Human-derived nicotinamide riboside kinase mutants, methods of making and uses thereof

Abstract

The application provides a human nicotinamide riboside kinase mutant and a preparation method and application thereof, and relates to the technical field of biological engineering. The nicotinamide riboside kinase mutant is mainly obtained by further modifying and optimizing the amino acid sequence of wild-type Nrk1 with the accession number NP_060351.1 in the NCBI database, and by performing unit point mutation on the wild-type Nrk1, a beneficial mutation site 45 is obtained, and it is proved that the mutation of the 45th amino acid of Nampt from aspartic acid residue to glutamic acid residue can effectively improve the relative activity of the enzyme, and the application of the enzyme in the process of catalyzing the generation of beta-nicotinamide mononucleotide can effectively improve the yield of nicotinamide mononucleotide, and a new purifiable, high-catalytic-activity, high-efficiency and stable engineering enzyme for the production of beta-nicotinamide mononucleotide is provided.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a human nicotinamide ribokinase mutant, its preparation method, and its application. Background Technology

[0002] In recent years, research by David Sinclair's team at Harvard University and other scholars has shown that β-NMN supplementation can rapidly increase the body's NAD+ levels, which gradually decline with age, thereby effectively activating the Sirtuins ("longevity proteins") family and delaying the aging process. Multiple animal model studies have further confirmed that β-NMN intake can significantly improve metabolic function and extend healthy lifespan. β-NMN (β-nicotinamide mononucleotide) has the molecular formula C0. 11 H 15 N2O8P has a molecular weight of 334.2192 g / mol and is composed of phosphate groups, ribose, and nicotinamide groups.

[0003] The salvage synthesis pathway of NAD+ includes the nicotinamide riboside (NR) phosphorylation pathway. This pathway uses NR as a substrate and ATP (adenosine triphosphate) as a phosphate donor to generate β-NMN and ADP (adenosine diphosphate) under the catalysis of nicotinamide ribokinase (NRK). Because this pathway is single-enzyme catalysis and has a short reaction path, NRK can be recycled with the energy donor ATP in industrial production. Therefore, the NR substrate route has become a research hotspot in the biocatalytic synthesis of NMN and is the mainstream industrial route. NRK is a class of phosphotransferases found in various organisms, including plants, fungi, animals, and bacteria, and is the most critical rate-limiting enzyme in the NR substrate route enzyme reaction. Existing literature reports that NRK sources include Kluyveromyces marxianus, humans, and Trichoderma asperellum. However, the catalytic activity of these NRKs is affected by NR concentration, ATP supply, and enzyme stability, leading to incomplete reactions and low purity of β-NMN products in industrial production. Summary of the Invention

[0004] Nicotinamide ribokinase (NRK) exists in two isoforms: Nrk1 (EC 2.7.1.173) and Nrk2 (EC 2.7.1.159), which exhibit significant differences in substrate specificity, co-substrate utilization, and kinetic properties. Nrk1, encoded by the NMRK1 gene, catalyzes the phosphorylation of NR, recognizing ATP or GTP as phosphate donors and providing energy. When ATP is used as a substrate, its catalytic efficiency for NR (kcat / km) is 6800 s⁻¹. -1 ·mol -1 ·L, at this point, its kcat / km for NaR is 4100 s -1 ·mol -1 The presence of ·L indicates that Nrk1 exhibits stronger substrate preference and higher catalytic efficiency for NR. When GTP is used as a substrate, its catalytic efficiency for NR, kcat / km, is 5000 s⁻¹. -1 ·mol -1 •L, slightly lower than ATP. Nrk2 is encoded by the NMRK2 gene and can only recognize ATP as a substrate; its catalytic efficiency for NR is 3900 kcat / km. -1 ·mol -1 The efficiency of ·L is lower compared to Nrk1. When NaR is used as the substrate, the catalytic efficiency kcat / km is 5400 s. -1 ·mol -1 The ·L indicates a preference for NaR. Therefore, compared to Nrk2, Nrk1, derived from mammalian tissues, is more suitable for NMN synthesis. In current Nrk1 research, Cheng et al. (Cheng F, Wu XH, Li H, et al. Streamlining design, engineering, and applications of nicotinamide ribosidekinase for sustainable biosynthesis of nicotinamide mononucleotide in flow[J]. ACS Sustain Chem Eng, 2023, 11(42): 15218-15227.) modified human NRK, ultimately achieving a specific enzyme activity as high as 8.11 U·mg. -1 This represents the highest level reported in the literature. Therefore, this invention uses unmodified human NRK as a wild-type template and further modifies and optimizes its sequence to obtain a purifiable NRK mutant with higher catalytic activity.

[0005] In a first aspect, the present invention provides a human nicotinamide ribokinase mutant, which is obtained by mutating the aspartic acid residue (D) at position 45 of the amino acid sequence of wild-type nicotinamide ribokinase to a glutamic acid residue (E). The wild-type nicotinamide ribokinase is Nrk1 derived from Human, and its amino acid sequence has the accession number NP_060351.1 in the NCBI database. The above-mentioned NRK mutant in the present invention is hereinafter referred to as the D45E mutant.

[0006] It is understood that those skilled in the art can select appropriate gene editing systems and methods to construct the mutants described above, based on the specific circumstances. Preferably, the mutants are obtained using whole-plasmid PCR.

[0007] Secondly, the present invention also provides a biomaterial comprising any one of the following A1)-A6): A1) A nucleic acid molecule that encodes the human nicotinamide ribokinase mutant; A2) An expression cassette, wherein the expression cassette contains the nucleic acid molecule described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1) and / or the expression cassette described in A2); A4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3); A5) Recombinant cells containing the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3); A6) A whole-cell catalyst, wherein the whole-cell catalyst contains the nucleic acid molecule described in A1), the expression cassette described in A2), the recombinant vector described in A3), the recombinant microorganism described in A4), and / or the recombinant cell described in A5).

[0008] The expression cassette described in this invention may also include functional elements such as promoters, terminators, and marker genes. Those skilled in the art can make conventional selections according to the actual situation, as long as the expression of the nucleic acid molecule described in A1) can be completed. No further restrictions are placed on the structure and composition of the expression cassette here.

[0009] The vector described in this invention refers to a vector capable of delivering exogenous DNA or a target gene into a host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), or viral vectors. Specifically, it can be the pET28a plasmid, pET30a plasmid, etc.

[0010] The cells described in this article can be plant cells or animal cells, and can be any biological cell capable of synthesizing the target β-NMN.

[0011] Further, the recombinant microorganism is one or more selected from Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. Preferably, the recombinant microorganism is Escherichia coli. More preferably, the Escherichia coli is Escherichia coli. E. coli BL21(DE3).

[0012] Thirdly, the present invention also provides a method for preparing the human nicotinamide ribokinase mutant, comprising the steps of: Step 1: Ferment and culture the biological material, then centrifuge to collect the precipitate; wherein, the biological material is recombinant microorganisms or recombinant cells; Step 2: Resuspend the precipitate, sonicate to break it up, and centrifuge to obtain the supernatant, which is the crude enzyme solution of nicotinamide ribokinase mutant; Step 3: Elute and perform chromatography on the crude enzyme solution of the nicotinamide ribokinase mutant to obtain the pure nicotinamide ribokinase mutant.

[0013] To further ensure enzyme activity and stability, preferably, step one includes: transferring the seed culture of the recombinant microorganism to a resistant fermentation medium at an inoculum size of 1-5%, and culturing it at 15℃-45℃ and 100-300 rpm for 4-24 h, and at OD... 600 When the concentration is 0.5-0.7, add IPTG to a final concentration of 0.1-0.5 mM to obtain the fermentation product; centrifuge the fermentation product and collect the bacterial cells.

[0014] Fourthly, this application also provides the use of the human nicotinamide ribokinase mutant or the biological material in the preparation of β-nicotinamide mononucleotide.

[0015] Preferably, the human nicotinamide ribokinase mutant is added to the substrate solution and reacted to obtain β-nicotinamide mononucleotide.

[0016] The present invention has the following beneficial effects: In this invention, the amino acid sequence of wild-type Nrk1 derived from Humam was further modified and optimized. By performing a single point mutation on wild-type NRK, a beneficial mutation site at position 45 was obtained. It was demonstrated that mutating the 45th amino acid of Nampt from an aspartic acid residue to a glutamic acid residue can effectively improve the relative activity of the enzyme. When applied to the catalytic production of β-nicotinamide mononucleotide, it can effectively improve the yield of nicotinamide mononucleotide, providing a new, purifiable, highly catalytically active, efficient, and stable engineered enzyme for the production of β-nicotinamide mononucleotide. Attached Figure Description

[0017] Figure 1 The image shows an SDS-PAGE analysis of the wild-type Nrk1 and the mutant NRK-D45E obtained from Example 1 after separation and purification. Figure 2 Experimental results of optimized conditions for the synthesis of β-NMN by mutant strain D45E (in each figure, "NMN" refers to "β-NMN"); Figure 3 SDS-PAGE protein electrophoresis images of crude enzyme solutions of mutant strain D45E at different induction times; Figure 4 Comparison of HPLC peaks of the fermentation broth used to synthesize β-NMN before and after purification of NRK-D45E crude enzyme solution. Detailed Implementation

[0018] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution studies.

[0019] Expression cassette: An expression cassette is a set of DNA sequences that consists of promoters, target genes, and reporter genes, and can be expressed in specific tissues and is easily detected.

[0020] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0021] Recombinant vectors: Recombinant vectors are vectors into which the target gene is transferred based on the basic framework of a cloning vector, thereby enabling the target gene to be expressed.

[0022] Recombinant microorganisms: bacterial cell lines in which foreign genes are expressed efficiently using genetic engineering methods.

[0023] Recombinant cells: The term "recombinant cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "recombinant cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0024] Whole-cell catalysts: Whole-cell biocatalysis refers to the process of using a complete biological organism (i.e., whole cell, tissue, or even individual) as a catalyst for chemical transformation. The complete biological organism that participates in this catalytic process is called a whole-cell catalyst.

[0025] Inoculation: Inoculation volume refers to the ratio of the volume of seed culture to the volume of culture medium after inoculation.

[0026] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0027] In this invention, there are no special requirements for the type of expression plasmid. It can be assumed that the construction method for expressing the target gene in Escherichia coli can adopt various methods commonly used in the field, such as ligating the target gene into a vector after enzyme digestion, replacing the promoter, knocking out the gene, gene mutation, etc., which will not be described in detail hereafter.

[0028] Unless otherwise specified in the examples, follow standard conditions, such as those described in J. Sambrook et al., eds., *Molecular Cloning: A Laboratory Manual (3rd Edition)*, Science Press, 2002; or D.L. Spector et al., eds., *Cellular Laboratory Manual*, Science Press, 2001; or follow the manufacturer's recommended conditions.

[0029] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0030] The plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products. The specific operations were performed according to the kit instructions.

[0031] Unless otherwise stated, the experimental methods, detection methods, preparation methods, and purification methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Specifically, they can be performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0032] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0033] In the following examples Detection method of β-NMN in fermentation productsThe NMN content in the fermentation broth was determined by high-performance liquid chromatography (HPLC). Detection conditions: 5 μm, 250 × 4.6 mol / L C18 column, flow rate 0.7 mL / min, wavelength 254 nm, injection volume 10 μL, column temperature 30℃, gradient elution. Mobile phase A was a mixture of tetrabutylammonium hydroxide, glacial acetic acid, methanol, and water in a volume ratio of 0.6:1:3:97; mobile phase B was pure methanol. The chromatographic peak was detected at 254 nm, and the retention times were compared with those of NR and β-NMN standards to calculate the β-NMN content in the sample. The gradient elution program is shown in Table 1. Table 1 Gradient elution program 0 100 0 20 40 60 25 20 80 35 100 0 40 100 0 In the following examples NRK enzyme heterologous expression First, wild-type NrK1 was heterologously expressed using the pET-30a plasmid as a vector. The coding sequence of the NrK gene was codon-optimized. NdeⅠ and NotⅠ restriction enzyme sites were introduced upstream and downstream of the gene sequence, respectively, and a 6×His tag was added to the N-terminus. The modified synthetic gene was cloned into the pET-30a(+) vector between the NdeⅠ and NotⅠ sites, and the resulting recombinant plasmid was named pET-30a(+)-NrK1. Double enzyme digestion confirmed the correct construction of the recombinant plasmid. Agarose gel electrophoresis showed that the target gene NrK1 was consistent, indicating that pET-30a(+)-NrK1 was successfully constructed. The verified recombinant plasmid was transformed into E. coli BL21(DE3) competent cells and cultured overnight at 37°C inverted on kanamycin-resistant LB agar plates. Single clones were screened, and after expansion culture, plasmids were extracted and verified by double enzyme digestion. Finally, the correctly identified strain E. coli BL21(DE3) / NrK1 was stored in glycerol tubes at -80°C. The heterologous expression method of the NRK mutant was the same as that of the wild-type NrK1, and the E. coli BL21(DE3) / NRK mutant strain was finally obtained. Example 1 Construction and preparation of NRK mutants

[0034] This embodiment uses the wild-type Nrk1 from the human population as the target, whose amino acid sequence accession number in the NCBI database is NP_060351.1, and whose nucleotide sequence is shown in SEQ ID NO. 1. A combination of semi-rational mutagenesis and site-directed mutagenesis was used to construct the NRK mutant. A homology model obtained from the Alphafold2 server was used for molecular docking with nicotinamide riboside (NR) molecules in Autodockvina to obtain a ligand-protein complex. After analyzing the obtained docking model, amino acids within a 6 Å radius of NR were selected as the screening range. Based on spatial occupancy and amino acid charge, nearby amino acids were selected for virtual mutation to enhance ligand-protein binding affinity, and this was named virtual library a. Next, a mutation design strategy that could indirectly affect enzyme-substrate binding and reduce binding energy was obtained using bioinformatics methods, and this was named virtual library b. Virtual library c was established based on a structure-guided consensus method. Virtual library d was constructed using RosettaDesign, where important sites obtained in the previous screening were again subjected to virtual mutation and random full-sequence mutation. The results of virtual libraries a, b, c, and d are superimposed. Mutation strategies that occur more than twice are subjected to molecular dynamics simulations in the Simulation section. Single-point mutation models with increased binding forces and hydrogen bond numbers are selected and named the final virtual library.

[0035] The final virtual library obtained from all 346 virtual mutation sites contains 7 single mutation forms: D45A, D45E, D45P, D45V, I88Q, I88T, I88R, and E189A. Mutation primers were designed using Primer5 software. The specific primer names and corresponding nucleotide sequence numbers are shown in Table 2. Using plasmid pET-30a-Nrk as a template, seven single-point mutant strains, E. coli BL21(DE3) / NRK-D45A, E. coli BL21(DE3) / NRK-D45P, E. coli BL21(DE3) / NRK-D45V, E. coli BL21(DE3) / NRK-I88Q, E. coli BL21(DE3) / NRK-I88T, E. coli BL21(DE3) / NRK-I88R, and E. coli BL21(DE3) / NRK-E189A, were constructed using reverse PCR.

[0036] Table 2. Nucleotide sequence list of primers for mutation sites D45A-F SEQ ID NO.2 I88Q-R SEQ ID NO.9 D45A-R SEQ ID NO.3 I88T-F SEQ ID NO.10 D45P-F SEQ ID NO.4 I88T-R SEQ ID NO.11 D45P-R SEQ ID NO.5 I88R-F SEQ ID NO.12 D45V-F SEQ ID NO.6 I88R-R SEQ ID NO.13 D45V-R SEQ ID NO.7 E189A-F SEQ ID NO.14 I88Q-F SEQ ID NO.8 E189A-R SEQ ID NO.15 D45E-F SEQ ID NO.16 D45E-R SEQ ID NO.17 The method for preparing wild-type Nrk1 and NRK mutants includes the following steps: Step 1: Inoculate each single-point mutant strain and the control strain E. coli BL21(DE3) / Nrk1 into 10 mL centrifuge tubes containing kanamycin-resistant LB medium. Incubate in a shaker at 37°C and 180 rpm for 8 h to obtain seed culture. Transfer the seed culture to 1 L LB liquid medium containing kanamycin at a 2% inoculation ratio. Incubate in a shaker at 37°C and 140 rpm for 8 h. Measure the OD. 600 When the concentration reached 0.6-0.8, the temperature was lowered to 16℃, IPTG was added at a concentration of 0.2 mM, and expression was induced for 16 h to obtain the fermentation broth; the fermentation broth was centrifuged at 4℃ and the wet cells were collected. Step 2: Re-vortex the collected wet bacteria with 20 mM Tris and 150 mM NaCl (pH=7.4), add 1 mM MPMSF and then sonicate to disrupt the cells; then centrifuge again at 12000 rpm for 10 min. The supernatant obtained is the crude enzyme solution containing the NRK mutant (the supernatant is denoted as CS and the precipitate as CP). Step 3: Using the 6×His tag embedded in the NRK gene, the crude enzyme solution was separated and purified by Ni-NTA affinity chromatography. The loading buffer was 20 mM phosphate buffer (pH 7.4) containing 300 mM NaCl and 20 mM imidazole. Elution was performed using a 20-500 mM linear imidazole gradient (20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 mM), corresponding to NRK protein being eluted within the 150-250 mM imidazole concentration range. The purification status was verified by SDS-PAGE. The purified enzyme solution was concentrated to a protein concentration of 20 mg / mL by ultrafiltration, and after adding 10% glycerol and 1 mM DTT as a stabilizer, it was stored at -80℃. The obtained pure enzyme solutions of wild-type Nrk1 and NRK mutant can be used for enzyme activity determination and NMN preparation, respectively.

[0037] The SDS-PAGE analysis results of wild-type Nrk1 and mutant NRK-D45E after isolation and purification are as follows: Figure 1 As shown. Figure 1 Both lanes showed a single band at 27.3 kDa, consistent with the theoretical value, indicating that nickel column affinity chromatography can effectively separate and purify wild-type Nrk1 from mutant NRK-D45E. Example 2: Determination of enzyme activity

[0038] This experiment first used pure NRK enzyme solutions to catalyze the substrates NR and ATP to prepare fermentation broth containing β-NMN using an enzyme-based catalysis method. Then, the content of β-NMN in the reaction solution was determined by HPLC, and the specific enzyme activity of each NRK enzyme was calculated.

[0039] This embodiment provides a method for preparing β-NMN, comprising: preparing 50 μL each of a stock solution of 1.2 mol / L NR, 1.2 mol / L ATP, and 0.5 mol·mol / L MgCl2; adding 800 μL of ddH2O and 50 μL of 0.02 g / L pure enzyme solution; shaking at 1500 rpm at 37℃ for 10 min; terminating the reaction by adding 100 μL of 25% HCl; and detecting the β-NMN content using HPLC-UV method. The pure enzyme solution refers to the pure enzyme solutions of wild-type Nrk1 and NRK mutant obtained in Example 1. The relative enzyme activity was calculated using the enzyme activity of wild-type enzyme strain E. coli BL21(DE3) / Nrk1 as 100%, and the results are shown in Table 3.

[0040] The calculation method for relative activity includes: relative activity = (activity of the tested enzyme / activity of the standard enzyme) × 100%.

[0041] Table 3. Comparison of β-NMN production by various enzymes Wild type Nrk1 7.87 NRK-I88Q 6.85 NRK-D45A 6.62 NRK-I88T 6.04 NRK-D45P 6.40 NRK-I88R 6.52 NRK-D45V 7.23 NRK-E189A 3.51 NRK-D45E 9.46 Table 3 shows that all seven mutant strains were active and capable of producing β-NMN. Among them, only the mutant strain D45E (E. coli BL21(DE3) / NRK-D45E) constructed based on NRK-D45E showed a 20.2% increase in β-NMN production compared to the control strain.

[0042] Estimate the Michaelis constant k using the double reciprocal plot (also known as the Lineweaver-Burk plot). m The maximum rate, Vmax, was determined. Wild-type Nrk1 and the optimal mutant NRK-D45E were added to reaction systems containing different concentrations of substrate, and their enzyme activities were measured. The data were summarized, and a graph was plotted with the reciprocal of the substrate concentration as the independent variable and the reciprocal of the corresponding reaction rate as the dependent variable. The results are shown in Table 4.

[0043] Table 4 Comparison of kinetic parameters between wild-type Nrk1 and mutant NRK-D45E Wild-type Nrk1 72±0.12 21±0.54 7.88±0.22 mutant NRK-D45E 37±0.36 62±0.59 22.95±0.31 The specific enzyme activity of wild-type Nrk1 was determined to be approximately 7.88 U / mg, while that of the optimal mutant NRK-D45E was approximately 22.95 U / mg, which is 2.91 times that of wild-type Nrk, showing a significant increase. The kcat / km value of NRK was also 0.51 times lower than that of the wild-type, indicating a stronger substrate affinity. Compared to the wild-type, its kcat / km value increased by 1.88 times, and compared with the 1.27-fold increase in kcat / km reported by Cheng et al. in previous literature. Example 3: Optimization of conditions for β-NMN synthesis by mutant strain D45E

[0044] The enzyme activity and stability of the mutant NRK-D45E are significantly affected by the induction timing, IPTG concentration, induction temperature, and induction time of the mutant strain D45E (E. coli BL21(DE3) / NRK-D45E). Based on the "Preparation Method of Wild-type Nrk1 and NRK Mutants" in Example 1, the induction timing, IPTG concentration, induction temperature, and induction time conditions of the mutant strain D45E in step one were optimized. Simultaneously, combined with the β-NMN preparation method and content measurement method in Example 2, the yield of β-NMN synthesized by NRK prepared under each optimized condition was detected.

[0045] 3.1 Optimization of Induction Timing The timing of inducer addition is the turning point for recombinant *E. coli* to transition from a growth state to expressing exogenous proteins. After the addition of the inducer, the promoter on the recombinant vector begins transcription and translation to express the exogenous target protein. Therefore, the appropriate induction timing is crucial for efficient expression of the target protein. In this experiment, induction was initiated when the bacterial cells reached the OD of the bacterial culture. 600 IPTG was added to the bacterial culture at induction times of 0.3, 0.4, 0.5, 0.6, and 0.7 mM to investigate the effect of induction timing on NRK expression levels.

[0046] The optimization results for the induction timing are shown in Figure 2 As the induction timing changes from OD 600 Increasing the concentration of IPTG from 0.3 to 0.6 consistently increased the content of β-NMN generated in the reaction system. This may be because adding IPTG at a short culture time can inhibit the growth of microorganisms in the early logarithmic growth stage due to the addition of the inducer, resulting in low biomass and poor NRK expression levels. 600 At a concentration of 0.6, β-NMN production reached its maximum value of 45.836 mmol / L. Cells were cultured further until OD... 600When the OD value was 0.7, the content of β-NMN generated from the conversion of crude NRK enzyme solution in the reaction system decreased to 209.99 μmol / L. This is presumably because the bacteria had entered a growth equilibrium phase at this point, and the ability of aging bacteria to synthesize proteins was weakened, resulting in a lower NRK expression level. In summary, the experimental conclusion is that at the OD value of the bacterial solution... 600 The highest NRK activity was obtained by adding IPTG to a final concentration of 0.2 mM at a concentration of 0.6 and inducing expression for 12 h in a constant-temperature shaking incubator at 30℃ and 200 rpm. This can be converted to produce 45.836 mmol / L of β-NMN.

[0047] 3.2 Optimization of IPTG Concentration as an Inducer IPTG is an inducer that is not metabolized by cells and has a sustained and stable inducing effect. It binds to repressor proteins, causing them to dissociate from the operator sequence, thereby specifically inducing the expression of exogenous proteins. The concentration of IPTG is particularly critical for the expression level of exogenous active proteins. In preliminary experiments, adding IPTG to the culture medium at a final concentration exceeding 0.4 mM resulted in poor growth of the engineered bacteria. Therefore, this experiment optimized the inducer concentration of IPTG in the engineered bacteria E. coli BL21(DE3)-pET-28a-NRK within the range of 0–0.35 mM. Figure 2 As shown, when the final concentration of added IPTG increased from 0.1 mM to 0.35 mM, the content of β-NMN generated by NRK conversion in the reaction system first increased and then decreased. The β-NMN yield reached its maximum of 47.098 mmol / L when the IPTG concentration was 0.15 mM. This may be because when the added IPTG concentration was 0.1 mM, a large amount of repressor protein failed to bind to IPTG, resulting in the lac operon not being fully activated. When the IPTG concentration increased from 0.15 mM to 0.35 mM, the β-NMN yield gradually decreased. This may be because excessively high IPTG concentrations increased the metabolic burden on the recombinant E. coli bacteria, and the physiological toxicity of IPTG itself inhibited bacterial growth, thus preventing the expression of a high level of the target protein. Furthermore, high concentrations of IPTG can induce excessively rapid protein expression, leading to the formation of insoluble inclusion bodies. In conclusion, the experimental findings suggest that at the OD of the bacterial culture... 600 When the concentration of IPTG was 0.6, a final concentration of 0.15 mM was added, and NRK expression was induced for 12 h in a constant-temperature shaking incubator at 30 ℃ and 200 rpm. The highest NRK expression level was observed. It can be converted into 47.098 mmol / L of β-NMN.

[0048] 3.3 Optimization of Induction Temperature During induction, temperature not only affects the growth and reproduction of recombinant E. coli, but also the expression of the target gene and the activity of the expression product. Appropriate induction temperature is crucial for high-level expression of exogenous active target proteins. Generally, induction at relatively low temperatures can prevent the formation of inclusion bodies. This experiment investigated the optimal induction temperature for NRK synthesis using five relatively low temperature gradients: 15℃, 20℃, 25℃, 30℃, and 37℃. The results are as follows: Figure 2 As shown.

[0049] As the induction temperature increased from 15℃ to 37℃, the content of β-NMN generated by NRK conversion in the reaction system showed a trend of first increasing and then decreasing. At an induction temperature of 20℃, the NMN yield reached its maximum of 51.06 mmol / L. At an induction temperature of 15℃, the β-NMN yield was only 43.138 mmol / L. This may be because the induction temperature was too low, inhibiting the growth rate of the recombinant engineered *E. coli* bacteria, and consequently affecting the protein expression level. A temperature of 20℃ was most favorable for the engineered bacteria to absorb and utilize the nutrients in the culture medium to express the protein. Further increasing the induction temperature from 20℃ to 37℃, the β-NMN yield gradually decreased to 41.428 mmol / L. The reason for this is that the closer the induction temperature is to the optimal growth temperature of *E. coli* (37℃), the faster the synthesis rate of the exogenous protein. The target protein is prone to overfolding during rapid synthesis, forming non-biologically active inclusion bodies, thus reducing its catalytic activity. In summary, the experimental conclusion is that at the optimal OD of the bacterial culture... 600 The highest NRK activity was obtained by adding IPTG to a final concentration of 0.15 mM at a concentration of 0.6 and inducing expression for 12 h in a constant-temperature shaking incubator at 20℃ and 200 rpm. This can be converted to produce 51.06 mmol / L β-NMN.

[0050] 3.4 Optimization of Induction Time Induction time is also a crucial factor affecting the expression of the target protein, and different induction temperatures correspond to different optimal induction times. In particular, low-temperature induction slows down cell metabolism, requiring a longer time for complete protein folding, necessitating a longer induction period. Previous experiments have determined the optimal induction temperature for NRK to be 20℃. This experiment fixed the induction temperature at 20℃ and set six induction times: 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h, to investigate the optimal induction time.

[0051] The results of the optimized induction time are shown in Figure 2. With increasing induction time, the content of β-NMN generated by NRK conversion in the reaction system showed a trend of first increasing and then decreasing. The expression level of the target protein increased rapidly in the first 12 hours of induction, suggesting that the bacteria were rapidly absorbing nutrients and synthesizing proteins during this stage. The NMN production reached a peak of 56.812 mmol / L at 20 hours of induction, at which point the exogenous protein had essentially completed its folding. Further increasing the induction time to 24 hours resulted in a decrease in β-NMN production, presumably due to the prolonged induction time causing bacterial aging and autolysis, leading to the degradation of the target protein by other proteases released from the cell.

[0052] The protein electrophoresis images of the crude enzyme solution of mutant strain D45E at different induction times (refer to Example 1) are shown below. Figure 3 As shown. From Figure 3 It can be seen that the protein began to show signs of overexpression from 8 h, and the protein expression level was the highest when the induction time was increased to 20 h. After further induction to 24 h, the expression level did not increase significantly. The trend of its protein expression level is consistent with the production of β-NMN.

[0053] In summary, the experimental conclusion can be drawn that: at the bacterial culture OD... 600 The highest NRK activity was obtained by adding IPTG at a final concentration of 0.15 mM at a concentration of 0.6 and inducing expression in a constant temperature shaking incubator at 20℃ and 200 rpm for 20 h. It could be converted to produce 56.812 mmol / L β-NMN, or 18.99 g / L, with a conversion rate of 56.81%, which is the highest level reported in the literature to date. Example 4: Preparation of β-NMN

[0054] Referring to the "Preparation Method of Wild-Type Nrk1 and NRK Mutants" in Example 1, the mutant strain D45E was induced and cultured under the optimal induction conditions provided in Example 3 to obtain a crude NRK-D45E enzyme solution with a protein concentration of 750 μg / mL. Referring to Example 2, the protein samples before and after purification of the NRK-D45E crude enzyme solution were mixed with substrate of 1000 μmol / L and reacted for 5 min according to the same transformation reaction method. The peak chromatograms before and after purification obtained by HPLC are shown below. Figure 4As shown in the figure. Comparing the two: the peak area of ​​β-NMN generated from the crude enzyme solution was 21.92, while the peak area of ​​β-NMN generated from the crude enzyme solution diluted 2 times and purified for the 5th time was 54.45. The β-NMN yield was approximately 2.5 times that before purification. This indicates that the purity of the NRK enzyme protein was significantly improved after purification, and the impurities in the crude enzyme solution were washed away during the purification process, reducing the degradation of the β-NMN product by these impurities. In addition, the peak area of ​​the crude enzyme solution conversion product at 10 min was relatively high, suggesting that the peak sample at this point was other byproducts generated by the conversion of other impurities in the crude enzyme solution using the substrate ATP, while the peak area of ​​the purified protein at this point was significantly reduced. Furthermore, the peak chromatogram was flatter in the later 12-15 min of the elution process, indicating that a large amount of impurities had not been eluted, which shows that the product in the sample was relatively pure. This further proves that the purified sample was relatively purified. Using the HPLC-UV method, the purity of the β-NMN sample synthesized by the NRK process is about 95%. To ensure that the purity of β-NMN exceeds 90%, multiple chromatographic purifications are required, with an overall yield of 40-60%.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A human nicotinamide ribokinase mutant, characterized in that, It is a mutant obtained by mutating the aspartic acid residue at position 45 of the amino acid sequence of wild-type nicotinamide ribokinase to a glutamic acid residue. The wild-type nicotinamide ribokinase is Nrk1 derived from Human, and its amino acid sequence has accession number NP_060351.1 in the NCBI database.

2. The human nicotinamide ribokinase mutant according to claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

1.

3. A biomaterial, characterized in that, Includes any one of the following: A1)-A6) A1) A nucleic acid molecule, said nucleic acid molecule encoding the human nicotinamide ribokinase mutant as described in claim 1 or 2; A2) An expression cassette, wherein the expression cassette contains the nucleic acid molecule described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1) and / or the expression cassette described in A2); A4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3); A5) Recombinant cells containing the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3); A6) A whole-cell catalyst, wherein the whole-cell catalyst contains the nucleic acid molecule described in A1), the expression cassette described in A2), the recombinant vector described in A3), the recombinant microorganism described in A4), and / or the recombinant cell described in A5).

4. The biomaterial according to claim 3, characterized in that, The recombinant microorganism is one or more of Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae.

5. The biomaterial according to claim 4, characterized in that, The recombinant microorganism is Escherichia coli. E. coli BL21(DE3).

6. A method for preparing the human nicotinamide ribokinase mutant, comprising the steps of: Step 1: Fermentate and cultivate the biological material as described in any one of claims 3-5, and collect the precipitate by centrifugation; wherein, The biomaterial is a recombinant microorganism or a recombinant cell; Step 2: Resuspend the precipitate, sonicate to break it up, and centrifuge to obtain the supernatant, which is the crude enzyme solution of nicotinamide ribokinase mutant; Step 3: Elute and perform chromatography on the crude enzyme solution of the nicotinamide ribokinase mutant to obtain the pure nicotinamide ribokinase mutant.

7. The preparation method according to claim 6, characterized in that, Step one includes: transferring the seed culture of the recombinant microorganism to a resistant fermentation medium at an inoculation rate of 1-5%, and culturing it at 15℃-45℃ and 100-300 rpm for 4-24 h, and at OD... 600 When the concentration is 0.5-0.7, add IPTG to a final concentration of 0.1-0.5 mM to obtain the fermentation product; centrifuge the fermentation product and collect the bacterial cells.

8. The use of a human nicotinamide ribokinase mutant as described in claim 1 or 2, or the biomaterial described in any one of claims 3-5, in the preparation of β-nicotinamide mononucleotide.

9. The application according to claim 8, characterized in that, The human nicotinamide ribokinase mutant was added to the substrate solution and reacted to obtain β-nicotinamide mononucleotide.

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