buffer (50mM, pH 7.5), react at 30°C for 8h . After the reaction, 1 mL was sampled and heated at 70°C for 15 minutes to inactivate the enzyme. Centrifuge at 12,000 ⁇ g for 10 minutes to collect the supernatant for HPLC analysis.
  • the instrument for HPLC detection of G6P is Agilent 1260 Infinity II (Agilent Technologies Co., Ltd., USA), equipped with a Dionex ED40 detector.
  • the mobile phase was aqueous sodium hydroxide solution at a rate of 1 mL/min.
  • the gradient elution method is used: 25mM sodium hydroxide aqueous solution within 0-10 minutes, the sodium hydroxide aqueous solution is raised from 25mM to 100mM within 12-15 minutes, and replaced with 25mM sodium hydroxide aqueous solution in the last 17-21 minutes.
  • the standard curve was used to calculate the G6P yield catalyzed by HK crude enzyme solution. As shown in Figure 9, the production of G6P was 18 mM after 8 h.
  • Reaction 2 (100mM ATP): Change the ATP concentration in Reaction 1 to 100mM, and the other operations are the same. The results are shown in Figure 9. When the input ATP reaches 100mM, the output of G6P increases to 78mM after 8 hours.
  • Reaction 3 25mM ATP+wild-type ChPPK: Add 4mg/mL wild-type ChPPK crude enzyme solution prepared by the method in Example 2 and 4.8g/L PPA to reaction 1, and other operations are the same. As shown in Figure 9, when the ATP regeneration system containing wild-type ChPPK was introduced into G6P synthesis, the G6P production increased to 38mM after 8 hours.
  • Reaction 4 (25mM ATP+ mutant ChPPK/A79G/S106C/I108F/L285P): Add 4mg/mL mutant ChPPK/A79G/S106C/I108F/L285P crude enzyme solution prepared by the method in Example 6 and 4.8g/mL to reaction 1
  • the PPA of L the other operations are the same, the results are shown in Figure 9, when the ATP regeneration system containing the mutant ChPPK/A79G/S106C/I108F/L285P was introduced into the G6P synthesis, the G6P production increased to 82mM after 8 hours. It shows that the ATP regeneration system composed of the introduced mutant can save 75% of ATP input without affecting the final G6P output.

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Abstract

提供一种多聚磷酸激酶突变体、工程菌及其应用，所述多聚磷酸激酶突变体是将SEQ ID NO：2所示氨基酸序列第79位、第106位、第108位、第111位或第285位进行单突变或多突变获得的。提供多种来源于Cytophaga hutchinsonii的多聚磷酸激酶突变体，这些突变体的比酶活较母本多聚磷酸激酶提升2.7-17.9倍，这些突变体构成的ATP再生体系能减少70％以上ATP依赖的生物催化合成反应中ATP的消耗，具有广泛的工业应用前景。

Description

一种多聚磷酸激酶突变体、工程菌及其应用 (一)技术领域
本发明属于生物工程技术领域，特别涉及一种多聚磷酸激酶(PPK)突变体、工程菌及其应用，开发高效廉价的ATP再生体系。
(二)背景技术
腺嘌呤核苷三磷酸(ATP)是生物体中调节能量代谢、RNA和DNA合成以及信号转导等多种生物过程的关键分子。大量潜在的生物催化剂的活性，包括连接酶、激酶和合成酶，也依赖ATP。在这些生物转化过程中导入ATP再生系统可以显著降低ATP的消耗。大多数ATP再生系统包括磷酸供体以及催化ADP和磷酸供体之间的磷酸转移酶。然而很多有价值的产品的生物合成需要由AMP直接生成ATP的再生系统，如土四环素、1,6-六甲二胺和β-卡碱酰胺等。为了从AMP再生ATP，可以采用将AMP合成ADP的再生系统导入到从ADP开始生成ATP的再生系统。
值得注意的是，多种酶和磷酸供体的使用会使原本的生物转化过程更加复杂。考虑到这些限制，人们对聚磷酸激酶2-III类(PPK2-III)酶进行了深入研究，因为它们可以将AMP转化为ATP。磷酸供体的可及性和稳定性对ATP再生系统应用至关重要。对于PPK2-III酶构成的ATP再生系统，无机聚磷酸盐(polyP)是一种稳定的磷酸供体。在很多研究中，长链polyP被作为磷酸供体。但是，使用短链polyP作为磷酸供体可以使ATP生成系统更具吸引力，因为短链polyP比长链polyP更廉价。最容易获得的polyP是的聚磷酸(PPA)，它是含有不同长度线性多聚磷酸的混合物，大量用于颜料、石油催化剂、香料和阻火剂的制造。在此，我们旨在开发一种基于PPK2-III酶并能以AMP和PPA为底物的的高效廉价ATP再生系统。我们对来自Cytophaga hutchinsonii的多聚磷酸酶(ChPPK)进行了分子改造，以提高其酶学性质，用于构建廉价高效的ATP再生体系。
(三)发明内容
本发明目的是针对现有多聚磷酸激酶活性不高的问题，提供一种多聚磷酸激酶突变体及利用该多聚磷酸激酶突变体基因重组菌或其粗酶液作为生物催化剂，用于ATP再生系统的构建，以解决现有ATP再生系统效率较低的问题。
本发明采用的技术方案是：
本发明提供一种多聚磷酸激酶突变体，所述多聚磷酸激酶突变体是将SEQ ID NO：2所示氨基酸序列第79位、第106位、第108位、第111位或第285位进行单突变或多突变获得的；所述SEQ ID NO：2所示氨基酸序列编码基因的核苷酸序列如SEQ ID NO：1所示。
优选的，所述多聚磷酸激酶突变体是将SEQ ID NO：2所示氨基酸序列突变为下列之一：(1)第79位丙氨酸突变为甘氨酸(A79G)；(2)第106位丝氨酸突变为半胱氨酸(S106C)；(3)第108位异亮氨酸突变为苯丙氨酸、天冬酰胺或酪氨酸(I108F、I108N、I108Y)；(4)第111位丝氨酸突变为谷氨酸或赖氨酸或丙氨酸(S111E、S111K、S111A)；(5)第285位亮氨酸突变为脯氨酸(L285P)；(6)第79位丙氨酸突变为甘氨酸、第108位异亮氨酸突变为苯丙氨酸(A79G/I108F)；(7)第79位丙氨酸突变为甘氨酸、第106位丝氨酸突变为半胱氨酸、第108位异亮氨酸突变为苯丙氨酸(A79G/S106C/I108F)；(8)第79位丙氨酸突变为甘氨酸、第106位丝氨酸突变为半胱氨酸、第108位异亮氨酸突变为苯丙氨酸、第111位丝氨酸突变为丙氨酸(A79G/S106C/I108F/S111A)；(9)第79位丙氨酸突变为甘氨酸、第106位丝氨酸突变为半胱氨酸、第108位异亮氨酸突变为苯丙氨酸、第285位亮氨酸突变为脯氨酸(A79G/S106C/I108F/L285P)。
本发明还提供一种所述多聚磷酸激酶突变体的编码基因、编码基因构建的重组载体以及重组载体转化宿主菌制备的重组基因工程菌；所述载体为pET表达载体，pCW表达载体或pPIC表达载体，优选质粒pET-28a(+)；所述的宿主细胞为大肠杆菌、枯草芽孢杆菌、链霉菌、酿酒酵母、毕赤酵母或哺乳动物细胞，优选大肠杆菌Escherichia coli(E.coli)BL21(DE3)。
本发明还提供一种所述多聚磷酸激酶突变体在构建ATP再生体系中的应用，所述应用是用多聚磷酸激酶突变体和聚磷酸(PPA)替换部分ATP，所述多聚磷酸激酶突变体以多聚磷酸激酶突变体基因工程菌经发酵培养获得的湿菌体提取的粗酶液或纯酶形式作用。本发明所述多聚磷酸激酶突变体能够替换所有ATP依赖的生物转化反应中部分ATP。
本发明还提供一种所述多聚磷酸激酶突变体在合成烟酰胺单核苷酸(NMN)中的应用，所述应用的方法为：以所述多聚磷酸激酶突变体基因工程菌和烟酰胺核糖激酶(NRK)基因工程菌分别经诱导培养获得的湿菌体用缓冲液重悬并采用超声破碎后的 上清液为催化剂，以腺嘌呤核苷三磷酸(ATP)和烟酰胺核糖(NR)为底物，加入氯化镁和聚磷酸(PPA)，以pH6.5的缓冲液为反应介质，在37℃进行反应(优选反应6h)，获得烟酰胺单核苷酸(NMN)。
所述腺嘌呤核苷三磷酸(ATP)加入量以缓冲液体积计为10-100mM，优选25mM；所述NR加入量以缓冲液体积计为50-200mM，优选100mM；所述氯化镁加入量以缓冲液体积计为5-20mM，优选10mM；所述PPA加入量以缓冲液体积计为1-10g/L，优选4.8g/L；所述多聚磷酸激酶突变体上清液加入量以破碎前湿菌体量计为2-30mg/mL缓冲液，优选4mg/mL；所述烟酰胺核糖激酶上清液加入量以破碎前湿菌体量计为5-30mg/mL缓冲液，优选8mg/mL。
优选的，所述缓冲液为pH 6.5、50mM磷酸钾缓冲液。
优选的，所述催化剂按如下方法制备：将多聚磷酸激酶突变体基因工程菌接种至含有50μg/mL卡那霉素抗性的LB液体培养基，37℃，200rpm下培养12h，再以1％(v/v)接种量接种至新鲜的含有50μg/mL卡那霉素抗性的LB液体培养基中，于37℃，150rpm下培养至菌体OD600达0.6，加入终浓度为0.1mM的IPTG，28℃下诱导培养12h后，4℃、8000rpm离心10min，弃去上清液，收集湿菌体沉淀；收集的湿菌体(优选按40g/L的量)用pH为7.2的50mM磷酸钾缓冲液(PBS)重悬，并使用超声波细胞粉碎机破碎，破碎功率为50W，每工作1s，间歇2s，总共破碎20min；收集细胞裂解液，在12000g下离心1min，取上清液，即为粗酶液。
优选的，所述烟酰胺核糖激酶(NRK)基因工程菌的上清液制备方法同多聚磷酸激酶突变体基因工程菌的上清液。所述NRK氨基酸序列如SEQ ID NO：4所示，核苷酸序列如SEQ ID NO：3所示，构建工程菌的载体为pET-28a(+)，宿主菌为E.coli BL21(DE3)。
本发明还提供一种所述多聚磷酸激酶突变体在合成葡萄糖6磷酸(G6P)中的应用，所述应用的方法为：以所述多聚磷酸激酶突变体基因工程菌和己糖激酶(HK)基因工程菌分别经诱导培养获得的湿菌体用缓冲液重悬并采用超声破碎后的上清液为催化剂，以腺嘌呤核苷三磷酸(ATP)和葡萄糖为底物，加入氯化镁和聚磷酸(PPA)，以pH 7.2的50mM磷酸钾缓冲液为反应介质，在37℃进行反应(优选8h)，获得G6P。
所述腺嘌呤核苷三磷酸(ATP)加入量以缓冲液体积计为10-100mM，优选25mM；所述葡萄糖加入量以缓冲液体积计为20-150mM，优选100mM；所述氯化镁 加入量以缓冲液体积计为5-20mM，优选10mM；所述PPA加入量以缓冲液体积计为1-10g/L，优选4.8g/L；所述多聚磷酸激酶突变体上清液加入量以破碎前湿菌体量计为2-30mg/mL缓冲液，优选4mg/mL；所述HK上清液加入量以破碎前湿菌体量计为5-30mg/mL缓冲液，优选12mg/mL。
优选的，所述缓冲液为pH 7.2、50mM磷酸钾缓冲液。
优选的，所述己糖激酶(HK)基因工程菌上清液的制备同多聚磷酸激酶突变体基因工程菌的上清液。所述HK氨基酸序列如SEQ ID NO：6所示，核苷酸序列如SEQ ID NO：5所示，构建工程菌的载体为pET-28a(+)，宿主菌为E.coli BL21(DE3)。
与现有技术相比，本发明有益效果主要体现在：
本发明提供了多种来源于Cytophaga hutchinsonii的多聚磷酸激酶突变体，这些突变体的比酶活较母本多聚磷酸激酶提升2.7-17.9倍，这些突变体构成的ATP再生体系在合成NMN与G6P的反应中能减少70％以上ATP的使用量而不影响最终产率。因此，本发明具有广泛的工业应用前景。
(四)附图说明
图1为实施例4方法制备的含突变体的粗酶液相对酶活。
图2为实施例5方法制备的突变体相对酶活。
图3为实施例6方法制备的突变体相对酶活。
图4为实施例7中温度对突变体ChPPK/A79G/S106C/I108F/L285P和野生型酶活的影响。
图5为实施例7中pH对突变体ChPPK/A79G/S106C/I108F/L285P和野生型酶活的影响。
图6为实施例7中AMP浓度对突变体ChPPK/A79G/S106C/I108F/L285P和野生型酶活的影响。
图7为实施例7中PPA浓度对突变体ChPPK/A79G/S106C/I108F/L285P和野生型酶活的影响。
图8为实施例9中不同反应体系中NMN产量曲线图。
图9为实施例10中不同反应体系中G6P产量柱状图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例中，未注明具体实验条件的实验方法，通常按照常规条件，如分子克隆实验指南(第三版,J.萨姆布鲁克等著)中所述的条件进行。
LB平板组成：10g/L胰蛋白胨、10g/L氯化钠、5g/L酵母提取物、15g/L琼脂，溶剂为水，pH自然。
LB液体培养基组成：10g/L胰蛋白胨、10g/L氯化钠、5g/L酵母提取物，溶剂为水，pH自然。
实施例1：野生型Ecoli.BL21(DE3)-ChPPK的构建
根据GenBank中来自哈氏嗜纤维菌(Cytophaga hutchinsonii)的PPK蛋白序列(ChPPK，GenBank号：ABG57400.1)，针对大肠杆菌密码子偏好性优化，并在序列C端融合一个6His标签，由北京擎科生物公司(中国北京)合成长度为930bp的重组基因ChPPK序列，其核苷酸序列如SEQ ID NO：1所示，编码蛋白的氨基酸序列为SEQ ID NO：2所示。
将重组基因ChPPK插入到pET-28a(+)的T7启动子下，得到表达质粒pET28-ChPPK。将该表达质粒转化至大肠杆菌E.coli BL21(DE3)中，涂布于含有50μg/mL卡那霉素抗性的LB平板，37℃下培养8-12h，挑取阳性克隆，即为野生型Ecoli.BL21(DE3)-ChPPK，用于表达重组ChPPK。
SEQ ID NO：1
Figure PCTCN2022082772-appb-000001
Figure PCTCN2022082772-appb-000002
SEQ ID NO：2
Figure PCTCN2022082772-appb-000003
实施例2：野生型Ecoli.BL21(DE3)-ChPPK的诱导表达及野生型多聚磷酸激酶的提取
(1)粗酶液：将实施例1获得的野生型Ecoli.BL21(DE3)-ChPPK接种至含有50μg/mL卡那霉素抗性的LB液体培养基，37℃，200rpm下培养12h，再以1％(v/v)接种量接种至新鲜的含有50μg/mL卡那霉素抗性的LB液体培养基中，于37℃，150rpm下培养至菌体OD600＝0.6，加入终浓度为0.1mM的IPTG，28℃下诱导培养12h后，4℃、8000rpm离心10min，弃去上清液，收集沉淀，即获得含有表达重组ChPPK的湿菌体。收集的湿菌体按40g/L的量用pH为7.2的50mM磷酸钾缓冲液(PBS)重悬成菌悬液，并使用超声波细胞粉碎机破碎，破碎功率为50W，每工作1s，间歇2s，总共破碎20min。收集细胞破碎液，在12000rpm下离心1min，取上清，即为粗酶液，后续粗酶液的用量以对应破碎前菌悬液中菌体量计。
(2)纯酶：取5mL粗酶液稀释到40mL磷酸钾缓冲液(20mM，pH 7.2)中，然后上样到GE Healthcare公司的HisTrap HP纯化柱中(10mL柱体积，预先用含500mM氯化钠的pH 7.2、20mM磷酸钾缓冲液冲洗)。上样后的纯化柱用100mL清洗缓冲液(500mM氯化钠+50mM咪唑的pH7.2、20mM磷酸钾缓冲液)以0.5mL/min的速度洗脱，除去结合在纯化柱上的杂蛋白。然后用洗脱缓冲液(500mM氯化钠+250mM咪唑的pH 7.2、20mM磷酸钾缓冲液)以0.5mL/min的速度洗脱，收集含目的蛋白的洗脱液，再用pH7.2的20mM磷酸钾缓冲液在透析袋(截留分子量为14KDa)中透析48h，取截留液，即为纯酶，纯酶用浓度用碧云天BCA蛋白浓度试剂盒(P0012)测定，后续纯酶的用量以蛋白含量计。
实施例3：酶活的测定
0.4mg实施例2方法制备的粗酶液或0.05mg纯酶、终浓度1.6g/L的聚磷酸(PPA)，终浓度2.25mM的磷酸腺苷(AMP)，终浓度10mM的MgCl 2加入到10mL 50mM的磷酸钾缓冲液中(pH 7.5)。反应液在37℃孵育5min后加入10mL 0.2M磷酸水溶液终止反应。最终溶液中的ATP含量用HPLC方法测定。
HPLC所用的仪器为Agilent 1260 Infinity Ⅱ(安捷伦科技有限公司，美国)，配置Agilent 2414紫外检测器，Agilent 1525泵，Agilent 717进样器。色谱柱为XBridge C18 column(C18,5μm,4.6×250mm,Waters,California,USA)。流动相速率为1mL/min，紫外检测波长为254nm，流动相为磷酸钾缓冲液(50mM，pH 7.0)，进样量为10μL，检测时间为9min。以不同浓度的ATP(0.125mM、0.25mM、0.5mM、1.0mM、2.0mM和4.0mM)进样得到的峰面积数据，得到ATP浓度与峰面积标准曲线，曲线方程为y＝(x+189.78)/3847(R 2＝0.998)，其中y为ATP浓度(mM)，x为液相得到ATP峰面积。
酶活定义：一个酶活定义为上述条件下每分钟产生1μmol ATP所需的酶量(前5分钟)。
实施例2中0.4mg粗酶液与0.05mg纯酶液的酶活分别是0.0029U与0.0932U。
实施例4：丙氨酸突变鉴定ChPPK中关键位点
1、突变位点的筛选
用含有5个磷酸的polyP代表底物PPA。使用Autodock vina 1.1.2进行ChPPK与底物的模拟(AMP与5个磷酸的polyP)对接。根据酶与底物结合的结果，选取大部分与底物距离在
Figure PCTCN2022082772-appb-000004
(D77、G80、K81、D82、F102、K103、V104、P105、R117、R133、E137、N138、V141和R208)以及部分与底物距离在
Figure PCTCN2022082772-appb-000005
(S106、I108、S111)的氨基酸。采用实施例1中的pET28-ChPPK质粒为模板，采用Quick-change的突变方法，利用表1中列举的引物，将各个位点氨基酸突变成丙氨酸。突变PCR体系含有25μl 2×Phanta Max混合液，上下游引物各0.4μM，约10ng模板，最后补纯水到50μl。PCR条件为：95℃1min。然后20个扩增循环，每个扩增循环包括95℃ 10s，55℃ 30s，72℃ 5min。最后72℃ 5min。
表1、突变位点及引物
Figure PCTCN2022082772-appb-000006
Figure PCTCN2022082772-appb-000007
2、突变工程菌及粗酶液
将步骤1突变的质粒转化宿主菌E.coli BL21(DE3)，采用实施例2方法制备粗酶液，按实施例3方法测定相对酶活。结果如图1所示，以野生型粗酶液的活力为100％，D77、D82、R133、E137和R208残基突变成丙氨酸后，含有ChPPK突变体的裂解液上清彻底失去活力，说明这些位点为保守氨基酸，不适合进行进化研究。G80、K81、F102、K103、P105、S106、I108、S111和R117突变后酶活发生了一定程度的改变， 证明这些位点对ChPPK活力影响较大，所以这些位点在实施例5中进行了饱和突变研究。
实施例5：饱和突变提高ChPPK的活力
1、定点饱和突变
除了实施例4的候选位点外，A79与L285也被选择用于饱和突变，理由如下：在ChPPK与底物结合模型中，A79位点与polyP的距离在
Figure PCTCN2022082772-appb-000008
范围之内。由于本身序列为丙氨酸，所以在实施例4中没有进行丙氨酸突变。在实施例5中，直接对A79进行了饱和突变。另外，L285是决定ChPPK盖子结构的关键位点，所以在实施例5中也进行了饱和突变。
采用Quick-change的突变方法，以实施例1方法构建的pET28-ChPPK质粒为模板，采用表2引物，选取位点A79、G80、K81、F102、K103、P105、S106、I108、S111、R117、R208和L285进行饱和突变。
表2、突变位点及引物
Figure PCTCN2022082772-appb-000009
表2中N＝A，T，G，C；K＝G，T；M＝A，C。
2、突变工程菌及粗酶液
将步骤1突变的质粒转化宿主菌E.coli BL21(DE3)，采用实施例2方法制备纯酶，按实施例3方法测定酶活。
结果如图2所示，含有A79G、S106C、I108F、I108N、I108Y、S111E、S111K、S111A或L285P的单残基突变体酶活有显著提升。
实施例6：有益突变体组合提高ChPPK的活力
1、双突变、三突变
单残基突变酶活提高的位点位于ChPPK氨基酸序列的79、106、108、111以及285位上。根据酶与底物对接结果，79、106、108和111位于底物结合口袋，而285位点距离这些底物结合位点较远。所以285位点与其他有益突变产生相互影响的可能性较小。因此首先对79、106、108和111位进行了组合突变。为了将79、106、108和111位点的有益突变进行组合，我们采用PCR的方法得到一段含有79、106、108和111位所有有益突变可能的片段。该PCR采用pET28-ChPPK质粒为模板，PPK-M-b primer F、PPK-M-b primer R1、PPK-M-b primer R2为引物。其中PPK-M-b primer F为上游引物，PPK-M-b primer R1与PPK-M-b primer R2的混合物(PCR反应中加入摩尔比1：1)为下游引物。上下游引物中包含了兼并碱基，可以产生所有可能的有益突变。
PPK-M-b primer F：TCAGGCAATGGATGCAGSAGGTAAAGATGGTA；
PPK-M-b primer R1：ACAGATAATCATGCTYCAGTTCAWWTTTASACGGAAC。PPK-M-b primer R2：ACAGATAATCATGTGMCAGTTCAWWTTTASACGGAAC
兼并碱基(S＝G，C；Y＝C，T；W＝A，T；M＝A，C)
为了将这个含有79、106、108和111位所有可能有益突变的片段重新连接到质粒中，用PCR扩增了不包含这个片段的pET28-ChPPK质粒。引物如下：
pET-PPK primer F：CATGATTATCTGTGGCGTCATTATGTG；
pET-PPK primer R：TGCATCCATTGCCTGAAAAACAATCAG。
将这个质粒片段与含有79、106、108和111位所有有益突变可能的片段链接后转化宿主菌E.coli BL21(DE3)，采用实施例2方法制备纯酶，按实施例3方法测定酶活。结果如图3所示，带有A79G/S108F的二突变ChPPK和带有A79G/S106C/I108F的三突变ChPPK有着较高的酶活。带有I108F/S111E的二突变和带有S106C/I108Y/S111K的三突变的活力较低。
2、四突变
将带有A79G/S106C/I108F的三位点突变的pET-ChPPK进行进一步突变，引入有益突变S111A突变。采用Quick-change的突变方法，以含有A79G/S106C/I108F的三位点突变的pET28-ChPPK质粒为模板，采用表1中111位点丙氨酸突变扩增引物进突变。将上述突变的质粒转化宿主菌E.coli BL21(DE3)后，如实施例1、2和3进行筛选、表达和纯化。结果如图3所示，带有A79G/S106C/I108F/S111A的四位点突变ChPPK活力较带有A79G/S106C/I108F的三位点突变的ChPPK明显下降。为了检测S111位点其他有益突变叠加对A79G/S106C/I108F的三位点突变的pET28-ChPPK的影响，采用Quick-change的突变方法，以含有A79G/S106C/I108F的三位点突变的pET28-ChPPK质粒为模板，采用如下引物将111位点突变成谷氨酸。
将111位的丝氨酸突变为谷氨酸所用的引物：
正向引物：TTGAACTGGAACATGATTATCTGTGGC；
反向引物：TAATCATGTTCCAGTTCAATTTTGGACG。
产生带有A79G/S106C/I108F/S111K的四突变ChPPK较带有A79G/S106C/I108F/的三突变ChPPK活力明显下降。说明把79、106、108和111位所有有益突变叠加并不能产生最佳效果。
将带有A79G/S106C/I108F的三位点突变的pET-ChPPK进行进一步突变，引入L285P突变。采用Quick-change的突变方法，以含有A79G/S106C/I108F的三位点突变的pET28-ChPPK质粒为模板，采用下列引物进突变。将上述突变的质粒转化宿主菌BL21(DE3)后，如实施例1、2和3进行筛选、表达和纯化。结果图3所示，带有A79G/S106C/I108F/L285P的四位点联合突变体(ChPPK/A79G/S106C/I108F/L285P)具有最高的活性。
285位点突变
正向引物：ACCGTGAGCCCAGAACAGAAAGCGG；
反向引物：TTCTGTTCTGGGCTCACGGTCGGAAA。
实施例7：突变体ChPPK/A79G/S106C/I108F/L285P的特性分析
我们比较了温度、pH和底物浓度对野生型ChPPK和突变型ChPPK/A79G/S106C/I108F/L285P活性的影响。
1、温度
将实施例1和6方法构建的工程菌E.coli BL21(DE3)-ChPPK和E.coli BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P，采用实施例2方法制备纯酶。采用实施例3方法测酶活，测酶活的温度分别改为25℃、30℃、35℃、37℃、40℃、45℃、50℃、55℃或60℃。
结果见图4所示，虽然37℃是野生型ChPPK和突变型ChPPK/A79G/S106C/I108F/L285P的最佳反应温度，但突变型ChPPK/A79G/S106C/I108F/L285P在42-50℃温度下表现出显著更高的相对活性。例如，突变体ChPPK/A79G/S106C/I108F/L285P在45℃时保留了82％的活性，而野生型ChPPK在这种条件下失去了96％的活性。
2、pH
将实施例1和6方法构建的工程菌E.coli BL21(DE3)-ChPPK和E.coli BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P采用实施例2方法制备纯酶。采用实施例3方法测酶活，并将缓冲液pH分别改为5.0-6.0(50mM柠檬酸-柠檬酸钠缓冲液)，6.0-8.0(50mM磷酸钾缓冲液)，8.0-9.0(50mM硼砂-硼酸缓冲液)或9.0-10.0(50mM甘氨酸-NaOH缓冲液)。
结果见图5所示，野生型ChPPK和突变型ChPPK/A79G/S106C/I108F/L285P的相对活性在pH值为7.5时达到最大值。然而，突变体ChPPK/A79G/S106C/I108F/L285P在酸性条件下具有较高的相对活性。
3、AMP浓度
将实施例1和6方法构建的工程菌E.coli BL21(DE3)-ChPPK和E.coli BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P采用实施例2方法制备纯酶。采用实施例3方法测酶活，并将AMP浓度分别改为0.25mM、0.50mM、0.75mM、1.00mM、1.50mM、2.00mM、2.50mM、3.00mM、3.50mM、4.00mM、4.50mM或5.00mM。
结果见图6所示，野生型ChPPK和突变型ChPPK/A79G/S106C/I108F/L285P在AMP浓度为2.0-2.5mM时达到了最高的相对活性。
4、PPA浓度
将实施例1和6方法构建的工程菌E.coli BL21(DE3)-ChPPK和E.coli BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P采用实施例2方法制备纯酶。采用实施例3方法测酶活，并将PPA浓度分别改为0.32g/L、0.64g/L、0.96g/L、1.28g/L、1.60g/L、1.92g/L、2.24g/L、2.56g/L、2.88g/L或3.20g/L。
结果见图7所示，野生型ChPPK的最佳PPA浓度为1.6g/L，而突变型ChPPK/A79G/S106C/I108F/L285P在2.24g/L PPA存在下表现出最高的相对活性，表明突变型ChPPK/A79G/S106C/I108F/L285P具有更高的PPA耐受性。
实施例8：用纯酶测定野生型ChPPK和突变型A79G/S106C/I108F/L285P的动力学参数
将实施例1构建的野生型Ecoli.BL21(DE3)-ChPPK和实施例6方法构建的工程菌Ecoli.BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P，采用实施例2方法分别制备野生型ChPPK和突变型ChPPK/A79G/S106C/I108F/L285P的纯酶。采用pseudo-one-substrate动力学模型计算Km值与Kcat值。为了分别计算酶对双底物的动力学，采用实施例3方法测试酶活，但反应在固定PPA浓度(1.6g/L)的条件下调整AMP浓度(0、0.25、0.5、0.75、1.0、1.5、2.0、2.5、3.0、3.5、4.0、4.5、5.0mM)或在固定AMP浓度(5mM)的条件下调整PPA浓度(0.32、0.64、0.96、1.28、1.6、1.92、2.24、2.56、2.88、3.2g/L)条件下进行。
野生型ChPPK对AMP和PPA的Km值与突变型ChPPK/A79G/S106C/I108F/L285P的Km值相似(表3和表4)。突变型ChPPK/A79G/S106C/I108F/L285P的酶活性的提高可以解释为其对AMP和PPA的周转数(kcat值)的增加。突变型ChPPK/A79G/S106C/I108F/L285P对AMP和PPA的催化效率(kcat/Km)分别是野生型ChPPK的16倍和18倍。
表3、野生型ChPPK和突变型ChPPK/A79G/S106C/I108F/L285P对底物AMP的动力学参数
Figure PCTCN2022082772-appb-000010
表4、野生型ChPPK和突变型ChPPK/A79G/S106C/I108F/L285P对底物PPA的动力学参数
Figure PCTCN2022082772-appb-000011
实施例9：突变体ChPPK/A79G/S106C/I108F/L285P在NMN生物合成中的应用1、烟酰胺核糖激酶粗酶液
由于烟酰胺核糖激酶可以催化烟酰胺核糖(NR)生物合成烟酰胺单核苷酸(NMN)，因此以来自棕硬尾鸭(Oxyura jamaicensis)的烟酰胺核糖激酶(NRK，GenBank号：XP_035204248.1)氨基酸序列为模板，人工合成针对大肠杆菌密码子优化了的烟酰胺核糖激酶基因，核苷酸序列如SEQ ID NO:3所示，氨基酸序列如SEQ ID NO:4所示。
SEQ ID NO:3
Figure PCTCN2022082772-appb-000012
SEQ ID NO:4
Figure PCTCN2022082772-appb-000013
将针对大肠杆菌密码子优化了的重组基因NRK(SEQ ID NO:3所示)插入到pET-28a(+)的T7启动子下，得到表达质粒pET28-NRK。将该表达质粒转化至大肠杆菌Ecoli.BL21(DE3)中，涂布于含有50μg/mL卡那霉素抗性的LB平板，37℃下培养8-12h，挑取阳性克隆，即为野生型E.coli BL21(DE3)-NRK，用于表达重组NRK。与实施例2中相同条件制备粗酶液，其中的NRK的使用量以对应破碎前菌体量计。
2、生物合成NMN
反应1(25mM ATP)：在1mL磷酸钾缓冲液(50mM，pH 6.5)中加入终浓度25mM的ATP、终浓度100mM的NR、终浓度10mM氯化镁和8mg/mL的NRK粗酶液，在37℃反应6h，在0.5h、1.5h、3h和6h取样，采用实例3中HPLC方法检测(NMN保留时间为2.9分钟)。NMN浓度与峰面积标准曲线获得方法：以不同浓度的NMN(0.0625mM、0.125mM、0.25mM、0.5mM、1.0mM和2.0mM)进样得到的峰面积数据，得到NMN浓度与峰面积标准曲线，曲线方程为y＝(x+254.3)/4587(R 2＝0.995)，其中y为NMN的浓度(mM)，x为NMN的峰面积。反应1生成的NMN浓度变化如图8所示，6小时后NMN的产量为34mM。
反应2(100mM ATP)：将反应1中ATP浓度改为100mM，其他操作相同，结果见图8所示，当投入的ATP达到100mM时，6小时后NMN的产量提升到85mM。
反应3(25mM ATP+野生型ChPPK)：在反应1中加入4mg/mL的实施例2方法制备的野生型ChPPK粗酶液和4.8g/L的PPA，其他操作相同，NMN浓度变化如图8所示，当含有野生型ChPPK ATP再生系统被引入NMN合成，6小时后NMN的产量提升到55mM。
反应4(25mM ATP+突变体ChPPK/A79G/S106C/I108F/L285P)：在反应1中加入4mg/mL的实施例6方法制备的突变体ChPPK/A79G/S106C/I108F/L285P粗酶液和4.8g/L的PPA，其他操作相同，结果见图8所示，当含有突变体ChPPK/A79G/S106C/I108F/L285P的ATP再生系统被引入NMN合成，6小时后NMN的产量提升到86mM。说明导入突变体构成的ATP再生系统，可以节省75％的ATP投入量而不影响最终NMN的产量。
实施例10：突变体ChPPK/A79G/S106C/I108F/L285P在G6P生物合成中的应用1、己糖激酶粗酶液
由于己糖激酶可以催化葡萄糖生物合成葡萄糖6磷酸(G6P)，因此以来自酿酒酵母(Saccharomyces cerevisiae)的己糖激酶(HK，GenBank号：NP_013551.1)氨基酸序列为模板，人工合成针对大肠杆菌密码子优化了的己糖激酶基因，核苷酸序列如SEQ ID NO:5所示，氨基酸序列如SEQ ID NO:6所示。
SEQ ID NO:5
Figure PCTCN2022082772-appb-000014
Figure PCTCN2022082772-appb-000015
SEQ ID NO:6
Figure PCTCN2022082772-appb-000016
将针对大肠杆菌密码子优化了的重组基因HK(核苷酸序列如SEQ ID NO:5所示)插入到pET-28a(+)的T7启动子下，得到表达质粒pET28-HK。将该表达质粒转化至大肠杆菌Ecoli.BL21(DE3)中，涂布于含有50μg/mL卡那霉素抗性的LB平板，37℃下培养8-12h，挑取阳性克隆，即为野生型E.coli BL21(DE3)-HK，用于表达重组HK。与实施例2中相同条件制备粗酶液，其中HK粗酶液的使用量以对应破碎前菌体量计。
2、生物合成G6P
反应1(25mM ATP)：在5mL磷酸钾缓冲液(50mM，pH 7.5)中加入终浓度25mM ATP、终浓度100mM葡萄糖、终浓度10mM氯化镁和12mg/mL的HK粗酶液，在30℃反应8h。反应结束后取样1mL，70℃加热15分钟使酶失活。12,000×g离心10分钟取上清进行HPLC分析。
HPLC检测G6P的仪器为Agilent 1260 Infinity Ⅱ(安捷伦科技有限公司，美国)，配置Dionex ED40 detector检测器。Agilent 1525泵，Agilent 717进样器，色谱柱为Dionex IonPac AS11-HC，检测温度为30℃。流动相为氢氧化钠水溶液，速率为1mL/min。采用梯度洗脱的方法：0-10分钟内为25mM氢氧化钠水溶液，12-15分钟内氢氧化钠水溶液从25mM提升到100mM，最后17-21分钟换成25mM氢氧化钠水溶液。G6P浓度与峰面积标准曲线获得方法：以不同浓度的G6P水溶液(0.125mM、0.25mM、0.5mM、1.0mM、2.0mM和4.0mM)进样得到的峰面积数据，得到G6P浓度与峰面积标准曲线，曲线方程为y＝(x+544.7)/3856(R 2＝0.997)，其中y为G6P的浓度(mM)，x为G6P的峰面积。用该标准曲线计算HK粗酶液催化生成的G6P产量。如图9所示，8h后G6P的产量为18mM。
反应2(100mM ATP)：将反应1中ATP浓度改为100mM，其他操作相同，结果见图9所示，当投入的ATP达到100mM时，8小时后G6P的产量提升到78mM。
反应3(25mM ATP+野生型ChPPK)：在反应1中加入4mg/mL实施例2方法制备的野生型ChPPK粗酶液和4.8g/L的PPA，其他操作相同。如图9所示，当含有野生型ChPPK ATP再生系统被引入G6P合成，8小时后G6P的产量提升到38mM。
反应4(25mM ATP+突变体ChPPK/A79G/S106C/I108F/L285P)：在反应1中加入4mg/mL实施例6方法制备的突变体ChPPK/A79G/S106C/I108F/L285P粗酶液和4.8g/L的PPA，其他操作相同，结果见图9所示，当含有突变体ChPPK/A79G/S106C/I108F/L285P的ATP再生系统被引入G6P合成，8小时后G6P的产量提升到82mM。说明导入突变体构成的ATP再生系统，可以节省75％的ATP投入量而不影响最终G6P的产量。

Claims (10)

  1. 一种多聚磷酸激酶突变体，其特征在于，所述多聚磷酸激酶突变体是将SEQ ID NO：2所示氨基酸序列第79位、第106位、第108位、第111位或第285位进行单突变或多突变获得的。
  2. 如权利要求1所述的多聚磷酸激酶突变体，其特征在于，所述多聚磷酸激酶突变体是将SEQ ID NO：2所示氨基酸序列突变为下列之一：(1)第79位丙氨酸突变变为甘氨酸；(2)第106位丝氨酸突变为半胱氨酸；(3)第108位异亮氨酸突变为苯丙氨酸、天冬酰胺或酪氨酸；(4)第111位丝氨酸突变为谷氨酸或赖氨酸或丙氨酸；(5)第285位亮氨酸突变为脯氨酸；(6)第79位丙氨酸突变为甘氨酸、第108位异亮氨酸突变为苯丙氨酸；(7)第79位丙氨酸突变为甘氨酸、第106位丝氨酸突变为半胱氨酸、第108位异亮氨酸突变为苯丙氨酸；(8)第79位丙氨酸突变为甘氨酸、第106位丝氨酸突变为半胱氨酸、第108位异亮氨酸突变为苯丙氨酸、第111位丝氨酸突变为丙氨酸；(9)第79位丙氨酸突变为甘氨酸、第106位丝氨酸突变为半胱氨酸、第108位异亮氨酸突变为苯丙氨酸、第285位亮氨酸突变为脯氨酸。
  3. 一种权利要求1所述多聚磷酸激酶突变体的编码基因。
  4. 一种包含权利要求3所述编码基因的重组基因工程菌。
  5. 一种权利要求1所述多聚磷酸激酶突变体在构建ATP再生体系中的应用。
  6. 一种权利要求1所述多聚磷酸激酶突变体在合成烟酰胺单核苷酸中的应用，其特征在于，所述应用的方法为：将所述多聚磷酸激酶突变体基因工程菌和烟酰胺核糖激酶基因工程菌分别经诱导培养获得的湿菌体用缓冲液重悬并采用超声破碎，以破碎后的上清液为催化剂，以腺嘌呤核苷三磷酸和烟酰胺核糖为底物，加入氯化镁和聚磷酸，以pH6.5的缓冲液为反应介质构成反应体系，在37℃进行反应，反应液分离纯化，获得烟酰胺单核苷酸。
  7. 如权利要求6所述的应用，其特征在于，所述腺嘌呤核苷三磷酸加入量以缓冲液体积计为10-100mM；所述烟酰胺核糖加入量以缓冲液体积计为50-200mM；所述氯化镁加入量以缓冲液体积计为5-20mM；所述聚磷酸加入量以缓冲液体积计为1-10g/L；所述多聚磷酸激酶突变体加入量以破碎前湿菌体量计为2-30mg/mL缓冲液；所述烟酰胺核糖激酶加入量以破碎前湿菌体量计为5-30mg/mL缓冲液。
  8. 如权利要求6所述的应用，其特征在于，所述催化剂按如下方法制备：将多聚磷酸激酶突变体基因工程菌接种至含有50μg/mL卡那霉素抗性的LB液体培养基，37℃，200rpm下培养12h，再以体积浓度1％接种量接种至新鲜的含有50μg/mL卡那霉素抗性 的LB液体培养基中，于37℃，150rpm下培养至菌体OD600达0.6，加入终浓度为0.1mM的IPTG，28℃下诱导培养12h后，4℃、8000rpm离心10min，弃去上清液，收集湿菌体沉淀；收集的湿菌体用pH为7.5的50mM磷酸钾缓冲液重悬，并使用超声波细胞粉碎机破碎，破碎功率为50W，每工作1s，间歇2s，总共破碎20min；收集细胞裂解液，在12000g下离心1min，取上清液，即为粗酶液；所述烟酰胺核糖激酶基因工程菌的上清液制备方法同多聚磷酸激酶突变体基因工程菌。
  9. 一种权利要求1所述多聚磷酸激酶突变体在合成葡萄糖6磷酸中的应用，其特征在于所述应用的方法为：将所述多聚磷酸激酶突变体基因工程菌和己糖激酶基因工程菌分别经诱导培养获得的湿菌体用缓冲液重悬并采用超声破碎，取破碎后的上清液为催化剂，以腺嘌呤核苷三磷酸和葡萄糖为底物，加入氯化镁和聚磷酸，以pH 7.2的缓冲液为反应介质构成反应体系，在37℃进行反应，反应液分离纯化，获得葡萄糖6磷酸。
  10. 如权利要求9所述的应用，其特征在于，所述腺嘌呤核苷三磷酸加入量以缓冲液体积计为10-100mM；所述葡萄糖加入量以缓冲液体积计为20-150mM；所述氯化镁加入量以缓冲液体积计为5-20mM；所述聚磷酸加入量以缓冲液体积计为1-10g/L；所述多聚磷酸激酶突变体入量以破碎前湿菌体量计为2-30mg/mL缓冲液；所述己糖激酶加入量以破碎前湿菌体量计为5-30mg/mL缓冲液。
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Publication number Priority date Publication date Assignee Title
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20050111454A (ko) * 2004-05-21 2005-11-25 (주) 헬릭스 팜스 마이코박테리움 투버쿨로시스 유래의 폴리포스페이트키나아제 단백질과 이의 변이 단백질 및 이를 코딩하는유전자
CN105861598A (zh) * 2016-04-27 2016-08-17 深圳市古特新生生物科技有限公司 一种酶法再生atp的方法及其应用
CN111254129A (zh) * 2020-03-24 2020-06-09 浙江华睿生物技术有限公司 一种多聚磷酸激酶突变体及其应用
US20210024912A1 (en) * 2016-12-30 2021-01-28 Ntxbio, Llc Cell-Free Expression System Having Novel Inorganic Polyphosphate-Based Energy Regeneration
CN112795606A (zh) * 2021-04-14 2021-05-14 深圳瑞德林生物技术有限公司 一种β-烟酰胺单核苷酸的酶催化合成方法
CN113025592A (zh) * 2021-04-28 2021-06-25 上海邦林生物科技有限公司 一种高性能多聚磷酸激酶突变体及其应用
CN113265382A (zh) * 2021-06-24 2021-08-17 洛阳华荣生物技术有限公司 多聚磷酸激酶突变体

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112280762B (zh) * 2020-11-13 2022-11-01 中山俊凯生物技术开发有限公司 一种烟酰胺核糖激酶突变体及其编码基因和应用
CN112553178B (zh) * 2020-12-25 2023-03-10 中山俊凯生物技术开发有限公司 热稳定性和活性增强的烟酰胺核糖激酶突变体及其编码基因和应用
CN113604454B (zh) * 2021-07-27 2023-10-20 浙江工业大学 一种磷酸酶突变体及在催化麦芽糊精制备果糖中的应用
CN113583997B (zh) * 2021-07-27 2023-10-20 浙江工业大学 一种磷酸酶突变体及在催化麦芽糊精制备甘露糖中的应用
CN113832125B (zh) * 2021-10-19 2023-09-26 中山百灵生物技术股份有限公司 一种烟酰胺核糖激酶突变体及其编码基因和应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20050111454A (ko) * 2004-05-21 2005-11-25 (주) 헬릭스 팜스 마이코박테리움 투버쿨로시스 유래의 폴리포스페이트키나아제 단백질과 이의 변이 단백질 및 이를 코딩하는유전자
CN105861598A (zh) * 2016-04-27 2016-08-17 深圳市古特新生生物科技有限公司 一种酶法再生atp的方法及其应用
US20210024912A1 (en) * 2016-12-30 2021-01-28 Ntxbio, Llc Cell-Free Expression System Having Novel Inorganic Polyphosphate-Based Energy Regeneration
CN111254129A (zh) * 2020-03-24 2020-06-09 浙江华睿生物技术有限公司 一种多聚磷酸激酶突变体及其应用
CN112795606A (zh) * 2021-04-14 2021-05-14 深圳瑞德林生物技术有限公司 一种β-烟酰胺单核苷酸的酶催化合成方法
CN113025592A (zh) * 2021-04-28 2021-06-25 上海邦林生物科技有限公司 一种高性能多聚磷酸激酶突变体及其应用
CN113265382A (zh) * 2021-06-24 2021-08-17 洛阳华荣生物技术有限公司 多聚磷酸激酶突变体

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 28 January 2014 (2014-01-28), ANONYMOUS : "Polyphosphate:AMP phosphotransferase [Cytophaga hutchinsonii ATCC 3340 ", XP093082372, retrieved from NCBI Database accession no. ABG57400.1 *
NOCEK BOGUSLAW P., KHUSNUTDINOVA ANNA N., RUSZKOWSKI MILOSZ, FLICK ROBERT, BURDA MALGORZATA, BATYROVA KHORCHESKA, BROWN GREG, MUCH: "Structural Insights into Substrate Selectivity and Activity of Bacterial Polyphosphate Kinases", ACS CATALYSIS, AMERICAN CHEMICAL SOCIETY, US, vol. 8, no. 11, 2 November 2018 (2018-11-02), US , pages 10746 - 10760, XP093082371, ISSN: 2155-5435, DOI: 10.1021/acscatal.8b03151 *

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