A nicotinamide riboside kinase mutant, encoding gene and application

Abstract

The present application relates to a kind of nicotinamide riboside kinase mutant, coding gene, vector containing coding gene, genetically engineered bacteria and application.The present application is aimed at wild type NrK, the three-dimensional structure of NrK is simulated by macromolecular modeling technology, and one or more sites possibly related to catalysis are predicted using energy minimum principle and molecular docking technology.The nicotinamide riboside kinase mutant constructed in the present application is compared with wild type enzyme, and enzyme activity is increased by 1.26-2.98 times, can significantly reduce the amount of enzyme, reduce fermentation volume and cost;And can greatly shorten reaction time, improve NMN yield, can meet the demand of large-scale industrial production of NMN prepared by biological enzyme method, has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a nicotinamide riboside kinase mutant, a coding gene, a vector containing the coding gene, a genetically engineered bacterium and an application. BACKGROUND

[0002] Nicotinamide mononuclotide (NMN) is a naturally occurring bioactive nucleotide. NMN has two irregular forms, alpha isomer and beta isomer. Among them, the beta isomer is the active form of NMN, and the molecular weight is 334.221 g / mol. NMN is an important intermediate in the synthesis pathway of nicotinamide adenine dinucleotide (NAD + , also known as coenzyme I) in mammals. In recent years, relevant research reports in international authoritative academic journals such as Science, Nature and Cell have shown that supplementing NMN can effectively increase and restore the level of coenzyme I in the body, significantly delay aging and prevent various neuronal degenerative diseases such as Alzheimer's disease, and thus fundamentally regulate and improve various symptoms of aging. Therefore, functional health care food with NMN as the active ingredient has great development potential and market prospects. At present, NMN has been approved as a health food raw material in Europe, the United States, Japan and other developed countries, and various health care products have been developed with NMN as the main component, such as HERBALmax in the United States, GeneHarbor NMN9000 in Japan, and MIRAI LAB NMN3000 capsules.

[0003] There are three main production methods for NMN at present: solid-state yeast fermentation process, in-vitro enzyme catalysis process and chemical synthesis process. Among them: (1) The solid-state yeast fermentation process is complex and has low yield, so the product price is high. (2) The chemical synthesis process uses nicotinamide ribose as raw material and phosphoric acid is obtained by using phosphorus oxychloride. Although the technology is easy to control, the product has too many impurities, and it is difficult to separate and purify and the overall yield is very low; at the same time, the amount of organic solvent used is large, and the environmental pollution cannot be ignored. (3) Enzyme as a class of complementary and efficient biological catalysts to chemical synthesis has been widely used in new drug research and development, food, chemical industry and other fields. The mainstream NMN production process at present is to use safe and green in-vitro enzyme catalysis process.

[0004] There are two main routes for the production of NMN by biological enzyme method. The first route is to use D-ribose and nicotinamide as starting materials, and under the action of ribokinase, phosphoribosyl pyrophosphate synthetase and nicotinamide ribosyltransferase, NMN is obtained through three-step catalytic reaction. The substrate conversion rate of this route is not high, and there are many intermediate products, which are difficult to separate and purify, so the overall yield is low, resulting in high production cost. The second route is to use nicotinamide riboside (NR) as the starting material, and under the action of nicotinamide ribosyl kinase (NR kinase, NrK) and ATP, NMN is obtained through one-step reaction, with high yield and high product purity, which will become the mainstream production method of NMN in the future.

[0005] However, the current research on nicotinamide ribosyl kinase is still less, which limits the application of one-step reaction method for preparing NMN in industrial production. It will be very helpful to improve the enzyme activity of nicotinamide ribosyl kinase by directed mutation method, which will greatly help to reduce the amount of enzyme and reduce the production cost in industry. SUMMARY

[0006] The purpose of the present application is to provide a nicotinamide ribosyl kinase mutant with improved activity, a coding gene, a vector containing the coding gene, a genetically engineered bacterium and an application, in order to solve the problems of high raw material cost and unsatisfactory activity of wild-type nicotinamide ribosyl kinase in the existing NMN preparation method, which makes it difficult to realize industrial production.

[0007] The technical scheme adopted by the present application is:

[0008] A nicotinamide ribosyl kinase mutant obtained by single mutation or multiple point mutation of the 10th, 12th, 38th, 55th and 102nd positions of the amino acid sequence shown in SEQ ID NO. 2.

[0009] The amino acid sequence of wild-type nicotinamide ribosyl kinase derived from Homo sapiens is shown in SEQ ID NO. 2, and the nucleotide sequence of its coding gene is shown in SEQ ID NO. 1.

[0010] The present application is directed to wild type NrK, the three-dimensional structure of NrK is simulated by macromolecular modeling technology, and one or more sites possibly related to catalysis are predicted by energy minimum principle and molecular docking technology. After selecting the site of site-directed mutation and the type of amino acid after mutation, the corresponding primer is synthesized by Hangzhou Genesee Biotechnology Co., Ltd., and the mutant DNA fragment is amplified by PCR (Polymerase Chain Reaction) using the NrK-pET 28a (+) vector plasmid as a template, and then purified. The obtained fragment is amplified into a full-length mutant gene by PCR. The full-length mutant gene is cloned into the pET28a (+) vector and transformed into the DH5a host bacteria, and the positive clones with mutant gene vectors are screened by culture. Finally, the plasmid DNA is extracted from the positive clones, and DNA sequence determination analysis is performed to determine the introduced mutation. The positive plasmid screened is transformed into BL21 (DE3) host bacteria for induction and expression, and the mutant with significantly improved activity is screened. Specifically, the present application is directed to wild type NRK, the free energy of the overall structure of the protein is reduced by macromolecular modeling technology and site-directed mutation to increase the stability of the protein, and five sites are predicted by calculation of the free energy of the protein structure, which may be related to the improvement of stability.

[0011] Specifically, the mutation is one of the following (single-point mutation) or a combination of two or more (multi-point mutation): (1) the 10th methionine (M) is mutated to glutamic acid (E); (2) the 12th asparagine (N) is mutated to serine (S); (3) the 38th phenylalanine (F) is mutated to tryptophan (W); (4) the 55th aspartic acid (D) is mutated to alanine (A); (5) the 102nd phenylalanine (F) is mutated to tryptophan (W).

[0012] Preferably, the mutant amino acid sequence is shown in SEQ ID NO. 3 (mutant N12S / F38W) or SEQ ID NO. 4 (mutant F38W / F102W).

[0013] The present application also relates to a gene encoding the nicotinamide riboside kinase mutant.

[0014] Preferably, the encoding gene nucleotide sequence is shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0015] The present application also relates to a recombinant vector containing the gene encoding the nicotinamide riboside kinase mutant and a genetically engineered bacterium containing the gene encoding the nicotinamide riboside kinase mutant. Specifically, the vector can be any of various expression vectors, including but not limited to a pET expression vector, a pCW expression vector, a pUC expression vector or a pPIC9k expression vector. The host cell of the genetically engineered bacterium can be any suitable host cell, including but not limited to E. coli, B. subtilis, Streptomyces or Pichia pastoris.

[0016] The present application also relates to the use of the nicotinamide riboside kinase mutant in the microbial catalytic preparation of beta-nicotinamide mononucleotide.

[0017] Specifically, the method for preparing beta-nicotinamide mononucleotide can comprise the following steps:

[0018] S1. Formulate the reaction system, comprising: 10 g / L of the nicotinamide riboside kinase mutant, 50 mM pH7.0 potassium phosphate buffer, 100 mM ATP, 10-100 g / L nicotinamide riboside, 20 mM anhydrous magnesium chloride; control the temperature of the reaction system at 40℃, and perform oscillation reaction;

[0019] S2. After 2h of reaction, perform HPLC detection; and after purification, obtain beta-nicotinamide mononucleotide.

[0020] The reaction product is detected by HPLC, and the conversion rate of the reaction is >99%, and the yield of beta-nicotinamide mononucleotide is >90%.

[0021] The present application has the following beneficial effects:

[0022] The nicotinamide riboside kinase mutant constructed by the present application has an enzyme activity 1.26-2.98 times higher than that of the wild-type enzyme, can significantly reduce the amount of enzyme used, reduce the fermentation volume and cost, greatly shorten the reaction time, improve the yield of NMN, meet the demand of large-scale industrial production of NMN prepared by the biological enzyme method, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a high performance liquid chromatography (HPLC) spectrum analysis diagram of NMN, ATP and NR samples.

[0024] Figure 2 It is a reaction schematic diagram of NMN generated by the nicotinamide riboside pathway.

[0025] Figure 3SDS-PAGE of the supernatant and precipitate of the broken NRK1 and NRK2 cells. Lane 2: supernatant of NRK001; Lane 3: supernatant of NRK002; Lane 4: precipitate of NRK001; Lane 5: precipitate of NRK002. DETAILED DESCRIPTION

[0026] The application will be further described in conjunction with specific examples. However, the application is not limited to the following examples.

[0027] Unless otherwise specified, the experimental methods used in the examples were performed according to conventional conditions. The materials and reagents used in the following examples were commercially available unless otherwise specified.

[0028] Unless otherwise specified, the experimental methods used in the examples were performed according to conventional conditions. The materials and reagents used in the following examples were commercially available unless otherwise specified.

[0029] Example 1: Construction of the prokaryotic expression system of Nrk

[0030] The Nrk gene fragment (SEQ ID NO. 1) was synthesized by Beijing Genki Biological Technology Co., Ltd. and recombined into a PUC57 vector. After double enzyme digestion with restriction endonucleases NdeI and HindIII (purchased from New England Biolabs Co., Ltd., NEB) at 37°C for 4 h, 1% agarose gel electrophoresis was performed for separation and gel recovery (gel recovery kit was purchased from Hangzhou Haofeng Biological Technology Co., Ltd.). Subsequently, the same double enzyme-digested expression vector pET28a(+) (Novage Co.) was connected under the action of T4 DNA ligase (purchased from Takara Co.) in a low-temperature ligation instrument overnight. The ligation liquid was transformed into E. coli BL21 (DE3) competent cells, and colony PCR screening and sequencing verification were performed, thereby obtaining the positive recombinant plasmid NrK-pET28a(+). The positive transformant containing the Nrk gene was recorded as the engineering bacteria Nrk001, which was stored at -80°C.

[0031] Example 2: Verification of the engineering bacteria Nrk001 synthesizing β-nicotinamide mononucleotide

[0032] The expression strain NrK-pET28a (+) / BL21 (DE3) constructed in Example 1 was inoculated into 100 mL LB liquid medium containing a final concentration of 50 μg / mL kanamycin sulfate at a ratio of 2% (V / V) after being cultured overnight at 37°C and 200 rpm in 10 mL LB liquid medium (10 g / L tryptone (OXIOD), 5 g / L yeast powder (OXIOD), 10 g / L sodium chloride (National Pharmaceutical Reagent)) with a final concentration of 50 μg / mL kanamycin sulfate. The culture was incubated at 37°C and 200 rpm. When the OD600 was between 0.6 and 0.8, 0.1 mM of the inducer IPTG (isopropyl-β-D-thiogalactoside) was added, and the culture was induced overnight at 28°C. The bacterial cells were collected by centrifugation at 4°C and 8000 rpm, then suspended in 50 mM pH 7.0 sodium phosphate buffer, and the bacterial suspension of the engineered bacteria Nrk001 was subjected to ultrasonic cell disruption (ultrasonic cell disruption program: 1 s of work, 2 s of stop, 250 W, 99 cycles of ultrasonic waves). The obtained cell disruption mixture was NRK free enzyme solution.

[0033] Example 3: Construction and screening of mutants

[0034] Construction of mutants: The possible beneficial mutation sites N12, F38, F102, M10, and D55 and their pairwise combination mutations were predicted by using macromolecular modeling technology. Then, using the NrK-pET28a (+) recombinant plasmid as the template, the corresponding synthesized primers were used to amplify the mutant DNA fragments by first PCR, and then the obtained fragments were used as the template for second PCR to amplify the full-length mutant gene of NrK.

[0035] wherein:

[0036] 12-site mutation:

[0037] Forward primer (SEQ ID NO. 7): 5'-ATGACCAGCGGTGGTAAAACCAC-3',

[0038] Reverse primer (SEQ ID NO. 8): 5'-TTTACCACCGCTGGTCATACCAC-3'

[0039] 38-site mutation:

[0040] Forward primer (SEQ ID NO. 9): 5'-CAGGATGATTTTTGGAAACCTCAGGATC-3',

[0041] Reverse primer (SEQ ID NO: 10): 5'-CTGATCCTGAGGTTTCCAAAAATCATCC-3';

[0042] 102 site mutation:

[0043] Forward primer (SEQ ID NO. 11): 5'-CTGGAAGGTTGCCTGCTGTATAGCT-3',

[0044] Reverse primer (SEQ ID NO. 12): 5'-TATACAGCAGTTCACCTTCCAGCAG-3';

[0045] 10 site mutation:

[0046] Forward primer (SEQ ID NO. 13): 5'-TGGGTATCGGTGGTGAAACCAACG-3',

[0047] Reverse primer (SEQ ID NO. 14): 5'-TTACCACCGTTGGTTTCACCACCG-3';

[0048] 55 site mutation:

[0049] Forward primer (SEQ ID NO. 15): 5'-TAAACAGTGGTGGGTGCTGGAAAG-3',

[0050] Reverse primer (SEQ ID NO. 16): 5'-ATATCCAGGCTTTCCAGCACCCAC-3';

[0051] Mutant culture: After the above-mentioned mutation of the plasmid to transform BL21 (DE3) host bacteria, spread on LB solid medium containing 50 μg / mL kanamycin, 37°C inverted culture overnight, then pick single clone from the plate, placed in 5 mL LB liquid medium containing 30 μg / mL kanamycin sulfate for culture. The overnight culture was inoculated in 100 mL LB liquid medium containing 30 μg / mL kanamycin sulfate at a ratio of 1% (V / V), and then induced by adding IPTG with a final concentration of 0.1 mM after 4 h of 37°C, 200 rpm shaking culture. The bacteria were cultured at 28°C overnight. The bacteria were collected by centrifugation at 4°C, 8000 rpm for 10 min, suspended with 50 mM pH 7.0 potassium phosphate buffer, and then broken by ultrasonic (250 W, 1 s / 2 s, 15 min). The bacteria were centrifuged at 4°C, 8000 rpm for 10 min, and the supernatant was used for unit enzyme activity determination.

[0052] Screening of mutants: substrate NR concentration 25 g / L, 100 mM ATP, 20 mM anhydrous magnesium chloride, add appropriate amount of supernatant prepared above, supplement with 50 mM pH 7.0 potassium phosphate buffer to 1 mL, place in a 40°C constant temperature metal bath reactor under shaking reaction. After 10 min of reaction, sample for HPLC detection.

[0053] The experimental results show that the mutant enzyme activity of the clones with significantly improved mutant sites is as follows:

[0054] comprising a methionine at position 10 mutated to glutamic acid, an asparagine at position 12 mutated to serine, a phenylalanine at position 38 mutated to tryptophan, a phenylalanine at position 102 mutated to tryptophan, and an aspartic acid at position 55 mutated to alanine.

[0055] The specific relative enzyme activity values are shown in Table 1 below.

[0056] Table 1: Enzyme activity of different mutant combinations

[0057]

[0058] Therefore, when the amino acid sequence shown in SEQ ID NO. 2 has an asparagine at position 12 mutated to serine and a phenylalanine at position 38 mutated to tryptophan, the amino acid sequence of the nicotinamide riboside kinase mutant is shown in SEQ ID NO. 3. Accordingly, the nucleotide sequence of the gene encoding the nicotinamide riboside kinase mutant is shown in SEQ ID NO. 5.

[0059] When the amino acid sequence shown in SEQ ID NO. 2 has a phenylalanine at position 38 mutated to tryptophan and a phenylalanine at position 102 mutated to tryptophan, the amino acid sequence of the nicotinamide riboside kinase mutant is shown in SEQ ID NO. 4. Accordingly, the nucleotide sequence of the gene encoding the nicotinamide riboside kinase mutant is shown in SEQ ID NO. 6.

[0060] Example 4: Biosynthesis of nicotinamide mononucleotide by wild-type nicotinamide riboside kinase

[0061] The final concentrations of the reaction solution components are as follows: nicotinamide ribose 100 mM, ATP 100 mM, anhydrous magnesium chloride 20 mM, and the whole-cell enzyme amount of wild-type nicotinamide riboside kinase is 10 g / L in 100 mL potassium phosphate buffer (50 mM, pH 7.0), under constant temperature magnetic stirring at 40°C, shaking reaction, after 2 h of reaction, HPLC detection; after purification, β-nicotinamide mononucleotide is obtained. The substrate conversion rate is 64%, and the yield of β-nicotinamide mononucleotide is 60% based on the converted substrate.

[0062] Example 5: Biocatalytic synthesis of NMN by mutant N12S

[0063] The final concentrations of each component of the reaction solution were as follows: nicotinamide riboside 100 mM, ATP 100 mM, anhydrous magnesium chloride 20 mM, and the whole-cell enzyme amount of nicotinamide riboside kinase mutant Nrk001-N12S was 10 g / L. The reaction was carried out in 100 mL of potassium phosphate buffer (50 mM, pH 7.0) under constant temperature magnetic stirring at 40°C, and the reaction was oscillated. After 2 h of reaction, HPLC detection was performed. After purification, β-nicotinamide mononucleotide was obtained, with a substrate conversion rate of 70% and a yield of the product β-nicotinamide mononucleotide of 63%.

[0064] Example 6: Biosynthesis of NMN by mutant F38W-F102W

[0065] The final concentrations of each component of the reaction solution were as follows: nicotinamide riboside 100 mM, ATP 100 mM, anhydrous magnesium chloride 20 mM, and the whole-cell enzyme amount of nicotinamide riboside kinase mutant F38W-F102W was 10 g / L. The reaction was carried out in 100 mL of potassium phosphate buffer (50 mM, pH 7.0) under constant temperature magnetic stirring at 40°C, and the reaction was oscillated. After 2 h of reaction, HPLC detection was performed. After purification, β-nicotinamide mononucleotide was obtained, with a substrate conversion rate of 99% and a yield of the product β-nicotinamide mononucleotide of 90%.

[0066] Example 7: Biosynthesis of NMN by mutant F38W-F102W under different substrate and ATP concentrations

[0067] The final concentrations of each component of the reaction solution were as follows: nicotinamide riboside 200 mM, ATP 200 mM, anhydrous magnesium chloride 20 mM, and the whole-cell enzyme amount of nicotinamide riboside kinase mutant Nrk001-F38W-F102W was 10 g / L. The reaction was carried out in 100 mL of potassium phosphate buffer (50 mM, pH 7.0) under constant temperature magnetic stirring at 40°C, and the reaction was oscillated. After 2 h of reaction, HPLC detection was performed. After purification, β-nicotinamide mononucleotide was obtained, with a substrate conversion rate of greater than 99% and a yield of the product β-nicotinamide mononucleotide of 90%.

[0068] The final concentrations of each component of the reaction solution were as follows: nicotinamide riboside 400 mM, ATP 400 mM, anhydrous magnesium chloride 20 mM, and the whole-cell enzyme amount of nicotinamide riboside kinase mutant Nrk001-F38W-F102W was 10 g / L. The reaction was carried out in 100 mL of potassium phosphate buffer (50 mM, pH 7.0) under constant temperature magnetic stirring at 40°C, and the reaction was oscillated. After 2 h of reaction, HPLC detection was performed. After purification, β-nicotinamide mononucleotide was obtained, with a substrate conversion rate of 98% and a yield of the product β-nicotinamide mononucleotide of 90%.

[0069] Example 8: Biocatalysis of mutant F38W-F102W under ATP cycle system

[0070] The final concentrations of each component of the reaction solution were as follows: substrate nicotinamide riboside 200 mM, ATP 20 mM, anhydrous magnesium chloride 20 mM, whole-cell enzyme amount of nicotinamide riboside kinase mutant Nrk001-F38W-F102W 10 g / L, whole-cell enzyme amount of acetate kinase (acetate kinase can convert by-product adenosine diphosphate ADP into adenosine triphosphate ATP) 10 g / L, 30 mM acetyl phosphate (provides phosphate groups for ATP cycle reaction), oscillation reaction under constant temperature magnetic stirrer at 40°C, HPLC detection after 3 h of reaction; after purification, β-nicotinamide mononucleotide was obtained, the substrate conversion rate was 99%, and the yield of β-nicotinamide mononucleotide was greater than 95%.

[0071] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A nicotinamide riboside kinase mutant, the amino acid sequence of which is shown as SEQ ID NO.

4.

2. A coding gene of the nicotinamide riboside kinase mutant of claim 1.

3. The genetic code of claim 2, wherein The nucleotide sequence of the coding gene is shown as SEQ ID NO.

6.

4. A recombinant vector containing the coding gene of the nicotinamide riboside kinase mutant of claim 3.

5. A genetically engineered bacterium containing the coding gene of the nicotinamide riboside kinase mutant of claim 3.

6. Use of the nicotinamide riboside kinase mutant of claim 1 in the microbial catalytic preparation of β-nicotinamide mononucleotide.

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