Saccharomyces cerevisiae strain, construction method and application thereof

Abstract

The application belongs to the field of synthetic biology and genetic engineering, and particularly relates to a Saccharomyces cerevisiae strain and a construction method and application thereof. The strain is Saccharomyces cerevisiae WZ 214, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20251331, and the preservation date is June 10, 2025. A fusion gene is constructed by fusing a key enzyme gene (Nampt and PRPS) for synthesizing β-NMN and a yeast surface anchoring protein gene, and a recombinant strain displaying the key enzyme on the surface is obtained by transforming the original Saccharomyces cerevisiae. Then, the FYV5 gene of the recombinant strain is knocked out to optimize the cell wall structure and reduce the product secretion resistance. The application effectively solves the problems of intracellular product accumulation, great separation difficulty and low production efficiency in the prior art, and provides new technical support for efficient and green production of β-NMN.

Description

Technical Field

[0001] This application belongs to the fields of synthetic biology and genetic engineering, and specifically relates to a strain of Saccharomyces cerevisiae, its construction method, and its application. Background Technology

[0002] Nicotinamide mononucleotide (NMN) belongs to the B group of derivatives and is characterized by high polarity, low volatility, intramolecular salt formation, high water solubility, and poor solubility in organic solvents. In nature, it mainly exists in two isomers, α and β. β-NMN is a naturally occurring and biologically active nucleotide. As a major source of cellular energy, it has antioxidant, oxidative stress reduction, and anti-inflammatory effects. β-NMN is closely related to NAD+ metabolism. It is a key intermediate in the NAD+ salvage pathway of coenzyme I in mammals and an important metabolite for maintaining normal NAD+ biosynthesis. Its physiological function can only be achieved through conversion into NAD+ within the body. NAD+, in turn, precisely and comprehensively regulates and controls energy metabolism, catalytic reactions, and cellular activity throughout the organism. As we age, the level of NAD+ in the human body gradually decreases, leading to abnormal bodily functions and triggering some diseases, such as Alzheimer's disease and Parkinson's disease. However, NAD+ cannot be directly absorbed by the human body, so it is necessary to supplement its precursor β-NMN to increase the level of NAD+ in the body.

[0003] The main ways to obtain β-NMN include autologous synthesis and intake from food. However, with age, the body's absorption rate decreases, and the total amount of β-NMN obtained through these two methods is insufficient to maintain normal cellular function. Studies have shown that β-NMN plays a crucial role in various physiological metabolic processes, and has potential application value, especially in anti-aging, improving degenerative and metabolic diseases.

[0004] Currently, among the methods for synthesizing β-NMN, biosynthesis has become the preferred approach due to its significant advantages such as high stereoselectivity, mild reaction conditions, and few byproducts. Surface display technology, by immobilizing key enzymes on the cell surface, enables direct utilization of extracellular substrates, reducing the energy consumption of transmembrane transport. Simultaneously, the orderly arrangement of enzyme molecules on the cell surface increases local enzyme concentration and enhances catalytic efficiency. Furthermore, the product can be directly secreted extracellularly, effectively avoiding degradation and feedback inhibition problems caused by intracellular product accumulation, providing a new technological direction for the efficient synthesis of β-NMN. The core reaction in β-NMN synthesis involves the reaction of nicotinamide (NAM) and phosphoribosyl pyrophosphate (PRPP) under the catalysis of nicotinamide phosphoribosyltransferase (NAMPT) to produce β-NMN and pyrophosphate. The synthesis of PRPP (catalyzed by the PRPP synthase PRS) and the capture of NAM are key auxiliary steps ensuring the efficient execution of this reaction. Because of its ability to efficiently utilize multiple carbon sources for growth and protein synthesis, and its excellent nitrogen accumulation capacity in biomanufacturing (protein content can reach 50%), Saccharomyces cerevisiae is widely used in the production of single-cell proteins and bacterial proteins, making it an ideal host for genetic engineering.

[0005] However, existing technologies for producing β-NMN using Saccharomyces cerevisiae still have many shortcomings: on the one hand, when expressing key enzymes intracellularly, the substrate transmembrane transport efficiency is low, the product is prone to accumulate intracellularly leading to feedback inhibition, and subsequent separation and purification are difficult and costly; on the other hand, the cell wall structure of Saccharomyces cerevisiae is dense, and even with surface display technology, there are problems such as insufficient space for enzyme protein embedding and high resistance to product secretion, which limit the production efficiency of β-NMN. Summary of the Invention

[0006] To address the aforementioned issues, this application utilizes systematic genetic engineering modification combined with fermentation process optimization to develop a *Saccharomyces cerevisiae* strain capable of efficiently producing β-NMN, along with a corresponding production method. Specifically, the following technical solutions are employed: In the first aspect, this application discloses a strain of Saccharomyces cerevisiae, namely Saccharomyces cerevisiae WZ 214, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO: M20251331 and deposit date of June 10, 2025.

[0007] Secondly, this application proposes a method for constructing a *Saccharomyces cerevisiae* strain for constructing the surface-displayed *Saccharomyces cerevisiae* strain, comprising the following steps: The key enzyme gene for β-NMN synthesis was fused with the anchoring protein gene to construct a fusion gene. The fusion gene was inserted into the expression vector and transformed into the original Saccharomyces cerevisiae strain to obtain recombinant Saccharomyces cerevisiae with Nampt and PRPS on the surface. Construct a knockout cassette containing an upstream homologous arm of the FYV5 gene, a downstream homologous arm of the FYV5 gene, and a KanMX4 selection marker gene; The knockout cassette was transferred into the recombinant Saccharomyces cerevisiae using the lithium acetate conversion method. After G418 resistance screening and PCR verification, the FYV5 gene of the recombinant Saccharomyces cerevisiae was knocked out to obtain the Saccharomyces cerevisiae strain.

[0008] Furthermore, the anchoring protein gene is the C-terminal coding region of the AGα1 gene, and its amino acid sequence is shown in SEQ ID: 1; The key enzyme genes for β-NMN synthesis are nicotinamide phosphoribosyltransferase gene and phosphoribosyl pyrophosphate synthase gene; wherein, the core sequence of the coding region of the homologous gene sequence of the Nampt gene is shown in SEQ ID: 2.

[0009] Furthermore, in the fusion gene, the anchoring protein gene, the nicotinamide phosphoribosyltransferase gene, and the phosphoribosyl pyrophosphate synthase gene are linked in series via a flexible linker peptide (Gly4Ser)3. The 5' end of the fusion gene is also connected to the coding sequence of the Saccharomyces cerevisiae α factor signal peptide, and the amino acid sequence encoded by the Saccharomyces cerevisiae α factor signal peptide is shown in SEQ ID: 5.

[0010] Furthermore, the expression vector is a high-copy YEp plasmid, and the expression of the fusion gene is driven by an inducible GAL1p promoter; The original Saccharomyces cerevisiae strain was Saccharomyces cerevisiae S288C, with the preservation number ATCC 204508.

[0011] Furthermore, the GenBank accession number for the FYV5 gene is NM_001178702.1.

[0012] Thirdly, this application proposes the use of the described Saccharomyces cerevisiae strain or the Saccharomyces cerevisiae strain constructed according to the method in the production of β-nicotinamide mononucleotide.

[0013] Fourthly, this application proposes a method for producing β-NMN, wherein the method uses the aforementioned Saccharomyces cerevisiae strain and selects a corresponding culture medium for fermentation production according to the fermentation method.

[0014] Furthermore, if the fermentation method is shake flask fermentation, the culture medium components are: glucose 30-70 g / L, yeast extract 10-30 g / L, peptone 10-30 g / L, nicotinamide 4-10 g / L, potassium dihydrogen phosphate 2-6 g / L, and magnesium sulfate 1-3 g / L; the shaking speed during shake flask fermentation is 150-250 r / min, and the culture time is 24-60 h; the preferred culture medium components are glucose 50 g / L, yeast extract 18 g / L, peptone 22 g / L, nicotinamide 6 g / L, potassium dihydrogen phosphate 4 g / L, and magnesium sulfate 1.5 g / L.

[0015] Furthermore, if the fermentation method is fermentation in a fermenter, the components of the culture medium include: glucose 30-70 g / L, casein hydrolysate 20-50 g / L, and nicotinamide 10-30 g / L; preferably: glucose 50 g / L, casein hydrolysate 35 g / L, and nicotinamide 20 g / L.

[0016] Compared with the prior art, this application has the following advantages: This application utilizes surface display technology to localize key enzymes (Nampt and PRPS) for β-NMN synthesis onto the surface of *Saccharomyces cerevisiae* cells, enabling direct utilization of extracellular substrates and avoiding the energy consumption associated with transmembrane transport. Simultaneously, the orderly arrangement of enzyme molecules on the cell surface increases local enzyme concentration and enhances catalytic efficiency. Building upon this, the yeast cell wall structure is optimized by knocking out the FYV5 gene. The FYV5 gene encodes a cell wall-associated membrane protein involved in the assembly of the cell wall glucan network; knocking it out reduces cell wall thickness, decreasing resistance to product secretion and potentially loosening the cell wall structure, providing more space for the embedding of large enzyme proteins. This modification also induces an intrinsic cellular stress response, upregulating the expression of protein translation and secretion-related genes, indirectly promoting the production of displayed proteins, and further enhancing the synthesis and secretion capacity of β-NMN. Experimental results show that the maximum enrichment of β-NMN in the shake-flask fermentation of the *Saccharomyces cerevisiae* WZ 214 constructed in this application reached 4322.13 mg / L, significantly higher than that of the original *Saccharomyces cerevisiae* S288C (1312.53 mg / L) and other commonly used microbial hosts (*Escherichia coli* N18 at 579.24 mg / L and *Bacillus subtilis* 168 at 321.46 mg / L), achieving a leapfrog improvement in production efficiency. Furthermore, the flexible linker peptide (Gly4Ser)3 used in the modification process avoids spatial structural interference between enzyme proteins, the α-factor signal peptide ensures efficient secretion and surface localization of the fusion protein, and the knockout of the FYV5 gene not only loosens the cell wall structure but also upregulates the expression of protein translation and secretion-related genes.

[0017] Other features and advantages of this application will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the application. The objectives and other advantages of this application may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The results of β-NMN content expressed by different modified strains in Example 2 of this application are shown; Figure 2 The response surface diagram in Embodiment 3 of this application is shown. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0021] Example 1 This embodiment utilizes genetic engineering technology to fuse a key enzyme for β-NMN synthesis with a yeast surface anchoring protein, achieving extracellular display of the enzyme. The specific steps are as follows: 1. Construction of the fusion gene: Using Saccharomyces cerevisiae genomic DNA as a template, the C-terminal coding region of the anchoring protein gene AGα1 was cloned (its amino acid sequence is: MSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS, SEQ ID: 1); the β-NMN synthesis key enzyme genes nicotinamide phosphoribosyltransferase gene (Nampt) and phosphoribosyl pyrophosphate synthase gene (PRPS) were cloned respectively.

[0022] The Nampt gene was derived from the Chitinophaga pinensis model strain UQM2034T. Homologous gene sequences were obtained from the NCBI database using the strain's whole genome data (Genome Accession Number CP001646.1). The coding region sequence is: ATGAAACCGCTGCTCGCCGCCATCGCCGCCGCTCTCGCCGCCGCTGCCGCTGCCGCTGCCGCTGCCGCTGCCGCTGCTG ...

[0023] The PRPS genes selected from the Saccharomyces cerevisiae S288C strain are PRS1 and PRS3 genes. These two genes are particularly crucial for the supply of PRPP in the metabolism of Saccharomyces cerevisiae and the synthesis of β-nicotinamide mononucleotide. The EMBL database accession number of the PRS1 gene is X70069, which corresponds to the core PRPS encoding gene of the Saccharomyces cerevisiae S288C strain. The EMBL database accession number of the PRS3 gene is X74415, which is an important gene regulating PRPP synthesis.

[0024] The anchoring protein gene, Nampt gene, and PRPS gene were tandemly linked using overlap extension PCR, and a flexible linker peptide (sequence (Gly4Ser)3) was inserted between each gene to avoid spatial structural interference between the proteins. The yeast α-factor signal peptide coding sequence MRFLQFFTAVFIRA SSA LAAPSN TTT EDET AQFRLKLIGYSD LKGFDVAVL PFPTAK (SEQ ID: 5) was added to the 5' end of the fusion gene to guide the newly synthesized fusion protein into the secretion pathway and finally to the cell surface, thus constructing the fusion gene.

[0025] The above overlap extension PCR reaction system (50 μL) consists of: 10×PCR Buffer (containing Mg²⁺) +5 μL of reagents, 4 μL of dNTP mix (2.5 mM each), 1 μL of upstream primer (20 μM), 1 μL of downstream primer (20 μM), 0.5 μL of Taq DNA polymerase (5 U / μL), 1 μL of template DNA, and sterile distilled water to a final volume of 50 μL (all reagents were purchased from Shanghai Sangon Biotech Co., Ltd.); the PCR reaction was performed in a Bio-Rad T100 thermal cycler.

[0026] 2. Vector Construction and Transformation: A high-copy-count YEp plasmid was selected as the expression vector. The fusion gene expression was driven by an inducible GAL1p promoter. The constructed fusion gene was inserted into the multiple cloning site of the YEp plasmid to construct the recombinant expression vector. The recombinant expression vector was transformed into competent *Saccharomyces cerevisiae* S288C (Novagen, ATCC204508) using the lithium acetate transformation method. A blank control group (untransformed yeast) and an empty vector transformation control group were also set up. The empty vector YEp was transformed into the same batch of original *Saccharomyces cerevisiae* strains using the same method. The transformation procedure was as follows: after heat shock at 30℃, YPD resuscitation medium was added and cultured for 2 hours to restore cell viability.

[0027] 3. Screening and Validation: The revived bacterial cultures of each experimental group were spread onto SD / -Ura defective plates and YPD plates (YPD plates served as growth controls). The plates were incubated upside down in a 30℃ incubator for 48 hours, and colony growth was observed: only successfully transformed yeast (recombinant vector group and empty vector group) could grow on SD / -Ura plates, while untransformed yeast and other bacteria could not grow, and no colonies grew in the blank control group.

[0028] Single colony purification: Pick single colonies from SD / -Ura plates (20-30 colonies for the experimental group and 10-15 colonies for the control group), streak them on new SD / -Ura plates for purification, and incubate at 30℃ for 24-48 hours to obtain homozygous strains.

[0029] The empty vector YEp, identical to the recombinant vector, was selected and transformed into the same batch of original Saccharomyces cerevisiae strains using the same transformation method as the experimental groups. The empty vector control group and the Nampt and PRPS overexpression experimental groups were simultaneously inoculated onto the same YPD and SD / -Ura plates and cultured concurrently at 30°C. The β-NMN production was compared between the empty vector control group and the Nampt and PRPS overexpression experimental groups to eliminate the influence of the empty vector itself on yeast metabolism.

[0030] The formulation for the SD / -Ura defective plate is as follows: Minimal SD Base 26.7g + 0.77g Ura defective amino acid mixture + 20g agar, dissolved in distilled water and brought to a final volume of 1000mL, pH adjusted to 5.8, and sterilized at 121℃ for 15min.

[0031] The YPD plate formulation is as follows: 10g yeast extract, 20g peptone, 20g glucose, and 20g agar, diluted with distilled water to a final volume of 1000mL, with a natural pH.

[0032] Example 2 Surface visualization of the Saccharomyces cerevisiae cell wall synthesis pathway: Construction of Saccharomyces cerevisiae strain: This embodiment optimizes cell wall structure and reduces product secretion resistance by knocking out the FYV5 gene (GenBank accession number: NM_001178702.1) that synthesizes the cell wall of Saccharomyces cerevisiae. The specific steps are as follows: 1. Construction of cassette knockout: Using Saccharomyces cerevisiae genomic DNA as a template, the upstream homologous arm of the FYV5 gene was amplified using primer pair FYV5-Up-F (SEQ ID: 6) / FYV5-Up-R (SEQ ID: 7), and the downstream homologous arm of the FYV5 gene was amplified using primer pair FYV5-Down-F (SEQ ID: 8) / FYV5-Down-R (SEQ ID: 9); The primer sequences are shown in Table 1: Table 1

[0033] The FYV5 gene (GenBank accession number: NM_001178702.1) encodes the FYV5 protein, a cell wall-associated membrane protein involved in the assembly of the cell wall dextran network. Its amino acid sequence (Uniprot P25585) is: MSKLFVSLIFLFLSSLVFFVVVYVYHGFGGGGF ... GFGGGGFGGGFGGGGFGGGFGGGGFGGGFGGGGFGGGGGGFGFGGGGFGGGFGGGGFGGGFGGGGFGGGFGGGGGGFGGGGGGFGGGGGGFGGGGGGFGGGGGGFGFGGGGFGGGFGGGGFGGGFGGGGFGGGFGGGGFGGGFGGGGFGGGFGG (SEQ ID: 10).

[0034] Using a plasmid containing the kanamycin resistance gene (KanMX4) as a template, the KanMX4 selection marker gene was amplified, and 15-20 bp adapter sequences complementary to the ends of the upstream homologous arm and the downstream homologous arm of the FYV5 gene were added to both ends of the primers.

[0035] The coding region sequence of the KanMX4 gene is as follows (SEQ ID: 11): ATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTCCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA Overlap extension PCR was used to seamlessly connect the upstream homologous arm of the FYV5 gene, the KanMX4 gene, and the downstream homologous arm of the FYV5 gene to form a complete linear knockout cassette (Up-FYV5-KanMX4-Down-FYV5, a linear DNA fragment assembly for knocking out the FYV5 gene in Saccharomyces cerevisiae). The fragment size (the target fragment is approximately 2kb) was verified by agarose gel electrophoresis, and the target fragment was recovered using a DNA purification kit.

[0036] 2. Yeast Transformation: The cassette knockout strain was transformed into the Saccharomyces cerevisiae strain with Nampt and PRPS surface display obtained in Example 1 using the lithium acetate (LiAc) transformation method. The specific operation is as follows: Yeast cells were cultured to an OD600 of 0.8–1.0, collected by centrifugation, and washed twice with sterile water. The cells were resuspended in transformation buffer containing 100 mM LiAc (pH 7.5), and 15 μg of knockout cassette was added. The cells were incubated at 30°C for 30 min, followed by heat shock at 42°C for 15 min. To improve transformation efficiency, 5% dimethyl sulfoxide (DMSO) was added to the transformation buffer. The cells were collected by centrifugation, plated on YPD selection plates containing 200 μg / mL G418, and incubated at 30°C for 48–72 h until single colonies appeared.

[0037] 3. Positive clone verification: During initial screening, the cultured bacterial solution was plated on YPD plates (KanMX labeled) or SD-URA plates (URA3 labeled) containing 200 μg / mL G418 and incubated at 30℃ for 2-3 days. Single colonies were picked for PCR verification. PCR amplification was performed using primers for the FYV5 gene (SEQ ID: 12 and SEQ ID: 13) and primers for the KanMX resistance marker (SEQ ID: 14 ​​and SEQ ID: 15). If a target band of approximately 1.5 kb was amplified, and the wild-type strain did not exhibit this band, it was considered a positive clone, thus obtaining a *Saccharomyces cerevisiae* strain exhibiting the key enzyme on its surface and with the FYV5 gene knocked out, named *Saccharomyces cerevisiae* WZ 214.

[0038] The primer sequences are shown in Table 2: Table 2

[0039] 4. Verification of β-NMN expression capacity of modified strains: Single colonies were picked from the preservation plates of the original Saccharomyces cerevisiae S288C strain, the surface-displayed Nampt and PRPS strains obtained in Example 1, and the Saccharomyces cerevisiae WZ 214 strain obtained in this example, and inoculated into 2 mL of liquid culture medium. The colonies were activated twice by shaking at 28 °C. The next day, the colonies were transferred to 50 mL of fresh culture medium with an initial inoculation OD600 value of 0.2. After culturing at 28 °C for 48 h, the reaction was terminated by centrifugation. The supernatant was used to detect the β-NMN yield by high performance liquid chromatography (HPLC) using the determination method in Example 3.

[0040] The results are as follows Figure 1 As shown, the surface of Nampt and PRPS strains showed a 138.13% increase in β-NMN production compared to the original strain (S288C strain in the figure), and Saccharomyces cerevisiae WZ 214 ( Figure 1 The β-NMN production of strain WZ 214 was 38.28% higher than that of surface-displayed Nampt and PRPS strains (surface-displayed strains in the figure), demonstrating that knockout of the FYV5 gene can significantly enhance the ability of Saccharomyces cerevisiae to synthesize β-NMN.

[0041] The brewer's yeast WZ 214 was deposited at the China Center for Type Culture Collection on June 10, 2025, with accession number CCTCC NO: M20251331.

[0042] Example 3 Using Box-Behnken design from response surface methodology, the fermentation process parameters for β-NMN production by Saccharomyces cerevisiae WZ 214 were systematically optimized to increase the yield of β-NMN. The specific steps are as follows: 1. Screening of key influencing factors: Single-factor experiments were conducted to screen the factors affecting β-NMN production. Only one factor was changed each time (including culture medium composition: carbon source type and concentration, nitrogen source type and concentration, precursor NAM concentration; environmental parameters: temperature, pH value, inoculum size, dissolved oxygen content), while other influencing factors were kept constant. The influence trend of each factor on β-NMN production (response value) was observed. Finally, glucose (carbon source), casein hydrolysate (nitrogen source), and NAM (precursor) were identified as the main influencing factors.

[0043] 2. Experimental design and level setting: Based on the main influencing factors identified in section 1, three levels were set, and the levels were reasonably distributed with the center point as the reference: glucose concentration 30-70 g / L, casein hydrolysate concentration 20-50 g / L, and NAM concentration 10-30 g / L.

[0044] The Box-Behnken experimental design was created using Design-Expert software, and the fermentation experiment was conducted in a 5L fermenter (Shanghai Baoxing Biotechnology, BIOTECH-2020). The fermentation temperature was controlled at 26-30℃, the pH at 4.5-5.5 (to avoid β-NMN degradation), and the dissolved oxygen level was maintained.

[0045] 3. Experimental Implementation and Data Acquisition: Fermentation experiments were completed for all experimental groups according to the design scheme. Three parallel samples were set up for each experimental group to ensure data reliability. After fermentation, the β-NMN yield (response value) of each group was determined by HPLC.

[0046] The method for determining β-NMN content is as follows: β-NMN was separated using a C18 column (4.6 mm × 250 mm, 5 μm) by taking advantage of its polarity. Quantitative analysis was performed using a UV detector or a diode array detector. The instrument used for detection was a Shimadzu LC-16 high-performance liquid chromatograph, and the β-NMN standard (purity 99.90%) was purchased from MedChemExpress LLC.

[0047] Sample pretreatment and detection conditions: Place the fermentation broth in a centrifuge tube, centrifuge at 8000-10000 r / min for 10 min to remove the bacterial cells, and filter the supernatant through a 0.22 μm filter membrane to obtain the sample solution to be tested.

[0048] The HPLC detection conditions were as follows: injection volume 20 μL, mobile phase using methanol-water or acetonitrile-water system (volume ratio 10:90 to 30:70) for gradient elution, flow rate 1.0 mL / min, column temperature 30-35℃, detection wavelength 266 nm (characteristic absorption peak of β-NMN), and detection time 15-35 min.

[0049] Content Calculation: Inject the treated sample solution into the HPLC system, record the chromatographic peak area of ​​β-NMN, substitute it into the pre-plotted β-NMN standard curve to obtain the concentration of the sample solution, and then calculate the actual content of β-NMN in the fermentation broth using the following formula: β-NMN content (mg / L) = C × D × (V / V_s) Where: C is the concentration of the sample solution obtained from the standard curve (mg / L); D is the sample dilution factor; V is the final volume of the sample (mL); V_s is the volume of the original fermentation broth sample (mL).

[0050] 4. Model Establishment and Analysis of Variance: Multiple regression analysis was performed on the experimental data using Design-Expert software to establish a quadratic polynomial model between the response value (β-NMN yield) and each factor (A: glucose, B: casein hydrolysate, C: NAM). The model equation is as follows: Y = β0+ β1A + β2B + β3C + β 12 AB + β 13 AC + β 23 BC + β 11 A² + β 22 B² + β 33 C², Where Y is the predicted response value, β0, β1, β2, β3, β 12 β 13 β 23 β 11 β 22 β 33 These are the coefficients for each item.

[0051] Analysis of variance (ANOVA) was performed on the model to test its significance and goodness of fit. The results are shown in Table 3. Table 3. Analysis of variance of the Plackett-Burman experiment results

[0052] As can be seen, the model's F-value is 3628.06, and the P-value is <0.0001, indicating that the model is highly significant; the coefficient of determination R² = 0.9995, indicating that the model has a very high good fit and can accurately predict β-NMN production. Among them, glucose (A), casein hydrolysate (B), NAM (C), and each interaction term (AB, AC, BC) and quadratic term (A², B², C²) all have significant effects on β-NMN production (all P values ​​<0.01).

[0053] 5. Model Validation and Optimal Process Determination: Insignificant terms are removed from the model (in this embodiment, there are no significant terms to remove), and the model is used to predict the optimal combination of factors: Y = -1.4732 + 0.1217A + 0.0433B + 0.1585C + 8.333AB + 0.00028AC + 0.00091BC - 0.00116A² - 0.00095B² - 0.00447C², and the consistency between the actual output and the predicted value was confirmed through verification experiments.

[0054] The optimal fermentation conditions predicted by the model are: glucose 50 g / L, casein hydrolysate 35 g / L, and NAM 20 g / L (e.g., ...). Figure 2 ).

[0055] To verify the reliability of the model, three parallel fermentation experiments were conducted under the optimal conditions. The actual measured β-NMN yield deviated from the model prediction by less than 2%, indicating that the optimal process conditions are stable and reliable.

[0056] In addition, the fed-batch production strategy was determined as follows: the initial carbon source concentration in the culture medium was 30 g / L, and 50% glucose solution was added after 24 h of fermentation to maintain the residual sugar concentration in the fermentation broth at 10-15 g / L; nicotinamide was added in three batches (0 h, 12 h, 24 h) with a cumulative concentration of 8-10 g / L.

[0057] To verify the reliability of the model, parallel fermentation experiments were conducted in shake flasks and 5L fermenters under the above optimal conditions. The actual measured β-NMN yields deviated from the model predictions by less than 2%, indicating that the optimal process conditions are stable and reliable. The optimal process conditions for different fermentation scenarios were obtained through model prediction: (1) Shake flask fermentation (liquid culture medium): The optimal culture medium formula is 50 g / L glucose + 18 g / L yeast extract + 22 g / L peptone + 6 g / L nicotinamide + 4 g / L potassium dihydrogen phosphate + 1.5 g / L magnesium sulfate. The fermentation temperature is 28℃, the rotation speed is 150 r / min, the initial pH is 5.0-6.0, and the culture time is 48 h. (2) Large-scale production in 5L fermenters: The special culture medium formula is glucose + casein hydrolysate + NAM, and the optimal fermentation culture medium composition is glucose 50g / L, casein hydrolysate 35g / L, and NAM 20g / L; The accompanying fed-batch strategy was as follows: the initial carbon source concentration in the culture medium was 30 g / L, and 50% glucose solution was added after 24 h of fermentation to maintain the residual sugar concentration in the fermentation broth at 10-15 g / L; nicotinamide was added in three batches (0 h, 12 h, and 24 h) with a cumulative concentration of 8-10 g / L. During the fermentation process, the temperature was controlled at 26-30℃, the pH at 4.5-5.5, and the dissolved oxygen level was maintained at an appropriate level.

[0058] Example 4 Comparative experiment on the fermentation production capacity of various strains of β-NMN: Using *Saccharomyces cerevisiae* WZ 214 as the experimental group, and *Saccharomyces cerevisiae* S288C, *Escherichia coli* N18 (laboratory-preserved), and *Bacillus subtilis* 168 (laboratory-preserved) as the control groups, the ability of each strain to produce β-NMN was compared. The specific steps are as follows: S1. Fermentation culture: In the experimental group, Saccharomyces cerevisiae WZ 214 was inoculated into liquid culture medium (formulation: glucose 50g / L + yeast extract 18g / L + peptone 22g / L + nicotinamide 6g / L + potassium dihydrogen phosphate 4g / L + magnesium sulfate 1.5g / L) and cultured at 28℃ and 150r / min for 48h with shaking. The initial pH value was controlled at 5.0-6.0, and the culture medium was sterilized at 121℃ for 30 minutes. During the fermentation process, samples were taken at 0h, 6h, 12h, 24h, 36h, and 48h to monitor the OD600 value (measured cell density and biomass) and the pH value of the fermentation broth (monitored in real time by an online pH probe).

[0059] Control group 1 (Saccharomyces cerevisiae S288C): The culture conditions were 25-30℃ and an aerobic environment. The same liquid culture medium as the experimental group was used, and the operation steps were the same as S1.

[0060] Control group 2 (E. coli N18): culture temperature 37℃, using the same liquid culture medium as the experimental group, and operation steps as S1.

[0061] Control group 3 (Bacillus subtilis 168): cultured at 30-37℃ in an aerobic environment, using the same liquid culture medium as the experimental group, and the operation steps were the same as S1.

[0062] S2. Sample processing: After fermentation, the fermentation broth of each experimental group and control group 1-3 was centrifuged to remove the bacterial cells, and the supernatant was filtered through a 0.22μm filter membrane.

[0063] S3. Content detection: The β-NMN content in each sample was detected by the RP-HPLC method described in Example 3, and the results are shown in Table 4.

[0064] Table 4 Production of each strain

[0065] As shown in Table 4, the maximum enrichment of β-NMN in Saccharomyces cerevisiae WZ 214 reached 4322.13 mg / L, which was significantly higher than that of the control groups (Saccharomyces cerevisiae S288C: 1312.53 mg / L, Escherichia coli N18: 579.24 mg / L, Bacillus subtilis 168: 321.46 mg / L), proving that the Saccharomyces cerevisiae WZ 214 constructed in this application has a significant high-efficiency β-NMN synthesis ability.

[0066] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A strain of *Saccharomyces cerevisiae*, characterized in that, The strain is Saccharomyces cerevisiae WZ 214, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20251331 and deposit date of June 10, 2025.

2. A method for constructing a strain of *Saccharomyces cerevisiae*, characterized in that, To construct the *Saccharomyces cerevisiae* strain of claim 1, the following steps are included: The key enzyme gene for β-NMN synthesis was fused with the anchoring protein gene to construct a fusion gene. The fusion gene was inserted into the expression vector and transformed into the original Saccharomyces cerevisiae strain to obtain recombinant Saccharomyces cerevisiae with Nampt and PRPS on the surface. Construct a knockout cassette containing an upstream homologous arm of the FYV5 gene, a downstream homologous arm of the FYV5 gene, and a KanMX4 selection marker gene; The knockout cassette was transferred into the recombinant Saccharomyces cerevisiae using the lithium acetate conversion method. After G418 resistance screening and PCR verification, the FYV5 gene of the recombinant Saccharomyces cerevisiae was knocked out to obtain the Saccharomyces cerevisiae strain.

3. The construction method according to claim 2, characterized in that, The anchoring protein gene is the C-terminal coding region of the AGα1 gene, and its amino acid sequence is shown in SEQ ID: 1; The key enzyme genes for β-NMN synthesis are nicotinamide phosphoribosyltransferase gene and phosphoribosyl pyrophosphate synthase gene.

4. The construction method according to claim 2, characterized in that, In the fusion gene, the anchoring protein gene, the nicotinamide phosphoribosyltransferase gene, and the phosphoribosyl pyrophosphate synthase gene are linked together by a flexible linker peptide (Gly4Ser)3. The 5' end of the fusion gene is also connected to the coding sequence of the yeast α factor signal peptide, the amino acid sequence of which is shown in SEQ ID:

5.

5. The construction method according to claim 2, characterized in that, The expression vector is a high-copy YEp plasmid, and the expression of the fusion gene is driven by an inducible GAL1p promoter. The original brewer's yeast strain was brewer's yeast S288C.

6. The construction method according to claim 2, characterized in that, The GenBank accession number for the FYV5 gene is NM_001178702.

1.

7. The use of the Saccharomyces cerevisiae strain according to claim 1 or the Saccharomyces cerevisiae strain constructed by the construction method according to any one of claims 2-6 in the production of β-nicotinamide mononucleotide.

8. A method for producing β-NMN, characterized in that, The brewing yeast strain described in claim 1 is used, and the corresponding culture medium is selected according to the fermentation method for fermentation production.

9. The method according to claim 8, characterized in that, If the fermentation method is shake flask fermentation, the culture medium components are: glucose 30-70g / L, yeast extract 10-30g / L, peptone 10-30g / L, nicotinamide 4-10g / L, potassium dihydrogen phosphate 2-6g / L and magnesium sulfate 1-3g / L.

10. The method according to claim 8, characterized in that, If the fermentation method is fermentation in a fermenter, the components of the culture medium include: glucose 30-70g / L, casein hydrolysate 20-50g / L and nicotinamide 10-30g / L.

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