Acid-resistant enhanced nicotinamide riboside kinase mutant and application thereof

Abstract

The present application relates to the technical field of enzyme catalysis, and particularly relates to a nicotinamide riboside kinase mutant with enhanced acid resistance and application, an amino acid sequence of which is shown as SEQ ID NO: 3, and the mutant has stronger acid resistance compared with wild-type nicotinamide riboside kinase. The nicotinamide riboside kinase mutant of the present application can maintain high catalytic activity at low pH, effectively improve substrate conversion rate; a low-pH environment ensures the stability of the product, effectively slows down the degradation of the product; in large-scale production, the yield of the product can be obviously increased, thereby reducing the production cost of the product and improving market competitiveness.

Description

Technical Field

[0001] The present invention relates to the field of enzyme catalysis technology, and specifically relates to a nicotinamide ribokinase mutant with enhanced acid resistance. Background Art

[0002] NAD+ (β-nicotinamide adenine dinucleotide) plays an indispensable role in cellular metabolism, acting as a coenzyme in numerous biochemical reactions. Recent research, particularly the review article "NAD Metabolism: Role in Aging Regulation and Senescence" published by the Mayo Clinic in the journal Aging Cell on July 9, 2023, has further elucidated the relationship between NAD+ and anti-aging. The study indicates that changes in NAD+ levels and its metabolism are closely associated with multiple biological hallmarks of aging, including but not limited to cellular senescence.

[0003] β-Nicotinamide mononucleotide (NMN), as a direct precursor of NAD and NADP, has attracted widespread attention and may open up new horizons for modern therapeutics. This biomolecule has demonstrated numerous beneficial pharmacological activities in several preclinical disease models, including those for cardiac and cerebral ischemia, neurodegenerative diseases such as Alzheimer's disease, and diabetes. Recent discoveries of its anti-aging and lifespan-extending properties in mouse models have made NMN even more attractive as a potential therapeutic candidate. In recent years, multiple research and development institutions and hospitals have conducted safety tests on NMN, further expanding its application scenarios as a dietary supplement and functional nutritional product.

[0004]

[0005]

[0006] At present, the main production processes of NMN are as follows: (1) Chemical synthesis. Because it involves raw materials that cause gene aberrations, it has low market acceptance; (2) ATP provides phosphate donors. Because ATP can only be used for one cycle, the raw material consumption is too high. With the sharp drop in product prices, it is no longer practical; (3) Acetyl phosphate cyclic ATP method, which provides phosphate groups through ATP; (4) Polyphosphate cyclic ATP method; (5) Fermentation method. The fermentation concentration is low, there are many impurities, the separation cost is too high, and the product quality is poor, resulting in low market acceptance.

[0007] Acetyl phosphate as the first generation of phosphate donor for ATP cycle reaction has a history of more than 40 years. In 1985, Japan Daiso published the patent US4753757 Process for preparing solid acetylphosphate salt, which disclosed the preparation method of lithium acetyl phosphate. Crans et al. published a synthesis method of acetyl phosphate in 1983 (Aconvenient synthesis of disodium acetyl phosphate for use in in-situ ATP cofactor regeneration). However, due to the preparation process of acetyl phosphate involves acetic anhydride, which is a precursor of illicit drugs, and acetyl group is wasted as a byproduct, making the utilization rate low and the finished product extremely unstable, it is difficult to continuously produce as a stable raw material, so the production cost of acetyl phosphate is expensive, and other acetyl phosphates can no longer meet the industrialization needs. Qian et al. also used nicotinamide riboside kinase from Kluyveromyces marxianus in 2022, combined with acetyl kinase from Bacillus stearothermophilus, and used acetyl phosphate to cycle ATP to prepare NMN. 100 g / L of NR can be completely phosphorylated to NMN in 8 h, with a molar yield of 84.2%. Similarly, due to the participation of acetyl phosphate, this process is not competitive (Qian, X.L., Dai, Y.S., Li, C.X. et al. Enzymatic synthesis of high-titer nicotinamide mononucleotide with a new nicotinamide riboside kinase and an efficient ATP regeneration system. Bioresour. Bioprocess. 9, 26. 2022). CN110373398 first disclosed the application of Kluyveromyces marxianus-derived nicotinamide riboside kinase in NMN preparation in 2019, which cooperated with E. coli-derived PPK2, and used polyphosphate as a phosphate donor, ultimately achieving a substrate concentration of 5% and a conversion rate of 80%. However, there are still disadvantages such as low substrate concentration and low conversion rate.

[0008] NMN is unstable in structure, and can be easily degraded in aqueous solutions in neutral and alkaline environments, and the possible degradation products are nicotinamide, ribose, phosphate, nicotinamide ribose, etc., but it is relatively more stable in acidic environment (such as pH 3.5-4.5), and can effectively slow down the degradation rate. In addition, as a compound with a phosphate group, NMN can be degraded by various phosphatases in host cells, such as ushA, cdh, aphA, etc. Acidic conditions can effectively inhibit the activity of various proteins or inactivate them, thereby protecting the target product. Due to the natural properties of the nicotinamide riboside kinases that have been discovered, the current NMN biological preparation process mainly focuses on neutral environment (pH 6.0-6.5), and a small amount of product will still be unstable and degraded, but it is very challenging and difficult to find nicotinamide riboside kinases with acid resistance.

[0009] Therefore, finding a nicotinamide riboside kinase mutant with stronger acid resistance has become one of the problems to be solved in the field. SUMMARY

[0010] The purpose of the present application is to provide a nicotinamide riboside kinase mutant with enhanced acid resistance and application.

[0011] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0012] A nicotinamide riboside kinase mutant with enhanced acid resistance, the amino acid sequence of which is shown in SEQ ID NO: 3.

[0013] The nicotinamide riboside kinase mutant of the present application has stronger acid resistance compared with the wild-type nicotinamide riboside kinase, and the amino acid sequence of the wild-type nicotinamide riboside kinase is shown in SEQ ID NO: 1.

[0014] The present application also provides a polynucleotide which can encode the above-mentioned nicotinamide riboside kinase mutant.

[0015] Further, the polynucleotide sequence is shown in SEQ ID NO: 4.

[0016] The nicotinamide riboside kinase mutant with enhanced acid resistance of the present application can be used to catalyze the generation of beta-nicotinamide mononucleotide from nicotinamide ribose, and the method for using it is as follows: taking crude nicotinamide ribose, MgCl2, sodium hexametaphosphate, AMP, and putting them into the crude enzyme solution of the nicotinamide riboside kinase mutant and polyphosphokinase, and reacting at pH 5.0-6.5 and 40℃.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] (1) Compared with wild-type nicotinamide ribokinase, the nicotinamide ribokinase mutant of the present invention exhibits stronger acid resistance, and the activity of the nicotinamide ribokinase mutant of the present invention at pH 4-5 is more sustained.

[0019] (2) The nicotinamide ribokinase mutant of the present invention can maintain high catalytic activity at low pH, effectively improving substrate conversion rate.

[0020] (3) The low pH environment suitable for the nicotinamide ribokinase of the present invention ensures the stability of the product and effectively slows down the degradation of the product.

[0021] (4) The nicotinamide ribokinase of the present invention is used to produce nicotinamide mononucleotide in a more economical route, which can significantly increase the yield of the product, thereby reducing the production cost of the product and improving market competitiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a gel image of the mutant protein.

[0023] Figure 2 This is the HPLC spectrum of the standard product of nicotinamide riboside, with a peak time of 4.9 min.

[0024] Figure 3 This is the HPLC spectrum of the standard product of nicotinamide mononucleotide, with a peak time of 4.5 minutes.

[0025] Figure 4 The HPLC results of the mutant in Example 6 at pH 5.0 are shown.

[0026] Figure 5 This is the HPLC result of the mutant in Example 6 at pH 6.5.

[0027] Figure 6 This is the HPLC result of the wild-type protein control reaction at pH 5.0 in Example 6, with more substrate remaining. DETAILED DESCRIPTION

[0028] Below in conjunction with the embodiment of the present invention and accompanying drawing, the technical scheme in the embodiment of the present invention is clearly and completely described. Obviously, the embodiment described is only a part of embodiment of the present invention, rather than all embodiments. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise belong to the scope of protection of the present invention. The instruments and reagents used in the present embodiment are commercially available products unless otherwise specified.

[0029] Example 1 Obtaining the wild-type nicotinamide ribokinase gene sequence

[0030] Based on the examples of the previous patent CN202211736710.1, the nicotinamide ribokinase gene from Zygosaccharomyces sp. was synthesized using whole-genome synthesis. PrimerPremier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) were used for design, ensuring that the annealing temperature (Tm) difference was within 3°C and the primer length was within 60 bases. The resulting primers were dissolved in double-distilled water and added to the following reaction system, ensuring a final concentration of 30 nM for each primer and 0.6 μM for the first and last primers.

[0031] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5 μl <![CDATA[ddH2O]]> Make the total volume of the reaction system to 50 μl

[0032] The prepared PCR reaction system was placed in a Biori XP cycler gene amplification instrument and amplified according to the following program: 98°C for 30s, 55°C for 45s, 72°C for 120s, 35x.

[0033] The DNA fragment obtained by PCR was gel-purified and cloned into the NdeI / XhoI sites of pET30a using homologous recombination. A single clone was selected for sequencing. The successfully sequenced DNA sequence is SEQ ID NO: 2, designated ZYKwt, and its corresponding amino acid sequence is SEQ ID NO: 1.

[0034] Example 2 Mutation of wild-type nicotinamide ribokinase

[0035] Nicotinamide ribokinase mutants are derived from the wild-type nicotinamide ribokinase of Example 1. The goal is to obtain mutants with enhanced acid resistance, exhibiting greater acid resistance than the wild-type nicotinamide ribokinase. Nicotinamide ribokinase mutants and polynucleotides encoding such mutants can be prepared using methods commonly used by those skilled in the art. Mutants can be obtained by in vitro recombination of enzymes encoding the enzyme, mutagenesis of polynucleotides, DNA shuffling, error-prone PCR, and directed evolution.

[0036] Through whole-genome synthesis, the secondary structure and codon preference of the gene were adjusted to achieve high expression in E. coli. Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) were used for design, ensuring that the annealing temperature (Tm) difference was within 3°C and the primer length was within 60 bases. The resulting primers were dissolved in double-distilled water and added to the following reaction system, achieving a final concentration of 30 nM for each primer and 0.6 μM for the first and last primers.

[0037] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5 μl <![CDATA[ddH2O]]> Make the total volume of the reaction system to 50 μl

[0038] The prepared PCR reaction system was placed in a Biori XP cycler gene amplification instrument and amplified according to the following program: 98°C for 30s, 55°C for 45s, 72°C for 120s, 35x.

[0039] The DNA fragment obtained by PCR was gel-purified and cloned into the NdeI / XhoI sites of pET30a using homologous recombination. A single clone was selected for sequencing. The successfully sequenced DNA sequence is SEQ ID NO:4, designated ZYKa, and its corresponding amino acid sequence is SEQ ID NO:3. Compared to the sequence of ZYKwt, it has five mutations: Y40F, E45Q, E149G, Y167FD, and 181S.

[0040] Example 3 Shake flask expression test

[0041] A single colony of E. coli harboring the expression vector was inoculated into 10 mL of autoclaved culture medium containing 10 g / L tryptone, 5 g / L yeast extract, 3.55 g / L sodium hydrogen phosphate (NaHPO), 3.4 g / L potassium dihydrogen phosphate (KHPO), 2.68 g / L ammonium chloride, 0.71 g / L sodium sulfate, 0.493 g / L magnesium sulfate heptahydrate, 0.027 g / L ferric chloride hexahydrate, 5 g / L glycerol, and 0.8 g / L glucose. Kanamycin was added to the culture medium to a concentration of 50 mg / L. The culture was incubated overnight at 30°C, 250 rpm. The next day, a 1L Erlenmeyer flask was inoculated with 100mL of autoclaved culture medium at a 1:100 ratio: 10g / L tryptone, 5g / L yeast extract, 3.55g / L sodium hydrogen phosphate (DAP), 3.4g / L potassium dihydrogen phosphate (KDP), 2.68g / L ammonium chloride, 0.71g / L sodium sulfate, 0.493g / L magnesium sulfate heptahydrate, 0.027g / L ferric chloride hexahydrate, 5g / L glycerol, 0.3g / L glucose, and kanamycin supplemented to 50mg / L. Culture the cells at 30°C until the OD value reached 5-6. Immediately, the flask was placed in a shaker at 25°C, 250 rpm, and incubated for 1 hour. IPTG was added to a final concentration of 0.1mM and incubated for an additional 16 hours at 25°C, 250 rpm. After completion of the incubation period, the culture was centrifuged at 12,000g for 20 minutes at 4°C to collect the wet cells. The bacterial pellet was then washed twice with distilled water, and the cells were collected and stored at -70°C. A small amount of cells was taken for SDS-PAGE analysis.

[0042] Example 4 Fed-batch fermentation

[0043] Fed-batch fermentation was conducted in a computer-controlled 7-L pressure-resistant stainless steel bioreactor (Jiangsu B. Braun) with a working volume of 4 L. The culture medium used was a modified TB medium composed of 24 g / L yeast extract, 12 g / L peptone, 0.4% glucose, 2.31 g / L dihydrogen phosphate, and 12.54 g / L dipotassium phosphate, pH 7.0. Approximately 200 mL of primary seed culture in a shake flask was transferred to the fermentor when the OD reached 2.0. During the initial fermentation, the temperature was maintained at 37°C. Dissolved oxygen concentration was automatically controlled at 30% by agitation speed (rpm) and aeration cascade control, while the pH of the culture medium was maintained at 6.7-7.1 using 50% (v / v) orthophosphoric acid and 30% (v / v) ammonia. Feeding was initiated when a significant increase in dissolved oxygen occurred. The feed solution contained 9% w / v peptone, 9% w / v yeast extract, and 14% w / v glycerol. When OD600 reached approximately 15.0, the temperature was lowered to 25°C, and IPTG was added to a final concentration of 0.2 mM to induce expression.

[0044] Example 5 Stability test of NMN at different pH

[0045] Prepare 50mL of a low-pH simulated reaction system: final concentration 10g / L β-nicotinamide mononucleotide, 60mM MgCl2, 25g / L sodium hexametaphosphate, 0.3g / L AMP, adjust pH to 5.0, add 10g / L of crude enzyme solution, and adjust the volume to 50mL. Prepare 50mL of a neutral pH simulated reaction system: final concentration 10g / L β-nicotinamide mononucleotide, 60mM MgCl2, 25g / L sodium hexametaphosphate, 0.3g / L AMP, adjust pH to 5.0, add 10g / L of crude enzyme solution, and adjust the volume to 50mL. Incubate at 37°C overnight and test the remaining NMN in the system. The test results are as follows:

[0046] pH 5.0 pH 6.5 12 hours 9.7g / L 8.7g / L 24 hours 9.5g / L 7.5g / L

[0047] Example 6 Comparison of wild type and mutants at different pH

[0048] A 100 mL reaction system was prepared: a final concentration of 60 g / L crude nicotinamide riboside (approximately 70% content, 40 g / L dry substrate concentration), 60 mM MgCl2, 25 g / L sodium hexametaphosphate, and 0.3 g / L AMP. The pH was adjusted to 5.0 and the volume was brought to 99 mL. Excess crude enzyme solution and excess polyphosphate kinase LE496 (purchased from Nanjing Longen Biotechnology) were added. The reaction was started and timed, with the water bath temperature maintained at 40°C. A control reaction was also performed using the same system but with the pH controlled at 6.5. Samples were taken after 9 hours for HPLC analysis. The results, shown in the table below, show that the mutant efficiently catalyzes substrate catalysis at low pH with no significant degradation products, while the wild-type protein produced a significant amount of substrate at pH 5.0, indicating that the wild-type protein was strongly inhibited or denatured at acidic pH. Comparison of the mutant's reactivity at different pH levels reveals significantly lower product yields at high pH, ​​due to significant degradation caused by product instability at medium to high pH. The test results are shown in the following table and Figure 4-6 shown.

[0049] pH 5.0 pH 6.5 mutant 40.77g / L 35.48g / L wild type 13.32g / L 19.51g / L

[0050] Example 7 Reaction of increasing substrate concentration

[0051] A 100 mL reaction system was prepared: 120 mM MgCl2, final concentration 25 g / L sodium hexametaphosphate, final concentration 50 g / L nicotinamide riboside, 1 g / L AMP, pH 5.0, constant volume to 90 mL, 10 mL of crude enzyme solution, and an appropriate amount of polyphosphokinase LE496 (purchased from Nanjing Langen Biological Technology Co., Ltd.), and the reaction was started and timed. The water bath temperature was controlled at 40°C, and the pH was controlled at 5.0. After 3 hours of reaction, 20 g / L of industrial sodium hexametaphosphate was added. After 9 hours of reaction, the sample was detected, and the results showed that the NMN concentration was 64.92 g / L. Due to the strong inhibition of high-concentration sodium hexametaphosphate on nicotinamide riboside kinase, in this example, the total concentration of sodium hexametaphosphate was added in batches, and the final concentration was 45 g / L. The substrate was successfully catalyzed.

[0052] While embodiments of the present application have been shown and described with reference to particular embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the application. The scope of the application is defined by the appended claims and their equivalents.

Claims

1. A nicotinamide ribokinase mutant with enhanced acid resistance, characterized in that: The amino acid sequence is shown in SEQ ID NO: 3, which contains Y40F, E45Q, E149G, Y167F, and D181S mutations.

2. A polynucleotide encoding the nicotinamide ribokinase mutant according to claim 1.

3. The polynucleotide according to claim 2, wherein: The sequence is shown in SEQ ID NO:

4.

4. Use of the acid-resistant nicotinamide ribokinase mutant according to claim 1 in catalyzing the production of β-nicotinamide mononucleotide from nicotinamide riboside.

5. Use of the acid-resistant nicotinamide ribokinase mutant according to claim 4 in catalyzing the production of β-nicotinamide mononucleotide from nicotinamide riboside, characterized in that: Nicotinamide riboside crude product, MgCl2, sodium hexametaphosphate, and AMP were added to the crude enzyme solution of the nicotinamide ribokinase mutant and polyphosphate kinase, and the mixture was reacted at pH 5.0-6.5 and 40°C.

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