Nicotinamide mononucleotide adenylyltransferase nmnat mutant and application thereof

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a nicotinamide mononucleotide adenosine transferase NMNAT mutant and its role in the synthesis of nicotinamide adenine dinucleotide (NAD). + The mutant is obtained by mutating at least one amino acid from the following positions: Gly (position 10), His (position 16), Ile (position 38), His (position 45), Ser (position 83), Thr (position 85), Trp (position 86), Ile (position 105), Ile (position 106), Gln (position 108), Ser (position 110), Thr (position 116), Asn (position 118), Cys (position 132), Arg (position 133), Phe (position 177), and Ile (position 179). Compared to wild-type nicotinamide mononucleotide adenosyltransferase, the NMNAT mutant of nicotinamide mononucleotide adenosyltransferase described in this invention exhibits higher catalytic efficiency, and can be used for whole-cell catalytic synthesis of nicotinamide adenine dinucleotide (NAD). + The reaction synthesis can be completed in just 4 hours, with a conversion rate of over 99%.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a nicotinamide mononucleotide adenosine transferase NMNAT mutant and its role in the synthesis of nicotinamide adenine dinucleotide (NAD). + Applications in ). Background Technology

[0002] Nicotinamide adenine dinucleotide (NAD) + NAD+ is an indispensable coenzyme in living organisms, widely involved in key physiological processes such as cellular energy metabolism, redox reactions, DNA repair, and signal transduction. Studies have shown that NAD+... + NAD+ levels are closely related to cellular senescence, neurodegenerative diseases, and metabolic disorders, possessing important physiological functions and potential applications. In the industrial field, NAD+... + As NAD + Essential cofactors for dependent enzymes (such as amino acid dehydrogenases and ketone reductases) are widely used in the biocatalytic synthesis of chiral drugs, high-value chemicals and food additives, and their market demand is growing.

[0003] Currently, NAD + The main production methods for NAD+ include chemical synthesis, microbial extraction, and enzymatic catalysis. Chemical synthesis involves cumbersome steps, demanding reaction conditions, and difficult separation and purification, and it uses large amounts of organic reagents, resulting in significant environmental pollution and high costs. Extraction methods from microorganisms such as yeast are mature, but they suffer from high energy consumption, low production efficiency, and difficulty in guaranteeing product purity, making them unsuitable for large-scale industrial production. In contrast, enzymatic catalysis uses nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP) as substrates to synthesize NAD+ under the catalysis of nicotinamide mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1). + It has significant advantages such as strong reaction specificity, mild conditions, few by-products, and environmental friendliness, making it a more promising green production process.

[0004] However, the industrial application of enzymatic catalysis still faces a core bottleneck: the catalytic efficiency, substrate tolerance, and stability of natural NMNAT are often insufficient, making it difficult to achieve efficient conversion at high substrate concentrations, resulting in high production costs. For example, the enzymatic synthesis of NAD reported in the prior art (CN103710321A) +The concentration of its substrate NMN is typically low (e.g., about 1.6 g / L), far below the high concentrations (above 100 g / L) required for ideal industrial production. Therefore, modifying NMNAT through protein engineering to obtain mutants with higher enzyme activity, stronger substrate tolerance, and better stability is crucial for enhancing NAD+ production. + Improving synthesis efficiency, reducing production costs, and promoting its industrial application are crucial.

[0005] In recent years, studies have explored the modification of NMNAT using techniques such as site-directed mutagenesis. For example, Chinese patent CN112574970A significantly improved enzyme activity by mutating specific sites (such as T59, Y119, and K149) of NMNAT derived from *Methanococcus maripaludis*. Chinese patent CN115873820A obtained a mutant capable of tolerating high concentrations of NMN substrate (100 g / L) through multi-site combined mutations of NMNAT derived from *Staphylococcus aureus*. Chinese patent CN119639715A achieved a mutant with significantly improved catalytic efficiency through directed evolution of NMNAT derived from *Methanococcus thermautotrophicus*. These studies demonstrate that rational design and directed evolution of NMNAT are effective ways to overcome its catalytic performance limitations.

[0006] Nevertheless, the catalytic efficiency, conversion capacity at high substrate concentrations, and overall performance suitable for industrial production of existing NMNAT mutants still need further improvement. In particular, the modification potential and application value of NMNAT derived from common industrial host bacteria such as *Escherichia coli* have not been fully explored. Therefore, developing novel NMNAT mutants with higher catalytic activity and better process adaptability is crucial for achieving NAD+ production. + The efficient and low-cost enzymatic synthesis method has important practical significance and application value. Summary of the Invention

[0007] The purpose of this invention is to provide a nicotinamide mononucleotide adenosine transferase NMNAT mutant and its role in the synthesis of nicotinamide adenine dinucleotide (NAD). + This technology is applied to address the problems of low enzyme activity and low catalytic efficiency of wild-type nicotinamide mononucleotide adenosine transferase, which makes it difficult to achieve efficient conversion at high substrate concentrations and leads to high production costs.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides a nicotinamide mononucleotide adenosine transferase (NMNAT) mutant, which is obtained by mutating at least one amino acid from the following positions: Gly at position 10, His at position 16, Ile at position 38, His at position 45, Ser at position 83, Thr at position 85, Trp at position 86, Ile at position 105, Ile at position 106, Gln at position 108, Ser at position 110, Thr at position 116, Asn at position 118, Cys at position 132, Arg at position 133, Phe at position 177, and Ile at position 179, based on SEQ ID NO:1.

[0010] Preferably, the mutant is based on SEQ ID NO:1, with its 10th position Gly mutated to Gly (synonymous mutation); or

[0011] Mutate its 16th His to His (synonymous mutation); or

[0012] Mutate its 38th Ile to Met; or

[0013] Mutate its 45th His position to Trp; or

[0014] Mutate its 83rd Ser position to Glu; or

[0015] Mutate its 85th Thr to Thr (synonymous mutation); or

[0016] Mutate its 86th Trp to Cys; or

[0017] Transform its 105th Ile into Leu; or

[0018] Mutate its 106th Ile to Met; or

[0019] Mutate its 108th Gln to His; or

[0020] Mutate its 108th Gln to Gly; or

[0021] Mutate its 108th Gln to Gln (synonymous mutation); or

[0022] Mutate its 108th Gln to Trp; or

[0023] Mutate its 110th Ser bit to Asp; or

[0024] Mutate its 116th Thr to Arg; or

[0025] Mutate its 118th Asn to Ala; or

[0026] Mutate its 132nd Cys position to Asn; or

[0027] Mutate its 132nd Cys position to Met; or

[0028] Mutate its 133rd Arg to Ser; or

[0029] Transform its 177th Phe into Val; or

[0030] Transform its 177th Phe into Trp; or

[0031] Transform its 179th Ile into Val; or

[0032] Transform its 179th Ile into Ile (synonymous mutation); or

[0033] Transform its 179th Ile into Met.

[0034] More preferably, the mutant is based on SEQ ID NO:1, with its 106th position Ile mutated to Met and its 10th position Gly mutated to Gly; or

[0035] Mutate its 106th Ile to Met and its 45th His to Trp; or

[0036] Mutate its 106th Ile to Met and its 85th Thr to Thr; or

[0037] Mutate its 106th Ile to Met and its 86th Trp to Cys; or

[0038] Mutate the 106th Ile to Met and the 105th Ile to Leu; or

[0039] Mutate Ile at position 106 to Met, and mutate Gln at position 108 to Gly; or

[0040] Mutate Ile at position 106 to Met, and mutate Gln at position 108 to Trp; or

[0041] Mutate Ile at position 106 to Met, and mutate Asn at position 118 to Ala; or

[0042] Mutate Ile at position 106 to Met, and mutate Cys at position 132 to Met; or

[0043] Mutate Ile at position 106 to Met, and mutate Arg at position 133 to Ser; or

[0044] Mutate Ile at position 106 to Met, and mutate Phe at position 177 to Trp; or

[0045] The 106th Ile was mutated to Met, and the 179th Ile was mutated to Met.

[0046] More preferably, the mutant is based on SEQ ID NO:1, with its 106th position Ile mutated to Met, its 86th position Trp mutated to Cys, and its 45th position His mutated to Trp; or

[0047] Mutate its 106th Ile to Met, its 86th Trp to Cys, and its 105th Ile to Leu; or

[0048] Mutate its 106th Ile to Met, its 86th Trp to Cys, and its 108th Gln to Trp; or

[0049] Mutate its 106th Ile to Met, its 86th Trp to Cys, and its 132nd Cys to Met; or

[0050] The 106th Ile was mutated to Met, the 86th Trp was mutated to Cys, and the 177th Phe was mutated to Trp.

[0051] More preferably, the mutant is based on SEQ ID NO:1, with its 106th Ile mutated to Met, its 86th Trp mutated to Cys, its 105th Ile mutated to Leu, and its 45th His mutated to Trp; or

[0052] Mutate its 106th Ile to Met, its 86th Trp to Cys, its 105th Ile to Leu, and its 108th Gln to Trp; or

[0053] Transform its 106th Ile into Met, its 86th Trp into Cys, its 105th Ile into Leu, and its 177th Phe into Trp.

[0054] The present invention also provides a gene encoding any of the above-mentioned nicotinamide mononucleotide adenosine transferase NMNAT mutants and a recombinant expression vector carrying the gene.

[0055] Furthermore, the recombinant expression vector uses pET-29a(+) as the original expression vector.

[0056] The present invention also provides recombinant microorganisms comprising the recombinant expression vector.

[0057] Preferably, the host bacterium of the recombinant microorganism is Escherichia coli, preferably Escherichia coli BL21(DE3).

[0058] Any of the above-mentioned nicotinamide mononucleotide adenosyltransferase NMNAT mutants in the production of nicotinamide adenine dinucleotide (NAD) + Applications in ).

[0059] A method for producing nicotinamide adenine dinucleotide (NAD) + The method includes the following steps:

[0060] Nicotinamide mononucleotide adenosine transferase (NMNAT) mutants or their recombinant microorganisms were added to a reaction system containing NMN, ATP, magnesium chloride hexahydrate, and manganese ions. The reaction was carried out at pH 5.0-6.0, 37°C, and 180 rpm to generate nicotinamide adenine dinucleotide (NAD). + ).

[0061] Preferably, the concentration of NMN in the reaction system is 30-50 g / L, the concentration of ATP is 80-100 g / L, the concentration of magnesium chloride hexahydrate is 200-250 mM, and the concentration of manganese ions is 2-5 mM.

[0062] The beneficial effects of this invention are as follows:

[0063] This invention utilizes directed evolution technology to modify the wild-type nicotinamide mononucleotide adenosyltransferase NMNAT (amino acid sequence shown in SEQ ID NO:1, nucleotide sequence shown in SEQ ID NO:2) derived from *Escherichia coli* (PDB protein sequence number 1k4m). The resulting mutant nicotinamide mononucleotide adenosyltransferase exhibits an enzyme activity 4.3 times higher than the wild-type nicotinamide mononucleotide adenosyltransferase. This mutant is then used for whole-cell catalytic synthesis of nicotinamide adenine dinucleotide (NAD). + This invention possesses advantages such as high conversion efficiency (achieving a conversion rate of over 99% in just 4 hours), tolerance to high substrate concentrations (120 g / L), and mild reaction conditions. This invention provides a new technical approach for the application of nicotinamide mononucleotide adenosine transferase. Attached Figure Description

[0064] Figure 1 Map of the nicotinamide mononucleotide adenosine transferase (NMNAT) expression vector.

[0065] Figure 2 This is the chromatogram of the standard sample. Detailed Implementation

[0066] The following examples are provided to further illustrate the present invention, but do not limit the invention in any way. Processes and methods not described in detail in the following examples are conventional methods known in the art, and the reagents used in the examples are commercially available or prepared by methods well known to those skilled in the art. The following examples all achieve the objectives of the present invention.

[0067] The culture media involved in the following examples are as follows:

[0068] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, sterilized at 121°C for 20 min.

[0069] LB solid medium: LB liquid medium with 2% agar added.

[0070] TB liquid culture medium: KH2PO4 2.31g / L, K2HPO4·3H2O 16.42g / L, yeast extract 24g / L, peptone 12g / L, glycerol 4g / L.

[0071] Example 1: Preparation of genetically engineered strains containing the wild-type nicotinamide mononucleotide adenosine transferase (NMNAT) gene

[0072] The nicotinamide mononucleotide adenosyltransferase mutant of the present invention was obtained by modifying the amino acid sequence of wild-type nicotinamide mononucleotide adenosyltransferase from Escherichia coli (PDB protein sequence number 1k4m) using directed evolution technology. The amino acid sequence of the wild-type nicotinamide mononucleotide adenosyltransferase is shown in SEQ ID NO:1, with a total length of 214 amino acids, and the nucleotide sequence is shown in SEQ ID NO:2, with a total length of 645 bases.

[0073] After designing primers, the gene of wild-type nicotinamide mononucleotide adenosyltransferase from *E. coli* was cloned into the pET29a vector using homologous recombination, constructing the recombinant vector pET29a-NMNAT. The NMNAT expression vector map is shown below. Figure 1 As shown in the figure; subsequently, the recombinant vector was transformed into E. coli BL21 (DE3) competent cells, plated on LB solid selective medium containing kanamycin (Kana), and incubated upside down for 12-16 h. Single colonies were picked for colony PCR identification, and the correctly identified positive transformants were the genetically engineered strains containing the wild-type nicotinamide mononucleotide adenosine transferase (NMNAT) gene.

[0074] Example 2: Construction of a unit point mutation library of the nicotinamide mononucleotide adenosine transferase (NMNAT) gene

[0075] 1. Based on the spatial structure of the wild-type nicotinamide mononucleotide adenosine transferase (NMNAT) gene and the spatial location of cofactor binding, 33 amino acid sites (F8, G9, G10, T11, F12, H16, G18, H19, P22, I38, N40, H45, R46, E76, S83, Y84, T85, W86, F104, I105, I106, G107, Q108, D109, S110, T116, W117, N118, C132, R133, R134, F177, I179) were selected for saturation mutation.

[0076] 2. Construct engineered strains of the nicotinamide mononucleotide adenosyltransferase (NMNAT) gene mutant expressing 33 nicotinamide mononucleotide adenosyltransferase (NMNAT) mutants.

[0077] The nicotinamide mononucleotide adenosine transferase (NMNAT) mutant was obtained by designing degenerate primers and constructing a mutant library for screening. Four forward mutagenesis primers were designed using the Tang method to uniformly and without redundancy introduce twenty natural amino acids into the mutant library. The designed degenerate primer sequences are shown below, and the specific construction method is as follows:

[0078] Using the recombinant vector pET29a-NMNAT constructed in Example 1 as a template, two rounds of PCR reactions were performed using primers corresponding to each mutant. The PCR reaction system for the first round is shown in Table 1, the PCR reaction system for the second round is shown in Table 2, and the PCR reaction procedure is shown in Table 3.

[0079] Primer names and sequences for constructing the nicotinamide mononucleotide adenosine transferase (NMNAT) gene unit point mutation library:

[0080] F8 mutation library:

[0081] NMNAT-F8-NDT-F1:TCTTTACAGGCTCTGNDTGGCGGCACCTTTGAT;

[0082] NMNAT-F8-VMA-F2:TCTTTACAGGCCTCTGVMAGGCGGCACCTTTGAT;

[0083] NMNAT-F8-ATG-F3:TCTTTACAGGCTCTGATGGGCGGCACCTTTGAT;

[0084] NMNAT-F8-TGG-F4: TCTTTACAGGCTCTGTGGGGCGGCACCTTTGAT;

[0085] NMNAT-8-86-WT-R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0086] G9 mutant library:

[0087] NMNAT-G9-NDT-F1: TTACAGGCTCTGTTTNDTGGCACCTTTGATCCG;

[0088] NMNAT-G9-VMA-F2: TTACAGGCTCTGTTTVMAGGCACCTTTGATCCG;

[0089] NMNAT-G9-ATG-F3: TTACAGGCTCTGTTTATGGGCACCTTTGATCCG;

[0090] NMNAT-G9-TGG-F4: TTACAGGCTCTGTTTTGGGGCACCTTTGATCCG;

[0091] NMNAT-8-86-WT-R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0092] G10 mutant library:

[0093] NMNAT-G10-NDT-F1: CAGGCTCTGTTTGGCNDTACCTTTGATCCGGTG;

[0094] NMNAT-G10-VMA-F2: CAGGCTCTGTTTGGCVMAACCTTTGATCCGGTG;

[0095] NMNAT-G10-ATG-F3: CAGGCTCTGTTTGGCATGACCTTTGATCCGGTG;

[0096] NMNAT-G10-TGG-F4: CAGGCTCTGTTTGGCTGGACCTTTGATCCGGTG;

[0097] NMNAT-8-86-WT-R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0098] T11 mutant library:

[0099] NMNAT-T11-NDT-F1: GCTCTGTTTGGCGGCNDTTTTGATCCGGTGCAC;

[0100] NMNAT-T11-VMA-F2: GCTCTGTTTGGCGGCVMATTTGATCCGGTGCAC;

[0101] NMNAT-T11-ATG-F3: GCTCTGTTTGGCGGCATGTTTGATCCGGTGCAC;

[0102] NMNAT-T11-TGG-F4: GCTCTGTTTGGCGGCTGGTTTGATCCGGTGCAC;

[0103] NMNAT-8-86-WT-R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0104] F12 mutant library:

[0105] NMNAT-F12-NDT-F1: CTGTTTGGCGGCACCNDTGATCCGGTGCACTAT;

[0106] NMNAT-F12-VMA-F2: CTGTTTGGCGGCACCVMAGATCCGGTGCACTAT;

[0107] NMNAT-F12-ATG-F3: CTGTTTGGCGGCACCATGGATCCGGTGCACTAT;

[0108] NMNAT-F12-TGG-F4: CTGTTTGGCGGCACCTGGGATCCGGTGCACTAT;

[0109] NMNAT-8-86-WT-R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0110] H16 mutant library:

[0111] NMNAT-H16-NDT-F1: ACCTTTGATCCGGTGNDTTATGGTCATCTTAAA;

[0112] NMNAT-H16-VMA-F2: ACCTTTGATCCGGTGVMATATGGTCATCTTAAA;

[0113] NMNAT-H16-ATG-F3: ACCTTTGATCCGGTGATGTATGGTCATCTTAAA;

[0114] NMNAT-H16-TGG-F4: ACCTTTGATCCGGTGTGGTATGGTCATCTTAAA;

[0115] NMNAT-8-86-WT-R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0116] G18 mutation library:

[0117] NMNAT-G18-NDT-F1: GATCCGGTGCACTATNDTCATCTTAAACCCGTG;

[0118] NMNAT-G18-VMA-F2: GATCCGGTGCACTATVMACATCTTAAACCCGTG;

[0119] NMNAT-G18-ATG-F3: GATCCGGTGCACTATATGCATCTTAAACCCGTG;

[0120] NMNAT-G18-TGG-F4: GATCCGGTGCACTATTGGCATCTTAAACCCGTG;

[0121] NMNAT-8-86-WT-R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0122] H19 mutation library:

[0123] NMNAT-H19-NDT-F1: CCGGTGCACTATGGTNDTCTTAAACCCGTGGAA;

[0124] NMNAT-H19-VMA-F2: CCGGTGCACTATGGTVMACTTAAACCCGTGGAA;

[0125] NMNAT-H19-ATG-F3: CCGGTGCACTATGGTATGCTTAAACCCGTGGAA;

[0126] NMNAT-H19-TGG-F4: CCGGTGCACTATGGTTGGCTTAAACCCGTGGAA;

[0127] NMNAT-8-86-WT-R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0128] P22 mutation library:

[0129] NMNAT - P22 - NDT - F1: TATGGTCATCTTAAANDTGTGGAAACGCTGGCG;

[0130] NMNAT - P22 - VMA - F2: TATGGTCATCTTAAAVMAGTGGAAACGCTGGCG;

[0131] NMNAT - P22 - ATG - F3: TATGGTCATCTTAAAATGGTGGAAACGCTGGCG;

[0132] NMNAT - P22 - TGG - F4: TATGGTCATCTTAAATGGGTGGAAACGCTGGCG;

[0133] NMNAT - 8 - 86 - WT - R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0134] I38 mutant library:

[0135] NMNAT - I38 - NDT - F1: ACGCGGGTCACAATCNDTCCTAATAATGTTCCT;

[0136] NMNAT - I38 - VMA - F2: ACGCGGGTCACAATCVMACCTAATAATGTTCCT;

[0137] NMNAT - I38 - ATG - F3: ACGCGGGTCACAATCATGCCTAATAATGTTCCT;

[0138] NMNAT - I38 - TGG - F4: ACGCGGGTCACAATCTGGCCTAATAATGTTCCT;

[0139] NMNAT - 8 - 86 - WT - R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0140] N40 mutant library:

[0141] NMNAT - N40 - NDT - F1: GTCACAATCATCCCTNDTAATGTTCCTCCGCAT;

[0142] NMNAT - N40 - VMA - F2: GTCACAATCATCCCTVMAAATGTTCCTCCGCAT;

[0143] NMNAT - N40 - ATG - F3: GTCACAATCATCCCTATGAATGTTCCTCCGCAT;

[0144] NMNAT - N40 - TGG - F4: GTCACAATCATCCCTTGGAATGTTCCTCCGCAT;

[0145] NMNAT - 8 - 86 - WT - R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0146] H45 mutant library:

[0147] NMNAT - H45 - NDT - F1: AATAATGTTCCTCCGNDTCGTCCCCAGCCGGAA;

[0148] NMNAT - H45 - VMA - F2: AATAATGTTCCTCCGVMACGTCCCCAGCCGGAA;

[0149] NMNAT - H45 - ATG - F3: AATAATGTTCCTCCGATGCGTCCCCAGCCGGAA;

[0150] NMNAT - H45 - TGG - F4: AATAATGTTCCTCCGTGGCGTCCCCAGCCGGAA;

[0151] NMNAT - 8 - 86 - WT - R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0152] R46 mutant library:

[0153] NMNAT - R46 - NDT - F1: AATGTTCCTCCGCATNDTCCCCAGCCGGAAGCG;

[0154] NMNAT - R46 - VMA - F2: AATGTTCCTCCGCATVMACCCCAGCCGGAAGCG;

[0155] NMNAT - R46 - ATG - F3: AATGTTCCTCCGCATATGCCCCAGCCGGAAGCG;

[0156] NMNAT - R46 - TGG - F4: AATGTTCCTCCGCATTGGCCCCAGCCGGAAGCG;

[0157] NMNAT-8-86-WT-R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT。

[0158] E76 mutant library:

[0159] NMNAT-E76-NDT-F1: ACTCTTGATGAACGCNDTCTAAAGCGCAATGCC;

[0160] NMNAT-E76-VMA-F2: ACTCTTGATGAACGCVMACTAAAGCGCAATGCC;

[0161] NMNAT-E76-ATG-F3: ACTCTTGATGAACGCATGCTAAAGCGCAATGCC;

[0162] NMNAT-E76-TGG-F4: ACTCTTGATGAACGCTGGCTAAAGCGCAATGCC;

[0163] NMNAT-8-86-WT-R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT。

[0164] S83 mutant library:

[0165] NMNAT-S83-NDT-F1: AAGCGCAATGCCCCCNDTTACACTTGGCAAACA;

[0166] NMNAT-S83-VMA-F2: AAGCGCAATGCCCCCVMATACACTTGGCAAACA;

[0167] NMNAT-S83-ATG-F3: AAGCGCAATGCCCCCATGTACACTTGGCAAACA;

[0168] NMNAT-S83-TGG-F4: AAGCGCAATGCCCCCTGGTACACTTGGCAAACA;

[0169] NMNAT-8-86-WT-R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT。

[0170] Y84 mutant library:

[0171] NMNAT-Y84-NDT-F1: CGCAATGCCCCCTCTNDTACTTGGCAAACACTG;

[0172] NMNAT-Y84-VMA-F2: CGCAATGCCCCCTCTVMAACTTGGCAAACACTG;

[0173] NMNAT-Y84-ATG-F3: CGCAATGCCCCCTCTATGACTTGGCAAACACTG;

[0174] NMNAT-Y84-TGG-F4: CGCAATGCCCCCTCTTGGACTTGGCAAACACTG;

[0175] NMNAT-8-86-WT-R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0176] T85 mutant library:

[0177] NMNAT-T85-NDT-F1: AATGCCCCCTCTTACNDTTGGCAAACACTGAAA;

[0178] NMNAT-T85-VMA-F2: AATGCCCCCTCTTACVMATGGCAAACACTGAAA;

[0179] NMNAT-T85-ATG-F3: AATGCCCCCTCTTACATGTGGCAAACACTGAAA;

[0180] NMNAT-T85-TGG-F4: AATGCCCCCTCTTACTGGTGGCAAACACTGAAA;

[0181] NMNAT-8-86-WT-R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0182] W86 mutant library:

[0183] NMNAT-W86-NDT-F1: GCCCCCTCTTACACTNDTCAAACACTGAAAGAG;

[0184] NMNAT-W86-VMA-F2: GCCCCCTCTTACACTVMACAAACACTGAAAGAG;

[0185] NMNAT-W86-ATG-F3: GCCCCCTCTTACACTATGCAAACACTGAAAGAG;

[0186] NMNAT-W86-TGG-F4: GCCCCCTCTTACACTTGGCAAACACTGAAAGAG;

[0187] NMNAT-8-86-WT-R: GATAAATTTTACCGGCAGGCTGAAGGTGAAGAT.

[0188] F104 mutant library:

[0189] NMNAT-F104-NDT-F1: GACGTGCCGCTGGCGNDTATTATTGGTCAGGAT;

[0190] NMNAT-F104-VMA-F2: GACGTGCCGCTGGCGVMAATTATTGGTCAGGAT;

[0191] NMNAT-F104-ATG-F3: GACGTGCCGCTGGCGATGATTATTGGTCAGGAT;

[0192] NMNAT-F104-TGG-F4: GACGTGCCGCTGGCGTGGATTATTGGTCAGGAT;

[0193] NMNAT-104-179-WT-R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0194] I105 mutant library:

[0195] NMNAT-I105-NDT-F1: GTGCCGCTGGCGTTTNDTATTGGTCAGGATTCA;

[0196] NMNAT-I105-VMA-F2: GTGCCGCTGGCGTTTVMAATTGGTCAGGATTCA;

[0197] NMNAT-I105-ATG-F3: GTGCCGCTGGCGTTTATGATTGGTCAGGATTCA;

[0198] NMNAT-I105-TGG-F4: GTGCCGCTGGCGTTTTGGATTGGTCAGGATTCA;

[0199] NMNAT-104-179-WT-R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0200] I106 Mutation Library:

[0201] NMNAT-I106-NDT-F1: CCGCTGGCGTTTATTNDTGGTCAGGATTCATTG;

[0202] NMNAT-I106-VMA-F2: CCGCTGGCGTTTATTVMAGGTCAGGATTCATTG;

[0203] NMNAT-I106-ATG-F3: CCGCTGGCGTTTATTATGGGTCAGGATTCATTG;

[0204] NMNAT-I106-TGG-F4: CCGCTGGCGTTTATTTGGGGTCAGGATTCATTG;

[0205] NMNAT-104-179-WT-R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0206] G107 Mutation Library:

[0207] NMNAT-G107-NDT-F1: CTGGCGTTTATTATTNDTCAGGATTCATTGCTG;

[0208] NMNAT-G107-VMA-F2: CTGGCGTTTATTATTVMACAGGATTCATTGCTG;

[0209] NMNAT-G107-ATG-F3: CTGGCGTTTATTATTATGCAGGATTCATTGCTG;

[0210] NMNAT-G107-TGG-F4: CTGGCGTTTATTATTTGGCAGGATTCATTGCTG;

[0211] NMNAT-104-179-WT-R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0212] Q108 Mutation Library:

[0213] NMNAT-Q108-NDT-F1: GCGTTTATTATTGGTNDTGATTCATTGCTGACC;

[0214] NMNAT-Q108-VMA-F2: GCGTTTATTATTGGTVMAGATTCATTGCTGACC;

[0215] NMNAT-Q108-ATG-F3: GCGTTTATTATTGGTATGGATTCATTGCTGACC;

[0216] NMNAT-Q108-TGG-F4: GCGTTTATTATTGGTTGGGATTCATTGCTGACC;

[0217] NMNAT-104-179-WT-R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0218] D109 mutant library:

[0219] NMNAT-D109-NDT-F1: TTTATTATTGGTCAGNDTTCATTGCTGACCTTT;

[0220] NMNAT-D109-VMA-F2: TTTATTATTGGTCAGVMATCATTGCTGACCTTT;

[0221] NMNAT-D109-ATG-F3: TTTATTATTGGTCAGATGTCATTGCTGACCTTT;

[0222] NMNAT-D109-TGG-F4: TTTATTATTGGTCAGTGGTCATTGCTGACCTTT;

[0223] NMNAT-104-179-WT-R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0224] S110 mutant library:

[0225] NMNAT-S110-NDT-F1: ATTATTGGTCAGGATNDTTTGCTGACCTTTCCG;

[0226] NMNAT-S110-VMA-F2: ATTATTGGTCAGGATVMATTGCTGACCTTTCCG;

[0227] NMNAT-S110-ATG-F3: ATTATTGGTCAGGATATGTTGCTGACCTTTCCG;

[0228] NMNAT - S110 - TGG - F4: ATTATTGGTCAGGATTGGTTGCTGACCTTTCCG;

[0229] NMNAT - 104 - 179 - WT - R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0230] T116 mutation library:

[0231] NMNAT - T116 - NDT - F1: TTGCTGACCTTTCCGNDTTGGAATGAATACGAA;

[0232] NMNAT - T116 - VMA - F2: TTGCTGACCTTTCCGVMATGGAATGAATACGAA;

[0233] NMNAT - T116 - ATG - F3: TTGCTGACCTTTCCGATGTGGAATGAATACGAA;

[0234] NMNAT - T116 - TGG - F4: TTGCTGACCTTTCCGTGGTGGAATGAATACGAA;

[0235] NMNAT - 104 - 179 - WT - R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0236] W117 mutation library:

[0237] NMNAT - W117 - NDT - F1: CTGACCTTTCCGACCNDTAATGAATACGAAACG;

[0238] NMNAT - W117 - VMA - F2: CTGACCTTTCCGACCVMAAATGAATACGAAACG;

[0239] NMNAT - W117 - ATG - F3: CTGACCTTTCCGACCATGAATGAATACGAAACG;

[0240] NMNAT - W117 - TGG - F4: CTGACCTTTCCGACCTGGAATGAATACGAAACG;

[0241] NMNAT - 104 - 179 - WT - R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0242] N118 mutant library:

[0243] NMNAT - N118 - NDT - F1: ACCTTTCCGACCTGGNDTGAATACGAAACGATA;

[0244] NMNAT - N118 - VMA - F2: ACCTTTCCGACCTGGVMAGAATACGAAACGATA;

[0245] NMNAT - N118 - ATG - F3: ACCTTTCCGACCTGGATGGAATACGAAACGATA;

[0246] NMNAT - N118 - TGG - F4: ACCTTTCCGACCTGGTGGGAATACGAAACGATA;

[0247] NMNAT - 104 - 179 - WT - R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0248] C132 mutant library: <S

[0249] NMNAT - C132 - NDT - F1: GCACATTTGATCGTCNDTCGGCGTCCAGGTTAC;

[0250] NMNAT - C132 - VMA - F2: GCACATTTGATCGTCVMACGGCGTCCAGGTTAC;

[0251] NMNAT - C132 - ATG - F3: GCACATTTGATCGTCATGCGGCGTCCAGGTTAC;

[0252] NMNAT - C132 - TGG - F4: GCACATTTGATCGTCTGGCGGCGTCCAGGTTAC;

[0253] NMNAT - 104 - 179 - WT - R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0254] R133 mutant library:<S

[0255] NMNAT - R133 - NDT - F1: CATTTGATCGTCTGTNDTCGTCCAGGTTACCCA;

[0256] NMNAT-R133-VMA-F2: CATTTGATCGTCTGTVMACGTCCAGGTTACCCA;

[0257] NMNAT-R133-ATG-F3: CATTTGATCGTCTGTATGCGTCCAGGTTACCCA;

[0258] NMNAT-R133-TGG-F4: CATTTGATCGTCTGTTGGCGTCCAGGTTACCCA;

[0259] NMNAT-104-179-WT-R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0260] R134 mutant library:

[0261] NMNAT-R134-NDT-F1: TTGATCGTCTGTCGGNDTCCAGGTTACCCACTT;

[0262] NMNAT-R134-VMA-F2: TTGATCGTCTGTCGGVMACCAGGTTACCCACTT;

[0263] NMNAT-R134-ATG-F3: TTGATCGTCTGTCGGATGCCAGGTTACCCACTT;

[0264] NMNAT-R134-TGG-F4: TTGATCGTCTGTCGGTGGCCAGGTTACCCACTT;

[0265] NMNAT-104-179-WT-R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0266] F177 mutant library:

[0267] NMNAT-F177-NDT-F1: GCTGAAACGCCGTGGNDTAACATCTCGGCGACC;

[0268] NMNAT-F177-VMA-F2: GCTGAAACGCCGTGGVMAAACATCTCGGCGACC;

[0269] NMNAT-F177-ATG-F3: GCTGAAACGCCGTGGATGAACATCTCGGCGACC;

[0270] NMNAT-F177-TGG-F4:GCTGAAACGCCGTGGTGGAACATCTCGGCGACC;

[0271] NMNAT-104-179-WT-R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0272] I179 mutation library:

[0273] NMNAT-I179-NDT-F1:ACGCCGTGGTTTAACNDTTCGGCGACCATCATC;

[0274] NMNAT-I179-VMA-F2:ACGCCGTGGTTTAACVMATCGGCGACCATCATC;

[0275] NMNAT-I179-ATG-F3: ACGCCGTGGTTTAACATGTCGGCGACCATCATC;

[0276] NMNAT-I179-TGG-F4: ACGCCGTGGTTTAACTGGTCGGCGACCATCATC;

[0277] NMNAT-104-179-WT-R: GCTAGTTATTGCTCAGCGGTGGCAGCAGCCAAC.

[0278] Table 1 First-round PCR reaction system

[0279]

[0280] Table 2 Second round PCR reaction system

[0281]

[0282] Table 3 PCR reaction procedure

[0283]

[0284] After completing two rounds of PCR reactions, add 2 μL of Dpn I enzyme to each reaction system, digest at 37°C for 3 hours, and take 1 μL to electroporate into E. coli BL21(DE3) competent cells. Incubate in an inverted incubator at 37°C for 12-16 hours. Once single clones have grown, the construction of the mutant library is complete.

[0285] Example 3: High-throughput screening of a mutant library of nicotinamide mononucleotide adenosyltransferase (NMNAT) gene and obtaining engineered strains of single-point mutants.

[0286] In this embodiment, the high-throughput screening method for the nicotinamide mononucleotide adenosine transferase (NMNAT) gene mutant library is ultraviolet spectrophotometry.

[0287] The specific methods and operating steps are as follows:

[0288] (1) Preparation of bacterial cells

[0289] The single colonies grown in Example 2 were picked up with a toothpick and cultured in 96-well plates at 37°C and 1200 rpm for 12 h with shaking. 120 μL of the overnight culture was transferred to a 96-well glycerol plate, and 80 μL of 50% glycerol was added and mixed thoroughly by pipetting. The plate was then capped and stored at -80°C. Simultaneously, 800 μL of TB+IPTG+Kana medium (0.1 M IPTG, 50 μg / mL Kana) was added to the 96-well plate and cultured at 25°C and 1200 rpm for 12 h for protein expression.

[0290] (2) Whole-cell catalytic reaction

[0291] Add 20 μL of bacterial culture, 20 μL of 50 mM ATP, 20 μL of 50 mM glucose, and 20 μL of 100 mM MgCl2 to a 96-well plate. Then add 10 μL of glucose dehydrogenase and 105 μL of prepared Tris-HCl buffer. Finally, add 20 μL of 50 mM NMN at room temperature to start the reaction immediately. Detect the absorbance at 340 nm using a UV spectrophotometer, reading the absorbance values ​​at 0 min and 5 min. Calculate the difference and screen for mutant strains with higher absorbance increases (i.e., relatively higher enzyme activity) for further screening.

[0292] (3) Obtaining single point mutant engineered strains

[0293] OD 340 Strains with a value higher than that of the wild type under nm were sent for sequencing. Transformants that were correctly sequenced were identified as engineered strains of the nicotinamide mononucleotide adenosine transferase (NMNAT) gene mutant.

[0294] Example 4: Preparation of engineered bacteria containing a two-site mutant of the nicotinamide mononucleotide adenosine transferase (NMNAT) gene

[0295] The double mutant in this embodiment is constructed by whole plasmid PCR based on the single mutant in Example 3.

[0296] Ultimately, 12 double mutants were obtained, which were named mutant I106M / G10G, mutant I106M / H45W, mutant I106M / T85T, mutant I106M / W86C, mutant I106M / I105L, mutant I106M / Q108G, mutant I106M / Q108W, mutant I106M / N118A, mutant I106M / C132M, mutant I106M / R133S, mutant I106M / F177W, and mutant I106M / I179M.

[0297] Example 5: Preparation of engineered bacteria containing three-site and four-site mutants of the nicotinamide mononucleotide adenosyltransferase (NMNAT) gene.

[0298] Based on the mutant I106M / W86C, a triple mutant was constructed by whole plasmid PCR. For details, please refer to Example 2.

[0299] The above method yields three mutants, which are named mutant I106M / W86C / H45W, mutant I106M / W86C / I105L, mutant I106M / W86C / Q108W, mutant I106M / W86C / C132M, and mutant I106M / W86C / F177W.

[0300] Based on the mutant I106M / W86C / I105L, a quadruple mutant was constructed by whole plasmid PCR. For details, please refer to Example 2.

[0301] Four mutants can be obtained using the above method, and they are named mutant I106M / W86C / I105L / H45W, mutant I106M / W86C / I105L / Q108W, and mutant I106M / W86C / I105L / F177W.

[0302] Example 6: Obtaining crude nicotinamide mononucleotide adenosine transferase enzyme solution and determining its activity.

[0303] (1) Obtaining crude enzyme solution

[0304] Recombinant bacterial transformants containing the above-mentioned nicotinamide mononucleotide adenosyltransferase (NMNAT) gene or the recombinant plasmids obtained in Examples 3-5 were picked and added to 5 mL of LB liquid medium containing 50 μg / mL kanamycin. The culture was incubated overnight at 37°C and 220 rpm for 12-16 hours. The transformants were then inoculated into LB liquid medium containing 50 μg / mL kanamycin at a 1% (volume percentage) inoculation rate and cultured at 37°C for 5 hours. IPTG was added to a final concentration of 0.1 mmol / L, and expression was induced at 25°C and 220 rpm for 18 hours. The cells were then centrifuged at 4°C and 7000 rpm for 10 minutes to collect the bacterial cells. The collected bacterial cells were resuspended in PBS buffer (50 mM, pH 7.4) to obtain whole cells of nicotinamide mononucleotide adenosyltransferase (NMNAT). The bacterial cells were then sonicated under ice bath conditions to obtain the sonicated sample. The sonicated sample was then centrifuged at 4°C and 7000 rpm for 10 minutes, and the supernatant (i.e., crude enzyme solution) was collected.

[0305] (2) Detection of nicotinamide mononucleotide adenosine transferase (NMNAT) enzyme activity

[0306] The enzymatic reaction system consisted of 1 mL of 900 μL of 56.4 mM nicotinamide ribochloride, 83.7 mM magnesium chloride hexahydrate, 84.6 mM ATP sodium salt solution, and 100 μL of crude enzyme solution; the reaction was carried out at pH 4.5, 30℃, and 220 rpm for 10 min.

[0307] Termination of reaction: After the reaction is complete, take 50 μL of the reaction solution and add it to 950 μL of pure water and mix well to terminate the enzyme reaction.

[0308] The sample was filtered through a 0.45 μm water membrane and detected by high-performance liquid chromatography (HPLC). Detection conditions: Shimadzu WondaCract ODS-2 column (250 mm × 4.6 nm 5 μm), flow rate 1.2 mL / min; detection wavelength 254 nm; mobile phase A: 0.05 M Na₂HPO₄ + 0.05 M KH₂PO₄ + 5 mM tetrabutylammonium bromide; mobile phase B: methanol; A:B = 80:20, v / v).

[0309] Enzyme activity is defined as the amount of enzyme required to convert 1 μmol of nicotinamide ribose into nicotinamide nucleotides per minute at 37℃ and pH 4.5, defined as 1 U.

[0310] Enzyme activity calculation formula:

[0311] Specific enzyme activity (U / mL) = (C2×S1×1000×V1×f) / (S2×334.22×T×V2)

[0312] Note: S1 represents the peak area of ​​nicotinamide nucleotide in the sample; S2 represents the peak area of ​​nicotinamide nucleotide in the standard; C2 represents the concentration of nicotinamide nucleotide in the standard (mg / mL); T represents the reaction time (min); V1 represents the volume of the reaction system (mL); V2 represents the volume of the original enzyme solution added to the reaction (mL); f represents the dilution factor of the reaction solution; 334.22 is the molecular weight of nicotinamide nucleotide.

[0313] Standard sample spectrum as shown Figure 2 As shown in Table 4, the enzyme activity of the single-site mutants with higher enzyme activity than wild-type nicotinamide mononucleotide adenosyltransferase is shown in Table 5.

[0314] Table 4. Enzyme activity assay results of wild-type and single-point mutant nicotinamide mononucleotide adenosyltransferase mutants.

[0315]

[0316] Table 5. Enzyme activity assay results of multi-site mutant nicotinamide mononucleotide adenosyltransferase mutants.

[0317]

[0318] Example 7: Screening for the optimal pH of the nicotinamide mononucleotide adenosine transferase (NMNAT) catalytic reaction system.

[0319] This example screened the pH values ​​for the reaction catalyzed by nicotinamide mononucleotide adenosine transferase (NMNAT). The results are shown in Table 6, with pH 5.5 being the optimal pH.

[0320] Table 6. Experimental conditions and results for the reaction system at pH 6-7.

[0321]

[0322] Example 8: Screening of the optimal magnesium ion concentration for the nicotinamide mononucleotide adenosine transferase (NMNAT) catalytic reaction system.

[0323] This example tested the magnesium ion concentration for the reaction catalyzed by nicotinamide mononucleotide adenosine transferase (NMNAT). The magnesium ion concentration gradient was from 25 mM to 250 mM, and the results are shown in Table 7. It can be seen that as the magnesium ion concentration increases, NAD... + The formation rate increased from 53.02% to 91.28%, indicating that 250 mM of magnesium ions is the optimal concentration for the reaction.

[0324] Table 7. Experimental conditions and results of magnesium ion concentration gradient in the reaction system.

[0325]

[0326] Example 9: Screening of the optimal NMN to ATP ratio in the nicotinamide mononucleotide adenosine transferase (NMNAT) catalytic reaction system.

[0327] The low conversion rate of NMN leads to waste of the substrate NMN. Therefore, this example tested the feed ratio of NMN to ATP in the nicotinamide mononucleotide adenosine transferase (NMNAT) catalytic reaction system. The results are shown in Table 8, which shows that 120 g / L of NMN and 120 g / L of ATP are the optimal experimental conditions.

[0328] Table 8. Experimental conditions and results of the NMN to ATP ratio in the reaction system.

[0329]

[0330] After continuous experimentation and refinement, the optimal reaction system was determined to be: NMN 120g / L, ATP 120g / L, magnesium chloride hexahydrate 250mM, manganese ions 5mM, nicotinamide mononucleotide adenosine transferase wet cells, pH 5.5, temperature 37℃, and 180rpm.

[0331] Example 10

[0332] Based on the experimental results of Examples 7-9, the optimal reaction conditions were determined as follows: For a 1L reaction system, the optimal conditions were: NMN 120g / L, ATP 120g / L, magnesium chloride hexahydrate 250mM, manganese ions 5mM, crude enzyme solution 2400U, pH 5.5, reaction temperature 37℃, and 180rpm. Under these conditions, using a mutant strain (mutant I106M / G10G), the conversion of NMN to NAD+ was completed after 4 hours of reaction. + The transformation reaction of NAD + The generation rate was 99.21%.

[0333] 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 nicotinamide mononucleotide adenosine transferase NMNAT mutant, characterized in that, The mutant is obtained by mutating at least one of the following amino acids from SEQ ID NO:1: Gly at position 10, His at position 16, Ile at position 38, His at position 45, Ser at position 83, Thr at position 85, Trp at position 86, Ile at position 105, Ile at position 106, Gln at position 108, Ser at position 110, Thr at position 116, Asn at position 118, Cys at position 132, Arg at position 133, Phe at position 177, and Ile at position 179.

2. The nicotinamide mononucleotide adenosine transferase NMNAT mutant according to claim 1, characterized in that, The mutant is based on SEQ ID NO:

1. Mutate its 10th Gly to Gly (synonymous mutation); or Mutate its 16th His to His (synonymous mutation); or Mutate its 38th Ile to Met; or Mutate its 45th His position to Trp; or Mutate its 83rd Ser position to Glu; or Mutate its 85th Thr to Thr (synonymous mutation); or Mutate its 86th Trp to Cys; or Transform its 105th Ile into Leu; or Mutate its 106th Ile to Met; or Mutate its 108th Gln to His; or Mutate its 108th Gln to Gly; or Mutate its 108th Gln to Gln (synonymous mutation); or Mutate its 108th Gln to Trp; or Mutate its 110th Ser bit to Asp; or Mutate its 116th Thr to Arg; or Mutate its 118th Asn to Ala; or Mutate its 132nd Cys position to Asn; or Mutate its 132nd Cys position to Met; or Mutate its 133rd Arg to Ser; or Transform its 177th Phe into Val; or Transform its 177th Phe into Trp; or Transform its 179th Ile into Val; or Transform its 179th Ile into Ile (synonymous mutation); or Transform its 179th Ile into Met.

3. The nicotinamide mononucleotide adenosine transferase NMNAT mutant according to claim 1, characterized in that, The mutant is based on SEQ ID NO:

1. Mutate its 106th Ile to Met and its 10th Gly to Gly; or Mutate its 106th Ile to Met and its 45th His to Trp; or Mutate its 106th Ile to Met and its 85th Thr to Thr; or Mutate its 106th Ile to Met and its 86th Trp to Cys; or Mutate its 106th Ile to Met and its 105th Ile to Leu; or Mutate its 106th position Ile to Met, and its 108th position Gln to Gly; or Mutate its 106th Ile to Met and its 108th Gln to Trp; or Mutate its 106th Ile to Met and its 118th Asn to Ala; or Mutate its 106th Ile to Met and its 132nd Cys to Met; or Mutate its 106th Ile to Met and its 133rd Arg to Ser; or Transform its 106th Ile into Met and its 177th Phe into Trp; or Transform its 106th Ile into Met, and its 179th Ile into Met.

4. The nicotinamide mononucleotide adenosine transferase NMNAT mutant according to claim 1, characterized in that, The mutant is based on SEQ ID NO:

1. Mutate its 106th Ile to Met, its 86th Trp to Cys, and its 45th His to Trp; or Mutate its 106th Ile to Met, its 86th Trp to Cys, and its 105th Ile to Leu; or Mutate its 106th Ile to Met, its 86th Trp to Cys, and its 108th Gln to Trp; or Mutate its 106th Ile to Met, its 86th Trp to Cys, and its 132nd Cys to Met; or Mutate its 106th Ile to Met, its 86th Trp to Cys, and its 177th Phe to Trp; or Mutate its 106th Ile to Met, its 86th Trp to Cys, its 105th Ile to Leu, and its 45th His to Trp; or Mutate its 106th Ile to Met, its 86th Trp to Cys, its 105th Ile to Leu, and its 108th Gln to Trp; or Transform its 106th Ile into Met, its 86th Trp into Cys, its 105th Ile into Leu, and its 177th Phe into Trp.

5. A gene encoding the nicotinamide mononucleotide adenosine transferase NMNAT mutant according to any one of claims 1-4.

6. A recombinant expression vector carrying the gene of claim 5.

7. Recombinant microorganisms comprising the recombinant expression vector as described in claim 6.

8. The use of the nicotinamide mononucleotide adenosyltransferase NMNAT mutant according to any one of claims 1-4 in the production of nicotinamide adenine dinucleotide.

9. A method for producing nicotinamide adenine dinucleotide, characterized in that, The method includes the following steps: adding the nicotinamide mononucleotide adenosine transferase NMNAT mutant of any one of claims 1-4 or the recombinant microorganism of claim 7 to a reaction system containing NMN, ATP, magnesium chloride hexahydrate, and manganese ions, and reacting under the conditions of pH 5.0-6.0, temperature 37°C, and rotation speed 180 rpm to generate nicotinamide adenine dinucleotide.

10. The method according to claim 9, characterized in that, The concentration of NMN in the reaction system is 30-50 g / L, the concentration of ATP is 80-100 g / L, the concentration of magnesium chloride hexahydrate is 20-250 mM, and the concentration of manganese ions is 2-5 mM.

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