A polyphosphate kinase mutant and its application in the synthesis of β-nicotinamide mononucleotide

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a polyphosphate kinase mutant and application thereof in synthesis of beta-nicotinamide mononucleotide, wherein the polyphosphate kinase mutant is obtained by single-point or multi-point mutation of amino acids at positions 99, 186, 190 and 204 of a polyphosphate kinase with an amino acid sequence shown in SEQ ID NO. 1. The polyphosphate kinase derived from Lampropedia Hyalina DSM 16112 is subjected to molecular modification, and a plurality of mutants are obtained. The enzyme activity of the mutants is 1.5-14 times higher than that of the wild-type polyphosphate kinase, and the preference for AMP is 1.08-4.88 times higher than that of the wild-type polyphosphate kinase. The ATP regeneration system constructed by using the mutants can significantly reduce the use amount of the enzyme, reduce the fermentation capacity and cost, greatly shorten the reaction time, reduce the influence of AMP on the enzyme activity, and meet the demand of large-scale industrial production of NMN prepared by using the biological enzyme method, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a polyphosphate kinase mutant and its application in the synthesis of β-nicotinamide mononucleotide. Background Technology

[0002] In the production of the functional product NMN (β-nicotinamide mononucleotide), ATP is a crucial cofactor. NMN is a precursor molecule of nicotinamide adenine dinucleotide (NAD+) and possesses important physiological functions such as anti-aging and metabolic regulation, leading to rapidly growing market demand. The biocatalytic synthesis of NMN is highly favored due to its environmental friendliness and high efficiency. A core step involves the catalytic reaction of nicotinamide phosphoribosyltransferase (NAMPT), a process requiring substantial ATP support. In this pathway, ATP consumption typically generates large amounts of AMP and small amounts of ADP. This accumulation not only reduces overall reaction efficiency but also increases subsequent processing costs. Therefore, developing an ATP regeneration system capable of efficiently utilizing AMP is key to solving these problems.

[0003] Polyphosphoryl kinases (PPKs) utilize polyphosphate (PolyP) to generate high-energy phosphorylated compounds, and are divided into two main categories: PPK1 and PPK2. PPK1 possesses bidirectional catalytic function, interconverting polyphosphate with triphosphate nucleotides, and is commonly used in classic ATP regeneration systems. PPK2 has more diverse functions, and is classified into three subtypes based on substrate preference: PPK2-I prefers AMP and generates ATP; PPK2-II prefers ADP and generates ATP; and PPK2-III has a broad substrate range, catalyzing the generation of ATP from AMP, ADP, and other substrates. This provides an important technological foundation for developing ATP regeneration systems that meet diverse biocatalytic needs.

[0004] The optimized PPK mutant was applied to an ATP regeneration system, working synergistically with NMN biosynthetic enzymes. The resulting engineered bacteria efficiently regenerated ATP and achieved AMP recycling. This system demonstrates significant advantages in both economy and efficiency, not only increasing NMN yield and purity but also significantly reducing the amount of exogenous ATP required, thus lowering overall production costs. In conclusion, developing highly efficient polyphosphate kinase mutants and using them to construct ATP regeneration systems can effectively address the problem of high cofactor costs in industrial production. Particularly in NMN biosynthesis, this system exhibits significant advantages, improving economic efficiency and promoting advancements in green and sustainable production technologies. This research direction provides crucial support for the large-scale production and application of functional molecules and has broad application prospects. Summary of the Invention

[0005] To address the need in existing technologies for developing highly efficient polyphosphate kinase mutants, this invention provides a polyphosphate kinase mutant and its application in the synthesis of β-nicotinamide mononucleotide. The specific technical solution is as follows:

[0006] In a first aspect, the present invention provides a polyphosphoric acid kinase mutant obtained by single-point or multi-point mutation of amino acids at positions 99, 186, 190, and 204 of the polyphosphoric acid enzyme as shown in SEQ ID NO.1.

[0007] Furthermore, the mutated form of the polyphosphatase mutant is one of the following:

[0008] (1) The alanine at position 99 is mutated to aspartic acid, glutamine, or serine;

[0009] (2) The glutamic acid at position 186 is mutated to alanine or glutamine;

[0010] (3) The 190th aspartic acid is mutated to alanine, valine, arginine or threonine;

[0011] (4) The aspartic acid at position 204 is mutated to alanine, cysteine, histidine or lysine.

[0012] (5) Glutamic acid at position 186 was mutated to alanine, and aspartic acid at position 204 was mutated to alanine;

[0013] (6) The alanine at position 99 was mutated to aspartic acid, the glutamic acid at position 186 was mutated to alanine, and the aspartic acid at position 204 was mutated to alanine.

[0014] (7) The 99th position of alanine is mutated to aspartic acid, the 186th position of glutamic acid is mutated to alanine, the 190th position of aspartic acid is mutated to arginine, and the 204th position of aspartic acid is mutated to alanine.

[0015] This invention targets the polyphosphate kinase (LhPPK) derived from Lampropedia Hyalina DSM 16112. It simulates the three-dimensional structure of LhPPK using macromolecular modeling technology and predicts one or more potential catalytically related sites using molecular docking technology. After selecting the site for site-directed mutation and the type of mutated amino acid, corresponding primers were synthesized by Hangzhou Qingke Biotechnology Co., Ltd. Using the pET 28a(+) vector plasmid as a template, the mutant DNA fragment was amplified by PCR (Polymerase Chain Reaction), isolated and purified, and then amplified into a full-length mutant gene by PCR. This full-length mutant gene was cloned into the pET-28a(+) vector and transformed into DH5α host bacteria. Positive clones with the mutant gene vector were selected through culture and screening. Finally, plasmid DNA was extracted from the positive clones and subjected to DNA sequencing analysis to determine the introduced mutation. The selected positive plasmids were transformed into BL21(DE3) host bacteria to induce expression, and mutants with significantly enhanced activity were screened. Using pET-28a(+)-LhPPK as a template, site-directed saturation mutagenesis was employed to modify it, and the selected beneficial mutations were combined together. Through enzyme activity assay, an LhPPK mutant with strong AMP preference was finally obtained, and the catalytic efficiency of this mutant for AMP was also improved.

[0016] Secondly, the present invention provides a gene encoding the above-mentioned polyphosphate kinase mutant.

[0017] Thirdly, the present invention provides a recombinant vector containing the aforementioned genes.

[0018] Fourthly, the present invention provides a genetically engineered bacterium containing the aforementioned genes.

[0019] Fifthly, the present invention provides an ATP regeneration system comprising an enzyme and a substrate;

[0020] The enzyme includes the above-mentioned polyphosphate kinase mutant;

[0021] The substrates include polyphosphates and adenosine monophosphate substrates.

[0022] Furthermore, the reaction temperature of the ATP regeneration system is 35–45°C.

[0023] Furthermore, the ATP regeneration system also includes a buffer solution.

[0024] Furthermore, the buffer solution is one of phosphate buffer, potassium phosphate buffer, citrate-sodium citrate buffer, and borax-boric acid buffer.

[0025] Furthermore, the pH of the buffer solution is 6.0 to 8.5.

[0026] In a sixth aspect, the present invention provides the application of the above-mentioned polyphosphate kinase mutant or the above-mentioned ATP regeneration system in the production of β-nicotinamide mononucleotide.

[0027] In a seventh aspect, the present invention provides an enzymatic method for preparing β-nicotinamide mononucleotide, using ribose, adenosine triphosphate, and nicotinamide as substrates, and ribokinase, phosphoribosyl pyrophosphate synthase, nicotinamide ribosyltransferase, and the above-mentioned polyphosphate kinase mutant as catalysts to form a reaction system for catalytic preparation of β-nicotinamide mononucleotide.

[0028] Furthermore, in the reaction system, the pH is 6.5–8.5 and the temperature is 35–45°C.

[0029] Furthermore, the reaction system has a pH of 7.5 and a temperature of 37°C.

[0030] Furthermore, the reaction system also includes a buffer solution.

[0031] Furthermore, the buffer solution is one of phosphate buffer, potassium phosphate buffer, citrate-sodium citrate buffer, and borax-boric acid buffer.

[0032] Furthermore, the ribokinase (RK) is derived from *Escherichia coli*, with GenBank accession number EFH2707387.1; the phosphoribosyl pyrophosphate synthase (PRS) is derived from *Bacillus amyloliquefaciens*, with GenBank accession number ADU02858.1; and the nicotinamide ribosyltransferase (Nampt) is derived from *Pinus pineensis*, with GenBank accession number WP_012788281.1.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This invention involves molecular modification of polyphosphokinase derived from Lampropedia Hyalina DSM 16112, resulting in various mutants. These mutants exhibit 1.5–14 times higher enzyme activity and 1.08–4.88 times greater preference for AMP compared to wild-type polyphosphokinase. The ATP regeneration system constructed using these mutants can significantly reduce enzyme usage, fermentation capacity, and costs. Furthermore, it can greatly shorten reaction time and reduce the impact of AMP on enzyme activity, meeting the needs of large-scale industrial production of NMN using enzymatic methods and possessing broad application prospects. Attached Figure Description

[0035] Figure 1A statistical graph showing the relative enzyme activity of polyphosphate kinase mutants A99S / E186A / D190R / D204A in catalyzing AMP to ATP at different temperatures.

[0036] Figure 2 A statistical graph showing the relative enzyme activity of polyphosphate kinase mutants A99S / E186A / D190R / D204A in catalyzing AMP to ATP production at different pH levels.

[0037] Figure 3 A schematic diagram of the reaction process for producing NMN using a reaction system containing polyphosphate kinase mutants A99S / E186A / D190R / D204A. Detailed Implementation

[0038] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0039] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0040] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0041] LB plate composition: 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract, 15 g / L agar, water as solvent, natural pH.

[0042] The LB liquid medium consists of 10 g / L tryptone, 10 g / L sodium chloride, and 5 g / L yeast extract, with water as the solvent and natural pH.

[0043] In the following examples, the types of enzymes used in the NMN production process are as follows:

[0044] PPK: Derived from Lampropedia Hyalina DSM 16112, GenBank No.: SHF67157.1, nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2;

[0045] RK: Derived from Escherichia coli, GenBank number: EFH2707387.1;

[0046] PRS: derived from Bacillus amyloliquefaciens, GenBank No.: ADU02858.1;

[0047] Nampt: Derived from *Chitino phagapinensis*, GenBank ID: WP_012788281.1.

[0048] SEQ ID NO.1:

[0049] ATGACTCGTAAAGAAAGTCCGCCGATGGAAGCATTCAAGAAATACCGCATCAGTCCGAAGAACACTCTGAAAGACATCGATCCAAGCGACAAACCGTTCAGCTTCGGCACCAAGAAAGACGAACTGCAACGTCTGGATGAACTGGCTGTTGAACTGGACGATCTGCAAAACACTCTGTACGCGGGTAAACGTCGTAAAGTTCTGCTGATTCTGCAAGGTCTGGACACCTCTGGTAAAGACGGTACTATCCGTTGGGTGTTCAGCCGTACCTCTCCGCTGGGTGTTCACGTTGCTGCATTCAAAGCACCAAGCGAACGTGAACGTGCGCACGACTTCTTGTGGCGTTGCCACGCGGTTGTACCGGCAAACGGTGAACTGACCGTTTGGAACCGTTCTCACTACGAAGATGTTCTGGTTCCGCCAGTGGAAGGTTGGATCGATAAAGCGGAAACTCAGCGTCGTTACGGTCACATCAACGACTTCGAACGTCTGCTGTCCGAAACCGGCACTACCATCGTTAAATGCATGCTGCACATCTCCAACGAAGAACAGCGTGAACGTCTGCAAGACCGTCTGAAAGATCCGGGCAAAAACTGGAAATTCGCAGCAGATGACCTGTCTACTCGTGCGAAATGGAACGACTACCAGCGTGCATACGAACAGGCACTGCAAGCAACTTCTACTCCGCACGCTCCGTGGTACGTTATTCCGGCGAACAGCAAACGTCACCGTAACCTGATGGTTGCTCAGCTGCTGGTTCAGACTCTGCGTGATATGAAACTGCGTCTGCCACCGCCAAACCTGGCATTCAAAGACCTGGTTGTTAAC

[0050] SEQ ID NO.2:

[0051] MTRKESPPMEAFKKYRISPKNTLKDIDPSDKPFSFGTKKDELQRLDELAVELDDLQNTLYAGKRRKVLLILQGLDTSGKDGTIRWVFSRTSPLGVHVAAFKAPSERERAHDFLWRCHAVVPANGELTVWNRSHYEDVL VPPVEGWIDKAETQRRYGHINDFERLLSETGTTIVKCMLHISNEEQRERLQDRLKDPGKNWKFAADDLSTRAKWNDYQRAYEQALQATSTPHAPWYVIPANSKRHRNLMVAQLLVQTLRDMKLRLPPPNLAFKDLVVN.

[0052] Example 1: Construction of wild-type E. coli BL21(DE3)-LhPPK

[0053] Based on the PPK protein sequence (LhPPK, GenBank ID: SHF67157.1) from *Lampropedia Hyalina* DSM 16112 in GenBank, a recombinant gene LhPPK sequence of 828 bp was synthesized by Hangzhou Qingke Biotechnology Co., Ltd., optimized for codon bias in *Escherichia coli*, and fused with a 6His tag at the C-terminus. Its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence encoding the protein is shown in SEQ ID NO.2.

[0054] The recombinant gene LhPPK was inserted between the NcoI and XhoI restriction sites of pET-28a(+) to obtain the expression plasmid pET28-LhPPK. This expression plasmid was transformed into E. coli BL21(DE3), plated on LB agar plates containing 50 μg / mL kanamycin, and incubated at 37°C for 8–12 h. Positive clones were selected, which were wild-type E. coli BL21(DE3)-LhPPK, for expression of recombinant LhPPK.

[0055] Example 2: Induction of wild-type E. coli BL21(DE3)-LhPPK expression and extraction of wild-type polyphosphate kinase

[0056] (1) Preparation of wet cells: Wild-type E. coli BL21(DE3)-LhPPK obtained in Example 1 was inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37°C and 180 rpm for 12 h. Then, 2% (v / v) inoculated into fresh LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37°C and 180 rpm until the cell OD600 = 0.6. IPTG was added to a final concentration of 0.1 mM and induced at 28°C for 12 h. After centrifugation at 4°C and 8000 rpm for 10 min, the supernatant was discarded and the precipitate was collected to obtain wet cells containing recombinant LhPPK expression.

[0057] (2) Preparation of crude enzyme solution: The wet bacterial cells collected in step (1) were resuspended in 50mM potassium phosphate buffer (PBS) at pH 7.5 at a concentration of 50g / L. The cells were then disrupted using an ultrasonic cell disruptor at a power of 150W, with a 1s operation followed by a 2s interval, for a total disruption time of 5min. The cell disruption fluid was collected and centrifuged at 8000rpm for 10min. The supernatant was collected as the crude enzyme solution. The amount of crude enzyme solution used subsequently was based on the amount of bacterial cells in the bacterial suspension before disruption.

[0058] (3) Preparation of pure enzyme solution: Rinse the nickel column (Ni-NTA) with distilled water, load the crude enzyme solution collected in step (2) with a loading volume of 5 column volumes, incubate with Ni affinity chromatography resin, and then rinse with washing buffer (50mM, pH 8.0 PBS buffer, containing 300mM NaCl and 50mM imidazole) until there are basically no impurities. Then elute with elution buffer (50mM, pH 8.0 PBS buffer, containing 300mM NaCl and 500mM imidazole) and collect the target protein. Then pass the protein through a desalting column (Sephadex G-25) and elute with buffer (pH 8.0, PBS buffer). The collected protein is the pure enzyme solution. The concentration of the purified enzyme is determined using the Beyotime BCA protein concentration kit. The purified enzyme used in subsequent examples is prepared according to the method of this example, and the amount of pure enzyme used is calculated according to the protein content.

[0059] Example 3: Determination of enzyme activity

[0060] Enzyme activity assay conditions: The total reaction system was 1 mL: final concentration of 10 mM AMP / ADP, 20 mM MgCl2, 20 mM PloyP, 50 μL of pure enzyme solution (protein concentration 0.02 mg / mL), and pH 7.5 Tris-HCl buffer. The reaction was carried out at 37℃ and 800 rpm for 5 min. 100 μL of the supernatant was taken and 10 μL of 0.2 M phosphate aqueous solution was added to terminate the reaction. The mixture was centrifuged at 12000 rpm for 5 min. The product concentration was determined by high-performance liquid chromatography (HPLC) of the supernatant. The ATP content in the final solution was determined by HPLC.

[0061] The concentration of ATP was quantitatively determined using a Thermo U3000 high-performance liquid chromatograph with a C18 column (5 μm, 4.6 mm × 250 mm). The mobile phase was 20 mM potassium phosphate buffer: 3.48 g of anhydrous K₂HPO₄ was dissolved in 900 mL of ultrapure water, the pH was adjusted to 7.0 with KH₂PO₄, and the volume was brought to 1 L. The solution was filtered through a hydrophilic microporous membrane and air bubbles were removed using an ultrasonic cleaner. The UV detection wavelength was 254 nm, the injection volume was 10 μL, the flow rate was 0.8 mL / min, and the column temperature was set to 40 °C. Peak area data from injections of different concentrations of ATP (0.05 mM, 0.1 mM, 0.5 mM, 1.0 mM, 2.0 mM, 5.0 mM, and 10.0 mM) were used to obtain a standard curve of ATP concentration versus peak area, with the equation y = y = 118.55x + 12.596 (R²). 2 =0.9993), where y is the ATP concentration (mM) and x is the ATP peak area obtained in the liquid phase.

[0062] Enzyme activity definition: An enzyme activity is defined as the amount of enzyme required to produce 1 μmol of ATP per minute (in the first 5 minutes) under the above conditions.

[0063] Example 4: Construction, screening, and enzyme activity detection of mutants

[0064] Macromolecular modeling techniques were used to predict potentially beneficial mutation sites. Primer designs are shown in Table 1. Using the vector pET28a(+)-E.coli-LhPPK from E.coli BL21(DE3) / pET28a(+)-E.coli-LhPPK as a template, the Quick-change mutagenesis method was employed, and amino acids at each site were mutated using the primers listed in Table 1. Recombinant plasmids pET28a-A99Q, pET28a-E186A, pET28a-D190A, and pET28a-D204H, containing single mutants A99Q, E186A, D190A, and D204H, respectively, were obtained. After completion, the bands were compared by gel electrophoresis. For the correctly identified PCR products, 1.5 μL of Dpn1 was added and digested in a shaker at 37℃ for at least 2 hours.

[0065] Table 1. Mutation sites and primers

[0066]

[0067]

[0068] PCR reaction system (total reaction volume 50 μL): 1×Phanta max Buffer 25 μL, 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP and dTTP) 1 μL, Phanta Max Super-FIDelity DNA Polymerase 0.5 μL, upstream and downstream primers (50 μM each) 2 μL each, recombinant vector pET28a(+)-E.coli-LhPPK 1 μL, ddH2O 18.5 μL.

[0069] The PCR reaction conditions were: 95℃ pre-denaturation for 3 min; followed by 98℃ denaturation for 10 s, 58℃ annealing for 5 s, and 72℃ extension for 3 min 30 s as one cycle, for a total of 32 cycles.

[0070] 2. Mutant engineered bacteria and crude enzyme solution

[0071] The plasmid mutated in step 1 was transformed into the host bacterium *E. coli* BL21(DE3), and crude enzyme solution was prepared using the method in Example 2. Enzyme activity was measured according to the method in Example 3. The results are shown in Table 2. When AMP was used as a substrate, the enzyme activities of the D190A and A99Q mutants were approximately 1-fold and 1.5-fold higher than those of the wild type, respectively. The enzyme activity of the E186A mutant was significantly increased, about 3-fold higher than that of the wild type. Furthermore, the E186A mutant significantly altered the substrate preference for AMP, with the relative enzyme activity of AMP / ADP increasing from 0.87 in the wild type to 1.5. This demonstrates that these sites have a significant impact on LhPPK activity and substrate preference. Therefore, these sites were studied for saturation mutation in Example 5.

[0072] Table 2. Enzyme activity of LhPPK and its mutants

[0073]

[0074]

[0075] Example 5: Saturation Mutation Enhances LhPPK Activity

[0076] 1. Site-directed saturation mutation

[0077] The Quick-change mutation method was used, with the pET28-LhPPK plasmid constructed by the method in Example 1 as a template, and primers in Table 3 were used to select sites A99, E186, D190, and D204 for saturation mutation.

[0078] Table 3. Mutation sites and primers

[0079] Primer Primer sequence (5'-3') 99-F TCACGTTGCTNNKTTCAAAGCACCA 99-R TTGAAMNNAGCAACGTGAACACC 186-F GAACAGCGTNNKCGTCTGCAAGAC 186-R AGACGMNNACGCTGTTCTTCGTTG 190-F ACGTCTGCAANNKCGTCTGAAAGAT 190-R AGACGNNKTTGCAGACGTTCACG 204-F CGCAGCAGNNKACCTGTCTACTCGTG 204-R AGGTCMNNTGCTGCGAATTTCCAGTTT

[0080] In the table above, N = A, T, G, C; K = G, T; M = A, C.

[0081] 2. Mutant engineered bacteria and crude enzyme solution

[0082] The mutated plasmid from step 1 was transformed into the host bacterium E. coli BL21(DE3), and the pure enzyme was prepared using the method of Example 2. The enzyme activity was then determined according to the method of Example 3.

[0083] The results are shown in Table 4. The enzyme activity of single-residue mutants containing A99D, A99S, D190A, D190T, D204A, D204C, and E186Q was significantly increased, which was 1.5 to 3.6 times that of wild-type pure enzymes. Furthermore, the D190R mutant and the D190T mutant significantly altered the preference for the substrate AMP.

[0084] Table 4. Enzyme activity of LhPPK and its mutants

[0085]

[0086]

[0087] Example 6: LhPPK-based combinatorial mutation

[0088] Based on the mutations in Example 5, some beneficial mutation sites were combined, and specific primers for each site were designed and used (primer sequences are shown in Tables 1 and 5). Stepwise overlap PCR was used to introduce the mutation sites stepwise. First, two-site mutant strains E186A / D190V (using E186A-F / R and D190A-F / R) and A99G / D190V (using A99Q-F / R and D190A-F / R) were constructed. Subsequently, by further introducing the D204A site, two-site mutant strains E186A / D204A (using E186A-F / R and D204A-F / R) and three-site mutant strains E186A / D190V / D204A (using E186A-F / R, D190A-F / R, and D204A-F / R) were constructed. Based on this, and in conjunction with A99S-F / R in Table 5, the A99 site was introduced, successfully constructing four multi-site mutant strains: A99D / E186A / D204A, A99S / E186A / D204A, A99S / E186A / D190A / D204A, and A99S / E186A / D190R / D204A. Mutants exhibiting significantly increased AMP or ADP activity were obtained. The mutation sites of these mutants and their effects on AMP and ADP enzymatic activities are shown in Table 6.

[0089] The mutant A99S / E186A / D190R / D204A significantly improved enzyme activity and substrate preference for the substrate AMP. Its nucleotide sequence is shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.4.

[0090] Table 5. Mutation sites and primers

[0091] Primer Primer sequence (5'-3') D204A-F CGCAGCAGCAGACCTGTCTACTCGTG D204A-R AGGTCTGCTGCTGCGAATTTCCAGTTT A99S-F GGTGTTCACGTTGCTAGCTTCAAAGCA A99S-R AGCAACGTGAACACCCAGCGGAGA D190R-F TTGCAGACGTGCACGCTGTTCTTCGTT D190R-R [[ID=2,6]]CGTCTGCAACGTCGTCTGAAAGATC

[0092] Table 6. Enzyme activity of LhPPK and its combined mutants.

[0093]

[0094]

[0095] Example 7 Characteristic Analysis of the Mutant LhPPK / A99S / E186A / D190R / D204A

[0096] 1. Temperature

[0097] The engineered bacteria E. coli BL21(DE3)-LhPPK and E. coli BL21(DE3)-LhPPK / A99S / E186A / D190R / D204A constructed in Examples 1 and 6 were used to prepare pure enzymes using the method in Example 2. The enzyme activity was measured using the method in Example 3, with the enzyme activity measurement temperatures changed to 25℃, 30℃, 35℃, 37℃, 40℃, 45℃, 50℃, 55℃, and 60℃, respectively.

[0098] The results are as follows Figure 1 As shown, although 37℃ is the optimal reaction temperature for both wild-type LhPPK and the mutant LhPPK / A99S / E186A / D190R / D204, the mutant LhPPK / A99S / E186A / D190R / D204A exhibits significantly higher relative activity for the substrate AMP at temperatures between 40 and 60℃. For example, the mutant LhPPK / A99S / E186A / D190R / D204A retained over 85% of its activity when catalyzing the substrate AMP at 45℃, while wild-type LhPPK lost 35% of its activity under these conditions.

[0099] 2. pH

[0100] The engineered strains E. coli BL21(DE3)-LhPPK and E. coli BL21(DE3)-LhPPK / A99S / E186A / D190R / D204A constructed using the methods in Examples 1 and 6 were purified into enzymes using the method in Example 2. Enzyme activity was measured using the method in Example 3, with the buffer pH adjusted to 5.0-6.0 (50mM citrate-sodium citrate buffer), 6.0-8.0 (50mM potassium phosphate buffer), 8.0-9.0 (50mM borax-boric acid buffer), or 9.0-10.0 (50mM glycine-NaOH buffer), respectively.

[0101] The results are as follows Figure 2 As shown, the relative activities of wild-type LhPPK and mutant LhPPK / A99S / E186A / D190R / D204A for AMP reached their maximum at pH 7.5. However, the mutant LhPPK / A99S / E186A / D190R / D204A exhibited higher relative activity for the substrate AMP under alkaline conditions.

[0102] 3. Half-life

[0103] Half-life (t) 1 / 2The time required for enzyme activity to decrease by 50% at a specific temperature is an important parameter characterizing enzyme thermostability. The purified enzymes of polyphosphoric acid kinase LhPPK and its mutants prepared by the method in Example 6 were diluted to a protein concentration of 1.0 mg / mL with pH 7.5 and 50 mM sodium phosphate buffer, and incubated at 30°C, 37°C, and 45°C, respectively. At regular intervals, 50 μL of the incubated enzyme solution was taken out, and its residual enzyme activity against the substrate AMP was determined according to the enzyme activity detection standard conditions in Example 7. The half-life was calculated based on the thermal inactivation equation (Et = E0e-K). d t) Calculate, in the thermal deactivation equation, K d For the non-dynamic constants, E0 and E t These represent the initial enzyme activity and the enzyme activity at time t, respectively. Let ln(E) represent the enzyme activity at time t. t Plot a graph with / E0) on the ordinate and incubation time t on the x-axis. Fit the experimental data to obtain kd, and use the formula ln2 / K. d The half-life t is calculated. 1 / 2 The results are shown in Table 7. Compared with the wild type, the half-life of the mutant LhPPK / A99S / E186A / D190R / D204A was significantly increased.

[0104] Table 7 Half-life of polyphosphoric acid kinase LhPPK and its mutants at 30℃, 37℃ and 45℃

[0105]

[0106] 4. Tm

[0107] Tm represents the melting temperature of a protein, referring to the temperature at which a protein unfolds to 50%. The thermal denaturation process of a protein is closely related to changes in its spatial conformation. The Tm value reflects the trend of conformational changes during temperature variations and is an important indicator of protein thermal stability. The melting temperatures of polyphosphokinase (LPK) and its mutants were analyzed using a Chirascan circular dichroism (CD) spectrometer. First, the purified enzymes of LhPPK and its mutants prepared according to the method in Example 4 were diluted to a protein concentration of 0.1 mg / mL. Then, 200 μL of the sample was loaded into a 10 mm quartz cuvette, and the Tm of LhPPK and its mutants was determined using a CD circular dichroism spectroscopy system. Melting curves of polyphosphate kinase LhPPK and its mutants were continuously collected at wavelengths of 180-260 nm and temperatures of 20-80 °C. The Tm values ​​of the mutants A99S / E186A / D190R / D204A were significantly increased, with an increase of 11.6 °C compared to the wild type.

[0108] Table 8 Melting temperatures of LhPPK polyphosphokinase and its mutants

[0109]

[0110] Example 8: NMN generated from ribose using a PPK-based ATP regeneration system.

[0111] (1) Preparation of expression plasmids

[0112] The engineered bacteria of the ribokinase EcRK gene underwent codon optimization using Escherichia coli as the host (the optimized sequence is shown in SEQ ID NO.3) and were synthesized by Qingke Biotechnology Co., Ltd. Then, the EcRK cDNA fragment was ligated to the TATACCAT site (after the NcoI restriction site) and CTCGAG site (before the XhoI restriction site) in pET-28a(+) to construct a recombinant expression vector. This expression vector was then transformed into E. coli BL21(DE3) to obtain E. coli BL21(DE3) / pET28a(+)-EcRK. The engineered strain of the phosphoribosyl pyrophosphate synthase BaPRS gene underwent codon optimization using Escherichia coli as the host (the optimized sequence is shown in SEQ ID NO.5), and was synthesized by Qingke Biotechnology Co., Ltd. Then, the BaPRS cDNA fragment was ligated after TATACCAT (after the NcoI restriction site) and CTCGAG (before the XhoI restriction site) in pET-28a(+) to construct a recombinant expression vector. This expression vector was then transformed into E. coli BL21(DE3) to obtain E. coli BL21(DE3) / pET28a(+)-BaPRS. The engineered strain of the nicotinamide phosphoribosyltransferase CpNampt gene underwent codon optimization using *E. coli* as the host (the optimized sequence is shown in SEQ ID NO. 7) and was synthesized by Qingke Biotechnology Co., Ltd. The Nampt cDNA fragment was then ligated to the TATACCAT site (after the NcoI restriction site) and CTCGAG site (before the XhoI restriction site) in pET-28a(+) to construct a recombinant expression vector. This expression vector was then transformed into *E. coli* BL21(DE3) to obtain *E. coli* BL21(DE3) / pET28a(+)-CpNampt. The preparation of the *E. coli* BL21(DE3)-LhPPK expression plasmid is shown in Example 1.

[0113] (2) Preparation, culture and cell-free extract of recombinant bodies

[0114] Wet cells of ribokinase EcRK, phosphoribosyl pyrophosphate synthase BaPRS, nicotinamide ribosyltransferase CpNampt, and polyphosphate kinase LhPPK were obtained according to the method in Example 1, and these four wet cells were then broken up.

[0115] (3) Synthesis of NMN

[0116] Add the following to 10 mL of potassium phosphate buffer (50 mM, pH 7.5): 100 mM ribose, 50 mM NAM, 5 mM ATP, 40 mM magnesium chloride, and 45 mM Polyphosphate. P, 2 g / L RK crude enzyme solution, 16 g / L PRS crude enzyme solution, 20 g / L Nampt crude enzyme solution, and 20 g / L mutant LhPPK / A99S / E186A / D190R / D204A crude enzyme solution were used as the control reaction system. The 20 g / L mutant LhPPK / A99S / E186A / D190R / D204A crude enzyme solution was adjusted to 20 g / L PPK crude enzyme solution. The reaction was carried out at a constant temperature of 37℃ with a magnetic stirrer for 6 h. Samples were taken at 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, and 6 h, and the NMN retention time was 2.9 min as described in Example 3. Method for obtaining the NMN concentration vs. peak area standard curve: Peak area data were obtained from injections of NMN at different concentrations (0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM) to obtain the NMN concentration vs. peak area standard curve. The curve equation is y = 38.081x + 2.3615 (R²). 2 =0.9998), where y is the concentration of NMN (mM) and x is the peak area of ​​NMN.

[0117] (4) Experimental Results

[0118] The results are as follows Figure 3 As shown, when the ATP regeneration system containing wild-type LhPPK was introduced into NMN synthesis, the NMN yield reached 20 mM after 6 hours. When the ATP regeneration system containing the mutant LhPPK / A99S / E186A / D190R / D204A was introduced into NMN synthesis, the NMN yield reached 38 mM after 6 hours. This indicates that in the NMN synthesis reaction containing only 5 mM ATP, introducing the ATP regeneration system composed of mutants can significantly save ATP input and increase NMN yield.

Claims

1. A mutant of polyphosphate kinase, characterized in that, The mutant form of the polyphosphatase mutant is one of the following: (1) the alanine at position 99 is mutated to aspartic acid, the glutamic acid at position 186 is mutated to alanine, and the aspartic acid at position 204 is mutated to alanine in the amino acid sequence shown in SEQ ID NO. 2; (2) the alanine at position 99 is mutated to aspartic acid, the glutamic acid at position 186 is mutated to alanine, the aspartic acid at position 190 is mutated to arginine, and the aspartic acid at position 204 is mutated to alanine in the amino acid sequence shown in SEQ ID NO.

2.

2. A gene encoding the polyphosphokinase mutant of claim 1.

3. A recombinant vector, characterized in that, comprising the gene of claim 2.

4. A genetically engineered bacterium, characterized by, comprising the gene of claim 2.

5. An ATP regenerating system characterized by comprising: including an enzyme and a substrate; the enzyme including the polyphosphokinase mutant of claim 1; the substrate including polyphosphate and adenosine phosphate.

6. Use of the polyphosphokinase mutant of claim 1 or the ATP regeneration system of claim 5 in the production of beta-nicotinamide mononucleotide.

7. A method for the enzymatic production of β-nicotinamide mononucleotide, characterized in that, A reaction system is formed with ribose, adenosine triphosphate, and nicotinamide as substrates, and ribokinase, ribose phosphate pyrophosphokinase, nicotinamide ribosyltransferase, and the polyphosphokinase mutant of claim 1 as catalysts to catalytically prepare beta-nicotinamide mononucleotide.

8. The method of claim 7, wherein, In the reaction system, the pH is 6.5-8.5, and the temperature is 35-45°C.

9. The method of claim 7, wherein, The reaction system further includes potassium phosphate buffer.

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