A nicotinamide phosphoribosyltransferase mutant, a recombinant strain and a construction method thereof

Abstract

This invention discloses a nicotinamide phosphoribosyltransferase mutant, a recombinant strain, and a method for constructing the same. The mutants are I457V, G344A, Q378A, T283G, or F182A, obtained by single-point mutation of the wild-type nicotinamide phosphoribosyltransferase shown in SEQ ID NO:1. This invention utilizes a method derived from... Schizotequatrovirus By rationally designing and site-directed mutagenesis of KVP40 nicotinamide phosphoribosyltransferase, combined with metabolic engineering to block the NMN degradation pathway and enhance substrate synthesis and product transport, the yield of β-NMN by the recombinant strain during shake-flask fermentation reached 4.82 g / L. Through optimization of fed-batch fermentation conditions, the highest yield of β-NMN during fed-batch fermentation in shake flasks reached 15.60 g / L.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and metabolic engineering, and more specifically to a nicotinamide phosphoribosyltransferase mutant, a recombinant strain, and a method for constructing the same. Background Technology

[0002] β-Nicotinamide mononucleotide (β-NMN) is a B vitamin derivative widely found in various organisms and is essential for the synthesis of nicotinamide adenine dinucleotide (NAD). + A direct precursor to NAD. + It plays an indispensable role in cellular energy metabolism and maintaining physiological homeostasis. Studies have shown that NMN can significantly increase intracellular NAD+. + It enhances DNA damage repair capabilities and effectively improves mitochondrial dysfunction, a mechanism believed to play a crucial role in delaying aging. Simultaneously, NMN demonstrates significant application potential in cardiovascular protection and the prevention of diseases such as diabetes, possessing broad market prospects in the fields of health supplement development and drug treatment.

[0003] Currently, NMN synthesis methods are mainly divided into chemical synthesis, in vitro enzymatic catalysis, and microbial fermentation synthesis. Chemical synthesis is a relatively mature process, but the organic solvents used in the production process can easily cause environmental pollution, and the product is prone to producing inactive chiral isomers, which are difficult to separate and purify, resulting in high product costs. In vitro enzymatic catalysis requires expensive enzyme preparations and substrates, leading to high production costs and making industrial-scale production difficult. Traditional whole-cell catalysis suffers from low enzyme activity and demanding reaction conditions, limiting catalytic efficiency.

[0004] In contrast, microbial fermentation synthesis can achieve efficient NMN synthesis using inexpensive raw materials. Escherichia coli (E. coli) Escherichia coli As a typical model microorganism, *E. coli* possesses advantages such as simple genetic manipulation, clear metabolic pathways, and mature industrial fermentation applications, making it an ideal engineered strain for constructing an NMN biosynthetic system. The NMN biosynthetic pathway using nicotinamide (NAM) as a raw material has low raw material costs and is currently the route with the greatest industrial potential. In *E. coli*, NAM and 5-phosphoribose-1-pyrophosphate (PRPP) are converted into NMN and pyrophosphate under the catalysis of nicotinamide phosphoribosyltransferase (NAMPT). Nicotinamide phosphoribosyltransferase is the key rate-limiting enzyme in this synthetic reaction, and its catalytic activity directly determines the NMN synthesis efficiency.

[0005] Despite the significant advantages of microbial fermentation, current technologies still face bottlenecks in NMN biosynthesis, primarily due to insufficient catalytic efficiency of key rate-limiting enzymes, inadequate optimization of metabolic pathways, and low product efflux and transport efficiency, thus hindering further increases in NMN production. Therefore, it is urgent to modify key enzymes and optimize synthetic pathways through enzyme and metabolic engineering techniques, and to construct engineered strains for efficient NMN synthesis, providing technical support for the industrial production of NMN. Summary of the Invention

[0006] The purpose of this invention is to provide a nicotinamide phosphoribosyltransferase mutant, a recombinant strain, and a method for constructing the same, thereby solving the problems of low catalytic efficiency of key enzymes, imperfect synthetic pathways, and low product yield in the production of NMN in the prior art.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: According to a first aspect of the present invention, a nicotinamide phosphoribosyltransferase mutant is provided. The mutant is obtained by single-point mutation using the wild-type nicotinamide phosphoribosyltransferase shown in SEQ ID NO:1 as a template. The single-point mutation is selected from: mutant I457V, formed by mutating isoleucine at position 457 to valine, with the amino acid sequence shown in SEQ ID NO:2; mutant G344A, formed by mutating glycine at position 344 to alanine, with the amino acid sequence shown in SEQ ID NO:3; mutant Q378A, formed by mutating glutamine at position 378 to alanine, with the amino acid sequence shown in SEQ ID NO:4; mutant T283G, formed by mutating threonine at position 283 to glycine, with the amino acid sequence shown in SEQ ID NO:5; and mutant F182A, formed by mutating phenylalanine at position 182 to alanine, with the amino acid sequence shown in SEQ ID NO:6.

[0008] The wild-type nicotinamide phosphoribosyltransferase is derived from Schizotequatrovirus KVP40, whose amino acid sequence is shown in SEQ ID NO:1.

[0009] Most preferably, the mutant is mutant G344A, which is formed by mutating glycine at position 344 to alanine using wild-type nicotinamide phosphoribosyltransferase as shown in SEQ ID NO:1 as a template, and its amino acid sequence is shown in SEQ ID NO:3.

[0010] This invention also provides a method for designing nicotinamide phosphoribosyltransferase mutants. The method includes homology modeling of the wild-type enzyme to obtain a three-dimensional structural model, predicting the active pocket and substrate channel of nicotinamide phosphoribosyltransferase, performing molecular docking of the three-dimensional structural model with nicotinamide and PRPP, and selecting amino acids within a 5 Å range of the enzyme-substrate docking site as candidate mutation sites for design.

[0011] According to a second aspect of the present invention, a nucleic acid molecule is provided, said nucleic acid molecule encoding the above-mentioned nicotinamide phosphoribosyltransferase mutant.

[0012] According to a third aspect of the present invention, a method for constructing a recombinant strain for producing β-nicotinamide mononucleotide is provided, comprising the following steps: 1) Starting with Escherichia coli C43 (DE3), the coding genes for endogenous UshA, PncC, NadR, and PurR were knocked out to obtain gene knockout strain C43 (DE3) ΔpurRΔnadRΔpncCΔushA; 2) The nucleic acid molecule encoding the nicotinamide phosphoribosyltransferase mutant, the phosphoribosyl pyrophosphate synthase gene, and the NMN transporter gene were introduced into the gene knockout strain to obtain the recombinant strain; The nicotinamide phosphoribosyltransferase mutant is as described above.

[0013] In step 1), the CRISPR / Cas9 system was used to knock out the endogenous UshA, PncC, NadR, and PurR coding genes to obtain gene knockout strains.

[0014] The nucleic acid molecule encoding the nicotinamide phosphoribosyltransferase mutant, the phosphoribosyl pyrophosphate synthase encoding gene, and the NMN transporter encoding gene described in step 2) were introduced into the gene knockout strain via the recombinant plasmid pACYC-nampt-prs-pnuc.

[0015] Preferably, the phosphoribosyl pyrophosphate synthase encoding gene is derived from Escherichia coli C43(DE3), whose nucleotide sequence is shown in SEQ ID No. 41, the NMN transporter encoding gene is derived from Bacillus mycoides Its nucleotide sequence is shown in SEQ ID No. 42.

[0016] According to a fourth aspect of the present invention, a recombinant strain for producing β-nicotinamide mononucleotide is provided, wherein the recombinant strain is obtained by using Escherichia coli C43 (DE3) with the endogenous UshA, PncC, NadR, and PurR coding genes knocked out as a host, and expressing the phosphoribosyl pyrophosphate synthase coding gene, the NMN transporter coding gene, and the nicotinamide phosphoribosyltransferase mutant.

[0017] According to a fifth aspect of the present invention, there is provided an application of a recombinant strain in the preparation of β-nicotinamide mononucleotide.

[0018] The recombinant strain was fermented to obtain the product β-nicotinamide mononucleotide. The fermentation culture includes: inoculating the recombinant strain into a seed culture medium to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium for shake-flask fermentation culture, or for fed-batch fermentation culture, to obtain the β-nicotinamide mononucleotide; The seed culture medium comprises 10 g / L sodium chloride, 10 g / L tryptone and 5 g / L yeast extract; The shake-flask fermentation includes: inoculating the recombinant strain into a seed culture medium and culturing for 12 h, then transferring 1% (v / v) of the seed culture to a fermentation culture medium and culturing for 24 h; the fermentation culture medium includes 8-12 g / L yeast extract, 15-25 g / L glucose, 0.8-1.2 g / L magnesium sulfate, 5 g / L ammonium sulfate and 6 g / L potassium dihydrogen phosphate; The fed-batch fermentation consists of 8 g / L glucose, 10 g / L yeast extract, 6 g / L potassium dihydrogen phosphate and 6 g / L ammonium sulfate, followed by fermentation culture, induced expression, and collection of fermentation broth containing β-nicotinamide mononucleotide.

[0019] Preferably, the fed-batch fermentation culture temperature is 35~37℃ and the pH is 6.8~7.3.

[0020] This invention, through analysis of a nicotinamide phosphoribosyltransferase resource library, found that existing technologies mostly use enzymes derived from *Manniella vulgaris* (…). Mannheimia varigena ) and pine chitinophytes ( Chitinophaga pinensis Nicotinamide phosphoribosyltransferase (NPT) has been disclosed, along with related enzyme molecules and modification strategies. This invention is the first to utilize nicotinamide phosphoribosyltransferase derived from Vibrio phage KVP40 (…). Schizotequatrovirus Using nicotinamide phosphoribosyltransferase (KVP40) as the research object, through rational design and enzyme engineering, the optimal single-point mutant G344A was obtained. This mutant mutates glycine at position 344 to alanine, which significantly increases the enzyme's catalytic activity to 1.3 times that of the wild type. When applied to engineered Escherichia coli, it can significantly enhance the synthesis and accumulation of β-nicotinamide mononucleotide.

[0021] This invention further modifies the recombinant strain through systematic metabolic engineering. Using *E. coli* C43 (DE3) as the starting strain, the degradation and metabolic bypass of β-nicotinamide mononucleotide (NMN) are blocked by knocking out the endogenous ushA, pncC, nadR, and purR genes. At the same time, overexpression of phosphoribosyl pyrophosphate synthase and NMN transporter enhances substrate supply and product efflux. In conjunction with a highly catalytically active NMN phosphoribosyltransferase mutant, efficient synthesis is achieved in *E. coli*. Fermentation verification shows that the recombinant strain can significantly increase the yield of β-nicotinamide mononucleotide and has the efficient and stable synthesis capability required for industrial production.

[0022] Compared with the prior art, the present invention has the following beneficial effects: By analyzing the sources Schizotequatrovirus Through rational design and site-directed mutagenesis of KVP40 nicotinamide phosphoribosyltransferase, the efficiency of enzyme-catalyzed synthesis of β-NMN from NAM and PRPP was significantly improved. Combined with metabolic engineering to block the NMN degradation pathway and enhance substrate synthesis and product transport, the yield of β-NMN by shake-flask fermentation of recombinant strain reached 4.82 g / L. Through optimization of fed-batch fermentation conditions, the highest yield of β-NMN by fed-batch fermentation in shake flasks reached 15.60 g / L. Attached Figure Description

[0023] Figure 1 A three-dimensional structural model of nicotinamide phosphoribosyltransferase; Figure 2 The pET28a-nampt plasmid map; Figure 3 The pACYC-nampt-prs-pnuc plasmid map; Figure 4 The results of shake-flask fermentation of the recombinant strain containing the mutant; Figure 5 Results of feed-in batch fermentation of recombinant engineered bacteria in shake flasks. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0025] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions, such as those described in *Molecular Cloning: A Laboratory Manual* (New York: Cold Spring Harbor Laboratory Press, 1989). Primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0026] Example 1: Genome Editing of Engineered Strains This embodiment uses the CRISPR / Cas9 gene editing system, with Escherichia coli C43 (DE3) as the starting base strain, and sequentially knocks out the NMN degradation pathway-related genes pncC, nadR, ushA, and the purR gene encoding the pyrimidine nucleoside metabolic repressor protein, ultimately constructing the gene knockout strain C43 (DE3) ΔpurRΔnadRΔpncCΔushA. The specific steps include: 1) Construction of pTarget plasmid containing sgRNA sequence: The gene knockout sgRNA sequence (PAM: 5'-NGG-3') was designed online using the CHOPCHOP online tool. Using pTarget plasmid as a template, PCR amplification was performed using the primers shown in Table 1 to obtain knockout plasmids containing the required sgRNA for knockout.

[0027] The PCR amplification reaction system was prepared according to the TOROBlue Flash KOD DyeMix instructions. The PCR program included: 98℃ pre-denaturation for 10 min; 98℃ denaturation for 10 s, 53℃ annealing for 30 s, 68℃ extension for 1 min, 30 cycles; 68℃ extension for 10 min (the same applies below).

[0028] Table 1

[0029] 2) Construction of homologous fragments required for gene knockout: Using Escherichia coli C42(DE3) bacterial culture as a template, PCR amplification was performed using the primers shown in Table 2 to obtain the upstream and downstream homologous arms of the target gene.

[0030] Table 2

[0031] Using the purified upstream and downstream homologous arms as templates, fusion PCR was performed using the primers shown in Table 3 to obtain the Donor DNA fragment required for gene knockout.

[0032] Table 3

[0033] (3) Construction of the deletion strain: The pEcCas plasmid was electroporated into the Escherichia coli C43(DE3) chassis strain to prepare electroporation competent cells. The pTarget plasmid containing sgRNA and the Donor DNA fragment constructed above were simultaneously electroporated into the above competent cells, plated on double antibiotic LB selection plates containing kanamycin and spectinomycin, and single colonies were picked after culture and sent for sequencing. Sequencing confirmed that the purR gene knockout was successful. The same method was used to knock out the nadR, pncC and ushA genes in sequence, and finally a gene knockout deletion strain C43(DE3) ΔpurRΔnadRΔpncCΔushA was obtained.

[0034] Example 2: Establishment of a three-dimensional structural model of nicotinamide phosphoribosyltransferase To obtain the three-dimensional structure of nicotinamide phosphoribosyltransferase, homology modeling was first performed. The crystal structure of human NAMPT enzymes is known in the PDB database, and... Schizotequatrovirus The sequence identity of the nicotinamide phosphoribosyltransferase derived from KVP40 was 41.53%. Using human NAMPT enzyme as a template, a predicted three-dimensional structural model was obtained. Figure 1 ).

[0035] Example 3: Construction of site-directed mutagenesis library The three-dimensional structural model was molecularly docked with nicotinamide and PRPP, and the amino acid at the 5 Å binding site between the enzyme and the substrate was selected as a candidate mutation point for design.

[0036] The gene encoding the NAMPT enzyme (Gene ID: 2546058) was synthesized by Beijing Qingke Biotechnology Co., Ltd. and cloned into the vector pET28a, named pET28a-nampt. Its plasmid map is shown below. Figure 2 As shown in Table 4, PCR amplification was performed using plasmid pET28a-nampt as a template, and site-directed mutagenesis was performed at sites 457, 344, 378, 283, and 182 of the NAMPT enzyme.

[0037] Mutation primers were designed. The forward and reverse primers are upstream and downstream PCR primers designed based on different mutation sites. Specific primer information is shown in Table 4.

[0038] Table 4

[0039] Among them, I457V-F and I457V-R can be used to obtain the mutant I457V; G344A-F and G344A-R can be used to obtain the mutant G344A; Q378A-F and Q378A-R can be used to obtain the mutant Q378A; T283G-F and T283GA-R can be used to obtain the mutant T283G; F182A-F and F182A-R can be used to obtain the mutant F182A; The PCR amplification reaction system is the same as that of TOROBlue Flash KOD DyeMix.

[0040] The target gene fragment obtained from PCR amplification was recovered using a Magen gel recovery kit. The specific steps are as follows: 1) Use agarose gel electrophoresis to separate the target DNA fragments, cut off the gel containing the target DNA fragments under ultraviolet light, and remove as much excess gel as possible.

[0041] 2) Weigh the gel block and transfer it to a clean 1.5 mL centrifuge tube. The melting ratio is 100 µL of GDP solution for every 100 mg of agarose gel. Place the centrifuge tube in a metal bath to melt the gel until it is completely dissolved. Transfer the solution to a HiPure DNA Column and attach it to the collection tube. Centrifuge at 12000 rpm for 30 s.

[0042] 3) Discard the filtrate and put the column back into the collection tube. Add 300 µL of GDP Buffer to the column and centrifuge at 12,000 rpm for 30 s.

[0043] 4) Discard the filtrate and reattach the column to the collection tube. Add 600 μL of DW2 Buffer to the column and centrifuge at 12000 rpm for 30 s. Repeat this step once. Note that DW2 Buffer should be diluted with anhydrous ethanol before use.

[0044] 5) Discard the filtrate, put the column back into the collection tube, and centrifuge at 12,000 rpm for 2 minutes.

[0045] 6) Place the column in a clean 1.5 mL centrifuge tube, open the cap, and allow it to dry for about 10 minutes to remove residual ethanol. After drying, add 20-40 µL of preheated double-distilled water and let it stand for about 2 minutes, then centrifuge at 12000 rpm for 2 minutes.

[0046] 7) The concentration of the obtained DNA solution was determined using Nanodrop, and the DNA solution was stored in a -20°C freezer.

[0047] The seamless cloning ligation reaction was carried out at 50 °C for 30 min. The ligation system consisted of 5 μL of Hieff clone enzyme and 5 μL of DNA fragment.

[0048] 10 μL of the ligation product was added to competent *E. coli* DH5α cells. The bacterial culture and plasmid were gently vortexed and incubated on ice for 30 min. Then, the thoroughly mixed bacterial culture and plasmid were heat-shocked in a 42°C water bath for 90 s. After heat shock, the cells were incubated on ice for 2 min, and 900 μL of antibiotic-free LB medium was added. The cells were then incubated at 37°C for 45 min. The bacterial culture was then plated onto LB agar plates containing kanamycin and incubated overnight at 37°C. Transformants from the plates were sent to BGI Genomics Co., Ltd. for sequencing verification.

[0049] Example 4 Construction of recombinant strains This example illustrates the construction of a recombinant strain C43(DE3)ΔpurRΔnadRΔpncCΔushA-nampt-prs-pnuc. Using the gene knockout strain C43(DE3)ΔpurRΔnadRΔpncCΔushA obtained in Example 1 as the base strain, the plasmid pACYC-nampt-prs-pnuc was introduced, specifically including: 1) Construction of recombinant plasmid pACYC-nampt-prs-pnuc: The pnuc gene (Gene ID: 66264729) was cloned into plasmid pACYC-Duet by Beijing Qingke Biotechnology Co., Ltd., and named pACYC-pnuc. Using the *E. coli* C43(DE3) genome as a template, the *prs* gene was amplified by PCR using the primers shown in Table 5. The *nampt* gene was amplified using pET28a-nampt as a template. The obtained PCR products were recovered, and homologous recombination ligation was performed on the *prs* gene fragment, the *nampt* gene fragment, or the mutant I457V, G344A, Q378A, T283G, and F182A gene fragments obtained in Example 3 with the vector pACYC-pnuc using a seamless cloning kit to obtain the recombinant plasmid pACYC-nampt-prs-pnuc. Its plasmid map is shown below. Figure 3 As shown.

[0050] Table 5

[0051] 2) Construction of recombinant strains: The recombinant plasmid pACYC-nampt-prs-pnuc obtained in step 1) was transformed into the deletion strain C43(DE3) ΔpurRΔnadRΔpncCΔushA, plated on solid plates containing chloramphenicol resistance, and cultured at 37℃ for 12-15 h to obtain recombinant strains C-0, C-1, C-2, C-3, C-4, and C-5, respectively.

[0052] Example 5: Shake-flask fermentation and product determination For shake-flask fermentation of *E. coli*, single colonies of the recombinant strain containing the recombinant plasmid pACYC-nampt-prs-pnuc constructed in Example 4 were picked from the corresponding solid medium, activated in seed medium at 37°C for 12 h, and then transferred to a 250 mL Erlenmeyer flask containing 25 mL of fermentation medium. Fermentation was carried out at 37°C and 220 rpm for 24 h. An inducer and 1.0 g / L precursor NAM were added 4 h into the first 4 h of fermentation.

[0053] HPLC analysis of fermentation products: Transfer 1 mL of fermentation broth to a centrifuge tube, centrifuge at 12000 rpm for 10 min, dilute the supernatant, filter through a 0.22 µm filter membrane, and transfer 200 µL of the solution to a liquid chromatography vial for HPLC detection.

[0054] Detection was performed using an Agilent SB AQ column. Mobile phase A was 100% methanol, mobile phase B was 0.1% TFA, flow rate was 1 mL / min, column temperature was 30℃, injection volume was 10 µL, and detection wavelength was 260 nm. Elution conditions: 0–5.3 min, 100% mobile phase B; 5.3–9 min, 80% mobile phase B and 20% mobile phase A; 9–12 min, 100% mobile phase B.

[0055] Fermentation results of nampt mutant and recombinant strains containing the nampt mutant gene are as follows: Figure 4 As shown in Table 6.

[0056] Table 6

[0057] As shown in Table 6, compared with the recombinant strains containing the wild-type nicotinamide phosphoribosyltransferase gene, the recombinant strains C-1 to C-5 all showed improved ability to synthesize β-NMN. In particular, the recombinant strain C-2, which contains the mutant G344A, produced the highest yield of β-NMN among all mutants.

[0058] Example 6: Feed-in-the-flask fermentation of recombinant strains This example illustrates the ability of recombinant strain C-2 to synthesize NMN in fed-batch shake-flask fermentation, specifically including: Seed culture preparation: Single colonies of recombinant strain C-2 were picked from solid agar plates and inoculated into seed culture medium and cultured for 12 h; then transferred to fermentation medium at an inoculation rate of 2% (v / v) and cultured at 37℃ and 220 rpm for 12 h to obtain seed culture.

[0059] NMN fermentation broth preparation: Inoculate 15% (v / v) into a 40 mL fermentation medium at 37°C and pH 6.9. After fermentation begins, OD... 600 IPTG at a final concentration of 0.5 mM was added between 6 and 8 pm. When the glucose in the initial culture medium was depleted, the feeding rate was controlled to keep the glucose concentration below 5 g / L. The glucose concentration and OD in the fermentation broth were monitored periodically. 600 Values ​​and NMN production, results as follows Figure 5 As shown.

[0060] Depend on Figure 5 It can be seen that the recombinant strain C-2 achieved a yield of 15.60 g / L after 48 h of fed-batch culture in shake flasks, showing good application prospects in industrial production.

[0061] The nucleotide sequences of the amino acids and primers involved in this invention are shown in Table 7 below: Table 7

[0062]

[0063]

[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A nicotinamide phosphoribosyltransferase mutant, characterized in that, The mutant was created using the wild-type nicotinamide phosphoribosyltransferase shown in SEQ ID NO:1 as a template. The mutant I457V is formed by mutating isoleucine at position 457 to valine, and its amino acid sequence is shown in SEQ ID NO:

2. The mutant G344A is formed by mutating glycine at position 344 to alanine, and its amino acid sequence is shown in SEQ ID NO:

3. The mutant Q378A is formed by mutating glutamine to alanine at position 378, and its amino acid sequence is shown in SEQ ID NO:

4. The mutant T283G is formed by mutating threonine at position 283 to glycine, and its amino acid sequence is shown in SEQ ID NO:5; or The mutant F182A is formed by mutating phenylalanine at position 182 to alanine, and its amino acid sequence is shown in SEQ ID NO:

6.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nicotinamide phosphoribosyltransferase mutant as described in claim 1.

3. A method for constructing a recombinant strain for producing β-nicotinamide mononucleotide, characterized in that, Includes the following steps: 1) Starting with Escherichia coli C43 (DE3) strain, the coding genes for endogenous UshA, PncC, NadR, and PurR were knocked out to obtain gene knockout strains; 2) The nucleic acid molecule encoding the nicotinamide phosphoribosyltransferase mutant, the phosphoribosyl pyrophosphate synthase gene, and the NMN transporter gene were introduced into the gene knockout strain to obtain the recombinant strain; The nicotinamide phosphoribosyltransferase mutant is as described in claim 1.

4. The construction method according to claim 3, characterized in that, In step 1), the endogenous UshA, PncC, NadR, and PurR coding genes were knocked out using the CRISPR / Cas9 system to obtain gene knockout strain C43 (DE3) ΔpurRΔnadRΔpncCΔushA.

5. The construction method according to claim 3, characterized in that, The nucleic acid molecule encoding the nicotinamide phosphoribosyltransferase mutant, the phosphoribosyl pyrophosphate synthase encoding gene, and the NMN transporter encoding gene described in step 2) were introduced into the gene knockout strain via the recombinant plasmid pACYC-nampt-prs-pnuc.

6. The recombinant strain according to claim 3, characterized in that, The phosphoribosyl pyrophosphate synthase encoding gene is derived from Escherichia coli C43(DE3), whose nucleotide sequence is shown in SEQ ID No. 41, the NMN transporter encoding gene is derived from Bacillus mycoides Its nucleotide sequence is shown in SEQ ID No.

42.

7. A recombinant strain for the production of β-nicotinamide mononucleotide, characterized in that, The recombinant strain was obtained by using Escherichia coli C43 (DE3) with the endogenous UshA, PncC, NadR, and PurR encoding genes knocked out as the host, and expressing the phosphoribosyl pyrophosphate synthase encoding gene, the NMN transporter encoding gene, and the nicotinamide phosphoribosyltransferase mutant as described in claim 1.

8. The use of a recombinant strain as described in claim 7 in the preparation of β-nicotinamide mononucleotide.

9. The application according to claim 8, characterized in that, The recombinant strain described in claim 8 was fermented to obtain the product β-nicotinamide mononucleotide. The fermentation culture includes: inoculating the recombinant strain into a seed culture medium to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium for shake-flask fermentation culture, or for fed-batch fermentation culture, to obtain the β-nicotinamide mononucleotide; The seed culture medium comprises 10 g / L sodium chloride, 10 g / L tryptone and 5 g / L yeast extract; The shake-flask fermentation includes: inoculating the recombinant strain into a seed culture medium and culturing for 12 h, then transferring 1% (v / v) of the seed culture to a fermentation culture medium and culturing for 24 h; the fermentation culture medium includes 8-12 g / L yeast extract, 15-25 g / L glucose, 0.8-1.2 g / L magnesium sulfate, 5 g / L ammonium sulfate and 6 g / L potassium dihydrogen phosphate; The fed-batch fermentation consists of 8 g / L glucose, 10 g / L yeast extract, 6 g / L potassium dihydrogen phosphate and 6 g / L ammonium sulfate, followed by fermentation culture, induced expression, and collection of fermentation broth containing β-nicotinamide mononucleotide.

10. The application according to claim 9, characterized in that, The fed-batch fermentation culture temperature is 35~37℃, and the pH is 6.8~7.3.

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