A nicotinamide riboside kinase mutant

Abstract

This invention relates to the field of enzyme catalysis technology, and in particular to a nicotinamide ribokinase mutant, the amino acid sequence of which is shown in SEQ ID NO: 3. Compared with wild-type nicotinamide ribokinase, it exhibits higher catalytic activity and thermal stability. The nicotinamide ribokinase mutant of this invention maintains longer activity at 45℃-50℃, effectively shortening the reaction cycle. This mutant has lower requirements for the cooling system, thus reducing energy consumption and costs. Furthermore, it can be transported and stored at room temperature, effectively extending shelf life. In addition, the high-temperature catalytic reaction conditions avoid the growth of contaminating microorganisms, thereby reducing bacterial metabolite contamination of the product, increasing substrate solubility, and improving production efficiency per unit volume.

Description

Technical Field

[0001] This invention relates to the field of enzyme catalysis technology, specifically to a nicotinamide ribokinase mutant. Background Technology

[0002] β-Nicotinamide mononucleotide (NMN) is an important natural compound widely found in organisms. As a direct precursor to NAD and NADP, NMN has attracted considerable attention, potentially opening new avenues for modern therapies. This biomolecule has demonstrated numerous beneficial pharmacological activities in several preclinical disease models, including those for cerebral ischemia, neurodegenerative diseases such as Alzheimer's disease, and diabetes. Recent findings of its anti-aging and life-extending properties in mouse models further enhance its appeal as a potential therapeutic candidate. Much of its pharmacological action occurs through promoting NAD synthesis, as direct administration of NAD at high doses can sometimes cause side effects such as insomnia, fatigue, and anxiety, and it has a less penetrating ability across the plasma membrane compared to NMN.

[0003] Currently, the main production processes for NMN are as follows: (1) Chemical synthesis. Due to the involvement of raw materials that can cause gene mutations, market acceptance is low; (2) ATP-based phosphate donor method. Because ATP can only be used for one cycle, the raw material consumption is too high, and with the significant drop in product price, it is no longer practical; (3) Acetyl phosphate cycling ATP method, which provides phosphate groups through ATP; (4) Polyphosphate cycling 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.

[0004] Acetyl phosphate has been used as a first-generation phosphate donor in the ATP cycle for over 40 years. For example, Daicel Chemicals of Japan published a patent in 1985, US4753757, "Process for preparing solid acetylphosphate salt," which disclosed a method for preparing lithium acetyl phosphate. Crans et al. published a method for synthesizing acetyl phosphate in 1983 ("A convenient synthesis of disodium acetyl phosphate for use in in-situ ATP cofactor regeneration"). However, due to its environmentally unfriendly nature, it is no longer widely used industrially. Because acetyl phosphate does not significantly inhibit biological enzymes, using high concentrations of acetyl phosphate as a phosphate donor does not have negative side effects on the system, thus allowing for relatively high product concentrations. Furthermore, its relatively high-energy phosphate bond allows for more thorough downstream reactions with minimal substrate residue. These are the two biggest advantages of acetyl phosphate. However, because the preparation process of acetyl phosphate involves acetic anhydride, a precursor to toxic substances, the acetyl group is wasted as a byproduct, resulting in low utilization and extremely unstable finished products, making it difficult to sustain large-scale production as a stable raw material. Furthermore, its production cost further limits its use to phosphorylation reactions for high-value-added products. With the price of NMN exceeding 500 yuan per kilogram becoming inevitable, all production processes using expensive substrates, such as acetyl phosphate, can no longer meet industrial demands.

[0005] CN110373398 first published the application of nicotinamide ribokinase derived from Kluyveromyces marxianus in the preparation of NMN in 2019. Combined with PPK2 derived from E. coli and using polyphosphate as the phosphate donor, it ultimately achieved a 5% substrate concentration and an 80% conversion rate, which is currently considered a superior production method. However, it still has drawbacks such as low substrate concentration and low conversion rate.

[0006] In 2022, Qian et al. also used nicotinamide ribokinase from Kluyveromyces marxianus, combined with acetylkinase from Bacillus thermophilus, to prepare NMN using acetyl phosphate cycling ATP. 100 g / L of NR could be completely phosphorylated to NMN within 8 hours, with a molar yield of 84.2%. However, the involvement of acetyl phosphate also made this process less competitive (Qian, XL., Dai, YS., Li, CX. et al. Enzymatic synthesis of high-titernicotinamide mononucleotide with a new nicotinamide riboside kinase and an efficient ATP regeneration system. Bioresour. Bioprocess. 9, 26, 2022).

[0007] Therefore, finding a nicotinamide ribokinase mutant with better thermal stability has become one of the urgent problems to be solved in this field. Summary of the Invention

[0008] The purpose of this invention is to provide a nicotinamide ribokinase mutant.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A nicotinamide ribokinase mutant, the amino acid sequence of which is shown in SEQ ID NO: 3.

[0011] The nicotinamide ribokinase mutant of the present invention has higher catalytic activity and thermal stability than wild-type nicotinamide ribokinase. The amino acid sequence of the wild-type nicotinamide ribokinase is shown in SEQ ID NO: 1.

[0012] The present invention also provides a polynucleotide that encodes the above-mentioned nicotinamide ribokinase mutant.

[0013] Furthermore, the polynucleotide sequence is shown in SEQ ID NO: 4.

[0014] Compared with the prior art, the beneficial effects of the present invention are:

[0015] (1) Compared with wild-type nicotinamide ribokinase, the nicotinamide ribokinase mutant of the present invention exhibits higher catalytic activity and thermal stability. The nicotinamide ribokinase mutant of the present invention has more sustained activity at 45℃-50℃.

[0016] (2) The nicotinamide ribokinase mutant of the present invention has many advantages: ① It accelerates the kinetic reaction and can effectively shorten the reaction cycle; ② It simplifies the enzyme extraction process, and the host cell protein denatures, coagulates and precipitates at high temperature, making it easy to separate from the target protein; ③ It does not require a high-performance cooling system for the reaction, thus reducing energy consumption and costs; ④ The protein with improved stability can be transported and stored at room temperature, and its shelf life is effectively extended; ⑤ Under the conditions of high-temperature catalytic reaction, the growth opportunity of contaminating bacteria is avoided, thereby reducing the contamination of the product by bacterial metabolites; ⑥ High temperature helps to increase the solubility of the substrate and improve the production efficiency per unit volume.

[0017] (3) The nicotinamide ribokinase of the present invention can be used to catalyze the generation of nicotinamide mononucleotide from nicotinamide ribose at high temperature. The reaction temperature is higher, which can effectively shorten the reaction time and effectively inactivate other enzymes, thereby reducing the degradation of the product.

[0018] (4) The route of the nicotinamide ribokinase of the present invention for the production of nicotinamide mononucleotide is more economical, has lower production costs, greater potential for scale-up and is easier to extract.

[0019] Sodium hexametaphosphate, polyphosphate, triphosphate, and tetraphosphate are all readily available in industrial production. Sodium hexametaphosphate, in particular, is widely used as a food additive, with commercially available food-grade sodium hexametaphosphate priced below 10 yuan per kilogram, and multiple manufacturers providing a stable supply. However, the main challenges in using sodium hexametaphosphate in the process are: ① Sodium hexametaphosphate has a very strong inhibitory effect on proteins; once the concentration exceeds 25 g / L, it easily causes protein denaturation and loss of enzyme activity; ② Sodium hexametaphosphate readily complexes with magnesium ions, reducing its concentration in the system, but all known nicotinamide ribokinases require sufficient magnesium ions as a cofactor; ③ Sodium hexametaphosphate requires the assistance of polyphosphokinase to function.

[0020] This invention finds a heat-resistant polyphosphokinase LE496 as an aid, and the cost of AMP is only 1 / 5 of the cost of ATP, further reducing the raw material cost of NMN. Attached Figure Description

[0021] Figure 1 The results are HPLC results for the mutant in Example 5 after 6 hours of reaction; the peak for NMN is at 6.2 minutes.

[0022] Figure 2 The results are HPLC results for the wild-type reaction in Example 5 after 6 hours. The peak at 6.1 minutes is NMN, and the peak at 7.3 minutes is the substrate peak.

[0023] Figure 3 The results are HPLC results for Example 6 after 7 hours of reaction.

[0024] Figure 4The results are HPLC results for Example 7 after 6 hours of reaction. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the instruments and reagents used in this embodiment are all commercially available products.

[0026] Example 1: Obtaining the wild-type nicotinamide ribokinase gene sequence

[0027] The secondary structure and codon bias of the nicotinamide ribokinase gene derived from *Zygosaccharomyces* sp. were modified using a whole-genome synthesis method to achieve high expression in *E. coli*. PrimerPremier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) were used for primer design, ensuring that the annealing temperature (Tm) difference was controlled within 3°C and the primer length was controlled within 60 bases. The obtained primers were dissolved in double-distilled water and added to the following reaction system, resulting in a final primer concentration of 30 nM and a final concentration of 0.6 μM for the first and last primers.

[0028] 10×Pfu buffer 5μl Pfu DNA polymerase (10 U / μl) 0.5μl <![CDATA[ddH2O]]> This brings the total volume of the reaction system to 50 μl.

[0029] Place the prepared PCR reaction system in the Bori XP cycler gene amplification instrument and amplify according to the following program: 98℃ for 30s, 55℃ for 45s, 72℃ for 120s, 35x.

[0030] The DNA fragment obtained by PCR was purified by gel extraction and cloned into the NdeI / XhoI site of pET30a using homologous recombination. Single clones were selected for sequencing. The successfully sequenced DNA sequence is SEQ ID NO: 2, named ZYKwt, and its corresponding amino acid sequence is SEQ ID NO: 1.

[0031] Example 2: Obtaining the gene sequence of the nicotinamide ribokinase mutant

[0032] The nicotinamide ribokinase mutant, derived from the wild-type nicotinamide ribokinase of Example 1, is capable of catalyzing the high-temperature conversion of nicotinamide ribose to nicotinamide mononucleotide (MNMN), and can be used for the high-temperature conversion of nicotinamide ribose to MNMN. The nicotinamide ribokinase mutant exhibits stronger thermostability compared to the wild-type nicotinamide ribokinase. The nicotinamide ribokinase mutant and the polynucleotide encoding this mutant can be prepared using methods commonly used by those skilled in the art. The mutant can be obtained through in vitro recombination of the enzyme, polynucleotide mutagenesis, DNA shuffling, error-prone PCR, and directed evolution methods.

[0033] High expression of the gene in *E. coli* was achieved by modifying its secondary structure and codon bias using whole-genome synthesis. Primers were designed using Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ), ensuring the annealing temperature (Tm) difference was controlled within 3°C and the primer length within 60 bases. The obtained primers were dissolved in double-distilled water and added to the following reaction system, resulting in a final primer concentration of 30 nM and a final concentration of 0.6 μM for the first and last primers.

[0034] 10×Pfu buffer 5μl Pfu DNA polymerase (10 U / μl) 0.5μl <![CDATA[ddH2O]]> This brings the total volume of the reaction system to 50 μl.

[0035] Place the prepared PCR reaction system in the Bori XP cycler gene amplification instrument and amplify according to the following program: 98℃ for 30s, 55℃ for 45s, 72℃ for 120s, 35x.

[0036] The DNA fragment obtained by PCR was purified by gel extraction and cloned into the NdeI / XhoI site of pET30a using homologous recombination. Single clones were selected for sequencing. The successfully sequenced DNA sequence is SEQ ID NO: 4, named ZYK, and its corresponding amino acid sequence is SEQ ID NO: 3.

[0037] Example 3: Shaking Bottle Expression Test

[0038] Single colonies of *E. coli* containing the expression vector were picked and inoculated into 10 mL of autoclaved medium containing: 10 g / L tryptone, 5 g / L yeast extract, 3.55 g / L disodium hydrogen phosphate, 3.4 g / L potassium dihydrogen phosphate, 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, 0.8 g / L glucose, and kanamycin to a final concentration of 50 mg / L. The medium was incubated overnight at 30°C and 250 rpm. The following day, take a 1L Erlenmeyer flask and inoculate it into 100mL of autoclaved culture medium at a 1:100 ratio: tryptone 10g / L, yeast extract 5g / L, disodium hydrogen phosphate 3.55g / L, potassium dihydrogen phosphate 3.4g / L, ammonium chloride 2.68g / L, sodium sulfate 0.71g / L, magnesium sulfate heptahydrate 0.493g / L, ferric chloride hexahydrate 0.027g / L, glycerol 5g / L, glucose 0.3g / L, and add kanamycin to a final concentration of 50mg / L. Incubate at 30℃ until the bacterial OD reaches 5-6, then immediately place the Erlenmeyer flask in a 25℃ shaker at 250rpm for 1 hour. Add IPTG to a final concentration of 0.1mM and continue incubating at 25℃ and 250rpm for 16 hours. After incubation, centrifuge the culture at 12000g for 20 minutes at 4℃ to collect the wet bacterial cells. The bacterial precipitate was then washed twice with distilled water, and the bacterial cells were collected and stored at -70℃. A small amount of the bacterial cells was also taken for SDS-PAGE analysis.

[0039] Example 4: Batch Feeding Fermentation

[0040] Fed-batch fermentation was conducted in a computer-controlled bioreactor (Shanghai Guoqiang) with a capacity of 15L and a working volume of 8L. The culture medium used consisted of 24g / L yeast extract, 12g / L peptone, 0.4% glucose, 2.31g / L dihydrogen phosphate enzyme, and 12.54g / L dipotassium hydrogen phosphate, at pH 7.0. A 200mL culture was prepared as the primary inoculum and inoculated at OD 2.0. Throughout the fermentation process, the temperature was maintained at 37℃. Dissolved oxygen concentration was automatically controlled at 30% by a cascade of agitation rate (rpm) and aeration. The pH of the culture medium was maintained at 7.0 by 50% (v / v) orthophosphate and 30% (v / v) ammonia. Fed-batch fermentation was initiated when a significant increase in dissolved oxygen was observed. The fed-batch solution contained 9% w / v peptone, 9% w / v yeast extract, and 14% w / v glycerol. When the OD600 was approximately 50.0 (wet weight approximately 100 g / L), the temperature was controlled at 28 °C, and expression was induced with 0.15 mM IPTG.

[0041] Example 5: Comparison of reaction rates

[0042] The 10 mL system was prepared as follows: 60 mM MgCl2, final concentration 30 g / L sodium hexametaphosphate, final concentration 45 g / L nicotinamide ribose, 0.3 g / L AMP, pH adjusted to 6.5, and the volume brought to 9.5 mL. 0.2 mL of crude enzyme solution and 0.2 mL of thermostable polyphosphokinase LE496 (purchased from Nanjing Langen Biotechnology Co., Ltd.) were added to each solution. The reaction was started and timed. The water bath temperature was controlled at 50℃, and the pH was maintained at 6.5. Samples were taken at 3 hours and 6 hours. The results are shown in the table below, indicating that the mutated solution can efficiently catalyze the substrate at 50℃. HPLC results are as follows. Figure 1-2 As shown.

[0043] 3 hours 49.4g / L 23.2g / L 6 hours 51.3g / L 25.3g / L

[0044] Example 6

[0045] The 10mL system was prepared as follows: 60mM MgCl2, final concentration 30g / L sodium hexametaphosphate, final concentration 50g / L nicotinamide ribose, 0.5g / L AMP, pH adjusted to 6.5, and the volume brought to 9.5mL. 0.3mL of crude enzyme solution and 0.2mL of thermostable polyphosphokinase LE496 (purchased from Nanjing Langen Biotechnology Co., Ltd.) were added, and the reaction was started and timed. The water bath temperature was controlled at 50℃, and the pH was maintained at 6.5. Samples were taken after 7 hours to detect the content; at this time, the product concentration was 53.97g / L. The HPLC results are as follows. Figure 3 As shown.

[0046] Example 7

[0047] The 10mL system was prepared as follows: 60mM MgCl2, final concentration 35g / L sodium hexametaphosphate, final concentration 55g / L nicotinamide ribose, 0.8g / L AMP, pH adjusted to 6.5, and the volume brought to 9.5mL. 0.5mL of crude enzyme solution and 0.1mL of thermostable polyphosphokinase LE496 (purchased from Nanjing Langen Biotechnology Co., Ltd.) were added, and the reaction was started and timed. The water bath temperature was controlled at 45℃, and the pH was maintained at 6.5. Samples were taken after 6 hours to detect the content. The results showed a product concentration of 58.37g / L. The HPLC results are as follows. Figure 4 As shown.

[0048] Example 8

[0049] The 10mL system was prepared as follows: 120mM MgCl2, final concentration of sodium hexametaphosphate 45g / L, final concentration of nicotinamide ribose 70g / L, 0.5g / L AMP, pH adjusted to 6.5, and the volume brought to 9.5mL. 0.5mL of crude enzyme solution and 0.1mL of thermostable polyphosphokinase LE496 (purchased from Nanjing Langen Biotechnology Co., Ltd.) were added, and the reaction was started and timed. The water bath temperature was controlled at 48℃, and the pH was maintained at 6.5. Samples were taken after 6 hours to detect the content. The results showed a product concentration of 61.96g / L, with a small amount of substrate remaining, suggesting that the excessively high concentration of sodium hexametaphosphate inhibited the reaction.

[0050] Example 9

[0051] The 10mL system was prepared as follows: 120mM MgCl2, final concentration of 35g / L sodium hexametaphosphate, final concentration of 70g / L nicotinamide ribose, 0.5g / L AMP, pH adjusted to 6.5, and the volume brought to 9.5mL. 0.5mL of crude enzyme solution and 0.1mL of thermostable polyphosphokinase LE496 (purchased from Nanjing Langen Biotechnology Co., Ltd.) were added, and the reaction was started and timed. The water bath temperature was controlled at 48℃, and the pH was maintained at 6.5. After 1.5 hours of reaction, 10g / L industrial sodium hexametaphosphate was added. Samples were taken after 8 hours to detect the NMN concentration, which showed to be 85.27g / L.

[0052] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A nicotinamide ribokinase mutant, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

3.

2. The nicotinamide ribokinase mutant according to claim 1, characterized in that: It exhibits higher catalytic activity and thermal stability than wild-type nicotinamide ribokinase, and the amino acid sequence of the wild-type nicotinamide ribokinase is shown in SEQ ID NO:

1.

3. A polynucleotide encoding a nicotinamide ribokinase mutant as described in any one of claims 1 or 2.

4. The polynucleotide according to claim 3, characterized in that: The sequence is shown in SEQ ID NO: 4.

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