Nicotinamide phosphoribosyltransferase mutants and uses thereof

Abstract

This invention discloses a nicotinamide phosphoribosyltransferase mutant and its applications, belonging to the field of bioengineering. The mutant is obtained by point mutation or multi-site mutation combination based on nicotinamide phosphoribosyltransferase from Filimonas lacunae. Compared with the wild type, the mutant can catalyze the production of NMN with higher efficiency. NMN yield detection results show that the mutant yield can reach up to 1.97 times that of the wild type under 5L fermentation conditions, and it also exhibits high yields across different temperature ranges, demonstrating good thermal stability.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to nicotinamide phosphoribosyltransferase mutants and their applications. Background Technology

[0002] β-Nicotinamide mononucleotide (NMN) is an important precursor of nicotinamide adenine dinucleotide (NAD+) and plays a vital biological role in cellular energy metabolism, DNA repair, and aging regulation. NAD+ is an important coenzyme for various intracellular metabolic reactions and is widely involved in life activities such as redox reactions, ATP synthesis, and gene repair.

[0003] With in-depth research, NMN, as a precursor to NAD+, has received widespread attention in recent years, especially for its potential in anti-aging, improving metabolic diseases, and enhancing immune function, sparking a large amount of research and application development. NMN can slow down the aging process by increasing intracellular NAD+ levels, activating deacetylases such as sirtuins (e.g., SIRT1, SIRT3), promoting DNA repair, antioxidant responses, and cell repair, while also helping to protect nerve cells and reduce the occurrence of neurodegenerative diseases. NMN can also improve insulin sensitivity, promote lipid and glucose metabolism, and alleviate obesity and metabolic syndrome. Furthermore, NMN has potential cardiovascular protective effects by maintaining vascular health, reducing inflammation, and improving endothelial function.

[0004] NMN can be synthesized in the body through various methods, primarily including enzymatic catalysis, chemical synthesis, and bio-fermentation. While chemical synthesis achieved large-scale production of NMN in the early stages, it suffers from high production costs and environmental pollution. Enzymatic catalysis has mitigated some of the drawbacks of chemical synthesis, but its reaction efficiency and cost still fall short of the requirements for industrial-scale production.

[0005] Bio-fermentation, utilizing microbial metabolic pathways to synthesize NMN, boasts advantages such as a wide availability of raw materials, mild conditions, high sustainability, and ease of scale-up, making it considered an ideal route for the industrial production of NMN. However, in existing fermentation systems, nicotinamide phosphoribosyltransferase (NAMPT), a key rate-limiting enzyme in the nicotinamide-to-NMN synthesis pathway, still suffers from insufficient natural catalytic activity, thermal stability, and substrate binding capacity, limiting the synthesis flux and final fermentation yield of NMN. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the problems of low catalytic efficiency, insufficient structural stability and limited fermentation adaptability of NAMPT, a key enzyme in the existing NMN bio-fermentation process, and to provide a rationally designed multi-site mutant of NAMPT, combined with engineered bacteria construction and fermentation process optimization, to achieve efficient bio-fermentation production of NMN.

[0007] The technical solution of this invention is: a nicotinamide phosphoribosyltransferase mutant, the amino acid sequence of which is obtained by any of the following mutations based on the wild type shown in SEQ ID No. 1:

[0008] 1) The glutamic acid at position 33 is mutated to leucine;

[0009] 2) Glutamic acid at position 33 is mutated to leucine, and serine at position 262 is mutated to alanine;

[0010] 3) Glutamic acid at position 33 is mutated to leucine, and glutamine at position 337 is mutated to methionine;

[0011] 4) Glutamic acid at position 33 is mutated to leucine, aspartic acid at position 180 is mutated to methionine, and serine at position 265 is mutated to isoleucine;

[0012] 5) Glutamic acid at position 33 is mutated to leucine, glycine at position 193 is mutated to leucine, and glutamine at position 337 is mutated to methionine.

[0013] The gene encoding the mutant described above.

[0014] Engineered bacteria expressing the genes described above.

[0015] The above-mentioned mutants or engineered bacteria are used in the production of β-nicotinamide mononucleotide.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention utilizes computer-aided prediction and screening of mutation sites to enhance protein structural stability. Through rational multi-site mutations, it significantly improves the catalytic efficiency and substrate affinity of NAMPT; expands the enzyme's temperature adaptability range, and enhances fermentation process stability. This significantly increases the final fermentation yield of NMN, making it suitable for industrial-scale production. Attached Figure Description

[0018] Figure 1 A comparison of NMN production under shake-flask fermentation conditions using different NAMPT mutant engineered bacteria.

[0019] Figure 2 A comparison chart of the final NMN yields of different engineered bacteria in a 5 L fermenter.

[0020] Figure 3 The graph shows the change in NMN accumulation over time during the fermentation of the E33L\G193L\Q337M mutant NMN. Detailed Implementation

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0022] Example 1: Computer-aided screening of NAMPT mutants

[0023] Based on the nicotinamide phosphoribosyltransferase (SEQ ID No. 1) from Filimonas lacunae, a computer-aided rational design method was used, along with analytical tools such as aMIc, BLAST, FoldX, DeepDDG, and MAESTROweb, to compare conserved sequences of NAMPT from different species and screen for key amino acid residues that may affect enzyme activity and structural stability. FoldX and MAESTROweb were used to predict the folding free energy of candidate mutants, and ΔΔG was screened. pred It is negative and is the same as C. pred Mutants with high confidence levels were selected as preferred candidates to enhance protein structural stability and expand the temperature adaptation range. The selected mutation sites are shown in Table 1.

[0024] Table 1: Screening for Nampt_fla mutants

[0025]

[0026] Note: ΔΔG pred Predicting stability changes, G pred <0 indicates a stable mutation. C pred For confidence level estimates, values ​​range from 0.0 (unreliable) to 1.0 (highly reliable).

[0027] Example 2: Construction of recombinant plasmid of pET28a(+)-Nampt_fla (E33L / S262A) mutant

[0028] This embodiment discloses a method for constructing a recombinant plasmid expressing the Nampt_fla(E33L / S262A) dual-site mutant gene. The method involves introducing the mutation site using reverse PCR amplification, followed by plasmid construction using enzyme digestion and ligation techniques. The specific steps are as follows:

[0029] 1. Experimental Materials

[0030] Template plasmid: pET28a (+) plasmid containing the wild-type Nampt_fla gene (SEQ ID No.2).

[0031] Primers: Specific forward and reverse primers were designed based on the E33L and S262A mutation sites. The primer sequences are shown in Table 2 below.

[0032] Vector and strain: pET28a(+) empty vector, Escherichia coli DH5α competent cells.

[0033] Reagents: Phanta Max Master Mix, Dpn I restriction endonuclease, EcoR I restriction endonuclease, Hind III restriction endonuclease, T4 DNA ligase, PCR product purification kit, agarose gel extraction kit, kanamycin, LB medium.

[0034] Table 2: Primer sequences for E33L and S262A mutation sites

[0035]

[0036] 2 Experimental Methods

[0037] 2.1 Reverse PCR amplification of mutant fragments

[0038] Using the pET28a (+) plasmid carrying the wild-type Nampt_fla gene as a template, reverse PCR amplification was performed using the E33L primer pair and the S262A primer pair, respectively, to obtain amplification products containing a single mutation site. The PCR reaction volume was 25 μL, and the specific components are shown in Table 3 below.

[0039] Table 3: Reverse PCR Reaction System

[0040]

[0041] PCR amplification program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 7 min, for a total of 30 cycles; 72℃ final extension for 10 min; 4℃ incubation.

[0042] 2.2 Dpn I enzyme treatment to remove template plasmid

[0043] After the PCR reaction was completed, 1 μL of Dpn I restriction endonuclease was added to each reaction system, mixed thoroughly, and incubated at 37°C for 2 h. This step is used to specifically degrade the methylated wild-type template plasmid while retaining the unmethylated PCR mutant product.

[0044] 2.3 Purification of PCR Products

[0045] The PCR products treated with Dpn I enzyme were analyzed by 1% agarose gel electrophoresis. The target bands were recovered using a PCR product purification kit, and the procedure was strictly performed according to the kit instructions. After purification, fragments containing the E33L mutation site and fragments containing the S262A mutation site were obtained, respectively.

[0046] 2.4 Double digestion of pET28a (+) vector

[0047] The pET28a(+) empty vector was double-digested with EcoR I and Hind III to obtain the linearized vector fragment. The double digestion reaction system was 50 μL: 1000 ng pET28a(+) empty vector, 1 μL EcoR I, 1 μL Hind III, 5 μL 10×Buffer, and ddH2O to a final volume of 50 μL. Reaction conditions: incubation at 37℃ for 120 min, storage at 4℃; after agarose gel electrophoresis, the linearized vector fragment was recovered by gel extraction.

[0048] 2.5 Connection Reaction

[0049] The purified E33L mutant fragment, S262A mutant fragment, and linearized pET28a (+) vector were mixed at a molar ratio of 3:3:1, and T4 DNA ligase was added for ligation. The ligation system consisted of 20 μL: 1 μL linearized vector fragment, 1 μL E33L mutant fragment, 1 μL S262A mutant fragment, 1 μL T4 DNA ligase, 2 μL 10×T4 Buffer, and 14 μL ddH2O. Reaction conditions: incubation at 22℃ for 2 h, storage at 4℃.

[0050] 2.6 Transformation and positive clone screening

[0051] The ligation product was added to 50 μL of *E. coli* DH5α competent cells and incubated on ice for 30 min; then heat-shocked at 42°C for 90 s, and rapidly cooled on ice for 5 min. 200 μL of antibiotic-free LB broth was added to the system, and the mixture was incubated at 37°C and 220 rpm for 1 h using a shaker. 100 μL of the bacterial culture was spread onto LB agar plates containing 50 mg / L kanamycin and incubated upside down at 37°C for 12–16 h.

[0052] 2.7 Sequencing Validation

[0053] Single colonies were picked from LB agar plates and inoculated into LB liquid medium containing 50 mg / L kanamycin, and cultured at 37°C and 220 rpm for 12 h. Plasmids were extracted from the bacterial culture and sent to a sequencing company for sequencing. The sequencing results were compared with the wild-type Nampt_fla gene sequence.

[0054] 3. Experimental Results

[0055] Sequencing results showed that the Nampt_fla gene in the recombinant plasmid underwent a base change from GAA to CTT at position E33, corresponding to a change from glutamic acid to leucine; and a base change from TCT to GCT at position S262, corresponding to a change from serine to alanine. No other random mutations were found in the gene sequence, indicating that the pET28a(+)-Nampt_fla(E33L / S262A) mutant gene expression recombinant plasmid was successfully constructed.

[0056] Example 3: Expression of the Nampt_fla mutant in Chameleon bacteria

[0057] After introducing the mutation according to the method in Example 2 above, it was ligated into the pET-28a(+) vector by enzyme digestion (EcoR I / Hind III) and placed downstream of the T7 promoter to achieve high-efficiency expression induced by IPTG (kanamycin resistance screening). Sequencing verification results showed that no random mutations appeared except for the required mutation site, so the mutant plasmid was successfully constructed.

[0058] Phosphoribose pyrophosphate synthase (BsPRS, gene sequence as shown in SEQ ID No. 3, amino acid sequence as shown in SEQ ID No. 4) and NMN transporter protein (BMpnuC, gene sequence as shown in SEQ ID No. 5, amino acid sequence as shown in SEQ ID No. 6) were tandemly cloned into the pCDF-1b vector and placed downstream of the tac promoter (for streptomycin resistance selection). The completed vector is: pCDF-BsPRS-BMpnuC.

[0059] The successfully constructed mutant plasmid and pCDF-BsPRS-BMpnuC were transferred into the basal bacteria, and positive clones were screened using a dual antibiotic regimen (kanamycin + streptomycin).

[0060] The engineered bacteria expressing the remaining mutants in Table 1 were constructed by referring to the methods of Examples 2 and 3.

[0061] Example 4: Shake-flask fermentation and HPLC detection of NMN

[0062] The modified BL21 Escherichia coli was cultured in shake flasks. The culture medium in the shake flasks consisted of Na2HPO4 (5.8 g / L), KH2PO4 (3 g / L), NaCl (0.5 g / L), NH4Cl (1 g / L) and MgSO4 (1 mM), with glucose (10 g / L) as the carbon source. The fed-batch culture medium contains 10 g glucose, 10 g xylose, 0.5 g NAM, 4 g (NH4)2HPO4, 13.3 g KH2PO4, 1.2 g MgSO4·7H2O, 1.7 g citric acid, and 10 mL of trace element solution (5 M HCl per liter containing 10 g FeSO4·7H2O, 2.25 g ZnSO4·7H2O, 1 g CuSO4·5H2O, 0.5 g MnSO4·5H2O, 0.23 g Na2B4O7·10H2O, 2 g CaCl2·2H2O, and 0.1 g (NH4)6Mo7O4 per liter). 24 The NMN yield was measured after culturing at 37℃ and 220 rpm for 72 hours, with the initial engineered bacteria transformed into wild-type Nampt_fla serving as a control.

[0063] NMN Detection Method: The content of NMN was detected by high performance liquid chromatography (HPLC). The chromatographic column was a Yuexu XB-C18 column (250 mm × 4.6 mm, 5 μm). Mobile phase: Phase A: 5 mM 1 g / L sodium heptanesulfonate dissolved in ultrapure water, with 30 μL of phosphoric acid added, followed by ultrasonication through a membrane; Phase B: pure methanol, followed by ultrasonication through a membrane. Detection conditions: Temperature: 30℃; Flow rate: 0.8 ml / min; Detection wavelength: 259 nm.

[0064] The comparison results are shown in Table 4. Among them, the E. coli expressing the three-point mutant (E33L / G193L / Q337M) had the highest shake flask yield and unit cell yield, reaching 1.12 g / L and 361.29 mg / g DCW.

[0065] Table 4: Comparison of NMN synthesis effects of engineered bacteria with different NAMPT forms after 48 h of shake-flask fermentation

[0066]

[0067] Example 5: 5L Fermentation Process Operation Flow and Parameter Control

[0068] The engineered strain expressing the superior NAMPT mutant enzyme from Example 4 was selected as the production strain. The engineered strain was activated and cultured in LB liquid seed medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl) containing 50 mg / L kanamycin and 50 mg / L streptomycin, followed by two-stage shake-flask culture to prepare the seed culture. After streaking the first-generation seed culture overnight on LB solid medium (containing 1.5% agar), single colonies were streaked into 50 mL shake flasks and cultured (37°C, 220 rpm, 10-12 h, until OD600 ≈ 3-4) to obtain the second-generation seed culture. Approximately 4% of the seed culture was inoculated into a fermenter, with an initial liquid volume of approximately 2.5 L.

[0069] The fermentation medium formula is as follows: ammonium sulfate 7.5 g / L, corn steep liquor 3.0 g / L, potassium dihydrogen phosphate 6.0 g / L, dipotassium hydrogen phosphate (trihydrate) 16.4 g / L, magnesium sulfate (heptahydrate) 1.0 g / L, glucose 10.0 g / L, yeast extract 10.0 g / L, plus 2 mL / L of metal ion solution and 0.2 mL / L of defoamer. The metal ion solution formula is: FeSO4·7H2O 10 g / L, CaCl2 1.53 g / L, ZnSO4·7H2O 2.2 g / L, MnSO4·4H2O 1.0 g / L, CuSO4·5H2O 1.0 g / L, (NH4)6Mo7O 24 • 4H₂O, 0.1 g / L; Na₂B₄O₇·10H₂O, 0.2 g / L; NiCl₂, 1.0 g / L; H₃BO₃, 1.0 g / L. Adjust with HCl and filter for sterilization. The saturated feed solution is 660 g / L glucose monohydrate stock solution and 10 g / L yeast extract (autoclaved at 121°C), used for feeding during fermentation. The substrate nicotinamide stock solution concentration is 100 g / L.

[0070] The fermentation process was controlled as follows: The fermentation started at 37°C, and after inoculation, the initial stirring speed was 200 rpm, with an aeration rate of 0.5 L / min. As the oxygen demand of the cells increased in the early stages of cultivation, the stirring speed (up to a maximum of 800 rpm) and aeration rate (gradually increasing to 3.0 L / min) were gradually increased to maintain dissolved oxygen (DO) at approximately 30%. When the residual sugar in the culture medium dropped to approximately 5 g / L after 4-5 hours of cultivation, sugar replenishment was initiated. During the replenishment period, the glucose concentration and OD600 of the culture medium were measured every hour (later changed to every 2 hours), and saturated glucose solution was added to maintain a stable glucose concentration at a low level (≤5 g / L). When the OD600 of the cells reached 10⁻¹⁵ (equivalent to entering the late logarithmic growth phase), pre-prepared IPTG was added to the fermenter to 0.5 mM to induce the expression of variant NAMPT, and nicotinamide substrate solution (100 g / L stock solution) was added in batches. Nicotinamide was added in batches, with the corresponding volume added every 2 hours to achieve a final concentration of 2.0 g / L. The feeding frequency was slightly increased initially, then gradually decreased according to the culture progress. In the later stages of fermentation, when the cell OD600 reached 40-50 and continued to grow, the DO (dissolved oxygen) was appropriately reduced (to approximately 10%) to slow the growth rate. Throughout the fermentation process, the pH was adjusted online to 6.9 using 50% ammonia. After fermentation, samples were centrifuged (14000 rpm, 5 min), the cells were discarded, and the supernatant was filtered through a 0.22 μm filter membrane. The NMN content was then determined by high-performance liquid chromatography (HPLC).

[0071] During the same fermentation cycle (72 h), the E. coli 5L expressing the three point mutants (E33L\G193L\Q337M) had the highest fermentation yield, reaching 12.8 g / L, which was significantly higher than that of the wild type (see Table 5).

[0072] Table 5: NMN production performance of different NAMPT engineered bacteria under batch feeding conditions in a 5 L fermenter

[0073]

[0074] Example 6: Fermentation Temperature Test and Effect Verification

[0075] Production was carried out using the fermentation process described in Example 5. Wild-type Nampt_fla and the engineered strain expressing the superior NAMPT mutant enzyme from Example 4 were selected as production strains. The fermentation temperature was controlled at 25–37°C to test the adaptability of the mutant strains to fermentation temperature. The results showed that at a fermentation temperature of 34°C, the mutant combination (E33L / S262A) still maintained a 95% relative yield, significantly higher than the wild-type's 67% relative yield (see Table 6).

[0076] Production was carried out using the 5 L fermenter fed-batch fermentation process described in Example 5. Wild-type Nampt_fla engineered bacteria and all NAMPT mutant engineered bacteria containing the E33L core mutation (including: single-site mutant E33L, two-site mutant E33L / S262A, two-site mutant E33L / Q337M, three-site mutant E33L / D180M / S265I, and three-site mutant E33L / G193L / Q337M) were selected as production strains. During fermentation, the culture temperature was controlled at 28℃, 34℃, and 37℃ to test the adaptability of each mutant strain to fermentation temperature. All other fermentation conditions and process parameters remained consistent with Example 5, and the fermentation cycle was 72 h. After fermentation, samples were taken and the final NMN yield was determined using the HPLC method described in Example 4. The NMN yield of each strain at 28℃ was taken as 100%, and the relative NMN yield at different temperatures was calculated. The results are shown in Table 6.

[0077] Table 6: Effects of different NAMPT mutants on fermentation temperature adaptability

[0078]

[0079] The results showed that, compared with wild-type Nampt_fla, all NAMPT mutant engineered bacteria containing the E33L core mutation of this invention exhibited a wider temperature adaptability range and superior thermal stability. At 34℃ fermentation, the relative NMN yield of the wild-type strain was only 67%, while the relative yield of the mutant strains of this invention could reach over 75%. Even at a higher fermentation temperature of 37℃, the mutant strains of this invention could still maintain a relative yield of over 62%, far superior to the 45% relative yield of the wild-type strain. These results confirm that the NAMPT mutants of this invention significantly improve protein thermal stability, broaden the temperature adaptability window of the fermentation process, and greatly enhance the stability and process tolerance of the fermentation process, making them more suitable for the production needs of large-scale industrial fermentation.

[0080] In the above embodiments, by introducing a rationally designed NAMPT mutant enzyme Nampt_fla and combining it with an optimized fed-batch fermentation process, a significant improvement in NMN yield and production efficiency was achieved. The results showed that, compared with engineered bacteria expressing wild-type NAMPT, engineered bacteria expressing the multi-site mutant NAMPT significantly increased the final NMN yield under shake-flask and 5 L fermenter conditions, while maintaining high enzyme activity over a wider temperature range and exhibiting significantly enhanced fermentation stability. This indicates that the mutant has significant advantages in industrial fermentation applications.

Claims

1. A nicotinamide phosphoribosyltransferase mutant, the amino acid sequence of which is obtained by any of the following mutations based on the wild type shown in SEQ ID No. 1: 1) The glutamic acid at position 33 is mutated to leucine; 2) Glutamic acid at position 33 is mutated to leucine, and serine at position 262 is mutated to alanine; 3) Glutamic acid at position 33 is mutated to leucine, and glutamine at position 337 is mutated to methionine; 4) Glutamic acid at position 33 is mutated to leucine, aspartic acid at position 180 is mutated to methionine, and serine at position 265 is mutated to isoleucine; 5) Glutamic acid at position 33 is mutated to leucine, glycine at position 193 is mutated to leucine, and glutamine at position 337 is mutated to methionine.

2. The gene encoding the mutant of claim 1.

3. An engineered bacterium expressing the gene of claim 2.

4. The application of the mutant of claim 1 or the engineered bacteria of claim 3 in the production of β-nicotinamide mononucleotide.

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