Saccharomyces cerevisiae strain and application thereof in efficient synthesis of beta-NMN

Abstract

The invention relates to a saccharomyces cerevisiae strain and application thereof in efficient synthesis of beta-NMN, and belongs to the technical field of bioengineering. The strain is saccharomyces cerevisiae Y1t, is classified and named as saccharomyces cerevisiae, and is preserved in China General Microbiological Culture Collection Center (CGMCC) on March 26, 2024, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.30138. The invention further discloses a preparation method of the saccharomyces cerevisiae. The strain can catalyze conversion of nicotinamide ribose into beta-NMN through one-step reaction, the purification step of enzyme liquid is omitted, and an enzymatic reaction system with higher stability and catalytic efficiency is realized by utilizing a surface immobilized enzyme technology; in addition, beta-NMN can be efficiently synthesized through the strain, powerful technical support is provided for industrial large-scale production of beta-NMN, and influences can be possibly generated on related health products, medicine and food industries. The production cost of the beta-NMN is reduced, and the application prospect of the beta-NMN in the fields of cell energy metabolism, anti-aging research and related medical treatment is widened.

Description

Technical Field

[0001] The present invention relates to a saccharomyces cerevisiae strain and application thereof in the efficient synthesis of β-NMN, belonging to the technical field of bioengineering. Background Art

[0002] Nicotinamide mononucleotide (NMN) is a natural active mononucleotide widely present in the human body and various vegetables and fruits. It exists in two forms, α and β. β-NMN is one of the key precursors of nicotinamide adenine dinucleotide.

[0003] Nicotinamide Adenine Dinucleotide (NAD + ) exists in all living cells and plays a vital role in maintaining thousands of biochemical reactions in the body. It plays a role in many key biological reactions, such as participating in the electron transfer of the respiratory chain as an electron carrier in the process of cellular energy transfer, helping cells to generate ATP, and serving as a substrate for PARP (poly ADP-ribose polymerase) family enzymes to repair DNA damage and maintain genome stability. In addition, its derivatives such as diphosphate ribose (ADPR) and cyclic ADP-ribose (cADPR) are also involved in intracellular signal transduction. Its active form also connects the tricarboxylic acid cycle and the glycolysis pathway. The SIRTs silencing protein family is also composed of NAD + Activated, this protein is associated with the regulation of cellular stress responses during aging.

[0004] As we age, NAD + The level of NAD decreases, which is considered to be one of the main causes of degenerative diseases in human aging. Therefore, exogenous supplementation of β-NMN has become a widely studied method, which can increase the level of NAD in the body. + Level. Compared to NAD which cannot be directly absorbed + β-NMN has a smaller molecular weight and can easily penetrate the cell membrane and enter the body to be converted into NAD. + Absorbed and utilized by the human body. Furthermore, exogenous supplementation of β-NMN can improve energy metabolism and reduce the risk of metabolic diseases such as obesity and diabetes, and has a good therapeutic effect on retinal damage and cardiovascular disease, as well as inhibiting melanin production in senile melanocytes.

[0005] At present, the highest yield in the chemical method is to use tetraacetyl ribose as the raw material, which is brominated with hydrobromic acid and then reacted with nicotinamide under sodium sulfate reaction conditions to undergo substitution reaction to obtain β-NMN, and then remove the acetyl group to obtain β-nicotinamide riboside bromide salt. Subsequently, after phosphorylation, separation and resin chromatography, β-NMN with a final yield of 78% is obtained (Tanimori S, Ohta T, Kirihata M. An efficient chemical synthesis of nicotinamide riboside (NAR)and analogues. Bioorg Med Chem Lett. 2002 Apr 22;12(8):1135–7). Although this method has a high yield, the resin chromatography method is too expensive and the process is cumbersome. In addition, a large number of chiral by-products will be produced in the process, and the chemical reagents used in the process will also cause environmental pollution, which is not the best production method.

[0006] Compared with chemical synthesis, bio-enzyme synthesis is safer, greener and more environmentally friendly, and is the best method for producing β-NMN. The biosynthesis route of β-NMN involves two key enzymes, Nicotinamide Phosphoribosyltransferase (NAMPT) and Nicotinamide Riboside Kinase (NRK). The former is widely present in mammals and produces β-NMN by catalyzing the substrate nicotinamide and phosphoribosyl pyrophosphate. The latter produces β-NMN by phosphorylation of nicotinamide riboside. Currently, the most common method is to use Escherichia coli to express heterologous nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide riboside kinase (NRK) in vivo, and then extract the enzyme solution for reaction (He JJ, Liu XX, Li Y, Wang Z, Shi HL, Kan YC, et al. High level expression of nicotinamide nucleoside kinase from Saccharomyces cerevisiaeand its purification and immobilization by one-step method. Front Bioeng Biotech. 2023 Feb 15;11:1134152;Shoji S, Yamaji T, Makino H, Ishii J, Kondo A. Metabolic design for selective production of nicotinamide mononucleotidefrom glucose and nicotinamide. Metab Eng. 2021 May 1;65:167–77). However, the complex enzyme extraction process and the easy inactivation of the enzyme are not conducive to large-scale industrial production. In addition, Escherichia coli does not have the glycosylation function, and the synthesized heterologous enzyme will be more easily degraded by the protease in the body, thereby reducing its stability and may even directly affect the active center of the enzyme and interfere with the signal transmission pathway within the cell. Summary of the invention

[0007] In view of the problems existing in the current bio-enzyme synthesis method for producing β-NMN, the present invention provides a Saccharomyces cerevisiae strain that can synthesize β-NMN using immobilized enzyme technology.

[0008] The technical solution of the present invention: A strain of saccharomyces cerevisiae, the strain is saccharomyces cerevisiae Y1t, classified and named saccharomyces cerevisiae Saccharomyces cerevisiae , and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 26, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNo.30138.

[0009] Another object of the present invention is to protect the use of the above-mentioned Saccharomyces cerevisiae strain in the efficient synthesis of β-NMN.

[0010] Another object of the present invention is to provide a method for efficiently synthesizing β-NMN using the above-mentioned Saccharomyces cerevisiae strain.

[0011] Preferably, the steps are included: placing in culture medium for cultivation after activation, and adding in reaction system for shaking culture after induction.

[0012] Furthermore, the reaction system includes adenosine-5'-triphosphate disodium salt, MgCl2, nicotinamide ribose chloride, and PBS buffer.

[0013] Another object of the present invention is to protect the use of β-NMN synthesized by the above method in the fields of health products, medicines, and foods.

[0014] The last purpose of the present invention is to protect the application of β-NMN synthesized by the above method in the fields of cellular energy metabolism and anti-aging medicine.

[0015] Description of biological deposit of Saccharomyces cerevisiae Y1t: Depository: China General Microbiological Culture Collection Center (CGMCC); Deposit number: CGMCC No. 30138; Deposit date: March 26, 2024; Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Taxonomic name: Saccharomyces cerevisiae Saccharomyces cerevisiae .

[0016] Beneficial effects of the present invention The strain of the present invention can efficiently synthesize β-NMN in one step, omitting the purification step of the enzyme solution. The method utilizes surface immobilized enzyme technology to achieve an enzymatic reaction system with higher stability and catalytic efficiency. Through molecular biological manipulation, the key nicotinamide ribokinase is fixed on the surface of Saccharomyces cerevisiae cells, which not only avoids the cumbersome enzyme solution purification step and reduces the industrial production cost, but also improves the stability of the enzyme and prolongs its service life.

[0017] The present invention uses response surface analysis to optimize the reaction conditions and designs the temperature, pH, Mg 2+ The concentration is regulated to maximize the efficiency of β-NMN synthesis. The construction of the strain of the present invention and the optimization of the system conditions provide strong technical support for the industrial large-scale production of β-NMN, and may have an impact on related health products, medicines and food industries. It helps to reduce the production cost of β-NMN and broaden its application prospects in cell energy metabolism, anti-aging research and related medical fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of β-NMN yield at different reaction times; Figure 2 Schematic diagram of β-NMN yield at different reaction temperatures; Figure 3 Schematic diagram of β-NMN yield at different reaction pH values; Figure 4 Schematic diagram of β-NMN production under different magnesium ion concentrations; Figure 5 Response surface and contour plots, where (a) is the response surface plot of pH and temperature, (b) is the response surface plot of magnesium ion concentration and temperature, (c) is the response surface plot of magnesium ion concentration and pH, (d) is the contour plot of pH and temperature, (e) is the contour plot of temperature and magnesium ion concentration, and (f) is the contour plot of magnesium ion concentration and pH; Figure 6 Schematic diagram of whole-cell catalytic amplification reaction yield. DETAILED DESCRIPTION

[0019] The present invention is described in detail below in conjunction with specific drawings and embodiments. The examples described below are only preferred embodiments of the present invention. It should be noted that the following description is only for the purpose of explaining the present invention and does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the embodiments according to the technical essence of the present invention are within the scope of the technical solution of the present invention.

[0020] In the following examples, the materials, reagents, etc. used were obtained from commercial sources unless otherwise specified.

[0021] Example 1 Construction of enzyme immobilized on the surface of Saccharomyces cerevisiae NRK 1. Construction of surface display plasmid NRK from thermotolerant Kluyveromyces was selected from the NCBI gene database (the amino acid sequence is shown in SEQ ID No. 1, specifically: MESPQVLLVGISGCSSSGKTTLAKLSAELLPTAVLIHQDDFFKHDEDIPFNDKYQIRDWDSPEALDMNLFGKELDHIKKTGSVSTTLIHNNNHDNSGAAEVRPETQKEVKTKFAKAFEGTNTKVVFVDGFMMFNDQNIKSKFDLKLLVRAPYGTLKKRRAARSGYQTLDSFWIDPPYYFDEFVYSSYARSHSQLFEDGNVEGVLKKGSGILDFINDDSTDINDALLWVCNCVVSMAQKK) was selected as the target gene, and the signal peptide SUC2 of endogenous sucrose hydrolase of Saccharomyces cerevisiae (base sequence as shown in SEQ ID No. 2, 5' -ATGCTTTTGCAAGCTTTCCTTTTCCTTTTGGCTGGTTTTGCAGCCAAAATATCTGCA-3') was added upstream of the thermotolerant Kluyveromyces NRK gene by PCR amplification. The upstream primer SUC2-F (base sequence as shown in SEQ ID No. 3, 5'-ATGCTTTTGCAAGCTTTCCTTTTCCTTTTGGCTGGTTTTGCAGCCAAAATATCTGCAATGGAATCTCCACAAGTTTTGTTGGT-3') and the downstream primer NRK-R (base sequence as shown in SEQ ID No. 4, 5'-GCTTTTTTTTGAGCCATAGAAACAACACAATTACAA -3') were designed to amplify the NRK expression cassette with the upstream signal peptide sequence using the NRK gene fragment as a template. The PCR amplification conditions were 95℃ pre-denaturation for 30s, 95℃ denaturation for 45s, 55℃ annealing for 15s, 72℃ extension for 30s, 35 cycles, and 72℃ final extension for 5min.

[0022] The upstream primer DAN4-F (base sequence as shown in SEQ ID No. 5, 5'-TATGGCTCAAAAAAAAGCTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTTCAGTCGCATCTTTTGCATCAAGTTCCC-3') and the downstream primer DAN4-R (base sequence as shown in SEQ ID No. 6, 5'-CCTGCAGGATTGAATTTTATAGCAGTAATAAAGCGACAACAGCAACGATA -3') with a flexible connecting peptide were designed. The Saccharomyces cerevisiae strain BY4741 purchased from Shanghai Maokang Biotechnology Co., Ltd. was used for rapid genome extraction. The genome was used as a template to amplify the GPI anchor region of the DAN4 protein with a flexible connecting peptide upstream (the amino acid sequence is shown in SEQ ID No. 7, specifically: SVASFASSSPLLVSSRSNCSDARSSNTISSGLFSTIENVRNATSTFTNLSTDEIVITSCKSSCTNEDSVLTKTQVSTVETTITSCSGGICTTLMSPVTTINAKANTLTTTETSTVETTITTCPGGVCSTLTVPVTTITSEATTTATISCEDNEEDITSTETELLTLETTITSCSGGICTTLMSPVTTINAKANTLTTTETSTVETTITTCSGGVCSTLTVPVTTITSEATTT The PCR amplification conditions were as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 45 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, 35 cycles, and final extension at 72°C for 5 min.

[0023] The NRK expression cassette gene fragment with a signal peptide sequence and the GPI anchor region gene fragment of the DAN4 protein were used as templates, and primers SUC2-F and DAN4-R were used for overlap extension PCR to construct a fusion anchored NRK gene fragment. The PCR amplification conditions were pre-denaturation at 95°C for 30s, denaturation at 95°C for 45s, annealing at 55°C for 15s, extension at 72°C for 30s, 35 cycles, and final extension at 72°C for 5min.

[0024] The template plasmid used was the pESC-URA vector purchased from UBO Biotech, which has a GAL inducible promoter and a URA selection marker. NotI , SacI The pESC-URA plasmid was linearized by two restriction endonucleases in a metal bath at 37°C for 1 hour. The electrophoresis conditions were 180V for 18 minutes. The correct length was verified by agarose gel electrophoresis and then purified and recovered.

[0025] The fusion anchored NRK gene fragment and the pESC-URA vector linear fragment were incubated in a 50°C constant temperature metal bath for 30 min using Gibson assembly seamless cloning enzyme. The construction of the recombinant surface display vector plasmid was completed in one step. After the plasmid connection was completed, it was transformed into the E. coli DH5α competent cells purchased by Sangon Biotechnology (Shanghai) Co., Ltd., coated with ampicillin-resistant LB plates, and cultured at 37°C for 12 hours. The transformants on the LB plates were selected as templates for E. coli colony PCR. The transformants with the correct bands were activated and sent to Shanghai Paisono Biotechnology Co., Ltd. for gene sequencing. After successful sequencing, the recombinant plasmid vector was named p1t.

[0026] 2. Construction of surface displayed NRK immobilized enzyme The electroporation method was used to construct the NRK immobilized enzyme displayed on the surface of Saccharomyces cerevisiae. The specific steps are as follows: Saccharomyces cerevisiae strain BY4741 was activated overnight and transferred to 100 mL YPD. The initial inoculation OD 600 =0.1, culture at 30℃ until OD 600 = about 2, place it on ice for 30 min, then centrifuge it at 4000 rpm in a 4°C precooled centrifuge for 5 min, wash the bacteria twice with precooled sterile water and centrifuge it, add 16 mL sterile water, 2 mL TE solution, 2 mL 1 M lithium acetate solution, incubate it at 30°C for 45 min, add 0.5 mL 1 M DTT and continue incubation, end the incubation after 15 min, wash the bacteria with precooled 1 M sorbitol solution, add 100 μL 1 M sorbitol solution and pipette it, and the preparation of Saccharomyces cerevisiae competent cells is now complete.

[0027] Take 50 μL of Saccharomyces cerevisiae BY4741 competent cells and add 1 μg of p1t plasmid DNA. After mixing by pipetting, transfer to a pre-cooled 2 mm sterile electroporation cup. After incubation on ice for 5 minutes, wipe off the residual moisture on the electroporation cup and immediately place it in the electroporation tank. The electroporation instrument equipment conditions are: 1500 v, 200 Ω. After the electroporation transformation step is completed, immediately add 800 μL of 1M sorbitol solution and mix well. Transfer to a sterile EP tube and incubate at 30°C for 1 h. After the incubation, centrifuge to remove the supernatant, add 100 μL of sterile water to resuspend the bacterial solution, and then apply it to the SC-URA nutrient deficiency plate. Incubate in an incubator at 30°C for 3 days. The correctly sequenced Saccharomyces cerevisiae strain was named Y1t and deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on March 26, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30138.

[0028] Example 2 Induction and catalysis of immobilized surface-displayed Saccharomyces cerevisiae strain Y1t 1. Qualitative and quantitative analysis of the product β-NMN. The specific technical steps are as follows: A Waters high performance liquid chromatograph was used, the chromatographic column was InertSustain C18 (4.6×250mm, 5μm), the mobile phase A was 75% 40 mM potassium dihydrogen phosphate and 5 mM tetramethylammonium hydrogen sulfate (1M potassium hydroxide titrated to pH 6.2), the mobile phase B was 25% methanol, the mobile phase A was filtered through a 0.22 μm microporous aqueous phase filter, the column temperature was set at 30°C, and the detector used a PDA photodiode array detector. The detection wavelength was 260 nm, the flow rate was 0.5 mL / min, and the injection volume was 10 μL. Take 1 mL of the fermentation broth, centrifuge at 13000 rpm for 1 min, and then aspirate the supernatant, filter it through a 0.22 μm microporous filter membrane for sterilization, and then inject it for detection. 2. Detection of enzyme activity of surface-displayed Saccharomyces cerevisiae strain Y1t (1) Activation of the strain: Pick a single colony of Saccharomyces cerevisiae Y1t on the plate and place it in 2 mL of SC-URA (glucose carbon source) liquid culture medium. Incubate it in a shaking incubator at 30°C overnight for 12 h and then transfer it to 2 mL of fresh culture medium.

[0029] (2) Cultivation of Y1t strain: Transfer the activated Y1t bacterial suspension twice to a triangular shake flask containing 20 mL SC-URA medium, and inoculate the initial OD 600 =0.2, and cultured in a shaking incubator at 30℃ for 24 h until mid-logarithmic phase.

[0030] (3) Induced expression of recombinant protein: All 20 mL of Y1t bacterial culture medium cultured to the mid-logarithmic phase was centrifuged and transferred to the same volume of SG-URA (galactose carbon source) liquid culture medium and induced overnight at 30°C and 200 rpm for 12 h.

[0031] (4) Catalytic reaction of Y1t Saccharomyces cerevisiae strain: 5 mL of bacterial liquid was centrifuged to collect bacterial sludge, 1 mL of sterile water was blown and mixed, and then centrifuged to remove the supernatant, and added to the prepared reaction system. The concentrations of each component of the system were 2 mM adenosine-5'-triphosphate disodium salt, 2 mM MgCl2, 500 μM nicotinamide riboside chloride, and PBS buffer (pH = 8.0), and cultured at 30°C with a shaker at 200 rpm. The fermentation broth was centrifuged at 2h, 4h, 6h, 8h, 10h, and 12h, and the supernatant was detected by HPLC. The results are shown in Figure 1 As shown in the figure, the substrate added after 12 h was almost completely consumed and converted into β-NMN. Therefore, 12 h was determined to be the optimal reaction time.

[0032] Example 3 Single factor experiment on the catalytic system of surface-displayed Saccharomyces cerevisiae strain Y1t (1) Determination of the optimal reaction temperature: It is known that Saccharomyces cerevisiae can still proliferate at 44°C. In order to explore the optimal reaction temperature of the immobilized cell recombinant enzyme system, the reaction steps of the surface display of the Saccharomyces cerevisiae strain Y1t enzyme activity detection in Example 2 were referred to. Under the condition of pH 8, the reaction was carried out at 20-60°C, with an interval of 10°C. 1 mL of the fermentation broth at different reaction temperatures was centrifuged, and the supernatant was retained for HPLC analysis as described in Example 2. The results are shown in Figure 2. Figure 2 As shown, the optimal temperature condition of the reaction system is between 20-40°C. (2) Determination of the optimal reaction pH: The pH of the reaction system was adjusted using prepared buffer solutions of different pH values. Referring to the reaction steps of the surface display of the enzyme activity detection of the cerevisiae yeast strain Y1t in Example 2, the culture was shaken at 30°C and 200 rpm for 12 h at a pH value of 5-10, and 1 mL of the supernatant was taken for HPLC analysis of the β-NMN content in the fermentation broth. The results were as follows: Figure 3 As shown, the optimal pH condition of the reaction system is between 8 and 10.

[0033] (3) Optimal Mg 2+ Concentration determination: The actual substrate of nicotinamide riboside kinase is a complex of Mg and ATP. 2+The activity of nicotinamide riboside kinase can be maintained, so the concentration of magnesium ions needs to be adjusted to an optimal ratio so that it can maintain the activity of the enzyme well and not waste industrial costs. Too high salt ion concentration is not conducive to the next step of industrial purification and separation. Referring to the reaction steps of the surface display of the enzyme activity detection of the cerevisiae yeast strain Y1t in Example 2, only the magnesium ion concentration in the reaction system is changed, and the results are as follows Figure 4 As shown, the optimal magnesium ion concentration of the reaction system is between 5-9.

[0034] Example 4 Optimization of surface display catalytic system of Saccharomyces cerevisiae strain Y1t by response surface experiment In order to obtain the optimal reaction conditions for the catalytic system, a Box-Behnken three-factor three-level response surface analysis experiment was carried out based on the results of the single-factor experiment. The specific design is shown in Table 1.

[0035] Table 1 Optimization scheme design and experimental results of the whole-cell catalytic system with Y1t displayed on the surface The binomial equation was fitted to the data in Table 1 using Design-Expert 10.0.1 software, and the quadratic polynomial regression equation was obtained, which is Y=123.58037+7.78A-0.95B-0.15C-0.59AB-0.31AC-1.38BC-13.22A 2 -2.04B 2 -1.89C 2 ; The results of the correlation analysis are shown in Table 2. The P value of the model is greater than the F value less than 0.01, indicating that the fitting accuracy is good and the response surface approximation model can be used for subsequent optimization design. The P value of the model lack of fit is 0.3586 (P>0.05), which is not significant, indicating that the model has a good fit, the model is meaningful, and the comprehensive model is accurate and reliable. From this table, the order of factors affecting β-NMN production is temperature>pH>magnesium ion concentration.

[0036] Table 2 Results of variance analysis according to Figure 5The response surface and contour analysis shown in the figure show that the interaction between different experimental factors has a significant effect on the yield of β-NMN produced by the whole cell catalysis of the Y1t strain. Among the factors investigated, temperature has been confirmed to be a key variable affecting the yield of β-NMN. According to data analysis and prediction, the yield of β-NMN produced by the whole cell catalysis of the Y1t strain can reach the optimal level under the conditions of 33°C, pH 8.7, and magnesium ion concentration of 7 mM, which is expected to be 124.884 mg / L. In order to verify the accuracy of this predicted value, this optimized condition was experimentally verified. The results show that under the optimal conditions set, the yield of β-NMN can be stabilized at around 123.9 mg / L, so this condition is determined to be the optimal process parameter for the whole cell catalytic production of β-NMN by the Y1t strain.

[0037] Example 5 Whole-cell catalytic amplification of the Saccharomyces cerevisiae strain Y1t on the surface (1) Pick a single colony of Y1t on the plate and activate it overnight in 2 mL SC-URA (glucose carbon source) liquid medium.

[0038] (2) Transfer the activated bacterial suspension to 40 mL SC-URA medium with an initial inoculum size of 0.2, 30°C, 200 rpm, and culture until the mid-logarithmic phase.

[0039] (3) All the bacterial culture medium cultured to the mid-logarithmic phase in step (2) was centrifuged and the supernatant was discarded. The bacterial sludge was washed with sterile ultrapure water and transferred to 40 mL of SG-URA (galactose carbon source) liquid culture medium and induced overnight at 30°C and 200 rpm for 12 h.

[0040] (4) Take 5 mL of bacterial liquid and centrifuge to collect the bacterial sludge, blow and pipet 1 mL of sterile water to mix, then centrifuge to remove the supernatant, add it to the prepared reaction system, the concentration of each component of the system is 80 mM adenosine-5'-triphosphate disodium salt, 100 mM MgCl2, 25 mM nicotinamide riboside chloride, PBS buffer (pH=8.7), and culture at 33°C and 200 rpm on a shaker. Take 1 mL of fermentation liquid and centrifuge at different time points to retain the supernatant. As described in Example 3, take 500 μL of the supernatant, blow and pipet mix it with 500 μL of sterile water, filter it through a 0.45 μm microporous filter membrane to sterilize it, and transfer it to a liquid phase vial for HPLC detection. The results are as follows Figure 6 As shown, 6.46±0.07 g / L β-NMN was obtained within 84 h of reaction time, which met the requirements of industrial production.

[0041] The above experiments obtained the optimal enzyme activity reaction conditions of the surface display immobilized enzyme catalytic system constructed by the present invention, which provides a reference for industrial production. In future research, it can be considered to rationally or irrationally transform the nicotinamide riboside kinase to make it more suitable for industrial production.

Claims

1. A strain of Saccharomyces cerevisiae, characterized in that: The strain is Saccharomyces cerevisiae Y1t, which is classified as Saccharomyces cerevisiae Saccharomyces cerevisiae , and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 26, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNo.30138.

2. Use of the Saccharomyces cerevisiae strain according to claim 1 in the efficient synthesis of β-NMN.

3. A method for efficiently synthesizing β-NMN, characterized in that: The saccharomyces cerevisiae strain according to claim 1 is used to optimize the whole-cell catalytic conditions, including temperature, pH, Mg 2+ concentration.

4. The method according to claim 3, characterized in that The method comprises the following steps: placing the activated cells in a culture medium for cultivation, and adding the cells in a reaction system for shaking culture after induction.

5. The method according to claim 4, characterized in that The reaction system comprises adenosine-5'-triphosphate disodium salt, MgCl2, nicotinamide ribose chloride, and PBS buffer.

6. Application of β-NMN synthesized by the method described in any one of claims 3 to 5 in the fields of health products, medicines and / or foods.

7. Application of β-NMN synthesized by the method described in any one of claims 3 to 5 in the field of cellular energy metabolism and / or anti-aging medicine.

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