Escherichia coli engineering strain capable of efficiently producing nicotinamide mononucleotide as well as NAMPT enzyme mutant, construction method and application of escherichia coli engineering strain

Abstract

The invention belongs to the technical field of enzyme engineering and gene engineering, and discloses an Escherichia coli engineering strain capable of efficiently producing nicotinamide mononucleotide as well as an NAMPT enzyme mutant, a construction method and application of the Escherichia coli engineering strain capable of efficiently producing nicotinamide mononucleotide, and the mutant is obtained by carrying out point mutation on a nicotinamide ribose phosphate transferase gene vni which is derived from vibrio mariticus and is represented by SEQ ID NO.1 in heterologous expression. The efficiency of the NAMPT mutant for catalyzing nicotinamide to generate NMN is remarkably improved on the premise of not depending on any metabolic pathway modification. An enzymatic activity detection result shows that the catalytic effect of the mutant is 2.56 times that of a wild type, the NMN generation amount of a part of mutation combination in an in-vitro reaction system can reach 3-5 times that of a control group, and the enzymatic reaction capacity exceeds the highest level which is publically reported at present.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of enzyme engineering and genetic engineering, and in particular to an engineered Escherichia coli strain for producing nicotinamide mononucleotide at high efficiency, a NAMPT enzyme mutant thereof, a construction method and application. BACKGROUND

[0002] Nicotinamide mononucleotide (NMN) is the direct precursor of nicotinamide adenine dinucleotide (NAD⁺) and is widely involved in a series of important life processes such as energy metabolism, DNA repair, and cell aging regulation. Studies have shown that NMN has a significant effect on delaying aging, improving insulin resistance, and enhancing mitochondrial function, and has been widely used in the development of health products, functional foods, and new drugs. With the increasing attention on anti-aging and health management, the market demand for NMN continues to grow, and higher requirements are placed on its efficient and low-cost green production methods.

[0003] Currently, the synthesis of NMN mainly includes chemical synthesis, enzymatic synthesis, and microbial fermentation. The chemical synthesis process is complex and highly polluting, making it difficult to meet the demand for green production; although enzymatic synthesis has high specificity, it is limited by substrate cost and enzyme stability. In contrast, microbial fermentation has become the mainstream technology due to the availability of substrates and controllable process. Among them, nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme for the synthesis of NMN from nicotinamide (NAM) and PRPP, and its expression level and catalytic performance directly affect the yield of NMN.

[0004] The expression activity of natural NAMPT in microbial chassis is limited, and it is easily affected by substrate concentration, pH fluctuation, and temperature under industrial fermentation conditions, making it difficult to meet the high-intensity fermentation requirements in terms of catalytic efficiency and stability. Currently, there have been studies on the co-expression of heterologous nicotinamide phosphoribosyltransferase (NAMPT) and PRPP synthesis-related enzymes in Escherichia coli to achieve the biosynthesis of nicotinamide mononucleotide (NMN), but the yield is low, about 15 mg / L, which is difficult to meet the demand of industrial application. Another technical solution is to regulate the expression of multiple genes related to NMN synthesis, which can achieve high yield under optimized conditions, but such solutions usually rely on complex multi-gene engineering or high-copy vector systems, which have problems such as complicated construction process, poor strain stability, and limited applicability.

[0005] Therefore, it is urgent to develop a NAMPT mutant with higher catalytic activity, better substrate adaptability, and thermal stability to achieve high-efficiency synthesis of nicotinamide mononucleotide.

[0006] Through retrieval, the following published patent documents related to the present application are found:

[0007] 1. A NAMPT enzyme derived from mammals and its application in improving NMN synthesis (CN116004489A), which discloses the use of mouse-derived NAMPT for heterologous expression in E. coli to improve NMN production, but does not involve mutation of the enzyme structure, nor does it achieve systematic optimization of its catalytic performance.

[0008] 2. A mutant NAMPT enzyme for NMN synthesis and its construction method (CN113073089A), which discloses a mutation strategy for a single site of NAMPT, which can improve enzyme activity to a certain extent, but the mutation effect depends on the specific host background, and the synergistic effect of multiple mutation combinations is not discussed.

[0009] Through comparison, the present application selects multiple site mutation combinations based on structure simulation and activity verification, systematically improves the catalytic efficiency and stability of NAMPT, and the mutant shows good enzymatic performance in multiple expression systems, which is suitable for industrialized NMN synthesis requirements. SUMMARY

[0010] The purpose of the present application is to overcome the deficiencies in the prior art, and to provide an E. coli engineering strain for efficient production of nicotinamide mononucleotide and its NAMPT enzyme mutant, construction method and application.

[0011] The technical scheme adopted by the present application to solve its technical problems is:

[0012] A NAMPT enzyme mutant for efficient production of β-nicotinamide mononucleotide, the mutant is obtained by heterologous expression of SEQ ID NO. 1 nicotinamide phosphoribosyltransferase (NAMPT) gene vni from Vibrio nigripulchritudo, and point mutation is performed thereon, the mutation site of the point mutation is selected from one of the following (1) to (15):

[0013] (1) the aspartic acid at position 55 is point mutated to leucine or isoleucine;

[0014] (2) the glutamic acid at position 79 is point mutated to methionine or phenylalanine;

[0015] (3) the threonine at position 114 is point mutated to isoleucine or valine;

[0016] (4) the serine at position 208 is point mutated to tryptophan or proline;

[0017] (5) glutamic acid at position 286 is mutated to serine or cysteine;

[0018] (6) aspartic acid at position 330 is mutated to proline or glutamine;

[0019] (7) aspartic acid at position 443 is mutated to histidine or asparagine;

[0020] (8) histidine at position 450 is mutated to tyrosine or tryptophan;

[0021] (9) the point mutations in (1) to (8) above are combined in a two-site mutation;

[0022] (10) the point mutations in (1) to (8) above are combined in a three-site mutation;

[0023] (11) the point mutations in (1) to (8) above are combined in a four-site mutation;

[0024] (12) the point mutations in (1) to (8) above are combined in a five-site mutation;

[0025] (13) the point mutations in (1) to (8) above are combined in a six-site mutation;

[0026] (14) the point mutations in (1) to (8) above are combined in a seven-site mutation;

[0027] (15) the point mutations in (1) to (8) above are combined in an eight-site mutation.

[0028] Further, the nicotinamide phosphoribosyltransferase gene vni from Vibrio nigripulchritudo has a nucleotide sequence as shown in SEQ ID NO. 1, or a nucleotide sequence obtained by substitution, deletion, or addition of one or more bases from the nucleotide sequence as shown in SEQ ID NO. 1, or a nucleotide sequence having at least 90% homology with the nucleotide sequence as shown in SEQ ID NO. 1; and the function is the same as or similar to that of the nucleotide sequence as shown in SEQ ID NO. 1;

[0029] Alternatively, the nicotinamide phosphoribosyltransferase gene vni from Vibrio nigripulchritudo, when constructing an engineered E. coli strain, contains or does not contain a His (6) -tag;

[0030] Alternatively, the nucleotide sequence of the nicotinamide phosphoribosyltransferase gene vni from Vibrio nigripulchritudo has been partially or fully optimized according to the codon preference of Escherichia coli.

[0031] A recombinant plasmid carrying the mutant gene as described above.

[0032] Microbial cells expressing the mutants described above or containing the recombinant plasmids as described in claim 3.

[0033] An engineered strain of Escherichia coli that produces nicotinamide mononucleotide efficiently, wherein the engineered strain uses Escherichia coli BL21 or MG1655 as a host and expresses the mutant described above.

[0034] The method for constructing engineered Escherichia coli strains as described above includes the following steps:

[0035] (1) Using the pET28a(+) plasmid containing the nicotinamide phosphoribosyltransferase gene vni from Vibrio nigripulchritudo as a template, forward and reverse primers containing the target mutation site were designed, and the target plasmid was amplified by reverse PCR using Phanta Max Master Mix.

[0036] (2) Add restriction endonuclease DpnI to the PCR product and react at 37 °C to remove the template plasmid. Then use a PCR purification kit to recover and purify the product.

[0037] (3) The recovered product was ligated using T4 DNA ligase and incubated at 22-25 °C to form a self-closing recombinant mutant plasmid;

[0038] (4) The ligation product was transformed into Escherichia coli DH5α competent cells, and the cells were heat-shocked at 42 °C for 90 seconds and then recovered. Single colonies were picked to extract plasmids and the sequences were verified.

[0039] (5) Transform the recombinant plasmid verified by sequencing into Escherichia coli BL21(DE3) or MG1655 strain to express the target nicotinamide phosphoribosyltransferase mutant.

[0040] The application of engineered E. coli strains as described above in the production of β-nicotinamide mononucleotide.

[0041] The method for producing β-nicotinamide mononucleotide by fermentation using the engineered strain of Escherichia coli as described above includes the following steps:

[0042] (1) The constructed Escherichia coli engineered strain was inoculated onto LB solid medium plates containing 50 μg / mL kanamycin and cultured at 35-38 °C for about 12-16 h to obtain single colonies;

[0043] (2) Pick a single colony and inoculate it into 3-5 mL of LB liquid medium containing 50 μg / mL kanamycin resistance, and incubate it at 35-38 °C and 180-220 rpm for 12-16 h to obtain the seed culture medium;

[0044] (3) Inoculate the seed culture medium into the fermentation medium at a volume ratio of 15%~25% (v / v), and culture at 35~38 °C and 180~220 rpm for 24 h. Then add IPTG to the final concentration of 0.1~1.0 mM to induce expression. After adding IPTG, the culture temperature is reduced to 28~30 °C. At the same time, nicotinamide is added in batches to a final concentration of 1 g / L. During the fermentation process, the pH is maintained between 7.0 and 7.3 by adding ammonia water to adjust the pH.

[0045] (4) Induced expression was further cultured at 20-30 °C and 220 rpm for 18-24 h;

[0046] (5) After fermentation, the fermentation broth is collected and the content of β-nicotinamide mononucleotide in the fermentation broth is detected by high performance liquid chromatography (HPLC).

[0047] Further, the fermentation medium is prepared at the following concentrations: potassium dihydrogen phosphate (KH₂PO₄) 5.0–6.0 g / L, dipotassium hydrogen phosphate (K₂HPO₄) 0.8–1.2 g / L, ammonium sulfate ((NH₄)₂SO₄) 1.8–2.2 g / L, disodium hydrogen phosphate (Na₂HPO₄) 0.8–1.2 g / L, citric acid monohydrate 1.0–1.2 g / L, magnesium sulfate (MgSO₄) 1.8–2.2 g / L, ferrous sulfate heptahydrate (FeSO₄·7H₂O) 15–25 mg / L, L-methionine 0.4–0.6 g / L, yeast extract 16–20 g / L, peptone 9–11 g / L, thiamine (VB1) 1.5–2.5 mg / L, and glucose is added before sterilization to a final concentration of 18–22 mg / L. g / L, and the initial pH of the culture medium was adjusted to 7.0–7.3.

[0048] Furthermore, the method for determining the yield of the β-nicotinamide mononucleotide includes the following steps:

[0049] (1) After fermentation, collect the fermentation broth sample, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant;

[0050] (2) The supernatant was filtered through a 0.22 μm PES microporous membrane and used directly for high performance liquid chromatography analysis;

[0051] (3) If it is necessary to detect the content of intracellular products, the fermentation liquid is centrifuged and precipitated, then resuspended in an equal volume of ultrapure water, and the mixture is broken up at 4 °C for 20 min with an ultrasonic disruptor, pulsed for 2 s / interval for 4 s, centrifuged again and filtered to obtain the supernatant;

[0052] (4) The filtrate was injected into a high performance liquid chromatography system for detection. The chromatographic conditions were as follows: the column was ChromCoreAQ C18 (4.6 mm × 250 mm), the column temperature was 30 ℃, the mobile phase was a mixture of 95% (v / v) 20 mmol / L ammonium acetate aqueous solution and 5% (v / v) acetonitrile, the flow rate was 1 mL / min, and the detection wavelength was 254 nm.

[0053] (5) Calculate the content of β-nicotinamide mononucleotide in the fermentation broth based on the NMN standard curve established by the standard.

[0054] The advantages and positive effects of this invention are as follows:

[0055] 1. Compared with existing technologies, the NAMPT mutant constructed in this invention significantly improves the efficiency of catalyzing the production of NMN from nicotinamide without relying on any metabolic pathway modification. Enzyme activity detection results show that the catalytic effect of the mutant is 2.56 times that of the wild type, and the NMN production of some mutant combinations in the in vitro reaction system can reach 3 to 5 times that of the control group. The enzymatic reaction capacity has exceeded the highest level reported to date.

[0056] 2. The mutant constructed in this invention is stably expressed in Escherichia coli, suitable for expression and application under conventional fermentation conditions, and has good pH tolerance and substrate adaptability. It is suitable for functioning under a wide range of process conditions, facilitating subsequent scale-up and industrial application.

[0057] 3. The mutants described in this invention can be rapidly constructed through conventional operations such as reverse PCR and enzyme digestion and ligation. The mutation strategy is highly systematic and suitable for batch combination screening and expression evaluation. It can be further extended to intelligent evolution or modular synthesis platforms to facilitate the industrial-grade NMN enzymatic synthesis.

[0058] 4. This invention first obtains an enzyme capable of catalyzing NMN production through screening, and then heterologously expresses it in E. coli. Subsequently, mutation sites are selected to mutate the enzyme. A total of 8 candidate sites were selected, namely D55, E79, T114, S208, E286, D330, D443, and H450. Single-point mutations or combination mutations were performed on these 8 sites. The catalytic ability of the mutant strains was improved compared to the wild-type strain. Among them, the most effective mutation increased the NMN production by 5 times compared to the wild type.

[0059] 5. The mutants provided by this invention not only have the advantages of high enzyme activity and stable reaction efficiency, but also have a simple construction method, are suitable for microbial expression systems, and have good industrial application potential, laying the foundation for the efficient synthesis and industrial production of NMN. Attached Figure Description

[0060] Figure 1 This is a spatial distribution diagram of the mutation sites in the NAMPT protein structure in this invention; it shows the relative positional relationship between multiple mutated residues (such as T114, S208, E286, D330, D443, etc.) and the ligand binding region.

[0061] Figure 2 The comparison of NMN production and biomass (OD) between the mutants and wild-type in this invention. 600 A comparison chart showing the relationship between NMN production and OD production; orange represents NMN production, and green represents OD production. 600 1 is the wild-type strain (WT), 2 is the double-mutant engineered strain (Vni-DM1), and 3 is the triple-mutant engineered strain (Vni-TM1).

[0062] Figure 3 This is a verification diagram of the NAMPT mutant recombinant plasmid in this invention; where M: DNA molecular weight standard; lane 1: verification of the double mutant Vni-NAMPT-E286S / S208P (DM1) recombinant plasmid, lane 2: verification of the triple mutant Vni-NAMPT-D55I / T114I / D330P (TM1) recombinant plasmid, and lane 3: verification of the wild-type Vni-NAMPT (WT) recombinant plasmid.

[0063] Figure 4 This is a verification diagram of the vni gene recombinant plasmid in this invention; where M: DNA molecular weight standard; lanes 1 and 2 are for enzyme digestion verification of the pET28a(+) recombinant plasmid containing the vni gene. Detailed Implementation

[0064] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0065] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0066] A mutant that efficiently produces β-nicotinamide mononucleotide, such as Figure 1 As shown, the mutant was obtained by heterologously expressing the nicotinamide phosphoribosyltransferase (NAMPT) gene vni from Vibrio nigripulchritudo (SEQ ID NO.1) and then performing point mutations on it. The mutation sites of the point mutations were selected from one of (1) to (15):

[0067] (1) The aspartic acid at position 55 is point-mutated to leucine or isoleucine;

[0068] (2) Mutate the glutamic acid at position 79 to methionine or phenylalanine;

[0069] (3) The threonine at position 114 is point-mutated to isoleucine or valine;

[0070] (4) Point mutation of serine at position 208 to tryptophan or proline;

[0071] (5) Point mutation of glutamate at position 286 to serine or cysteine;

[0072] (6) Mutate the aspartic acid at position 330 to proline or glutamine;

[0073] (7) Mutate the aspartic acid at position 443 to histidine or asparagine;

[0074] (8) Point mutation of histidine at position 450 to tyrosine or tryptophan;

[0075] (9) Combine the point mutations in (1) to (8) above with two site mutations;

[0076] (10) Combine the point mutations in (1) to (8) above with three-point point mutations;

[0077] (11) Combine the point mutations in (1) to (8) above with four site mutations;

[0078] (12) Combine the point mutations in (1) to (8) above with five site mutations;

[0079] (13) Combine the point mutations in (1) to (8) above with six site mutations;

[0080] (14) Combine the point mutations in (1) to (8) above with seven site mutations;

[0081] (15) Combine the point mutations in (1) to (8) above into an eight-site mutation combination.

[0082] Alternatively, the nicotinamide phosphoribosyltransferase gene *vni* from *Vibrio nigripulchritudo* has the nucleotide sequence shown in SEQ ID NO. 1, or a nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in SEQ ID NO. 1, or a nucleotide sequence that has at least 90% homology with the nucleotide sequence shown in SEQ ID NO. 1; and its function is the same as or similar to that of the nucleotide sequence shown in SEQ ID NO. 1.

[0083] Alternatively, the engineered Escherichia coli strain was constructed with or without the nicotinamide phosphoribosyltransferase gene vni from Vibrio nigripulchritudo, which may or may not contain the His(6)-tag.

[0084] The nucleotide sequence of the nicotinamide phosphoribosyltransferase gene vni from Vibrio nigripulchritudo underwent partial or complete codon optimization based on Escherichia coli codon preference.

[0085] This invention uses standard single-letter codes for amino acids, for example: H450W indicates that histidine (H) at position 450 is mutated to tryptophan (W). In specific experiments, the amino acid sequence needs to be converted into a nucleotide sequence.

[0086] The present invention also provides a recombinant vector comprising the nucleotide sequence. The recombinant vector is a pET28a(+) plasmid vector capable of maintaining replication in host cells, used to amplify or express the nucleotide sequence.

[0087] A highly efficient recombinant Escherichia coli engineered strain producing β-nicotinamide mononucleotide, using Escherichia coli BL21 or MG1655 as a host, expresses either of the mutants described above.

[0088] Furthermore, the method for constructing the recombinant Escherichia coli engineered strain includes the following steps:

[0089] (1) Using the pET28a(+) plasmid containing the nicotinamide phosphoribosyltransferase gene vni from Vibrio nigripulchritudo as a template, forward and reverse primers containing the target mutation site were designed, and the target fragment was amplified by reverse PCR using Phanta Max Master Mix.

[0090] (2) Add restriction endonuclease Dpn I to the PCR product and react at 37 °C to remove the template plasmid. Then use a PCR purification kit to recover and purify the product.

[0091] (3) The recovered product was ligated using T4 DNA ligase and incubated at 22-25 °C to form a self-closing recombinant mutant plasmid;

[0092] (4) The ligation product was transformed into Escherichia coli DH5α competent cells, heat-shocked at 42 °C for 90 seconds and then recovered. Single colonies were picked to extract plasmids and sent to a sequencing company for sequence verification.

[0093] (5) Transform the recombinant plasmid verified by sequencing into Escherichia coli BL21(DE3) or MG1655 strain to express the target nicotinamide phosphoribosyltransferase mutant.

[0094] The application of the recombinant Escherichia coli engineered strains described above in the production of β-nicotinamide mononucleotide.

[0095] The method for producing β-nicotinamide mononucleotide by fermentation using the recombinant Escherichia coli engineered strain described above comprises the following steps:

[0096] (1) The constructed recombinant Escherichia coli engineered strain was inoculated onto LB solid medium plates containing 50 μg / mL kanamycin and cultured at 35-38 °C for about 12-16 h to obtain single colonies;

[0097] (2) Pick a single colony and inoculate it into 3-5 mL of LB liquid medium containing kanamycin resistance, and incubate it at 35-38 °C and 180-220 rpm for 12-16 h to obtain the seed culture medium;

[0098] (3) Inoculate the seed culture solution into 50 mL of fermentation medium at 15%~25% (v / v), and culture at 35~38 °C and 180~220 rpm for 24 h. Then add IPTG to the final concentration of 0.1~1.0 mM to induce expression. After adding IPTG, the culture temperature is reduced to 28~30 °C. At the same time, nicotinamide is added in batches to the final concentration of 1 g / L. During the fermentation process, the pH is maintained between 7.0 and 7.3 by adding ammonia water to adjust the pH.

[0099] (4) Induced expression was further cultured at 20-30 °C and 220 rpm for 18-24 h;

[0100] (5) After fermentation, the fermentation broth is collected and the content of β-nicotinamide mononucleotide in the fermentation broth is detected by high performance liquid chromatography (HPLC).

[0101] Further, the fermentation medium is prepared at the following concentrations: potassium dihydrogen phosphate (KH₂PO₄) 5.0–6.0 g / L, dipotassium hydrogen phosphate (K₂HPO₄) 0.8–1.2 g / L, ammonium sulfate ((NH₄)₂SO₄) 1.8–2.2 g / L, disodium hydrogen phosphate (Na₂HPO₄) 0.8–1.2 g / L, citric acid monohydrate 1.0–1.2 g / L, magnesium sulfate (MgSO₄) 1.8–2.2 g / L, ferrous sulfate heptahydrate (FeSO₄·7H₂O) 15–25 mg / L, L-methionine 0.4–0.6 g / L, yeast extract 16–20 g / L, peptone 9–11 g / L, thiamine (VB1) 1.5–2.5 mg / L, and glucose is added before sterilization to a final concentration of 18–22 mg / L. g / L, and the initial pH of the culture medium was adjusted to 7.0–7.3.

[0102] The specific steps for determining the yield of the β-nicotinamide mononucleotide are as follows:

[0103] (1) After fermentation, collect the fermentation broth sample, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant;

[0104] (2) The supernatant was filtered through a 0.22 μm PES microporous membrane and used directly for high performance liquid chromatography analysis;

[0105] (3) If it is necessary to detect the content of intracellular products, the fermentation broth is centrifuged and precipitated, then resuspended in an equal volume of ultrapure water, and then broken up at 4 °C for 20 min (pulse 2 s / interval 4 s) using an ultrasonic disruptor. After centrifugation and filtration, the supernatant is collected.

[0106] (4) The filtrate was injected into a high performance liquid chromatography system for detection. The chromatographic conditions were as follows: the column was ChromCoreAQ C18 (4.6 mm × 250 mm), the column temperature was 30 °C, the mobile phase was a mixture of 95% 20 mmol / L ammonium acetate aqueous solution and 5% acetonitrile (V / V), the flow rate was 1 mL / min, and the detection wavelength was 254 nm.

[0107] (5) Calculate the content of β-nicotinamide mononucleotide in the fermentation broth based on the NMN standard curve established by the standard.

[0108] Specifically, the relevant preparation and testing methods are as follows:

[0109] Example 1: Construction of recombinant plasmids for gene expression

[0110] (1) Plasmid extraction

[0111] E. coli DH5α strain carrying the commercially available pET28a(+) expression plasmid and a strain containing a synthetically produced target gene fragment (the target gene fragment being the NAMPT gene synthesized by Genewiz and cloned into a commercial vector) were used to construct a recombinant expression strain. The strain was streaked in three zones on LB solid medium containing kanamycin (Kan, 50 μg / mL) and incubated overnight at 37°C for activation. Single colonies were then inoculated into test tubes containing 5 mL of LB liquid medium. Kanamycin (Kan, 50 μg / mL) resistance was added to the medium according to the instructions for the pET28a(+) plasmid. The culture was incubated at 37°C with shaking at 200 rpm for 10–12 h. Plasmid extraction was then performed according to the procedures specified in the Omega Plasmid Mini Kit. Both plasmids were then stored at -20°C for further use.

[0112] (2) Restriction endonuclease digestion

[0113] The enzyme digestion system is shown in the table below:

[0114]

[0115] The enzyme digestion system was prepared according to the table above. Both the pET28a(+) plasmid and the plasmid containing the target gene synthesized by Suzhou Genewise Co., Ltd. were double-digested. The reaction conditions were 37 ℃ and incubation in a metal bath for 120 min. Subsequently, the digestion products were purified using an Omega product purification kit.

[0116] (3) Spe I eliminates template plasmid

[0117] Because double digestion of the plasmid can result in incomplete digestion, Spe I is used to eliminate the template plasmid, thereby improving recombination efficiency in the subsequent enzyme ligation transformation process. The system ratio is shown below:

[0118]

[0119] After the system was prepared according to the proportions in the table above, it was briefly centrifuged using a small centrifuge to ensure that all the liquid was collected at the bottom of the tube. The tube was then incubated in a 37 ℃ water bath for 30 min, followed by product purification.

[0120] (4) Fragment connection

[0121] The DNA fragment ligation system is shown in the table below:

[0122]

[0123] The linearized pET28a(+) and the digested target gene fragment were ligated using T4 ligase and incubated in a metal bath at 25 °C for 120 min to form a recombinant plasmid.

[0124] (5) Chemical transformation of competent cells

[0125] The ligation product was transformed into competent E. coli DH5α cells, and positive transformants were obtained on LB liquid medium with 50 mg / L kanamycin resistance.

[0126] (6) Colony PCR verification

[0127] Single colonies were selected for colony PCR. The PCR products were then detected by agarose gel electrophoresis to verify the identity of the single colonies. Plasmid extraction and sequencing were subsequently performed for verification. Figure 4 As shown.

[0128] The PCR system is shown in the table below:

[0129]

[0130] The specific primers for verifying the mutation are: upper primer: tctcccttatgcgactcctgc; lower primer: ccggatatagttcctcctttcagca

[0131] The PCR program is set up as follows:

[0132]

[0133] Example 2 Construction of recombinant plasmid for expression of vni(E286S / S208P)-pET28a(+) mutant gene

[0134] Using the pET28a(+) plasmid containing the original NAMPT gene vni (i.e., the one obtained in the previous step) as a template, forward and reverse primers containing the mutation sites E286S and S208P were designed, and reverse PCR amplification was performed using the Phanta Max Master Mix system. The primer sequences for the E286S mutation site are as follows:

[0135] E286-F: 5'-GACGAATTAAAAGAAAAGGTTTCACAAAGCGGTGGTACTCTGG-3',

[0136] E286-R: 5'-ACCAGAGTACCACCGCTTTGTAGACCTTTTCTTTTAATTCGTCAC -3'; The primer sequence for the S208P mutation site is:

[0137] S208P-F: 5'-CTTTATGGGTACGGACACGGTGGCCGCACTCGTCTATGCGAGACG-3'

[0138] S208P-R: 5'-CGTCCTCGCATAGACGAGTGCGGCCACCGTGTCCGTACCCATAAA-3'

[0139] Reverse PCR was performed on plasmid vectors containing the vni gene using primers with different mutation sites. The PCR systems are shown in the table below:

[0140]

[0141] After the PCR reaction, Dpn I enzyme was added to remove the template plasmid. The PCR product was then detected by agarose gel electrophoresis and purified using a PCR product purification kit to obtain the target fragment.

[0142] The vector pET28a(+) was double-digested with Kpn I and BamH I. The reaction system consisted of 1000 ng of template plasmid, 1 μL each of Kpn I and BamH I, 5 μL of 10× Buffer, and ddH2O to a total volume of 50 μL. The reaction conditions were incubation at 37 ℃ for 90–120 min and storage at 4 ℃. The digestion products were detected by agarose gel electrophoresis and the linearized vector fragment was obtained by gel extraction.

[0143] The resulting E286S and S208P mutant fragments were mixed and ligated into the linearized pET28a(+) vector. The ligation reaction was performed using T4 DNA ligase at 22 °C for 2 hours. The ligation product was transformed into DH5α competent cells, heat-shocked at 42 °C for 90 seconds, and then screened on LB agar plates containing 50 mg / L kanamycin. Positive clones were picked, plasmids were extracted, and sent to a sequencing company for sequence verification.

[0144] Example 3 Construction of recombinant plasmid for expression of vni(T114I / D330P / D55I)-pET28a(+) mutant gene

[0145] This embodiment uses the same method as in Example 2 to construct a recombinant plasmid for the expression of the vni gene containing mutations at three sites: T114I, D330P, and D55I. Using the pET28a(+) plasmid carrying the original NAMPT gene vni as a template, forward and reverse primers for the corresponding mutation sites were designed and synthesized. Reverse PCR amplification was performed using the Phanta Max Master Mix system. After amplification, the template plasmid was removed by Dpn I enzyme treatment, and the target fragment was purified and recovered.

[0146] The primer sequence for the T114I mutation site is:

[0147] T114I-F: 5'-GTTCCGTGGTTCCGCCACGTTCCCCGCTGGTTCAGATA-3',

[0148] T114I-R: 5'-GTTTTGTATCTGAACCAGCGGGGAACGTGGCGGAACCAC -3';

[0149] The primer sequence for the D330P mutation site is: D330P-F: 5'-GGGCTTCCGTATGCTGCCGCATTGCGTTCGTGTCATTCA-3'.

[0150] D330P-R: 5'- CCTGAATGACACGAACGCAATGGGCAGCATACGGAAGCCCTT -3';

[0151] The primer sequence for the D55I mutation site is: 55D-IF: 5'-CTTCGGCCTCCAAATGTTTCTGAACCAGTACTTGTCGAAGCC -3'.

[0152] 55D-IR: 5'-GCGGCTTCGACAAGTACTGGTTCAGAAACATTTGGAGGCC-3'.

[0153] After constructing single-mutant and double-mutant recombinant plasmids, a triple-mutant plasmid was constructed by progressively stacking mutation sites. The PCR product containing the final T114I, D330P, and D55I mutation sites was ligated to a linearized pET28a(+) plasmid fragment digested with Kpn I and BamHI, using the same ligation reaction system as in Example 2. The ligation product was transformed into DH5α competent cells, heat-shocked at 42 °C for 90 seconds, and then screened on LB agar plates containing 50 mg / L kanamycin. Positive clones were picked, plasmids were extracted, and sent to a sequencing company for sequence verification.Figure 3 As shown.

[0154] Example 4 Construction of Escherichia coli engineered strains overexpressing mutants

[0155] The obtained recombinant plasmids vni(E286S / S208P)-pET28a(+) and vni(T114I / D330P / D55I)-pET28a(+) were transformed into *E. coli* BL21(DE3) to construct double-mutant and triple-mutant recombinant expression strains, respectively. The transformed bacterial cultures were plated on LB agar containing kanamycin (Kan, 50 μg / mL) and incubated at 37 °C for 12–16 hours to obtain engineered strains expressing the D55I / T114I double mutant and the D55I / T114I / D330P triple mutant, respectively. These strains were used for NMN yield determination in subsequent fermentation systems.

[0156] Example 5: Fermentation production of nicotinamide mononucleotide by engineered strains of Escherichia coli

[0157] 1. Activating bacterial strains

[0158] The engineered strain of Escherichia coli BL21(DE3) containing the expression plasmid of the target mutant was removed from the glycerol tube, streaked in three regions on LB solid medium plates containing 50 μg / mL kanamycin, and incubated at 37 °C for 12–16 h to obtain single colonies.

[0159] 2. Seed culture

[0160] Pick a single colony and inoculate it into 5 mL of LB liquid medium containing kanamycin resistance (Kan, 50 μg / mL), and incubate at 37 °C and 220 rpm for 10–12 h to obtain seed culture.

[0161] 3. Fermentation culture

[0162] The seed culture was inoculated into 50 mL of fermentation medium at a ratio of 4% (v / v). 0.5 mM IPTG was added to the fermentation medium to induce expression, and nicotinamide (NAM) was added four times, with a final concentration of 1 g / L. During fermentation, the pH was maintained between 7.0 and 7.3, and adjusted by adding ammonia. The culture was carried out at 37 °C and 220 rpm for 30 h, and samples were taken periodically for NMN yield determination and biomass analysis.

[0163] The fermentation medium was prepared with the following concentrations: potassium dihydrogen phosphate (KH2PO4) 6.0 g / L, dipotassium hydrogen phosphate (K2HPO4) 1.2 g / L, ammonium sulfate ((NH4)2SO4) 2.2 g / L, disodium hydrogen phosphate (Na2HPO4) 1.2 g / L, citric acid monohydrate 1.0 g / L, magnesium sulfate (MgSO4) 2.2 g / L, ferrous sulfate heptahydrate (FeSO4·7H2O) 20 mg / L, L-methionine 0.5 g / L, yeast extract 18 g / L, peptone 10 g / L, thiamine (VB1) 2 mg / L. Before sterilization, glucose was added to a final concentration of 20 g / L, and the initial pH of the medium was adjusted to approximately 7.0.

[0164] Example 6: HPLC determination of nicotinamide mononucleotide content in fermentation broth

[0165] After fermentation, take 1 mL of sample, centrifuge at 12000 rpm for 5 min, and collect the supernatant for extracellular NMN content detection. If intracellular NMN content needs to be measured, resuspend the bacterial pellet in an equal volume of ultrapure water, and disrupt the cells using an ultrasonic cell disruptor (2 s pulses / 4 s intervals) at 4 °C for 20 min. After centrifugation again, collect the supernatant, filter it through a 0.22 μm PES membrane, and use it for subsequent detection.

[0166] The concentration of NMN was determined by high-performance liquid chromatography (HPLC) under the following chromatographic conditions: ChromCore AQ C18 column (4.6 mm × 250 mm), column temperature 30 °C; mobile phase: a mixture of 95% 20 mmol / L ammonium acetate solution and 5% acetonitrile (V / V); flow rate 1.0 mL / min; detection wavelength 254 nm. A standard curve was established using the external standard method, and the NMN content in the sample was calculated.

[0167] Bacterial biomass was measured using a spectrophotometer to determine OD. 600 If the sample concentration is greater than 1.0, it needs to be diluted with ultrapure water to between 0.1 and 0.8. The measurement wavelength is 600 nm, with a blank culture medium as a reference and zero set. The measured OD value multiplied by the dilution factor is the actual biomass.

[0168] The results are as follows Figure 2As shown in the figure, after 24 h of fermentation, the yield of nicotinamide mononucleotide (NMN) in the engineered strain of this invention was significantly increased compared to the wild-type strain. Specifically, the NMN yield of the wild-type strain was approximately 180 mg / L; the yield of the double mutant Vni-DM1 increased to approximately 330 mg / L, an increase of approximately 2.2 times compared to the wild-type; and the yield of the triple mutant (Vni-TM1) reached approximately 460 mg / L, an increase of approximately 3.5 times compared to the wild-type. Compared to the wild-type, the engineered strain significantly increased the NMN yield, with the triple mutant showing the most significant increase, indicating that the mutation strategy has a clear advantage in improving the synthesis efficiency of the target product.

[0169] The wild-type amino acid sequence of the vni enzyme involved in this invention, SEQ NO. 1, is as follows: MDRYSFMNIILNTDSYKASHYLQYPPKTEFVSSYIESRGGEYPQGVYFGLQMFLDQYLSKPLSQEDIDEAKAVLTAHGEPFNEEGWQYILDTHNGYLPVEIQALPEGSVVPPRTPLVQIQNTDPKCAWLTSYLETALLRAVWYPTTVASKSFAIKSIIRRYLEETADSLDGLPFKLHDFGARGAASNEAASIGGLAHLLNFMGTDTVSALVYARRYYNEDIAGYSIPAA EHSTITAWGQNEKLAYQNMLEQFAKPGSLVAVVSDSYDLFHAIDHIWGDELKEKVEQSGGTLVVRPDSGNPVEIVAQTIERLMAKFGESRNSKGFRMLPDCVRVIQGDGISKTTIE SILSELKSKEISADNLAFGMGAELLQKVNRDTMKFAMKASAICIDGKWHDVYKDPATDPNKASRKGRLAVIEASNWETVREDELNGRENALQVVFKDGKVLKRHTLQDIRERIQTYV

[0170] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A NAMPT enzyme mutant for the efficient production of β-nicotinamide mononucleotide, characterized in that: The mutant was obtained by heterologously expressing the nicotinamide phosphoribosyltransferase (NAMPT) gene vni from Vibrio nigripulchritudo as shown in SEQ ID NO. 1 and then performing point mutations therein. The mutation sites of the point mutations were selected from one of the following (1) to (15): (1) The aspartic acid at position 55 is point-mutated to leucine or isoleucine; (2) Mutate the glutamic acid at position 79 to methionine or phenylalanine; (3) The threonine at position 114 is point-mutated to isoleucine or valine; (4) Point mutation of serine at position 208 to tryptophan or proline; (5) Point mutation of glutamate at position 286 to serine or cysteine; (6) Mutate the aspartic acid at position 330 to proline or glutamine; (7) Mutate the aspartic acid at position 443 to histidine or asparagine; (8) Point mutation of histidine at position 450 to tyrosine or tryptophan; (9) Combine the point mutations in (1) to (8) above with two site mutations; (10) Combine the point mutations in (1) to (8) above with three-point point mutations; (11) Combine the point mutations in (1) to (8) above with four site mutations; (12) Combine the point mutations in (1) to (8) above with five site mutations; (13) Combine the point mutations in (1) to (8) above with six site mutations; (14) Combine the point mutations in (1) to (8) above with seven site mutations; (15) Combine the point mutations in (1) to (8) above into an eight-site mutation combination.

2. The mutant according to claim 1, characterized in that: The nicotinamide phosphoribosyltransferase gene *vni* from *Vibrio gripulchritudo* has the nucleotide sequence shown in SEQ ID NO. 1, or a nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the nucleotide sequence shown in SEQ ID NO. 1, or a nucleotide sequence that has at least 90% homology with the nucleotide sequence shown in SEQ ID NO. 1; and its function is the same as or similar to that of the nucleotide sequence shown in SEQ ID NO.

1. Alternatively, the nicotinamide phosphoribosyltransferase gene *vni* from *Vibrio nigripulchritudo* may or may not contain His during the construction of the engineered *E. coli* strain. (6) -tag; Alternatively, the nucleotide sequence of the nicotinamide phosphoribosyltransferase gene vni from Vibrio nigripulchritudo has been partially or fully optimized according to the codon preference of Escherichia coli.

3. A recombinant plasmid carrying the mutant gene as described in claim 1 or 2.

4. Microbial cells expressing the mutant as described in claim 1 or 2 or containing the recombinant plasmid as described in claim 3.

5. A highly efficient engineered strain of *Escherichia coli* that produces nicotinamide mononucleotide, characterized in that: The engineered strain uses Escherichia coli BL21 or MG1655 as a host and expresses the mutant as described in claim 1 or 2.

6. The method for constructing engineered Escherichia coli strains as described in claim 5, characterized in that: Includes the following steps: (1) Using the pET28a(+) plasmid containing the nicotinamide phosphoribosyltransferase gene vni from Vibrio nigripulchritudo as a template, forward and reverse primers containing the target mutation site were designed, and the target plasmid was amplified by reverse PCR using PhantaMax Master Mix. (2) Add restriction endonuclease DpnI to the PCR product and react at 37 °C to remove the template plasmid. Then use a PCR purification kit to recover and purify the product. (3) The recovered product was ligated using T4 DNA ligase and incubated at 22-25 °C to form a self-closing recombinant mutant plasmid; (4) The ligation product was transformed into Escherichia coli DH5α competent cells, and the cells were heat-shocked at 42 °C for 90 seconds and then recovered. Single colonies were picked to extract plasmids and the sequences were verified. (5) Transform the recombinant plasmid verified by sequencing into Escherichia coli BL21(DE3) or MG1655 strain to express the target nicotinamide phosphoribosyltransferase mutant.

7. The use of the engineered Escherichia coli strain as described in claim 5 in the production of β-nicotinamide mononucleotide.

8. A method for producing β-nicotinamide mononucleotide by fermentation using the engineered strain of *Escherichia coli* as described in claim 5, characterized in that: The steps are as follows: (1) The constructed Escherichia coli engineered strain was inoculated onto LB solid medium plates containing 50 μg / mL kanamycin and cultured at 35-38 °C for about 12-16 h to obtain single colonies; (2) Pick a single colony and inoculate it into 3-5 mL of LB liquid medium containing 50 μg / mL kanamycin resistance, and incubate it at 35-38 °C and 180-220 rpm for 12-16 h to obtain the seed culture medium; (3) Inoculate the seed culture medium into the fermentation medium at a volume ratio of 15%~25% (v / v), and culture at 35~38 °C and 180~220 rpm for 24 h. Then add IPTG to the final concentration of 0.1~1.0 mM to induce expression. After adding IPTG, the culture temperature is reduced to 28~30 °C. At the same time, nicotinamide is added in batches to a final concentration of 1 g / L. During the fermentation process, the pH is maintained between 7.0 and 7.3 by adding ammonia water to adjust the pH. (4) Induced expression was further cultured at 20-30 °C and 220 rpm for 18-24 h; (5) After fermentation, the fermentation broth is collected and the content of β-nicotinamide mononucleotide in the fermentation broth is detected by high performance liquid chromatography.

9. The method according to claim 8, characterized in that: The fermentation medium was prepared with the following concentrations: potassium dihydrogen phosphate 5.0–6.0 g / L, dipotassium hydrogen phosphate 0.8–1.2 g / L, ammonium sulfate 1.8–2.2 g / L, disodium hydrogen phosphate 0.8–1.2 g / L, citric acid monohydrate 1.0–1.2 g / L, magnesium sulfate 1.8–2.2 g / L, ferrous sulfate heptahydrate 15–25 mg / L, L-methionine 0.4–0.6 g / L, yeast extract 16–20 g / L, peptone 9–11 g / L, thiamine 1.5–2.5 mg / L. Before sterilization, glucose was added to a final concentration of 18–22 g / L, and the initial pH of the medium was adjusted to 7.0–7.

3.

10. The method according to claim 8 or 9, characterized in that: The method for determining the yield of β-nicotinamide mononucleotide includes the following steps: (1) After fermentation, collect the fermentation broth sample, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant; (2) The supernatant was filtered through a 0.22 μm PES microporous membrane and used directly for high performance liquid chromatography analysis; (3) If it is necessary to detect the content of intracellular products, the fermentation liquid is centrifuged and precipitated, then resuspended in an equal volume of ultrapure water, and the mixture is broken up at 4 °C for 20 min with an ultrasonic disruptor, pulsed for 2 s / interval for 4 s, centrifuged again and filtered to obtain the supernatant; (4) The filtrate was injected into a high performance liquid chromatography system for detection. The chromatographic conditions were as follows: the column was ChromCore AQC18 (4.6 mm × 250 mm), the column temperature was 30 ℃, the mobile phase was a mixture of 95% (v / v) 20 mmol / L ammonium acetate aqueous solution and 5% (v / v) acetonitrile, the flow rate was 1 mL / min, and the detection wavelength was 254 nm. (5) Calculate the content of β-nicotinamide mononucleotide in the fermentation broth based on the NMN standard curve established by the standard.

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