Enzyme for catalyzing high-yield nicotinamide mononucleotide and application thereof

Abstract

The present application relates to the field of biotechnology, in particular to a catalytic enzyme for high-yield nicotinamide mononucleotide and application, and provides a mutant of nicotinamide riboside kinase, wherein the wild type is derived from a flying fox and has mutation sites G87A / E91P / D109Y, and the heat-stable nicotinamide riboside kinase NRK03-G87A / E91P / D109Y of the present application can withstand 50 DEG C heat shock, and is suitable for completing a catalytic reaction of 600 mM high substrate concentration at 45 DEG C, and the reported highest yield of NMN is 184 g / L, thereby reducing the manufacturing cost in the production process.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to enzymes for catalyzing nicotinamide mononucleotides and their applications. Background Technology

[0002] Nicotinamide adenine dinucleotide (NAD) and its reduced form NADH are major coenzymes for redox reactions in organisms. NAD is also a common substrate for histone deacetylase SIRT1, ADP-ribose cyclization hydrolysis bifunctional enzyme CD38, poly-ADP-ribose polymerase PARP, and axon mutant enzyme SARM1. These enzymes are involved in a variety of physiological processes, including cell proliferation and differentiation, cell senescence and death, tumorigenesis, synthesis of second messengers, DNA repair, axon maintenance and degeneration.

[0003] Nobel laureate Arthur Kornberg discovered in 1948 that nicotinamide mononucleotide (NMN) is a precursor for the synthesis of NAD (Kornberg A. 1948, Kornberg A. 1950), and later discovered the synthetic pathway of "nicotinamide (NAm) + 1-phosphoribose (R1P) → nicotinamide ribose (NR) → NMN → NAD" (Rowen JW, Kornberg A. 1951). In 1957, Jack Preiss and Philip Handler of Duke University discovered another NMN synthesis pathway: “5-phosphoribose (R5P)-phosphoribose pyrophosphate (PRPP) + NAm → NMN” (Preiss J., Handler P. 1957). Later, they discovered a pathway from nicotinic acid (Na) to NAD that does not involve NMN: “Na + PRPP → NaMN → NaAD → NAD” (Preiss J., Handler P. 1958a, Preiss J., Handler P. 1958b). In 1963, Osamu Hayaishi of Kyoto University discovered that quinolinic acid (QA) and phosphoribose pyrophosphate (PRPP) are precursors to nicotinic acid mononucleotide (NaMN) in the de novo NAD synthesis pathway (Andreoli AJ et al. 1963).

[0004] Giulio Magni of the University of Ancona first determined the sequence of nicotinamide mononucleotide adenosine transferase (NMNAT), which synthesizes NAD using NMN and adenosine triphosphate (ATP) as substrates, in thermophilic archaea, and resolved its structure (Raffaelli N. et al. 1997, D'Angelo I. et al. 2000). Homologous sequences were subsequently identified in cyanobacteria, Escherichia coli, Salmonella, yeast, nematodes, and humans (Raffaelli N. et al. 1999a, Raffaelli N. et al. 1999b, Emanuelli M. et al. 1999, Emanuelli M. et al. 2001, Raffaelli N. et al. 2002). Hong Zhang of Southwest Medical Center was the first to resolve the structure of human NMNAT (Zhou T. et al. 2002, Garavaglia S. et al. 2002, Werner E. et al. 2002) and discovered a third type of human NMNAT located in mitochondria (Zhang X. et al. 2003).

[0005] Regarding the first synthetic pathway, Andrei L. Osterman of Integrated Genomics reported that nicotinamide ribokinase NRK, which synthesizes NMN using NR and ATP as substrates, and NMNAT, which synthesizes NAD using NMN and ATP as substrates, fuse into a bifunctional protein NadR (Kurnasov OVetal. 2002). In collaboration with Hong Zhang of Southwestern Medical Center, the structure of NadR in Haemophilus influenzae was resolved (Singh S.K. et al. 2002). Meanwhile, Charles Brenner of Dartmouth Medical School identified independent NRKs in yeast and humans (Bieganowski P., & Brenner C. 2004) and resolved the structure of human NRK1 (Khan JA et al. 2007, Tempel W. et al. 2007). Meanwhile, the structure of NAMPT, the human nicotinamide phosphoribosyltransferase used in the second synthetic pathway to synthesize NMN using NAm and PRPP as substrates, was also resolved (Khan JA et al. 2006).

[0006] Beginning in 2007, Shin-ichiro Imai of Washington University School of Medicine and David A. Sinclair of Harvard University reported that intraperitoneal injection or oral administration of NMN could increase intracellular NAD levels, thereby promoting insulin secretion and anti-aging effects (Revollo J et al. 2007, Ramsey K et al. 2008, Yoshino J. 2011, Gomes AP et al. 2013, Mills K et al. 2016, Das A et al. 2018, Grozio A et al. 2019, Irie J. 2020, Yoshino M et al. 2021). This attracted companies such as Oriental Yeast and Shinshinwa in Japan to attempt industrial-scale production of NMN.

[0007] In current existing technologies, Merck's patent WO2011012270 discloses a method for synthesizing NMN using human NRK1 or NadR derived from Streptococcus sanguinis with NR and ATP as substrates. Metro Biotech's patent WO2016198948 discloses a method for synthesizing PRPP using human PRPP synthase RPPK with ribose-5-phosphate (R5P) and ATP as substrates, and then using NAMPT with PRPP and ATP as substrates to synthesize NMN. Shenzhen Bangtai's patent WO2017185549 discloses a method for first synthesizing R5P using ribokinase RK with ribose and ATP as substrates, and then synthesizing NMN using a method similar to Metro Biotech's patent.

[0008] However, using ribose, sodium hexametaphosphate, and ATP as substrates, NMN is synthesized under the catalysis of four enzymes: ribokinase RK, PRPP synthase RPPK, nicotinamide phosphoribosyltransferase NAMPT, and polyphosphokinase PPK. Among them, polyphosphokinase PPK plays a role in consuming sodium hexametaphosphate to regenerate ATP and significantly reducing its dosage (see reaction formula). Figure 1 This route involves a long reaction process and many types of enzymes, resulting in poor production stability after scale-up. Furthermore, it is limited by the low activity of NAMPT, leading to low product concentration.

[0009] An earlier method used NR, sodium hexametaphosphate, and ATP as substrates to synthesize NMN under the catalysis of nicotinamide ribokinase NRK and polyphosphokinase PPK (see reaction formula). Figure 2 The poor expression and thermal stability of eukaryotic NRK in Escherichia coli limit its industrial application. Summary of the Invention

[0010] In view of this, the present invention provides an enzyme for catalyzing high-yield nicotinamide mononucleotides and its application. The enzyme has high thermal stability and promotes high yield of the product generated by the reaction.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0012] This invention provides a mutant of nicotinamide ribokinase derived from the wild type of the flying fox (Pteropus alecto), with the mutation site E91P.

[0013] In some specific embodiments of the present invention, the nicotinamide ribokinase derived from foxes has the sequence shown in SEQ ID NO:7.

[0014] In some specific embodiments of the present invention, the nicotinamide ribokinase derived from foxes is encoded by the gene shown in SEQ ID NO:8.

[0015] In some specific embodiments of the present invention, the above-mentioned mutants further have any of the following mutation sites:

[0016] (i)D109Y; or

[0017] (ii)G87A; or

[0018] (iii)G87A and D109Y.

[0019] The present invention also provides a nucleic acid molecule having a nucleotide sequence encoding the above-mentioned mutant.

[0020] The present invention also provides an expression vector that expresses the above-mentioned mutant or contains the above-mentioned nucleic acid molecule.

[0021] The present invention also provides a cell comprising the above-described nucleic acid molecule or the above-described expression vector.

[0022] This invention also provides the application of the above mutant in the preparation of nicotinamide mononucleotide.

[0023] This invention also provides a method for preparing nicotinamide mononucleotide, comprising:

[0024] (a) The substrate and acceptable excipients and auxiliaries are mixed with the above mutant and reacted to obtain nicotinamide mononucleotide; or

[0025] (b) The substrate and acceptable excipients and auxiliaries are mixed with crude enzyme solution and reacted to obtain nicotinamide mononucleotide;

[0026] The crude enzyme solution contains the aforementioned mutant.

[0027] In some specific embodiments of the present invention, the above preparation method includes: mixing nicotinamide ribochloride, sodium hexametaphosphate, magnesium chloride, ATP-Na2 and water, adjusting the pH to 5.8, 5.9, 6.0, 6.1 or 6.2, and the temperature to 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C, and then mixing with crude enzyme solution I containing the above mutant and crude enzyme solution II containing ChPPK2 to obtain a mixture, reacting to obtain nicotinamide mononucleotide;

[0028] The concentration of nicotinamide ribochloride in the mixture is 580 mM, 585 mM, 590 mM, 595 mM, 600 mM, 610 mM, 615 mM or 620 mM;

[0029] The concentration of sodium hexametaphosphate in the mixture is 82 mM, 83 mM, 84 mM, 85 mM, 86 mM or 87 mM;

[0030] The concentration of magnesium chloride in the mixture is 58 mM, 59 mM, 60 mM, 61 mM or 62 mM;

[0031] The concentration of ATP-Na2 in the mixture is 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM or 2.5 mM;

[0032] The ChPPK2 content in the mixture is 7.0 U / mL, 7.05 U / mL, 7.1 U / mL, 7.15 U / mL or 7.2 U / mL, and it is a polyphosphoric acid kinase derived from Cytophaga hutchinsonii, whose amino acid sequence can be obtained by translating the nucleotide sequence shown in SEQ ID NO:4;

[0033] The mutant content in the mixture is 9.0 U / mL, 9.05 U / mL, 9.1 U / mL, 9.15 U / mL, 9.2 U / mL, 9.25 U / mL, 9.3 U / mL, 9.35 U / mL, or 9.4 U / mL.

[0034] In some specific embodiments of the present invention, the preparation method of the crude enzyme solution I in the above preparation method includes:

[0035] (I) The above nucleic acid molecules are linked to an expression vector and introduced into cells, induced to express, and then broken to obtain crude enzyme solution;

[0036] (II) The above expression vector was introduced into cells, expression was induced, and the cells were broken to obtain crude enzyme solution;

[0037] (III) Culture the above cells, induce expression, break them up, and obtain crude enzyme solution.

[0038] The thermostability-modified nicotinamide ribokinase NRK03-G87A / E91P / D109Y of this invention can withstand 50°C heat shock and is suitable for completing a high substrate concentration of 600mM at 45°C, generating NMN at 184g / L, which is the highest reported, thus reducing manufacturing costs in the production process. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0040] Figure 1 This illustrates the existing reaction route for synthesizing NMN;

[0041] Figure 2 This illustrates the existing reaction route for synthesizing NMN;

[0042] Figure 3 Electrophoresis results of NRK01-NRK06 and hNRK1 proteins expressed in shake flasks;

[0043] Figure 4 Electrophoresis results of AtRK, PcRPPK, XccNAMPT and ChPPK2 proteins expressed in shake flasks. Detailed Implementation

[0044] This invention discloses an enzyme for high-yield nicotinamide mononucleotide (NMN) catalysis and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0045] The Uniprot accession number for the ribokinase AtRK derived from Arabidopsis thaliana is A1A6H3, which is a truncated form (Δ1-47) in this application;

[0046] Nucleotide sequence (codon-optimized) of ribokinase AtRK derived from Arabidopsis thaliana:

[0047]

[0048] The Uniprot accession number for the PRPP synthase PcRPPK derived from the archaea Pyrobaculum calidifontis is A3MV85.

[0049] Nucleotide sequence (codon optimized) of the PRPP synthase PcRPPK from the archaea Pyrobaculum calidifontis:

[0050] ATGGATAAAATTAATTCGCGCGCACTTTACACGCCAATGCACATTTTGACATTTCAGAACGCTCTGGATATTGCTGAACATTTTGAGGGATTAGGCAAGGTTGTCCAGGTGGAGGAACGCACTTTCCCTGATGGGGAAGTCTTAGTGCGTGTCCCGGAGGCAGGCCCCGTGGTAGTCTTAGTAGCGCGCTTATATCCGGGCGTAAACGATTCAGTGTTTAAGCTTTTCTTGGCTTTGGACGCTCTGAACGATATGGGTGTTGGGCGCGTCGTAGTGGTGGCTCCTTATCTTCCGTATGCACGTCAAGACCGCCGCTTTCGTCCCGGAGAACCTATTTCGGCAAAAGCTTTGCTGAAAACTTTGGCGAATCTTAGCGTGGGCGCGTTAGTTGCGGTCGACCTGCACAAACCGTATATCGCAGATTACGTTCCGCGCGTCGCTGTTCGCAATGTATACCCCGCCGAGGAGTTCGCTGAGCGCCTGAAGGGAGTTGACGCTGTCGTGTCTCCTGACTTTGGCTCTTTACATCGTGCAGAAGCCGTGGCTCGTATCTTGGGTGTCCCTTATACCTATTTTGAAAAGTATCGTGATCGTGAAACTGGCGCTATCACCCTTATGCCACGCCGCGACCTTGAATTACGTGGGGCCCGCGTCGCAATCGTGGATGATATCCTTTCAACCGGTGGTACTCTGGTAGACGCTTGCAAAGCGGCACGCACGCTTGGAGCCTCGGAAGTGTACGCTGCGGTGACTCATTGCCAGTTGCTGAAGGACGCGCGCGAAAAAGCTAAGTCTTGTGTCGACCGCTTAATTTGTACTGATAGTATTCTTAATGAGTTCGCTGAAGTTAAAGTGGGACCCCTGTTACGCCGTGAAGTTGAGAAGCTTCTTTAA(SEQ ID NO:2);

[0051] The Uniprot accession number for the nicotinamide phosphoribosyltransferase XccNAMPT derived from Xanthomonas campestris pv. campestris is A0A0H2X5R2.

[0052] The nucleotide sequence (codon-optimized) of the nicotinamide phosphoribosyltransferase XccNAMPT from Xanthomonas campestris pv. campestris:

[0053]

[0054] The polyphosphoric acid kinase ChPPK2 derived from Cytophaga hutchinsonii has the Uniprot accession number A0A6N4SMB5, ​​and is a truncated form (Δ285-305) in this application;

[0055] Nucleotide sequence (codon optimized) of ChPPK2, a polyphosphoric acid kinase derived from Cytophaga hutchinsonii:

[0056] ATGGCCACCGACTTCTCGAAGCTGTCCAAATATGTAGAGACCCTGCGTGTTAAACCGAAACAGAGTATTGACTTGAAAAAAGATTTTGATACTGACTACGATCATAAAATGTTAACAAAAGAAGAAGGCGAGGAGCTGTTGAATCTGGGTATTTCCAAGCTGTCAGAGATTCAAGAGAAACTTTACGCGAGTGGAACGAAGTCTGTCCTGATCGTGTTTCAAGCGATGGATGCCGCGGGAAAGGACGGCACTGTTAAGCACATCATGACAGGGTTGAACCCACAGGGAGTGAAGGTGACCAGCTTTAAGGTACCTTCCAAGATTGAACTGAGCCACGATTATTTGTGGCGTCATTATGTAGCATTACCCGCAACGGGTGAGATCGGAATTTTTAATCGCTCCCATTACGAGAACGTGTTGGTGACTCGCGTCCACCCCGAATACCTGCTGAGCGAGCAAACCTCAGGGGTGACGGCAATTGAACAAGTTAATCAAAAATTCTGGGACAAGCGCTTTCAGCAGATCAATAATTTTGAGCAGCATATCAGTGAAAACGGAACGATTGTCTTGAAGTTCTTCCTGCATGTGTCAAAGAAGGAACAGAAGAAGCGCTTTATCGAACGCATTGAACTTGACACGAAAAATTGGAAATTCTCAACCGGCGATCTTAAAGAACGTGCTCATTGGAAGGACTATCGCAACGCTTACGAGGATATGTTGGCCAACACAAGCACTAAGCAGGCCCCGTGGTTCGTTATTCCCGCCGATGACAAGTGGTTTACACGCCTTTTGATCGCTGAGATCATTTGCACTGAATTAGAGAAGCTTAATCTTACTTTTCCTACGGTATCGTAA(SEQ ID NO:4);

[0057] Nucleotide sequence (codon-optimized) of nicotinamide riboside kinase NRK01 derived from Xenopus tropicalis:

[0058] ATGATGAAGCAATTTATTATTGGTATTTCCGGCATTACCAACGGAGGCAAGACAACACTGGCAAATCGTTTGTTGAAGCTGCTGCCAAACTGTAGTCTTATTTGCCAGGATGACTACTTCAAACCCGATTCTGATATTGAAACGGATGAGAATGGTTTTAAACAATACGACACCATTGAAGCGCTTGACATGGAAACTATGATCAAAGCCGTCCATTCCTGGATCAAGTTGTCTCAGGATGTGCTTGCTATGGAAGAAAAAAAGGAGATGTGTTCGACTTGTGAGGAAAAAGCGTACTTTCTTATCGTGGAGGGCTTCTTACTGTATCACTACAAGCCCCTTGAAAACGTACTTAATCGTAAATACTTCTTGTCTATTCCTTATGAAGAATCTAAACAGCGTCGCCGCCGTATTTACAATCCCCCGGACCCGCCAGGATACTTCGATGGTCATGTGTGGCCCATGTTTCTTAAACATAAGAAAGAAATGGAGGAGACTCACTCTGACATCGTCTACCTGGATGGGACGAAATCCGAAGATGAGATCCAGTCCCTTGTCTACTCAGACATCATCTCGTCCTTCTCGATCCATAAGTAA(SEQID NO:5);

[0059] Nucleotide sequence (codon-optimized) of nicotinamide riboside kinase NRK02 from the naked mole rat Heterocephalus glaber:

[0060] ATGAAGACTTTCGTAATCGGCATTGGCGGTGTAACAAACGGCGGAAAAACAACCCTTGCAAAAAATCTTCAAAAACATTTGACAAATTGCTCTGTTATTTCCCAAGACGACTTTTTTAAACCCGAGTCCGAGATCGAAAAAGACAAGAATGGTTTCCTGCAGTACGATGTCCTTGAAGCACTTAACATGGAGAAGATGATGGCCGCCATCTCGTGTTGGATGGAGAACCCAGGCCATTGCTTAGGCTCAACCGGGTCCCGCACTGAAGAGATTCCCATCTTGATTATCGAAGGCTTTCTGTTGTTCAACTACAAACCCTTAGACACTATTTGGAACCGTTCGTATTTCCTTACCATCCCCTATGAAGAATGTAAACGCCGTCGTTCCACCCGTGTTTATGAACCACCAGACCCTCCAGGCTATTTCGATGGGCACGTATGGCCAATGTATCTTAAACATCGCCGTGAGATGGACAGTGTGACCTGTGAGGTTGTTTATTTAGACGGAACTCGCTCGGAGGAGGATTTGTTCTTACAGGTTTATGAAGATTTAACCCAAGAACTTGCGAATCAGTCCGCTAGTACAATGGTAGCAAATGGTATGGTTAAAAATGGCGAGGCAGGTAAGTAA(SEQ ID NO:6);

[0061] Amino acid sequence of nicotinamide riboside kinase NRK03 derived from the flying fox Pteropus alecto:

[0062] MKTFIIGISGVTNGGKTTLAKNLQKHLPNCCVISQDDFFKPESEIEIDENGFLQYDVLEALNMEKMMSTISSW IESPSHSLVSTDQGNAEEIPILIIEGFLLFNYKPLDTIWNRSYFLTIPYEECKKRRSTRVYKPPDPPGYFDGHVWPM YLKHRREMEDITWEIVYLDGTKSEEELFSQVYEDLRQELAKRKY(SEQ ID NO:7);

[0063] Nucleotide sequence (codon-optimized) of nicotinamide ribokinase NRK03 derived from the flying fox Pteropus alecto:

[0064] (SEQ ID NO:8);

[0065] Nucleotide sequence (codon optimized) of nicotinamide ribokinase NRK04 from cynomolgus monkey Macaca fascicularis:

[0066] ATGAAAACATTCATTGTAGGTATCTCCGGCGTGACTAACGGCGGGAAAACCACGTTAGCGAAGAATCTTCAGAAACACCTTCCCAATTGTAGTGTGATTAGTCAGGATGACTTTTTTAAGCCAGAGAGCGAGATCGAGACTGACAAGAACGGGTTTCTTCAGTATGATGTTTTGGAAGCCCTTAATATGGAAAAAATGATGTCCACCATCTCATGTTGGATGGAATCCGCACGTCGTTCTGTGGTATCCACTGACCGCGAATCTGCCGAGGAGATTCCCATCCTTATTATCGAGGGTTTCCTGTTGTTCAACTACAAGCCCTTAGACACCATCTGGAATCGTTCATATTTCCTGACTATTCCCTACGAAGAATGCAAACGTCGTCGCAGCACGCGTGTCTATGAGCCTCCCGACTCCCCAGGATACTTCGATGGTCATGTGTGGCCGATGTACTTGAAACATCGTCAGGAAATGCAAGATATTACCTGGGAGGTAGTCTACTTGGATGGCACCAAGTCGGAAGAGGATCTTTTTTTGCAAGTGTACGAGGACTTAATCCAAGAATTGGCGAAACAGAAATGTTTGCAGGTCACCGCCTAA(SEQ ID NO:9);

[0067] Nucleotide sequence (codon-optimized) of nicotinamide riboside kinase NRK05 from the Arctic ground squirrel Urocitellus parryii:

[0068] ATGAAAACCTTCGTCATCGGTATCGGCGGAGTCACCAACGGAGGTAAAACCACATTAGCGAAAAACTTACAGAAGCACCTGCCAAATTGCAGCGTTATCAGTCAGGACGACTTCTTTAAACCCGAGTCTGAGGTGGAACGCGACAAGAACGGTTTCCTTCAGTATGACGTATTGGAGGCTTTGAATATGGAGGAAATGATGTCTGCCATCTCATGCTGGATGGAATCGCCAGGTCATTCTGTCGAATCTACTGATGGCACGTCAGCGGAAGAGATTCCCATTTTGATTATTGAGGGTTTCTTGTTATTCAACTACCGTCCGCTTGACACCGTGTGGAATCGCAGCTACTTTTTAACAATTCCGTATGAGGAGTGCAAACGCCGCCGCTCGACCCGTGTGTACAAACCTCCTGACCCACCGGGGTATTTCGACGGCCATGTCTGGCCTATGTACTTGAAATACCGTCGTGAGATGGAGGACATTACACGTGAGATCGTCTATCTGGATGGAACCAAGCCCGAGGAAGACTTGTTCGCTCAAGTCTACGAGGATGTTATTCAACAGCTTGCCAAGCAAAAGTGTTTGCAAGTCACAGCGTAA(SEQ ID NO:10);

[0069] Nucleotide sequence (codon-optimized) of nicotinamide riboside kinase NRK06 derived from Physeter macrocephalus:

[0070] ATGAAAACCTTCGTGATCGGTATTGGCGGGGTGACCAACGGTGGCAAAACTACTTTGGCTAAGAATCTGCAGAAACGCCTGCCCAACTGTAGCATCATTTCCCAAGACAACTTTTTCAAGCCGGAGAGTGAAATCGAAACGGACGAGAACGGTTTCCTTCAGTACGACGTGCTGGAAGCGTTAAATATGGAGGAGATGATGTCGGCCATTTCCTGCTGGATGGAGTCGGCGGCTCACCCCTTGGCGAGCACGGATCGTGGAAATACAGAAGAGATTCCGATCTTGATCATCGAAGGATTCTTACTGTATAACTATAAGCCATTAGACACAATCTGGAACCGCTCATATTTTCTTACCATTCCCTATGAAGAATGTAAGCGCCGCCGTTCGACTCGTATTTACGAGCCTCCCGACACCCCTGGCTATTTTGAAGGACACGTCTGGCCTATGTATCTTAAGCATCGCAAAGAGATGGAGAACATCTCGTGGGAAATTGTCTACCTTGACGGGACGAAGAGTGAGGAAGATCTGTTTAGTCAGGTGTACGAAGATCTGATCCAGGAATTAGCCAAGCAAAAGTGTCTTCAAGTGACAGCTTAA(SEQ ID NO:11);

[0071] Nucleotide sequence (codon-optimized) of nicotinamide riboside kinase hNRK1 derived from Homo sapiens:

[0072] (SEQ ID NO:12).

[0073] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0074] The present invention will be further illustrated below with reference to the embodiments.

[0075] Example 1: Screening for Nicotinamide Ribokinase (NRK)

[0076] In UniProt's BLAST module, the amino acid sequence of human nicotinamide ribokinase hNRK1 was input to retrieve homologous sequences. The sequences listed in Table 1 were selected and codon optimized to obtain the corresponding nucleotide sequences. The whole gene was then synthesized and constructed on the pET-28a vector.

[0077] Table 1

[0078] UniProt ID. Species origin enzyme name A0A6I8QYR6 African clawed frog Xenopus tropicalis NRK01 G5BHT8 Naked mole rat Heterocephalus glaber NRK02 L5K6B5 Flying Fox (Pteropus alecto) NRK03 A0A2K5TY09 Crab-eating macaques (Macaca fascicularis) NRK04 A0A8D2GN53 Arctic ground squirrel Urocitellus parryii NRK05 A0A2Y9T1G6 sperm whale Physeter macrocephalus NRK06 Q9NWW6 Homo sapiens hNRK1

[0079] The obtained plasmids were transformed into BL21(DE3) competent cells and cultured to 200 mL LB liquid medium (Kana). Expression was induced at 20℃ with 0.1 mM IPTG. Cells were collected by low-temperature centrifugation, and resuspended in lysis buffer (50 mM K₂HPO₄·3H₂O, 5 mM KH₂PO₄, 100 mM NaCl, pH 7.6) at a ratio of 1 g: 4 mL wet cells. The cells were then sonicated to obtain the corresponding crude enzyme solution. Protein electrophoresis analysis of expression results is shown below. Figure 3 NRK01 and NRK06 were expressed at low levels, while NRK02, NRK04 and NRK05 were expressed in the precipitate, and only NRK03 and hNRK were expressed in the supernatant.

[0080] Weigh 1.32 g of ATP-Na2 (120 mM, 20 mL of 2× reaction solution), 0.58 g of nicotinamide ribochloride (100 mM), and 0.33 g of magnesium chloride hexahydrate (80 mM) and dissolve them in 16 mL of pure water. Adjust the pH to 6.0 and bring the volume to 20 mL. Add 800 μL of pure water and 1000 μL of 2× reaction solution to 10 mL centrifuge tubes. After preheating at 38 °C, add 200 μL of crude enzyme solution of NRK01-06 / hNRK1 and incubate for 15 min. Take 50 μL of the sample, quench it with 950 μL of 25 mM hydrochloric acid, and then perform high performance liquid chromatography (HPLC) to detect the amount of NMN produced and calculate the enzyme activity. Referring to Table 2, only NRK03 and hNRK1 have the activity to synthesize NMN, and the enzyme activity of NRK03 is higher than that of hNRK1.

[0081] Table 2

[0082]

[0083]

[0084] Example 2: Synthesis of NMN using NR as a substrate

[0085] NRK03 glycerol bacteria were cultured to 2000 mL of TB liquid medium (Kana) and induced to express the enzyme at 20℃ under 0.1 mM IPTG conditions. The bacterial cells were collected by low-temperature centrifugation, and resuspended in lysis buffer (50 mM K2HPO4·3H2O, 5 mM KH2PO4, 100 mM NaCl, pH 7.6) at a ratio of 1 g: 4 mL of wet bacterial cells. The cells were then sonicated to obtain the corresponding crude enzyme solution.

[0086] Weigh out 8.72 g of nicotinamide ribochloride (300 mM, reaction volume 100 mL), 5.20 g of sodium hexametaphosphate (85 mM), 1.22 g of magnesium chloride hexahydrate (60 mM), and 0.11 g of ATP-Na2 (2 mM) and dissolve them in 80 mL of pure water. Adjust the pH to 6.0 and bring the volume to 95 mL. After preheating at 38 °C, add 4 mL of crude NRK03 enzyme solution (567.4 U / mL) and 1 mL of crude ChPPK2 enzyme solution (669 U / mL) to start the reaction.

[0087] Every 0.5 hours, 20 μL of sample was taken and quenched with 980 μL of 25 mM hydrochloric acid. The NMN production was then detected by high performance liquid chromatography (HPLC), and the conversion rate was calculated. Referring to Table 3, 278.4 mM of NMN was generated after 2 hours of reaction, with a final product concentration of 93 g / L.

[0088] Table 3

[0089] reaction time Conversion rate 0.5h 18.27％ 1h 55.97％ 1.5h 88.81％ 2h 92.80％ 2.5h 93.25％

[0090] Example 3: Thermal stability modification of nicotinamide ribokinase NRK

[0091] To further increase the product concentration, the energy changes of the NRK03 point mutation were calculated using the thermostable protein design tools FireProt and Pythia. Point mutations with lower ΔΔG values ​​were selected from those shown in Table 4, and primers were designed to construct the mutant plasmid using the NRK03 nucleotide sequence as a template and the QuickChange site-directed mutagenesis kit (Agilent).

[0092] The obtained plasmids were transformed into BL21(DE3) competent cells and cultured to 200 mL LB liquid medium (Kana). Expression was induced at 20℃ with 0.1 mM IPTG. The cells were collected by low-temperature centrifugation, and resuspended in lysis buffer (50 mM K2HPO4·3H2O, 5 mM KH2PO4, 100 mM NaCl, pH 7.6) at a ratio of 1 g: 4 mL of wet cells. The cells were then sonicated to obtain the corresponding crude enzyme solution.

[0093] Table 4

[0094]

[0095]

[0096] Using the same activity measurement method as in Example 1, the enzyme activity of crude enzyme solution and the enzyme activity of crude enzyme solution after incubation at 50℃ for 10 min were measured in parallel for two groups, and the average value was taken. See Table 5.

[0097] Table 5

[0098] mutant Normal enzyme activity (U / mL) Enzyme activity (U / mL) after incubation at 50℃ for 10 min wild type 177.8 0.8 D109M 279.0 4.1 D109Y 320.3 0.4 E91P 905.8 51.2 E91I 310.4 1.1 Q180M 192.7 0.6 Q180W 193.8 0.1 S71K 141.4 0.3 S71I 129.7 0.2 G87A 229.7 5.1 G87W 216.6 3.8

[0099] Based on the NRK03-E91P mutant, other mutation sites were added, and the mutant was constructed, cultured, expressed, and subjected to cell disruption and activity testing in the same manner as described above. The results are shown in Table 6, with NRK03-G87A / E91P / D109Y being the optimal mutation.

[0100] Table 6

[0101] mutant Normal enzyme activity (U / mL) Enzyme activity (U / mL) after incubation at 50℃ for 10 min E91P / D109M 724.4 239.9 E91P / D109Y 1599.4 1573.4 E91P / G87A 1391.9 1285.7 E91P / G87W 416.5 76.3 G87A / E91P / D109Y 2174.6 2110.4

[0102] Example 4: Synthesis of high concentrations of NMN using NR as a substrate

[0103] NRK03-G87A / E91P / D109Y glycerol bacteria were cultured in 2000 mL of TB liquid medium (Kana) and induced to express the enzyme at 20℃ under 0.1 mM IPTG conditions. The cells were collected by low-temperature centrifugation, and resuspended in lysis buffer (50 mM K2HPO4·3H2O, 5 mM KH2PO4, 100 mM NaCl, pH 7.6) at a ratio of 1 g: 4 mL of wet cells. The cells were then sonicated to obtain the corresponding crude enzyme solution.

[0104] Weigh out 17.5 g of nicotinamide ribochloride (600 mM, reaction volume 100 mL), 5.20 g of sodium hexametaphosphate (85 mM), 1.22 g of magnesium chloride hexahydrate (60 mM), and 0.11 g of ATP-Na2 (2 mM) and dissolve them in 80 mL of pure water. Adjust the pH to 6.0 and bring the volume to 95 mL. After preheating at 45 °C, add 4 mL of crude enzyme solution of NRK03-G87A / E91P / D109Y (2308.4 U / mL) and 1 mL of crude enzyme solution of ChPPK2 (710.3 U / mL) to start the reaction. At 0.5 h and 1 h of reaction time, add 2.6 g of sodium hexametaphosphate each time.

[0105] Every 0.5 hours, 20 μL of sample was taken and quenched with 980 μL of 25 mM hydrochloric acid. The NMN production was then detected by high performance liquid chromatography (HPLC), and the conversion rate was calculated. Referring to Table 7, 549.7 mM of NMN was generated after 2.5 hours of reaction, with a final product concentration of 184 g / L.

[0106] Table 7

[0107] reaction time Conversion rate 0.5h 21.97％ 1h 49.46％ 1.5h 83.54％ 2h 87.44％ 2.5h 91.62％

[0108] Comparative example: Synthesis of NMN using ribose as a substrate

[0109] Ribokinase AtRK (Δ1-47) from Arabidopsis thaliana, PRPP synthase PcRPPK from Pyrobaculum calidifontis, nicotinamide phosphoribosyltransferase XccNAMPT from Xanthomonas campestris pv. campestris, and polyphosphoric acid kinase ChPPK2 (Δ285-305) from Cytophaga hutchinsonii were selected. Codon optimization was performed on Escherichia coli and Saccharomyces cerevisiae using the Optipyzer online codon optimization tool. The optimized nucleotide sequences were synthesized and constructed into the pET-28a vector.

[0110] The obtained plasmids were transformed into BL21(DE3) competent cells and cultured to 2000 mL LB liquid medium (Kana). Expression was induced at 20℃ with 0.1 mM IPTG. Cells were collected by low-temperature centrifugation, and resuspended in lysis buffer (50 mM K₂HPO₄·3H₂O, 5 mM KH₂PO₄, 100 mM NaCl, pH 7.6) at a ratio of 1 g: 4 mL of wet cells. The cells were then sonicated to obtain the corresponding crude enzyme solution. Protein expression was detected by electrophoresis (see [link to relevant documentation]). Figure 4 All of these are expressed in the Shangqing.

[0111] Weigh 1.50 g of ribose (100 mM, reaction volume 100 mL), 5.20 g of sodium hexametaphosphate (85 mM), 1.22 g of magnesium chloride hexahydrate (60 mM), and 0.06 g of ATP-Na2 (1 mM) and dissolve them in 80 mL of pure water. Adjust the pH to 7.0 and bring the volume to 90 mL. After preheating at 38 °C, add 2 mL of AtRK crude enzyme solution (1508 U / mL), 2 mL of PcRPPK crude enzyme solution (1025 U / mL), 5 mL of XccNAMPT crude enzyme solution (61 U / mL), and 1 mL of ChPPK2 crude enzyme solution (669 U / mL) to start the reaction.

[0112] Separately dissolve 2.25g of ribose (150mM, reaction volume 100mL), 7.65g of sodium hexametaphosphate (125mM), 1.63g of magnesium chloride hexahydrate (80mM), and 0.06g of ATP-Na2 (1mM) in 75mL of pure water. Adjust the pH to 7.0 and bring the volume to 85mL. After preheating at 38℃, add 2mL of AtRK crude enzyme solution (1508U / mL), 2mL of PcRPPK crude enzyme solution (1025U / mL), 10mL of XccNAMPT crude enzyme solution (61U / mL), and 1mL of ChPPK2 crude enzyme solution (669U / mL) to start the reaction simultaneously.

[0113] Every hour, 50 μL of sample was taken, quenched with 950 μL of 25 mM hydrochloric acid, and then analyzed by high-performance liquid chromatography (HPLC) to determine the amount of NMN produced and calculate the conversion rate. Referring to Table 8, the 100 mM ribose group produced 83.96 mM of NMN after 4 hours of reaction. However, as the ribose concentration increased to 150 mM, only 89.96 mM of NMN was produced, and the conversion rate decreased from 83.96% to 59.97%. This indicates that the route for synthesizing NMN using ribose as a substrate may experience product inhibition, preventing further increases in product concentration.

[0114] Table 8

[0115]

[0116] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A wild-type mutant of nicotinamide ribokinase derived from the fox (Pteropus alecto), characterized in that, It has the mutation site E91P.

2. The mutant as described in claim 1, characterized in that, It also has a mutation site that contains any of the following: (i)D109Y; or (ii)G87A; or (iii)G87A and D109Y.

3. A nucleic acid molecule, characterized in that, It has a nucleotide sequence that encodes the mutant of claim 1 or 2.

4. An expression carrier, characterized in that, Expressing the mutant of claim 1 or 2 or comprising the nucleic acid molecule of claim 3.

5. A cell, characterized in that, It comprises the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. The use of the mutant as described in claim 1 or 2 in the preparation of nicotinamide mononucleotide.

7. A method for preparing nicotinamide mononucleotide, characterized in that, include: (a) Mixing the substrate and acceptable excipients and auxiliaries with the mutant of claim 1 or 2, reacting to obtain nicotinamide mononucleotide; or (b) The substrate and acceptable excipients and auxiliaries are mixed with crude enzyme solution and reacted to obtain nicotinamide mononucleotide; The crude enzyme solution contains the mutant described in claim 1 or 2.

8. The preparation method according to claim 7, characterized in that, include: Nicotinamide ribochloride, sodium hexametaphosphate, magnesium chloride, ATP-Na2 and water were mixed, and the pH was adjusted to 5.8-6.2 and the temperature to 40-50°C. The mixture was then mixed with crude enzyme solution I containing the mutant described in claim 1 or 2 and crude enzyme solution II containing ChPPK2 to obtain a mixture. The mixture was then reacted to obtain nicotinamide mononucleotide. The concentration of nicotinamide ribochloride in the mixture is 580–620 mM; The concentration of sodium hexametaphosphate in the mixture is 82–87 mM; The concentration of magnesium chloride in the mixture is 58–62 mM; The concentration of ATP-Na2 in the mixture is 1.5–2.5 mM; The ChPPK2 content in the mixture is 7.0-7.2 U / mL, and it is a polyphosphoric acid kinase derived from Cytophaga hutchinsonii; The mutant content in the mixture is 9.0–9.4 U / mL.

9. The preparation method according to claim 8, characterized in that, The pH is 6.0; The temperature is 45°C; The concentration of nicotinamide ribochloride in the mixture is 600 mM; The concentration of sodium hexametaphosphate in the mixture is 85 mM; The concentration of magnesium chloride in the mixture is 60 mM; The concentration of ATP-Na2 in the mixture is 2 mM; The content of ChPPK2 in the mixture was 7.103 U / mL; The mutant was present in a concentration of 92.336 U / mL in the mixture.

10. The preparation method according to claim 8, characterized in that, The preparation method of the crude enzyme solution I includes: (I) The nucleic acid molecule described in claim 3 is linked to an expression vector and introduced into cells, induced to express, and then broken to obtain a crude enzyme solution; (II) The expression vector according to claim 4 is introduced into cells, induced to express, and then broken to obtain crude enzyme solution; (III) Culturing the cells described in claim 5, inducing expression, cleaving, and obtaining crude enzyme solution.

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