Nicotinamide ribokinase mutant and its application

Abstract

The present invention discloses a series of nicotinamide ribokinase mutants and applications thereof, wherein the amino acid sequence of the nicotinamide ribokinase mutant is shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.10. The nicotinamide ribokinase mutant of the present invention can efficiently catalyze the substrate nicotinamide ribose (NR) to generate nicotinamide mononucleotide (NMN) under the action of ATP, and can significantly reduce the reaction time and the amount of enzyme used, reduce the production cost, and has good industrial application prospects.

Description

Technical Field

[0001] The invention belongs to the field of microbial gene technology recombination, and specifically relates to a recombinant bacterium expressing nicotinamide ribokinase (NRK), a preparation method thereof and practical application thereof. Background Art

[0002] β-Nicotinamide mononucleotide (NMN) is a substance naturally present in the human body, which is directly involved in the synthesis of NAD+ (nicotinamide adenine dinucleotide) in cells. As a coenzyme present in all living cells, NAD+ can control cell function and survival in response to environmental changes, such as nutrient intake, cell damage, etc. With age, the level of NAD+ in the human body continues to decline, which reduces the activity of cell mitochondria; at the same time, the reduction of NAD+ also impairs the ability of cells to produce energy, leading to aging and the occurrence of age-related diseases. Therefore, as a precursor of NAD+, NMN has a direct and fundamental anti-aging effect. In view of this, the NMN project has now become the object of competition for research and development by pharmaceutical and food companies around the world.

[0003] At present, there are two main methods for producing NMN in China: chemical synthesis and bio-enzyme catalysis. The chemical synthesis method is a direct NMN synthesis process, which uses nicotinamide riboside as the raw material and phosphorylates it with trichloride phosphorus. However, the chemical synthesis phosphorylation has many impurities, low production efficiency, low product purity, and the large amount of organic solvents used will cause a certain degree of pollution to the environment.

[0004] The bio-enzyme catalysis method of NMN mainly uses nicotinamide riboside (NR) as the starting material. In the presence of ATP, NMN is catalyzed by nicotinamide ribokinase (NRK). This pathway requires fewer reaction steps and has a high yield. It is currently the mainstream solution for NMN production. However, the current nicotinamide ribokinase (NRK) has a low enzyme activity, resulting in a low conversion rate in production. Therefore, it is of great significance to develop nicotinamide ribokinase with higher enzyme activity. Summary of the invention

[0005] In view of the problem that the enzyme activity of strains producing nicotinamide ribokinase is generally low in the existing NMN bio-enzyme catalysis process, the present invention constructs a nicotinamide ribokinase mutant encoding gene, and the bacterial cells obtained after induced expression of the recombinant engineered bacteria are used as an enzyme source for the production of nicotinamide mononucleotide (NMN), thereby improving the production efficiency.

[0006] The purpose of the present invention can be achieved by the following measures:

[0007] The present invention provides a series of nicotinamide ribokinase mutants, whose amino acid sequences are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.10.

[0008] The mutation sites of the amino acid residues of the nicotinamide ribokinase mutants of the present invention are mainly 108D deletion of mutant 1, S161T of mutant 2, E123L D143E of mutant 3, and L65F N90I of mutant 4.

[0009] The present invention also provides a gene encoding the nicotinamide ribokinase mutant of the present invention. In some embodiments, the gene is shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5.

[0010] The present invention also provides a recombinant vector comprising the coding gene of the present invention. The recombinant vector of the present invention can be obtained by recombining the coding gene of the present invention with a common expression plasmid according to conventional methods in the art. For example, in one embodiment, the plasmid pET-28a is digested with BamHI and HindIII and connected with the coding gene with T4 DNA ligase.

[0011] The present invention also provides competent cells comprising the coding gene or recombinant vector of the present invention. Specifically, the coding gene or recombinant vector of the present invention can be transferred into competent cells commonly used in the art according to conventional methods in the art, for example, the recombinant vector can be transferred into BL21 (DE3) competent cells by heat shock transformation method.

[0012] The present invention also provides the use of the nicotinamide ribokinase mutant, encoding gene, recombinant vector or competent cell of the present invention in the preparation of nicotinamide mononucleotide. The use of the present invention is to use nicotinamide riboside (NR) as a substrate to generate nicotinamide mononucleotide under the action of ATP.

[0013] The specific application methods and conditions of the present invention can be based on conventional methods in the art, for example:

[0014] In some embodiments, the reaction system used in the present invention is: 100-180 mol / L nicotinamide riboside, 100-150 mol / L ATP-2Na, 1-5 g / L magnesium chloride, PBS buffer, pH 6.5-7.5.

[0015] In some embodiments, in the application of the present invention, if a nicotinamide ribokinase mutant is used, the reaction concentration is 10-20 U / ml; if bacteria are used, the amount of bacteria is 20-30 g / L.

[0016] In some embodiments, in the application of the present invention, the reaction temperature is 25-30°C.

[0017] Beneficial effects of the present invention:

[0018] (1) The nicotinamide ribokinase mutant protein provided by the present invention can efficiently catalyze the substrate nicotinamide riboside (NR) to generate nicotinamide mononucleotide (NMN) under the action of ATP.

[0019] (2) The nicotinamide ribokinase mutant protein provided by the present invention can significantly reduce the reaction time and the amount of enzyme used, thereby reducing the production cost and having a good industrial application prospect. DETAILED DESCRIPTION

[0020] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0021] Unless otherwise specified, "NR" in the following examples represents nicotinamide riboside, and "NMN" represents β-nicotinamide mononucleotide.

[0022] Example 1 Screening of recombinant bacteria with high enzyme activity

[0023] Using the wild nicotinamide ribokinase Escherichia coli genome (SEQ ID NO.1) as a template, the error-prone PCR method was used to construct a gene random mutation plasmid library. The specific PCR amplification system and PCR program are shown in Table 1:

[0024] Table 1 PCR amplification system and PCR program

[0025]

[0026] The amplified fragments were used to recover the DNA products by agarose gel electrophoresis. The pET-28a empty vector was digested with BamHI and HindIII. The recovered DNA products were digested in the same way. The digested DNA fragments and the pET-28a vector were connected with T4 DNA ligase and reacted at 16°C overnight.

[0027] The plasmid library was transformed into BL21 (DE3) competent cells by heat shock transformation method, and spread on LB plates containing kanamycin at a final concentration of 50 mg / ml. Single clones were selected for bacterial detection. The target band was 0.8 kb. The bacterial detection system is shown in Table 2:

[0028] Table 2 Bacteria inspection system

[0029]

[0030] The monoclones with positive bacterial test results were randomly selected and inoculated into 24-well plates, each well contained 4 ml of liquid LB medium containing kanamycin with a final concentration of 50 mg / ml, and cultured at 37°C, 220 rpm / min on a shaking table for 16 h. The culture solution was inoculated into 50 ml of liquid TB medium containing 50 mg / ml kanamycin at a volume ratio of 3%, and cultured at 37°C, 220 rpm / min on a shaking table until OD600 = 0.4-0.6, and then the inducer: IPTG (final concentration of 50 umol / L) was added, and the culture was induced at 25°C, 220 rpm / min on a shaking table for 16 h. After enzyme activity and protein electrophoresis detection and shake flask test verification, the strains with soluble expression and higher enzyme activity were screened out, and the fermentation broth containing the strains with higher enzyme activity was centrifuged at 6000 rpm for 30 min in a high-speed refrigerated centrifuge at 4°C, and the wet bacteria were collected and stored in a -20°C refrigerator. Four high-activity mutant strains were obtained through this example, and the nicotinamide ribokinase gene was sequenced respectively. The amino acid sequences of the mutant nicotinamide ribokinase in the four mutants 1 to 4 are shown in SEQ ID NOs. 2 to 5. The mutation sites of the amino acid residues are mainly 108D deletion of mutant 1, mutant 2: S161T, mutant 3: E123LD143E, and mutant 4: L65F N90I.

[0031] Example 2 Determination of enzyme activity of the four high-activity mutant strains and the wild-type nicotinamide ribokinase strain screened in Example 1

[0032] The preparation method of the crude enzyme solution used for measuring enzyme activity of the present invention is as follows:

[0033] (1) Take 4 ml of the fermentation broth in the stable fermentation period (fermentation by conventional method) and centrifuge it at 4°C and 10,000 r / min for 5 min. Discard the supernatant, collect the bacterial cells, and resuspend them in 4 ml of PBS buffer.

[0034] (2) The resuspended liquid was ultrasonically crushed using a multi-channel ultrasonic crusher. Then, it was centrifuged at 4°C and 10,000 r / min for 2 min, the precipitate was discarded, and the collected supernatant was the crude enzyme solution.

[0035] The enzyme activities of the four high-activity mutant strains and the wild-type nicotinamide ribokinase strain were determined respectively, and the determination method was as follows:

[0036] 1. The reaction system for the test is 0.8 mL, including 0.2 mL of NR solution, 0.2 mL of ATP-2Na solution, 0.1 mL of magnesium chloride solution and 0.29 mL of PBS buffer solution;

[0037] 2. Add 0.01 mL of crude enzyme solution, react at 25°C for 10 minutes, and immediately add 0.2 mL of dilute sulfuric acid to terminate the reaction. Centrifuge at 12000 rpm for 3 minutes, remove the supernatant, and use high performance liquid chromatography (HPLC) to determine the content of nicotinamide mononucleotide in the supernatant.

[0038] 3. HPLC method uses Agilent high performance liquid chromatography (Agilent 1100, USA), and the detection conditions are as follows:

[0039] Chromatographic column: Eclipse Plus C18 5um 4.6*250mm;

[0040] Column temperature: 30°C;

[0041] Mobile phase: A: 80 mL water + 0.1 g sodium heptane sulfonate + 0.02 mL triethylamine + 0.1 mL glacial acetic acid; B: acetonitrile; A:B = 9:1;

[0042] Flow rate: 0.8 mL / min;

[0043] Detection wavelength: 265nm;

[0044] Injection volume: 20 μL;

[0045] 4. Nicotinamide ribokinase activity: The number of millimoles of nicotinamide mononucleotide generated after 1 mL of fermentation liquid reacts with 2.85 g of nicotinamide riboside at 25°C for 10 minutes. The number of millimoles is the total enzyme activity unit U. The results are shown in Table 3.

[0046] Table 3: Enzyme activity assay results

[0047] strain Enzyme activity (U / ml) wild type 20 Mutant 1 35 Mutant 2 48 Mutant 3 70 Mutant 4 56

[0048] Example 3: 50 ml reaction system shake flask catalytic experiment:

[0049] 1. Reaction system: prepare the reaction solution with a final concentration of NR of 100 mM, a final concentration of ATP of 110 mM, and a magnesium chloride content of 1%. Stir and dissolve. Adjust the pH of the reaction solution to 7.0 with 30% NaOH. Dispense into shake flasks, 30 ml of the reaction solution per bottle, and preheat in a 30°C incubator.

[0050] 2. Take out 1 g of bacterial slurry from 4 high-activity mutant strains and NRK wild strain frozen in a -20°C refrigerator for 24 hours, resuspend each tube with 20 ml PBS buffer, place in a 30°C incubator for preheating for 30 minutes, add the bacterial solution (20 ml) to the corresponding numbered shake flask reaction solution (30 ml) (bacterial density is 20 g / L), temperature 30°C, speed 220 rpm, start the reaction, take samples every 30 minutes, detect the contents of NMN, NR, and ATP, until NR is basically converted and the reaction is over.

[0051] 3. The experimental results are shown in Table 4. It can be seen that compared with the wild-type strain, the reaction time and NR conversion rate of the mutant strains are improved to a certain extent, especially mutant 3.

[0052] Table 4: Shake flask catalytic reaction results

[0053]

[0054]

[0055] Example 4: Catalytic experiment of 10L reaction system reactor:

[0056] The preparation method of the enzyme solution used in this example is as follows:

[0057] (1) Take the fermentation broth in the stable fermentation period (fermented according to the conventional method), use a high-speed refrigerated centrifuge to divide the fermentation broth into 500 ml bottles, centrifuge at 4°C and 4000 r / min for 30 min, discard the supernatant, collect the bacterial cells, and freeze them in a -20°C freezer for 24 h;

[0058] (2) Resuspend the frozen bacterial cells in PBS buffer at a ratio of 30 g / L, and use a homogenizer to break the resuspended bacterial liquid under high pressure. Then centrifuge it at 4°C and 10,000 r / min for 2 min, discard the precipitate, and collect the supernatant as the enzyme solution.

[0059] Prepare 20% NR reaction solution (purity 90%) 5L, add 30g of magnesium chloride, stir to dissolve, prepare 15% ATP sodium salt 5L, adjust the pH of ATP sodium salt to above 5 with 30% concentration of NaOH, first add 1L ATP sodium salt to NR, and the remaining 4L is added to NR at the beginning of the reaction; connect the reaction tank, adjust the pH to 7.0, the temperature is 30°C, add enzyme solution (final concentration 15U / ml), start the reaction, take samples at intervals, and after NR is basically converted, stop the reaction, collect the reaction solution, measure the NMN, NR, and ATP content during the reaction, and calculate the NR conversion rate. The results are shown in Table 5. The final mutant strain has a higher conversion rate than the wild-type nicotinamide ribokinase strain, especially the mutant strain 3 has a conversion rate of 97.09%, and shortens the time required for the reaction, improving production efficiency.

[0060] Table 5: 10L system catalytic test results

[0061]

Claims

1. A nicotinamide ribokinase mutant, characterized in that The amino acid sequence is shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.

10.

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

3. The gene according to claim 2, characterized in that The gene nucleotide sequence is shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.

5.

4. A recombinant vector comprising the gene according to claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that The plasmid pET-28a is digested with BamHI and HindIII, and connected with the gene described in claim 2 or 3 to obtain the plasmid.

6. A competent cell comprising the gene according to claim 2 or 3 or the recombinant vector according to claim 4 or 5.

7. The competent cell according to claim 6, characterized in that The recombinant vector according to claim 4 or 5 is transformed into BL21 (DE3) competent cells by heat shock transformation method to obtain the result.

8. Use of the nicotinamide ribokinase mutant of claim 1, the gene of claim 2 or 3, the recombinant vector of claim 4 or 5, or the competent cell of claim 6 or 7 in the preparation of nicotinamide mononucleotide.

9. The use according to claim 8, characterized in that: The reaction system used is: 100-180 mol / L nicotinamide riboside, 100-150 mol / L ATP-2Na, 1-5 g / L magnesium chloride, PBS buffer solution, pH 6.5-7.

5.

10. The use according to claim 9, characterized in that: If a nicotinamide ribokinase mutant is used, the reaction concentration is 10-20 U / ml; if bacteria are used, the amount of bacteria is 20g~30g / L.

11. The use according to claim 8, characterized in that: The temperature during the reaction is 25 to 30°C.

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