A nicotinamide mononucleotide high-yielding yeast strain and its application

Abstract

The present invention relates to a high-yield nicotinamide mononucleotide saccharomyces cerevisiae strain and its application, belonging to the technical fields of enzyme engineering and genetic engineering. The strain is saccharomyces cerevisiae YZH-01, which is classified as saccharomyces cerevisiae Saccharomyces cerevisiae , was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on March 28, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 30174. This strain can improve the efficiency of whole-cell catalytic production of NMN; combined with the optimization of the feeding process in the whole-cell catalytic reaction system and the precise control of the substrate concentration, the method of adding substrate in batches is adopted, which not only enhances the efficiency and selectivity of the catalytic reaction, but also greatly improves the economic benefits of production.

Description

Technical Field

[0001] The invention relates to a saccharomyces cerevisiae strain with high production of nicotinamide mononucleotide and application thereof, belonging to the technical fields of enzyme engineering and gene engineering. Background Art

[0002] Nicotinamide mononucleotide (NMN) is an important intermediate metabolite of NAD in the human body. + It is a precursor supplement of NMN, composed of ribose, phosphate group and nicotinamide group from vitamin B3. It is widely found in animals and plants in nature. For example, there are 0.47-1.88 mg NMN in every 100 grams of edamame and 0.22 g NMN in every 100 grams of raw shrimp. Studies have shown that due to NAD + Excessive polarity will inhibit its passive transport through the cell membrane, so direct oral supplementation of NAD + Failure to increase its levels in blood and tissues and direct administration of high doses of NAD + It can cause adverse reactions in the human body, such as insomnia, anxiety, etc. Studies have shown that NAD + Compared with other precursors NA and NAM, a daily dose of NA exceeding 50 mg will cause headaches and dizziness, stimulate skin immune cells, dilate skin capillaries, and cause spontaneous skin flushing reactions. + Dependent enzymes have feedback inhibition and consume methyl groups, resulting in reduced epigenetic methylation. Therefore, they are not the most ideal supplementary precursors. NMN is relatively + NMN is not only smaller in molecular weight, but also easier to penetrate the cell membrane and be absorbed by the human body. Studies have shown that a single oral dose of 500 mg of NMN is also safe for the human body. Therefore, it is a NAD with great application value. +Supplementation. There is some evidence (Campagna R, Vignini A. NAD+ Homeostasis and NAD+-Consuming Enzymes:Implications for Vascular Health. Antioxidants. 2023 Feb;12(2):376; Wang X,Hu X, Yang Y, Takata T, Sakurai T. Nicotinamide mononucleotide protectsagainst β-amyloid oligomer-induced cognitive impairment and neuronal death.Brain Res. 2016 Jul 15;1643:1–9; Lundt S, Zhang N, Polo-Parada L, Wang X,Ding S. Dietary NMN supplementation enhances motor and NMJ function in ALS.Exp Neurol. 2024 Apr 1;374:114698) that NMN administration can increase NAD in various peripheral tissues, such as liver, adipose tissue, heart, skeletal muscle, and kidney. + level.

[0003] Biosynthesis has gradually become the dominant method for NMN synthesis due to its environmentally friendly characteristics. Currently, there are two main biosynthesis strategies for NMN. One of them uses Escherichia coli as the chassis cell to construct the NMN biosynthesis pathway and produce NMN through fermentation or whole-cell catalytic processes. However, Escherichia coli is not a biosafe strain, and its synthesized products cannot be directly used in food and health products. Another strategy is to create an immobilized yeast strain by fixing the key catalytic enzymes on the cell surface of the food-safe strain Saccharomyces cerevisiae using surface display technology, and directly convert the substrate nicotinamide riboside (NR) into NMN through whole-cell catalysis. The NMN synthesized by this method can be directly used as a food additive with higher safety, but the yield is low. Although the method of using NRK kinase on the surface of Saccharomyces cerevisiae to produce NMN in a biocatalytic manner has significant advantages in environmental protection and food safety, this system limits the catalytic activity of the immobilized enzyme due to the low protein secretion capacity of Saccharomyces cerevisiae itself. Summary of the invention

[0004] In view of the problems existing in the current biosynthesis of NMN, combined with the characteristics of Saccharomyces cerevisiae, the present invention screened and constructed a Saccharomyces cerevisiae cell with high NRK kinase activity. The strain of the present application enhances the protein anchoring efficiency at the single cell level, thereby improving the activity of the immobilized enzyme. The efficiency of the whole-cell catalytic enzyme in the production of NMN is improved, and finally, combined with the optimization of the feed process in the whole-cell catalytic reaction system, a solution with practical prospects is provided for the industrial-scale production of NMN.

[0005] The technical solution of the present invention is as follows:

[0006] A cerevisiae yeast strain with high nicotinamide mononucleotide production, the strain is cerevisiae yeast YZH-01, classified and named cerevisiae yeast Saccharomyces cerevisiae The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection on March 28, 2024, with the deposit number CGMCC No.30174, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] Another object of the present invention is to protect the use of the above-mentioned Saccharomyces cerevisiae strain in the preparation of nicotinamide mononucleotide.

[0008] Preferably, a Saccharomyces cerevisiae strain is used as the strain for whole-cell catalytic production of NMN.

[0009] Preferably, the reaction system comprises nicotinamide riboside (NR), adenosine triphosphate (ATP), and MgCl2;

[0010] Preferably, the reaction system further comprises Tris-HCl buffer.

[0011] Another object of the present invention is to protect a method for high-yield nicotinamide mononucleotide, comprising the following steps: using the cerevisiae strain of claim 1, and a reaction system comprising nicotinamide riboside NR, adenosine triphosphate ATP, and MgCl2; and further comprising supplementing nicotinamide riboside NR multiple times during the reaction.

[0012] Description of biological preservation of Saccharomyces cerevisiae YZH-01:

[0013] Depository: China General Microbiology Center (CGMCC);

[0014] Deposit number: CGMCC No. 30174;

[0015] Deposit date: March 28, 2024;

[0016] Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0017] Taxonomic name: Saccharomyces cerevisiae ( Saccharomyces cerevisiae ).

[0018] Beneficial effects of the present invention

[0019] The present invention first successfully constructed a Saccharomyces cerevisiae cell line that maintains high activity of NRK kinase expression after surface fixation through screening experiments. Furthermore, two key cell wall synthesis genes in Saccharomyces cerevisiae were knocked out individually and in combination using molecular biology techniques, thereby effectively transforming the exogenous protein secretion pathway of Saccharomyces cerevisiae. This transformation significantly enhanced the protein anchoring efficiency at the single cell level, thereby improving the activity of the immobilized enzyme. The efficiency of the whole-cell catalytic enzyme in catalyzing the production of NMN was improved, and finally, combined with the optimization of the feeding process in the whole-cell catalytic reaction system, a solution with practical prospects was provided for the industrial-scale production of NMN. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of strain effect implementation;

[0021] Figure 2 Growth curve of knockout strain;

[0022] Figure 3 Schematic diagram of the standard curve of NMN standard peak area versus mass concentration;

[0023] Figure 4 Specific enzyme activity of knockout strains;

[0024] Figure 5 Schematic diagram of whole-cell catalytic amplification reaction yield;

[0025] Figure 6 Schematic diagram of the yield after optimized feeding in the amplified reaction; (a) is single feeding, and (b) is multiple feeding. DETAILED DESCRIPTION

[0026] 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.

[0027] In the following examples, the materials, reagents, etc. used were obtained from commercial sources unless otherwise specified.

[0028] Example 1 Construction of a highly active NRK kinase surface display strain

[0029] 1. Amplification of target gene fragments

[0030] Primers 1-F (base sequence as shown in SEQ ID No. 2) and 1-R (base sequence as shown in SEQ ID No. 3) were used to amplify the target gene fragment; the target gene was selected from the thermophilic Chaetomium NRK kinase gene (amino acid sequence as shown in SEQ ID No. 1, specifically:

[0031] MTASDSDSPQRAIVIAISGCSSSGKTTLARLLRDLLPETFIIHEDDFYKPEAELPIKDGLVDWDCPEAIAFDDLERALRHVREKGTYPVSGAGDVPPSPSSLAPSRPVPLSRRSDHGCVLPLPHPISVTSSSSSSSFNSIASTSVSSTHIPSSSTSTTVNSSLTIPPTLSPPGQQPFLDSKEDLNSVGPS PVTPDQIACCRAKINEWLSPGHPGSLIFGSPSSPDDSNPTNTKPRIRLCILDGFLLYTPTTTPPSESFFPLSKIQPLLDIKLFLTVSKSSATRRREARDGYVTLEGFWKDPPGYVEKIVWPNYVEAHKWMFEGGDVERGVVNTEVADREGVRVMDTQGRGDVEFGEVLKWAVGEVMGEVERLCLGDGK*).

[0032] The PCR reaction system was 25 μL 2 × Phanta Max Buffer, 1 μL dNTP Mix, 2 μL upstream primer (concentration of 10 μM), 2 μL downstream primer (concentration of 10 μM), 1 μL Phanta Max Super-Fidelity DNA Polymerase, 1 μL template DNA, and 19 μL ddH2O. The PCR amplification conditions were pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 1 min, 30 cycles, and final extension at 72°C for 10 min. After amplification, gel electrophoresis was performed, and the gel was cut and recovered after the length was correct. The concentration was measured by an ELISA reader and stored in a refrigerator.

[0033] Proteomic analysis revealed the roles of YRR1 deletion in enhancing the vanillin resistance of Saccharomyces cerevisiae strain CEN.PK2-1C (Cao W, Zhao W, Yang B, Wang X, Shen Y, WeiT, Qin W, Li Z, Bao X. Saccharomyces cerevisiae. Microb Cell Fact. 2021 Jul 23;20(1):142.) was used as a template to amplify the endogenous SED1 anchor protein gene fragment of Saccharomyces cerevisiae using primers SF (base sequence as shown in SEQ ID No. 4) and SR (base sequence as shown in SEQ ID No. 5); the base sequence of the endogenous SED1 anchor protein gene of Saccharomyces cerevisiae is shown in SEQ ID No. 6; (5'--3'); the PCR reaction system and conditions were the same as above, and after amplification, the gel was cut and recovered, and the concentration was measured by an ELISA reader and stored in a refrigerator.

[0034] Using the above-amplified thermophilic Chaetomium NRK kinase gene fragment and SED1 anchor protein gene fragment as templates, primers 1-F (base sequence as shown in SEQ ID No. 2) and SR (base sequence as shown in SEQ ID No. 5) were used for overlap extension PCR to splice the two genes together to form a fusion protein gene; the PCR reaction system was the same as above, and the DNA molar addition amount of the thermophilic Chaetomium NRK kinase gene fragment and the SED1 anchor protein gene fragment was 1:1. The PCR amplification conditions were pre-denaturation at 95℃ for 30 s, denaturation at 95℃ for 15 s, annealing at 56℃ for 15 s, extension at 72℃ for 1 min 30 s, 28 cycles, and final extension at 72℃ for 10 min. After amplification, gel electrophoresis was performed, and the gel was cut and recovered after the length was correct. After the concentration was determined by an enzyme marker, the fusion protein gene fragment was stored in a 4℃ refrigerator.

[0035] 2. Construction of Saccharomyces cerevisiae strain for surface display of NRK kinase

[0036] use Xgf The commercial yeast shuttle plasmid pDR196 provided by Beijing Zhongyuan Heju Biotechnology Co., Ltd. was treated with restriction endonucleases to achieve linearization of the plasmid. Subsequently, the linearized plasmid was purified by gel excision recovery, and the linearized vector and fusion protein gene fragment were assembled using Gibson seamless cloning, and the colon transformation experiment was carried out. The LB plate with ampicillin resistance was coated and placed in a 37°C incubator. After a single colony grows on the plate, the successfully verified colon transformants are selected for liquid activation and then the plasmid is extracted. Subsequently, the LiAc chemical transformation method is used to transfer it into the chassis strain CEN.PK2-1C, and the URA-deficient plate is coated and placed in a 30°C incubator. The Saccharomyces cerevisiae transformant carrying the fusion protein gene plasmid vector is temporarily named Ct-NRK. See the construction effect diagram. Figure 1 .

[0037] 4. Gene knockout and surface display of protein secretion pathways in Saccharomyces cerevisiae strains

[0038] (1) Construction through Cre / LoxP recombinase system FVV Single knockout strains CCW12 Single knockout strains and FVV , CCW12 Double knockout strain; FVVThe amino acid sequence of the gene is shown in SEQ ID No. 7, specifically: MQYHSALYVYIYVTFTTIPYKEKPDIISICFSMLSFVFDFSVRICSRTLESFSWSLISSSAFKVVSAFSLAGSCVLASRSSVGIIVSLLLFNFSTCNFVLFLSAVLIDLFFCTFLPTPTFLPTPFFFMLHLPIFSLLNALELLYLIIAGLHI*; CCW12 The amino acid sequence of the gene is shown in SEQ ID No. 8, specifically: MQFSTVASIAAVAAVASAAANVTTATVSQESTTLVTITSCEDHVCSETVSPALVSTATVTVDDVITQYTTWCPLTTEAPKNGTSTAAPVTSTEAPKNTTSAAPTHSVTSYTGAAAKALPAAGALLAGAAALLL*.

[0039] (2) FVV , CCW12 Construction of double knockout strains: FVV Based on the single knockout strain, the C re The plasmid of the gene (amino acid sequence is shown in SEQ ID No. 9, specifically: MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRTLDSETGAMVRLLEDGD*) was transformed again by the LiAc method after the antibiotic marker was eliminated. CCW12 Gene knockout integration cassette FVV Single knockout strain construction FVV , CCW12 Double knockout strains.

[0040] Example 2 Analysis of NRK surface displayed catalytic enzyme activity

[0041] 1. Growth assay

[0042] Pick a single colony from the storage plate and activate it in 2 mL of liquid culture medium, then transfer it to 20 mL of fresh culture medium to make its initial OD 600 The value was 0.2, and the OD was measured every 12 hours. 600 For specific results, see Figure 2 , as shown in the figure, single knockout gene FVV and CCW12 Both affected the growth of the strain. From the growth status of the double knockout strain, we can see CCW12 The knockout of the gene has a greater impact on the growth of the strain. Figure 2 It can be seen that the strain initially reached the stable growth period at 36 h, and the catalytic efficiency of the immobilized cell enzyme (i.e., bacterial sludge precipitation) at this time point was relatively high.

[0043] 2. Quantitative detection of NMN

[0044] (1) Detection conditions: Waters e2695 high performance liquid chromatograph was used, the chromatographic column was Waters XBridge BEHC18 (4.6×250 mm, 5 μm), and the mobile phase was 0.1 mol / L phosphate buffer-methanol (95:5 V / V ), detection wavelength 254nm, flow rate 1.2 mL / min, injection volume 5 μL, column temperature set to 30℃, and detector used was PDA photodiode array detector.

[0045] (2) Standard curve drawing: The NMN standard was prepared into 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, and 0.5 g / L solutions with ultrapure water. The peak area corresponding to each concentration was obtained after HPLC analysis. The regression equation and correlation coefficient were obtained by linear fitting. The peak shape diagram and regression curve are shown in Figure 2. Figure 3 As shown, the curve has a good linear relationship and can be used as an external standard curve for quantitative and qualitative detection of NMN.

[0046] (3) Determination of dry cell weight: After numbering the 0.22 μm filter membrane, place it in an 80°C oven and dry it until constant weight is reached. Then measure the weight. Centrifuge the bacterial solution to remove the supernatant and wash it twice with ultrapure water. Use the filtration system with the constant weight filter membrane to filter the bacterial suspension. The bacteria will remain on the filter membrane and the excess liquid will be removed. Then, place the constant weight filter membrane with the bacteria back into the 80°C oven overnight and measure the weight the next day. The dry weight of the bacteria is the total weight of the bacteria on the filter membrane minus the weight of the constant weight filter membrane.

[0047] 3. Enzyme activity determination of knockout strains

[0048] (1) From FVV Single knockout strains CCW12 Single knockout strains FVV , CCW12 Single colonies were picked from the storage plates of the double knockout strains and the Ct-NRK strain without gene knockout and activated twice in 2 mL of liquid culture medium on a shaking platform at 30°C.

[0049] (2) The next day, transfer the cells to 40 mL of fresh culture medium and set the initial inoculation OD to 600 The value is 0.2.

[0050] (3) After 36 h of incubation at 30°C in a shaking incubator, measure the OD 600 After washing twice with sterile ddH2O, the bacterial sludge was blown into the prepared enzyme activity reaction system. The components of the enzyme activity assay system were: 20 mM Tris-HCl (pH=8.0), 1 mM ATP, 1 mM MgCl2, and 0.6 mM NR. 600 The value is 18.

[0051] (4) After shaking at 30°C for 1 hour, the reaction solution was immediately removed and centrifuged to terminate the reaction. 1 mL of the supernatant was taken for high performance liquid chromatography to detect the NMN production. The amount of enzyme required to produce 1 μmol of NMN per minute under the reaction conditions was defined as 1 enzyme activity unit (IU). The results are shown in Figure 2. Figure 4 As shown, the enzyme activity of the double gene knockout strain increased by 87% compared with that of the strain without gene knockout, proving that the anchoring efficiency of the fusion protein of anchor protein and NRK kinase on the cell wall surface can be improved by modifying the secretion pathway of exogenous proteins in Saccharomyces cerevisiae. The Ct-NRK strain after double gene knockout was named YZH-01. It was deposited in the General Microbiology Center of China Microbiological Culture Collection on March 28, 2024, with the deposit number CGMCC No.30174, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0052] Example 3 Whole-cell catalytic amplification reaction of strain YZH-01

[0053] (1) Pick a single colony of YZH-01 strain on the plate and activate it twice in 2 mL of liquid culture medium. The next day, transfer it to 80 mL of fresh culture medium to make its initial inoculation OD 600 The value is 0.2.

[0054] (2) After 36 h of incubation at 30°C in a shaking incubator, measure the OD 600The value was about 5.5. After centrifugation, 20 mL of bacterial solution was taken and the supernatant was discarded. After washing twice with sterile ddH2O, the bacterial sludge was pipetted into the reaction system configured in advance. The concentrations of each component were increased to 23 mM NR, 150 mM ATP, 300 mM MgCl2, and 20 mM Tris-HCl buffer (pH 8.0), so that the OD of the immobilized cell enzyme was 600 The value was 36, and then cultured in a shaking incubator at 30°C.

[0055] (3) Every 12 h, 1 mL of the reaction solution was centrifuged and the supernatant was filtered through a 0.22 μm filter membrane and then tested by high performance liquid chromatography. The reported data were obtained based on three independent parallel experiments to ensure the reliability and repeatability of the experimental results. Figure 5 As shown, the highest NMN yield of 5.2 g / L was obtained after the reaction was carried out for 72 h. After 72 h, the NMN yield showed a downward trend. It is speculated that the lack of substrate nicotinamide riboside led to the decrease in NMN yield.

[0056] Example 4 Optimization of fed-substrate reaction conditions of strain YZH-01

[0057] In order to obtain a higher yield of NMN, the experimental design optimized the substrate addition in the whole-cell catalyzed reaction process and obtained a higher yield through feeding.

[0058] (1) Pick a single YZH-01 colony from the plate and activate it overnight. The next day, transfer it to 80 mL of fresh culture medium and set the initial inoculation OD to 600 The value is 0.2;

[0059] (2) After culturing at 30°C in a shaking incubator for 36 h, measure the OD 600 value, centrifuge to remove the supernatant, wash twice with sterile ddH2O, and then pipette the bacterial sludge into the amplified reaction system, and then culture on a shaking table at 30°C. The initial concentration of the reaction system refers to Example 3.

[0060] (3) After 24 h of reaction, the reaction system was fed once more and the concentration of nicotinamide riboside NR in the reaction system was replenished to 23 mM as determined by HPLC.

[0061] (4) Take 1 mL of the reaction solution every 24 hours and centrifuge it to obtain the supernatant for HPLC quantitative detection. The results are as follows: Figure 6 As shown in (a), under the condition of single feeding, the NMN yield of 8.1 g / L was obtained after 120 h of reaction. This result proves that the method of supplementing the substrate nicotinamide riboside is indeed beneficial to the production of NMN.

[0062] (5) Based on this single feeding, the steps (1) to (3) in Example 4 were repeated again. At 72h and 120h of the reaction, 1 mL of the reaction solution was centrifuged and the supernatant was taken for HPLC determination. The nicotinamide riboside NR in the system was replenished to 23 mM. During the entire reaction, samples were taken every 24h for quantitative detection of NMN. The results are shown in Figure 6 As shown in (b), the NMN production increased to 14.46 g / L at 168h. Substrate was added at a later time point, and it was found that the NMN production no longer increased. It was speculated that the NMN concentration of 14.46 g / L inhibited the enzymatic reaction. This yield is competitive for the whole-cell synthesis of NMN in Saccharomyces cerevisiae, which is conducive to further large-scale industrial production.

Claims

1. A cerevisiae yeast strain that produces high levels of nicotinamide mononucleotide, characterized in that: The strain is Saccharomyces cerevisiae YZH-01, which is classified as Saccharomyces cerevisiae Saccharomyces cerevisiae The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection on March 28, 2024, with the deposit number CGMCC No.30174, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the high-nicotinamide mononucleotide-producing Saccharomyces cerevisiae strain according to claim 1 in preparing nicotinamide mononucleotide.

3. The use according to claim 2, characterized in that: Saccharomyces cerevisiae strain was used as the strain for whole-cell catalytic production of NMN.

4. The use according to claim 3, characterized in that: The reaction system includes nicotinamide riboside NR, adenosine triphosphate ATP, and MgCl2.

5. A method for high production of nicotinamide mononucleotide, characterized in that: The method comprises the following steps: using the brewer's yeast strain according to claim 1, and a reaction system comprising nicotinamide riboside NR, adenosine triphosphate ATP, and MgCl2; and further comprising supplementing nicotinamide riboside NR multiple times during the reaction.

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