Phosphate dehydrogenase mutant, recombinant vector, recombinant genetically engineered bacterium and application thereof, and method for synthesizing reduced beta-nicotinamide mononucleotide

Abstract

The invention provides a phosphite dehydrogenase mutant, a recombinant vector, a recombinant genetically engineered bacterium and application thereof, and a method for synthesizing reduced beta-nicotinamide mononucleotide, and belongs to the technical field of biological enzyme engineering. The phosphite dehydrogenase is derived from a soil metagenome library, has extremely high alkali resistance and meets the characteristic of NMNH alkaline condition stability, multi-point combined mutagenesis is carried out on the basis of a wild type, the enzyme catalytic activity is finally improved by 87 times, the reaction is short-time and efficient, purification is easy, and industrial production is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of bioenzyme engineering technology, specifically relating to a phosphite dehydrogenase mutant, a recombinant vector, a recombinant genetically engineered bacterium, their applications, and a method for synthesizing reduced β-nicotinamide mononucleotide (NMNH). Background Technology

[0002] Reduced β-Nicotinamide mononucleotide (NMNH) is a novel NAD+. + As a precursor, it possesses significant biological activity and application potential. The core function of NMNH is to efficiently increase intracellular NAD+ levels. + (Nicotinamide adenine dinucleotide) levels, NAD + It is a key coenzyme for energy metabolism, DNA repair, and anti-aging; it has been reported that NMNH increases NAD at the same concentration. + The rate of NMNH production is 5-10 times faster than that of NMN (β-Nicotinamide Mononucleotide). Natural NMNH exists in the kidneys, but in very small amounts. No natural food has been found that can directly supplement NMNH; it is currently mainly obtained through chemical or biosynthetic methods.

[0003] Currently, reduced β-nicotinamide mononucleotide (NMN) can be synthesized via chemical or biological methods. Chemical synthesis uses NMN as a raw material and reduces it with thiourea dioxide (TDO) under alkaline conditions. This method has a conversion rate exceeding 90% and is a relatively mature synthesis scheme; however, incomplete conversion results in NMN residue, and the reaction solution is difficult to purify. Biological synthesis primarily targets NAD+. + Enzyme combinations of glucose dehydrogenase (CN119060974A), formate dehydrogenase (CN114752572A), and malate dehydrogenase (CN119060973A) were mutated to catalyze the conversion of NMN to NMNH using corresponding substrates as reducing agents. In vitro catalytic reactions were conducted at pH 7.0-8.0. Currently reported enzymes for NMNH synthesis are unstable or exhibit inhibited activity under strongly alkaline conditions, while NMNH exhibits high stability at pH 9-10. Therefore, it is not possible to guarantee the yield of NMNH synthesis under optimal conditions. Further research is needed to identify enzymes with stability comparable to NMNH for subsequent immobilization and industrial production.

[0004] Phosphite dehydrogenase (PTDH) generally uses NAD+ as its active ingredient. + As a cofactor, it catalyzes the conversion of phosphite to orthophosphate and converts NAD+. +It is reduced to NADH and is mainly found in some bacteria, such as Pseudomonas spp. ( Pseudomonas ), genus Rollston Ralstonia ) and Alcaligenes spp. Alcaligenes ) and others, such as Pseudomonas stearothermia ( Pseudomonas stutzeri PsPTDH, due to its high activity, is widely used in gene modification and coenzyme cycling, and exhibits strong alkali resistance, with no significant inhibition of enzyme activity in environments with pH 9-11. Existing literature has identified a mutant TsPTDH Triple (A155N-E175A-A176F) (same as PsPTDH) derived from *Pseudomonas stearothermiae* using a non-classical redox cofactor system, which can catalyze the conversion of NMN to NMNH. The screened TsPTDH Triple was used in an in vivo coenzyme cycling system, with Km and Kcat calculated under neutral conditions and used as a substitute for cofactor cycling in the enzyme catalysis reaction. However, in vitro enzyme activity testing and large-scale in vitro synthesis of NMNH were not performed.

[0005] It is essential to explore novel NMNH synthases and study their applications in synthesis. On one hand, to ensure the stability of NMNH, the selected enzyme needs to remain stable in alkaline solutions and possess high enzyme activity; on the other hand, the introduced substrate should facilitate the removal of impurities, thereby reducing the loss of NMNH during purification. Summary of the Invention

[0006] To address the problems existing in the current synthesis process of reduced β-nicotinamide mononucleotide, this invention discovers a strong base phosphite dehydrogenase that naturally uses β-nicotinamide mononucleotide as a non-characteristic cofactor for the synthesis of reduced β-nicotinamide mononucleotide. After multiple rounds of mutation, the enzyme activity is significantly improved, showing good prospects for industrial application.

[0007] This invention uses the original gene sequence of PsPTDH as a template to perform laboratory soil metagenomic sequence alignment, and identifies sequences originating from the genus *PsPTDH*. Janthinobacterium The phosphite dehydrogenase of 3,000 phosphates was named JaPTDH, and its nucleotide sequence is shown in SEQ ID NO.1.

[0008] Computer simulations were used to design single-point and multi-point mutants of JaPTDH, including single-point mutants G72A, A155N, D175Q, and R307K; double-point mutants G72A-A155N, A155N-D175Q, and A155N-R307K; triple-point mutants G72A-A155N-D175Q, G72A-A155N-R307K, A155N-D175Q-R307K; and quadruple-point mutant G72A-A155N-D175Q-R307K. All of these mutants significantly increased the synthesis rate of reduced β-nicotinamide mononucleotide compared to the wild type, with the triple-point mutant A155N-D175Q-R307K being the preferred choice.

[0009] Amino acid sequence of wild-type phosphite dehydrogenase: JaPTDH (SEQ ID NO.2): MKPQVVVTHWVHPEILELLESVAEVIPNNTRDTLSREEVLSRAKNAAAIMVFMPDSIDEDFLNACPKLRMVGAALKGYDNFDVEACTRRGIWFSIVPDLLTIPTAELTIGLLLGLTRHMLEGDRRIRTGQFQGWRPELYGAGLTGQTLGIIGMGAVGTAIAKRLAG FDMRLVYCDSVALDSAREQAWGLERVSLDALLQESDFVVPMLPMTPETFHLINDDSIARMKRGAYLINACRGSVVDEQAVSTALKAGHLSGYAADVYEMEEWRRADRPAGIPQSLLVNQAQTLFTPHLGSAVKEVRLEIERQAALNIVQALSGQRPVGAINNPVEPTLVTAGC The amino acid sequence of the single-point mutant: G72A(SEQ ID NO.3):MKPQVVVTHWVHPEILELLESVAEVIPNNTRDTLSREEVLSRAKNAAAIMVFMPDSIDEDFLNACPKLRMVAAALKGYDNFDVEACTRRGIWFSIVPDLLTIPTAELTIGLLLGLTRHMLEGDRRIRTGQFQGWRPELYGAGLTGQTLGIIGMGAVGTAIAKRLAGFDMRLVYCDSVALDSAREQAWGLERVSLDALLQESDFVVPMLPMTPETFHLINDDSIARMKRGAYLINACRGSVVDEQAVSTALKAGHLSGYAADVYEMEEWRRADRPAGIPQSLLVNQAQTLFTPHLGSAVKEVRLEIERQAALNIVQALSGQRPVGAINNPVEPTLVTAGC A155N(SEQ ID NO.4):MKPQVVVTHWVHPEILELLESVAEVIPNNTRDTLSREEVLSRAKNAAAIMVFMPDSIDEDFLNACPKLRMVGAALKGYDNFDVEACTRRGIWFSIVPDLLTIPTAELTIGLLLGLTRHMLEGDRRIRTGQFQGWRPELYGAGLTGQTLGIIGMGNVGTAIAKRLAGFDMRLVYCDSVALDSAREQAWGLERVSLDALLQESDFVVPMLPMTPETFHLINDDSIARMKRGAYLINACRGSVVDEQAVSTALKAGHLSGYAADVYEMEEWRRADRPAGIPQSLLVNQAQTLFTPHLGSAVKEVRLEIERQAALNIVQALSGQRPVGAINNPVEPTLVTAGC D175Q (SEQ ID NO.5): MKPQVVVTHWVHPEILELLESVAEVIPNNTRDTLSREEVLSRAKNAAAIMVFMPDSIDEDFLNACPKLRMVGAALKGYDNFDVEACTRRGIWFSIVPDLLTIPTAELTIGLLLGLTRHMLEGDRRIRTGQFQGWRPELYGAGLTGQTLGIIGMGAVGTAIAKRLAGFDMRLVYCQSVALDSAREQAWGLERVSLDALLQESDFVVPMLPMTPETFHLINDDSIARMKRGAYLINACRGSVVDEQAVSTALKAGHLSGYAADVYEMEEWRRADRPAGIPQSLLVNQAQTLFTPHLGSAVKEVRLEIERQAALNIVQALSGQRPVGAINNPVEPTLVTAGC R307K (SEQ ID NO.6): MKPQVVVTHWVHPEILELLESVAEVIPNNTRDTLSREEVLSRAKNAAAIMVFMPDSIDEDFLNACPKLRMVGAALKGYDNFDVEACTRRGIWFSIVPDLLTIPTAELTIGLLLGLTRHMLEGDRRIRTGQFQGWRPELYGAGLTGQTLGIIGMGAVGTAIAKRLAGFDMRLVYCDSVALDSAREQAWGLERVSLDALLQESDFVVPMLPMTPETFHLINDDSIARMKRGAYLINACRGSVVDEQAVSTALKAGHLSGYAADVYEMEEWRRADRPAGIPQSLLVNQAQTLFTPHLGSAVKEVRLEIEKQAALNIVQALSGQRPVGAINNPVEPTLVTAGC Amino acid sequence of the double point mutant: G72A-A155N(SEQ ID NO.7):MKPQVVVTHWVHPEILELLESVAEVIPNNTRDTLSREEVLSRAKNAAAIMVFMPDSIDEDFLNACPKLRMVAAALKGYDNFDVEACTRRGIWFSIVPDLLTIPTAELTIGLLLGLTRHMLEGDRRIRTGQFQGWRPELYGAGLTGQTLGIIGMGNVGTAIAKRLAGFDMRLVYCDSVALDSAREQAWGLERVSLDALLQESDFVVPMLPMTPETFHLINDDSIARMKRGAYLINACRGSVVDEQAVSTALKAGHLSGYAADVYEMEEWRRADRPAGIPQSLLVNQAQTLFTPHLGSAVKEVRLEIERQAALNIVQALSGQRPVGAINNPVEPTLVTAGC A155N-D175Q(SEQ ID NO.8):MKPQVVVTHWVHPEILELLESVAEVIPNNTRDTLSREEVLSRAKNAAAIMVFMPDSIDEDFLNACPKLRMVGAALKGYDNFDVEACTRRGIWFSIVPDLLTIPTAELTIGLLLGLTRHMLEGDRRIRTGQFQGWRPELYGAGLTGQTLGIIGMGNVGTAIAKRLAGFDMRLVYCQSVALDSAREQAWGLERVSLDALLQESDFVVPMLPMTPETFHLINDDSIARMKRGAYLINACRGSVVDEQAVSTALKAGHLSGYAADVYEMEEWRRADRPAGIPQSLLVNQAQTLFTPHLGSAVKEVRLEIERQAALNIVQALSGQRPVGAINNPVEPTLVTAGC A155N-R307K (SEQ ID NO.9): MKPQVVVTHWVHPEILELLESVAEVIPNNTRDTLSREEVLSRAKNAAAIMVFMPDSIDEDFLNACPKLRMVGAALKGYDNFDVEACTRRGIWFSIVPDLLTIPTAELTIGLLLGLTRHMLEGDRRIRTGQFQGWRPELYGAGLTGQTLGIIGMGNVGTAIAKRLAGFDMRLVYCDSVALDSAREQAWGLERVSLDALLQESDFVVPMLPMTPETFHLINDDSIARMKRGAYLINACRGSVVDEQAVSTALKAGHLSGYAADVYEMEEWRRADRPAGIPQSLLVNQAQTLFTPHLGSAVKEVRLEIEKQAALNIVQALSGQRPVGAINNPVEPTLVTAGC Amino acid sequence of the triple mutant: G72A-A155N-D175Q (SEQ ID NO.10): MKPQVVVTHWVHPEILELLESVAEVIPNNTRDTLSREEVLSRAKNAAAIMVFMPDSIDEDFLNACPKLRMVAAALKGYDNFDVEACTRRGIWFSIVPDLLTIPTAELTIGLLLGLTRHMLEGDRRIRTGQFQGWRPELYGAGLTGQTLGIIGMGNVGTAIAKRLAGFDMRLVYCQSVALDSAREQAWGLERVSLDALLQESDFVVPMLPMTPETFHLINDDSIARMKRGAYLINACRGSVVDEQAVSTALKAGHLSGYAADVYEMEEWRRADRPAGIPQSLLVNQAQTLFTPHLGSAVKEVRLEIERQAALNIVQALSGQRPVGAINNPVEPTLVTAGC[[ID=⑤]] G72A - A155N - R307K (SEQ ID NO.11): MKPQVVVTHWVHPEILELLESVAEVIPNNTRDTLSREEVLSRAKNAAAIMVFMPDSIDEDFLNACPKLRMVAAALKGYDNFDVEACTRRGIWFSIVPDLLTIPTAELTIGLLLGLTRHMLEGDRRIRTGQFQGWRPELYGAGLTGQTLGIIGMGNVGTAIAKRLAGFDMRLVYCDSVALDSAREQAWGLERVSLDALLQESDFVVPMLPMTPETFHLINDDSIARMKRGAYLINACRGSVVDEQAVSTALKAGHLSGYAADVYEMEEWRRADRPAGIPQSLLVNQAQTLFTPHLGSAVKEVRLEIEKQAALNIVQALSGQRPVGAINNPVEPTLVTAGC A155N - D175Q - R307K (SEQ ID NO.12): MKPQVVVTHWVHPEILELLESVAEVIPNNTRDTLSREEVLSRAKNAAAIMVFMPDSIDEDFLNACPKLRMVGAALKGYDNFDVEACTRRGIWFSIVPDLLTIPTAELTIGLLLGLTRHMLEGDRRIRTGQFQGWRPELYGAGLTGQTLGIIGMGNVGTAIAKRLAGFDMRLVYCQSVALDSAREQAWGLERVSLDALLQESDFVVPMLPMTPETFHLINDDSIARMKRGAYLINACRGSVVDEQAVSTALKAGHLSGYAADVYEMEEWRRADRPAGIPQSLLVNQAQTLFTPHLGSAVKEVRLEIEKQAALNIVQALSGQRPVGAINNPVEPTLVTAGC Amino acid sequences of the quadruple mutants: G72A-A155N-D175Q-R307K (SEQ ID NO.13): MKPQVVVTHWVHPEILELLESVAEVIPNNTRDTLSREEVLSRAKNAAAIMVFMPDSIDEDFLNACPKLRMVAAALKGYDNFDVEACTRRGIWFSIVPDLLTIPTAELTIGLLLGLTRHMLEGDRRIRTGQFQGWRPELYGAGLTGQTLGIIGMGNVGTAIAKRLAG FDMRLVYCQSVALDSAREQAWGLERVSLDALLQESDFVVPMLPMTPETFHLINDDSIARMKRGAYLINACRGSVVDEQAVSTALKAGHLSGYAADVYEMEEWRRADRPAGIPQSLLVNQAQTLFTPHLGSAVKEVRLEIEKQAALNIVQALSGQRPVGAINNPVEPTLVTAGC “G72A” indicates a mutation at position 72 where glycine (G) is replaced by alanine (A); “A155N” indicates a mutation at position 155 where alanine (A) is replaced by asparagine (N); “D175Q” indicates a mutation at position 175 where aspartic acid (D) is replaced by glutamine (Q); “R307K” indicates a mutation at position 307 where arginine (R) is replaced by lysine (K). “G72A-A155N” indicates a combined mutation where G at position 72 is replaced by A, and A at position 155 is replaced by N. The “-” indicates “and” or “as well” and is used to separate different mutation sites.

[0010] The phosphite dehydrogenase mutant plasmid is transferred to a host bacterium to obtain recombinant engineered bacteria. The recombinant engineered bacteria synthesize the phosphite dehydrogenase mutant through fermentation. In this invention, Escherichia coli BL21(DE3) is used for induction expression. Other host bacteria such as Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastoris, or animal and plant cells can also synthesize phosphite dehydrogenase.

[0011] This invention uses β-nicotinamide mononucleotide and phosphite as substrates, and under alkaline conditions, adds phosphite dehydrogenase to convert β-nicotinamide mononucleotide into reduced β-nicotinamide mononucleotide.

[0012] The phosphite dehydrogenase in this invention can be an enzyme solution of a phosphite dehydrogenase mutant, a lyophilized enzyme powder, an enzyme-containing wet bacterial cell, a lyophilized bacterial powder, an immobilized enzyme, or an immobilized cell, etc. In this invention, the enzyme solution is used to screen and detect the enzyme activity of wild-type and mutant phosphite dehydrogenases, and the reduced β-nicotinamide mononucleotide is synthesized in the form of wet bacterial cells.

[0013] The alkaline conditions of this invention preferably have a pH range of 9-11, with pH 9.5 being optimal. The buffer solution can be Tris-HCl buffer, carbonate buffer, glycine buffer, or direct NaOH solution to control the pH at 9-10, with direct NaOH solution being preferred.

[0014] The preferred reaction temperature of this invention is 10-40 °C, with an optimal reaction temperature of 30 °C.

[0015] The concentration of the substrate β-nicotinamide mononucleotide in this invention is 1-200 g / L, with the optimal concentration being 150 g / L.

[0016] The wet cell concentration of the present invention is 1-20 g / L, and the feeding is calculated according to the mass ratio of cell to β-nicotinamide mononucleotide of 1:10.

[0017] The optimized preferred conditions of this invention are as follows: the concentration of substrate β-nicotinamide mononucleotide is 150 g / L, sodium phosphite pentahydrate is 100 g / L, the solution pH is adjusted to 9.5±0.3, the temperature is 30 ℃, and 15 g / L of recombinant engineered bacteria wet cells of phosphite dehydrogenase mutant are added. The 100% conversion of substrate β-nicotinamide mononucleotide can be achieved in 2 h of reaction.

[0018] The present invention achieves the following beneficial effects: The phosphite dehydrogenase mutant of this invention ultimately increases the enzyme's catalytic activity by 87 times, exhibiting a short and efficient reaction time, easy purification, and suitability for industrial production. The enzyme used in this invention has high activity, requires a small amount of bacterial cells for the reaction, and the bacterial cells can be directly removed by filtration. The raw material, phosphite, is inexpensive, and the generated phosphate can be removed by nanofiltration. Compared to existing methods for synthesizing reduced β-nicotinamide mononucleotides, the production and purification process is simpler, demonstrating significant potential for industrial application.

[0019] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0020] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0021] Figure 1 This is a comparison diagram of the amino acid sequence of PTDH.

[0022] Figure 2 The reaction formula for the synthesis of NMNH from PTDH.

[0023] Figure 3 SDS-PAGE gel electrophoresis images of JaPTDH-WT and JaPTDH-M3 mutants.

[0024] Figure 4 The standard curve for NMNH microplate reader detection at 340 nm was obtained.

[0025] Figure 5 Liquid chromatography chromatograms of NMN (0.3025 g / L) and NMNH (0.3025 g / L) standards.

[0026] Figure 6 The liquid chromatogram of the reaction for synthesizing NMNH using the JaPTDH-M3 mutant. Detailed Implementation

[0027] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0028] Example 1: Gene mining and single-point mutation The specific process is as follows: This invention uses the PsPTDH gene sequence as a basis for laboratory soil metagenomic sequence alignment, and finds that it originates from the genus *Porphyromonas* (…). Janthinobacterium The gene with the identifier gene_540670 has a 53.69% amino acid sequence similarity to PsPTDH. The discovered phosphite dehydrogenase has been named JaPTDH. For detailed amino acid sequence alignment, see [link to gene sequence]. Figure 1 The nucleotide sequence is shown in SEQ ID NO.1.

[0029] SEQ ID NO.1: atgaaaccac aagtagtcgt cacccactgg gtacatccgg aaatcctcga attactggaaagcgttgcgg aagtcattcc caacaatacg cgcgacacct tgtcgcgcga ggaagtgctg agccgagcaaaaaacgccgc cgccatcatg gtgttcatgc cagatagcat cgatgaggat tttttgaatg cttgcccgaagctaaggatg gttggggcag ccctgaaggg ttacgacaac ttcgatgtgg aagcctgcac gaggcgcggcatctggtttt ccatcgtgcc ggatctgttg acgattccaa cggctgaatt gaccatcgga ttgctgctgggtctaacgcg ccacatgctt gaaggcgatc gccgcattcg cactgggcaa tttcaaggct ggcgcccggaattgtacggc gcaggattga ctggccaaac gcttggcatt attggaatgg gcgcggtcgg aacagcaatcgccaagcgct tggctggttt tgatatgcga ctggtgtatt gcgacagcgt tgcgctcgat tctgcacgggagcaagcctg gggattggag cgagtcagtc tggatgcgct tctgcaggaa agcgatttcg tggttcccatgctgccgatg acgccggaga cctttcattt gatcaatgac gattcgatcg ccaggatgaa acgcggcgcttacctgatca atgcatgccg cggctccgtg gtcgatgaac aagccgtgag taccgcatta aaagccggccatttatcagg ctacgccgcc gatgtatatg aaatggaaga atggcgccgc gccgaccggc cagcaggcatcccgcaatcg ctgctcgtta atcaagcgca gactttattt acaccccatt taggctcggc ggtaaaggaagtacgcctgg agatcgagcgacaagcggcg ctgaatattg ttcaggcact ctccgggcag agacctgttggcgccatcaa caatccggtt gagcctactt tggtgactgc aggatgctag.

[0030] Soil metagenomic libraries were created using the Tiangen Soil Metagenomic Extraction Kit. Primers F1: 5'-GTGCCGCGCGGCAGCCATATGAAACCACAAGTAGTCGTCACC-3' and R1: 5'-CTCGAGTGCGGCCGCAAGCCTAGCATCCTGCAGTCACCAAAG-3' were synthesized by Genewiz Suzhou. PCR was performed using the synthesized primers to obtain the target gene. Homologous recombination with the plasmid pET-28a fragment was performed using a one-step cloning kit and introduced into competent E. coli BL21(DE3) cells. After overnight culture, single colonies were selected for colony PCR verification, and Genewiz sequencing was used for verification. The recombinant E. coli BL21(DE3)-pET-JaPTDH glycerol tubes were preserved, and the plasmid pET-JaPTDH was extracted and preserved using a plasmid miniprep kit.

[0031] Using the recombinant plasmid pET-JaPTDH as a template, primers were designed with specific gene sequences shown in Table 1. The primers were synthesized by Genewiz Suzhou. The whole plasmid was amplified using the primers shown in Table 1. The amplified reaction solution was digested with Dpn I restriction enzyme. The digested fragments were transformed into Escherichia coli BL21(DE3) competent cells and cultured overnight. Single colonies were selected for culture and sequencing verification. The successfully mutated strains were preserved.

[0032] Table 1: Primer sequence listing for site-directed mutagenesis The literature (DOI:10.1038 / s41467-022-32727-w) discloses the discovery of a mutant TsPTDH Triple (A155N-E175A-A176F) (same as PsPTDH) from *Pseudomonas schlegelii* using a non-classical redox cofactor system. To compare the enzyme activity of the mutant constructed using metagenomic mining in this invention with the aforementioned mutant, a control experiment is also provided. The PsPTDH and PsPTDH-A155N-E175A-A176F mutant genes were synthesized by Suzhou Genewise, constructed on pET-28a, and transformed into *E. coli* BL21(DE3) competent cells. The glycerol tubes were cultured overnight and stored, and named BL21(DE3)-pET-PsPTDH and BL21(DE3)-PsPTDH-M3, respectively.

[0033] Fermentation of recombinant *Escherichia coli* BL21(DE3)-pET-JaPTDH, BL21(DE3)-pET-PsPTDH, BL21(DE3)-PsPTDH-M3, and their mutants G72A, A155N, D175Q, and R307K was carried out using TB medium. Single colonies were transferred to 50 ml of LB medium (containing 50 mg / L Kana) and cultured overnight. Then, 2% of the colonies were inoculated into 400 ml of TB medium (containing 25 mg / L Kana) and cultured at 37 °C and 200 rpm / min until the bacterial concentration reached OD500. 600 =0.8-1; cool to 20 ℃, add IPTG to a final concentration of 0.1 mM for overnight expression, induce for 20 h, centrifuge to recover the bacterial cells, and take a small amount of bacterial cells for SDS-PAGE gel electrophoresis to detect its soluble expression level.

[0034] Wild-type and mutant cells of BL21(DE3)-pET-JaPTDH (G72A, A155N, D175Q, R307K) and wild-type and mutant cells of BL21(DE3)-pET-PsPTDH (PsPTDH-M3) were dissolved at a concentration of 40 g / L in 50 mM pH 9.5 Tris-HCl buffer. Cell debris was removed by centrifugation, and the supernatant was retained. The supernatant was the crude enzyme solution of JaPTDH wild-type or mutant. A 50 mM pH 9.5 Tris-HCl buffer containing 2 mM NMN (0.668 g / L) and 1 g / L sodium phosphite pentahydrate was prepared for detecting the enzyme activity of JaPTDH wild-type or mutant.

[0035] Enzyme activity assay using an ELISA reader: Scanning with an ELISA reader revealed that NMNH, similar to NAD(P)H, exhibits characteristic absorption at 340 nm. Phosphite dehydrogenase catalyzes the synthesis of phosphate and NMNH from phosphite and NMN. The change in absorbance of NMNH at 340 nm reflects the enzyme activity of phosphite dehydrogenase in synthesizing NMNH. (See attached...) Figure 4 Enzyme activity was calculated using a standard curve. This invention primarily uses wet cell culture as the feedstock. One unit of enzyme activity (U / g fresh weight) is defined as 1 μmol of NMNH produced per minute per gram of wet cell culture. The added enzyme solution is also calculated based on the original fresh weight of the cell culture.

[0036] Table 2: Enzyme activity of PTDH wild-type and mutant strains The results showed that the wild-type JaPTDH enzyme activity was slightly higher than that of the wild-type PsPTDH, but much lower than that of the PsPTDH-M3 mutant, indicating that further genetic modification is needed to enhance the enzyme activity of JaPTDH. The four JaPTDH mutants—G72A, A155N, D175Q, and R307K—all showed significant improvements compared to the wild-type JaPTDH, with A155N exhibiting the highest enzyme activity, potentially representing the most critical site for reversing the NMN activity of PTDH.

[0037] Example 2: Multi-point combination mutation The specific process is as follows: To further enhance the activity of JaPTDH, plasmid pET-JaPTDH-A155N was extracted from *E. coli* strain BL21-pET-JaPTDH-A155N. Two-, three-, and four-point combination mutations were performed using pET-JaPTDH-A155N as a template. Full plasmid or fragment PCR amplification was performed according to the multi-point mutation PCR primer table (gene sequences corresponding to primer numbers are shown in Table 1). Fragment ligation was performed using a one-step cloning kit or a multi-fragment one-step cloning kit. The fragments were then transformed into *E. coli* BL21(DE3) competent cells for recovery and cloning. Single colonies were selected for sequencing, and successfully mutated strains were preserved.

[0038] Table 3: Primer list for multipoint mutation PCR Fermentation was carried out using TB medium for BL21(DE3)-pET-JaPTDH-WT, BL21(DE3)-pET-PsPTDH-WT, BL21(DE3)-pET-PsPTDH-M3, BL21(DE3)-pET-JaPTDH-A155N mutants and other multipoint mutants G72A-A155N, A155N-D175Q, A155N-R307K, G72A-A155N-D175Q, G72A-A155N-R307K, A155N-D175Q-R307K, and G72A-A155N-D175Q-R307K. Select corresponding single colonies and transfer them to 50 ml of LB medium (containing 50 mg / L Kana) for overnight culture. Then, inoculate 2% of the colonies into 400 ml of TB medium (containing 25 mg / L Kana) and culture at 37 ℃ and 200 rpm / min until the bacterial concentration reaches OD500. 600 =0.8-1; cool to 20 ℃, add IPTG to a final concentration of 0.1 mM for overnight expression, induce for 20 h, centrifuge to recover the bacterial cells, and take a small amount of bacterial cells for SDS-PAGE gel electrophoresis to detect its soluble expression level.

[0039] The cells of JaPTDH-WT, PsPTDH-WT, PsPTDH-M3, JaPTDH-A155N mutants and other multi-point mutants G72A-A155N, A155N-D175Q, A155N-R307K, G72A-A155N-D175Q, G72A-A155N-R307K, A155N-D175Q-R307K, and G72A-A155N-D175Q-R307K were dissolved at a concentration of 40 g / L in 50 mM pH 9.5 Tris-HCl buffer. The cells were centrifuged to remove cell debris, and the supernatant was retained. The supernatant was the crude enzyme solution of the JaPTDH mutant. Prepare a 50 mM Tris-HCl buffer solution (pH 9.5) containing 2 mM NMN and 1 g / L sodium phosphite pentahydrate for detecting the enzyme activity of the JaPTDH mutant. Figure 2 ).

[0040] Table 4: Activity of PTDH multipoint mutant enzyme The two-, three-, and four-point combination mutations of JaPTDH all showed significant improvements compared to JaPTDH-A155N. Among them, the enzyme activity of JaPTDH-A155N-D175Q-R307K was increased by 87.21 times compared to the wild-type JaPTDH and by 31.24 times compared to JaPTDH-A155N. Compared with the PsPTDH-M3 mutant strain disclosed in the literature (DOI:10.1038 / s41467-022-32727-w), the enzyme activity of JaPTDH-A155N-D175Q-R307K was also 125 U / g fresh weight higher, which is 1.72 times higher, indicating a significant mutation effect. Therefore, the three-point mutant with the highest enzyme activity, JaPTDH-A155N-D175Q-R307K, was selected for subsequent process scale-up and production, and named recombinant Escherichia coli BL21(DE3)-pET-JaPTDH-M3, or simply JaPTDH-M3.

[0041] Example 3: Optimization of Reaction pH The specific process is as follows: pH stability test of NMNH: A 50 g / L NMNH standard was accurately prepared, and the sample was dissolved in 100 mM Tris-HCl buffer solutions at pH 7.0, pH 8, pH 8.5, pH 9, pH 9.5, pH 10, and pH 11. Solutions containing NMNH standards at different pH values ​​were incubated at room temperature in the dark for 24 hours, and the residual amount of NMNH was calculated by liquid chromatography. According to the data in Table 5, NMNH exhibits the best stability at pH 9.5; pH values ​​below 9.0 or above 11.0 lead to significant degradation of NMNH.

[0042] Table 5: Stability test of NMNH at different pH values pH optimization for JaPTDH-NMNH synthesis: Accurately prepare 50 g / L NMN and 50 g / L Na2HPO3·5H2O solutions. Dissolve the samples in 400 mM Tris-HCl buffer solutions at pH 7.0, 8, 8.5, 9, 9.5, 10, and 11, respectively. Add 5 g / L wet JaPTDH-M3 cells to each solution and place in a 30℃ metal bath. Shake and react. Measure the NMN conversion rate and NMNH formation rate after 2 hours using liquid chromatography. Table 6 shows that NMN can be 100% converted within the pH range of 9.0-10.0 after 2 hours of reaction. However, considering the stability of NMNH, pH 9.5 is the optimal pH for JaPTDH-M3-NMNH synthesis, as its optimal reaction pH matches the optimal stability pH of NMNH. When the pH of the reaction solution is below 9.0, the product NMNH is easily degraded; when the pH of the reaction solution is above 10.0, the substrate NMN is significantly degraded, while the product NMNH is only slightly degraded. Testing showed that the optimal temperature for the enzymatic reaction was 30℃. Compared to room temperature, this temperature resulted in high overall enzyme activity and a short reaction time.

[0043] Table 6: Conversion rate of JaPTDH-M3 at different pH values Example 4, Production Case 1 The specific process is as follows: JaPTDH-M3 was fermented using TB medium and cultured at 37 ℃ and 200 rpm / min until the cell concentration reached OD500. 600 =0.8-1; cool to 20 ℃, add 0.1 mM IPTG for overnight expression, and after 20 h of induction, centrifuge to recover JaPTDH-M3 cells. Take a small amount of cells for SDS-PAGE gel electrophoresis to detect its soluble expression level. SDS-PAGE gel electrophoresis image is shown below. Figure 3 .

[0044] NMNH was synthesized using JaPTDH-M3. The total reaction volume was 100 mL. 15 g of NMN and 11.6 g of Na2HPO3·5H2O were weighed and added to 75 mL of pure water. The pH of the reaction solution was adjusted to 9.5±0.2 using 30% (w / v) NaOH solution, and the volume was brought to 90 mL. The temperature was adjusted to 30 ℃, and 15 g of JaPTDH-M3 cells were added. The mixture was slowly stirred for 2–3 h, during which the pH of the reaction solution was maintained at 9.5±0.2 using 10% (w / v) NaOH solution. Samples were taken regularly. After complete NMN conversion was confirmed by liquid chromatography, the cells were centrifuged to terminate the reaction. Liquid chromatography graphs are shown in Figures 5 and 6. The NMNH concentration was 148.5 g / L, and the molar conversion rate was 98.41%.

[0045] Example 5, Production Case 2 The specific process is as follows: JaPTDH-M3 was fermented using TB medium at 37 ℃ and 200 rpm / min until the cell concentration OD600 = 0.8-1. The temperature was then lowered to 25 ℃, and 0.1 mM IPTG was added for overnight expression. After induction for 20 h, the JaPTDH-M3 cells were recovered by centrifugation.

[0046] NMNH was synthesized using JaPTDH-M3. In a total reaction system of 1 L, 100 g of NMN and 77.6 g of Na2HPO3·5H2O were weighed and added to 800 ml of pure water. The pH of the reaction solution was adjusted to 9.5±0.2 using 30% (w / v) NaOH solution, and the volume was brought to 0.95 L. The temperature was adjusted to 30 ℃, and 10 g of JaPTDH-M3 cells were added. The mixture was slowly stirred for 2-3 h, during which the pH of the reaction solution was maintained at 9.5±0.2 using 30% (w / v) NaOH solution. Samples were taken regularly, and after complete NMN conversion was confirmed by liquid chromatography, the cells were centrifuged to terminate the reaction. The NMNH concentration was 95.23 g / L, and the molar conversion rate was 94.66%.

[0047] Example 6, Production Case 3 The specific process is as follows: NMNH was synthesized using JaPTDH-M3. In a total reaction system of 1 L, 150 g of NMN and 110 g of Na2HPO3·5H2O were weighed and added to 750 ml of pure water. The pH of the reaction solution was adjusted to 9.5±0.2 using 30% (w / v) NaOH solution, and the volume was brought to 0.9 L. The temperature was adjusted to 30 ℃, and 10 g of JaPTDH-M3 cells were added. The mixture was slowly stirred for 2-3 h, during which the pH of the reaction solution was maintained at 9.5±0.2 using 30% (w / v) NaOH solution. Samples were taken regularly, and after complete NMN conversion was confirmed by liquid chromatography, the cells were centrifuged to terminate the reaction. The NMNH concentration was 142.5 g / L, and the molar conversion rate was 94.43%.

[0048] In summary, this invention provides a phosphite dehydrogenase mutant, a recombinant vector, a recombinant genetically engineered bacterium, their applications, and a method for synthesizing reduced β-nicotinamide mononucleotide. The phosphite dehydrogenase of this invention is derived from a soil metagenomic library and exhibits strong alkali resistance, conforming to the characteristics of stability under NMNH alkaline conditions. This invention utilizes multi-point combined mutagenesis based on the wild type, ultimately increasing the enzyme's catalytic activity by 87 times. The reaction is short, efficient, easy to purify, and suitable for industrial production.

Claims

1. A mutant phosphite dehydrogenase enzyme, characterized in that, The phosphite dehydrogenase mutant is obtained by mutating wild-type phosphite dehydrogenase; the amino acid sequence of the wild-type phosphite dehydrogenase is shown in SEQ ID NO. 2, and the mutation is any single mutation or combined mutation as follows: Gly at position 72 is mutated to Ala, Ala at position 155 is mutated to Asn, Asp at position 175 is mutated to Gln, and Arg at position 307 is mutated to Lys.

2. The phosphite dehydrogenase mutant of claim 1, wherein, The amino acid sequence of the phosphite dehydrogenase mutant is shown in SEQ ID NO.

12.

3. A recombinant vector, characterized in that, The recombinant vector comprises the phosphite dehydrogenase mutant according to any one of claims 1-2.

4. A recombinant genetically engineered bacteria, characterized in that, The genetically engineered bacterium comprises the recombinant vector according to claim 3.

5. Use of the phosphite dehydrogenase mutant according to any one of claims 1-2, the recombinant vector according to claim 3, or the genetically engineered bacterium according to claim 4 in the synthesis of reduced β-nicotinamide mononucleotide.

6. A method of synthesizing a reduced form of β-nicotinamide mononucleotide, characterized by, The method comprises the following steps: mixing β-nicotinamide mononucleotide, phosphite, and water, adjusting the pH to 7.0-11.0, adding the phosphite dehydrogenase mutant according to any one of claims 1-2, and reacting to obtain reduced β-nicotinamide mononucleotide.

7. The method of claim 6, wherein, The phosphite dehydrogenase mutant is selected from any form of enzyme in enzyme solution, freeze-dried enzyme powder, enzyme-containing wet bacterial cells, freeze-dried bacterial powder, immobilized enzyme, or immobilized cells.

8. The method of claim 6, wherein, The mass ratio of the β-nicotinamide mononucleotide, phosphite, and phosphite dehydrogenase mutant is 1-20:1-20:1-2; the pH is 9-10; the reaction temperature is 10-40℃, and the reaction time is 1-5 hours.

9. The method of claim 8, wherein, The mass ratio of the β-nicotinamide mononucleotide, phosphite, and phosphite dehydrogenase mutant is 15:10:1.5; the pH is 9.5; the reaction temperature is 30℃, and the reaction time is 2-3 hours.

10. The method of claim 6, wherein, The phosphite is sodium phosphite pentahydrate.

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