NMN efficient synthesis strain and construction method and production application thereof

Abstract

The invention provides an NMN efficient synthesis strain and a construction method and production application thereof, and belongs to the technical field of engineering bacteria. According to the invention, firstly, an NMN synthesis route taking NAM as a substrate is constructed in escherichia coli, and specifically, NadV and PrsD128A are introduced into the escherichia coli, so that the NMN synthesis flux is improved; pnuC is introduced to promote the external arrangement of the NMN; the method comprises the following steps: knocking out PncC, NadR, UshA, NudC and MazG; pfkA and TalA are knocked out to improve the carbon flux of the pentose phosphate pathway. GDH1 and GDH2 are heterologously expressed in engineering bacteria to establish a reducing power balance system, the reducing power balance system is used for converting NADPH excessively accumulated in an NMN synthesis route into NADH, and meanwhile, an esaR-esaI group response element is introduced to dynamically regulate and control the system. The engineering strain constructed by the method can accumulate 3.0 g / L of NMN after 24-hour shake flask fermentation.

Description

Technical Field

[0001] The present invention belongs to the technical field of engineering bacteria, and specifically relates to an NMN efficient synthesis strain and a construction method and production application thereof. Background Art

[0002] Nicotinamide mononucleotide (NMN) is a + A key intermediate in biosynthesis, it has multiple physiological functions in mammals, such as anti-aging, anti-inflammatory and neuroprotection. The significant therapeutic effects of NMN on humans and mice make it a useful additive for health products and medicines. NMN is also used as a non-canonical redox cofactor in biocatalysis to circumvent inherent redox regulation and specifically transfer redox power to the target reaction. Due to the increasing demand for NMN, it is very important to construct efficient microbial cell factories to achieve high production of NMN.

[0003] Microbial synthesis of NMN mainly uses NA, NAM and other substances as substrates. In contrast, NAM as a substrate has the advantages of low cost and less substrate residue. According to existing reports, by screening the efficient nicotinamide phosphoribosyltransferase NAMPT with NMN transporter, NMN was synthesized by batch fermentation with NAM as substrate. Although research on NMN biosynthesis has achieved certain results, as an important precursor for NAD+ synthesis, the effect of enhancing NMN synthesis on redox balance, cell growth and strain productivity has been ignored. Strengthening the synthesis of NMN will affect the intracellular cofactor content, and usually increasing the carbon flux of the pentose phosphate pathway will also be accompanied by a large amount of NADPH output, further affecting the intracellular reducing power balance. Relevant studies have shown that fluctuations in the availability or ratio of redox equivalents will affect all redox reactions and change the distribution of cellular resources in catabolism and anabolism. Therefore, metabolic engineering to maximize NMN carbon flux may encounter the problem of redox imbalance, and it is necessary to develop methods to accurately regulate the redox balance of NMN engineering strains.

[0004] Constructing a redox balance system based on transhydrogenase is an effective strategy to overcome redox imbalance and has been used to promote the biosynthesis of many value-added chemicals with NAD(P)H as a cofactor. At the same time, the intracellular redox level changes dynamically with changes in growth and culture conditions, and it is necessary to fine-tune the expression of the redox balance system to maintain the optimal redox state for NMN synthesis. In recent years, the bacterial quorum sensing system (QS) has become a hot topic in the microbial community. Bacteria can monitor changes in the number of themselves or other bacteria in the surrounding environment based on specific signal molecules, thereby initiating the expression of related genes in the bacteria to adapt to changes in the environment. Many quorum response-related genes have been mined and used as regulatory elements for gene expression.

[0005] Based on the above background, constructing an engineered strain with an efficient NMN synthesis pathway and a reducing power balance system is beneficial to the industrial production of NMN. The intracellular reducing power imbalance produced by the engineered bacteria during the synthesis of NMN will limit its production capacity. Using a population response element to finely regulate the reducing power balance system is an effective strategy to optimize the synthesis capacity of NMN engineered strains. Summary of the invention

[0006] The purpose of the present invention is to improve the production efficiency of NMN production strains and use the strains to efficiently produce NMN, strengthen the E. coli NMN synthesis pathway; use genome editing technology to strengthen the NMN synthesis pathway, knock out key enzymes of central metabolism, so that the carbon source is concentrated on NMN synthesis; finally, use the GDH1-GDH2 reducing power system controlled by Esa population response to maintain the intracellular reducing power homeostasis of NMN production bacteria. The engineered bacteria obtained by the construction obtained 3.0g / LNMN yield in 24h shake flask fermentation, indicating that strains with central metabolic reconstruction and reducing power balance system have more advantages in NMN production.

[0007] In order to achieve the above-mentioned purpose, the present invention will provide one of the technical solutions: a method for constructing an NMN engineering strain, comprising the following steps:

[0008] (1) Nicotinamide phosphoribosyltransferase, phosphoribosyl pyrophosphokinase and nicotinamide mononucleotide transporter from Vibrio phage KVP40 were introduced into Escherichia coli to construct an Escherichia coli strain with high NMN production efficiency.

[0009] (2) Based on (1), the genes encoding PncC, NadR, UshA, NudC, MazG, PfkA, and TalA enzymes in the engineered bacteria were knocked out to drive carbon flow into the NMN synthesis pathway and enhance NMN synthesis.

[0010] (3) Introducing yeast transhydrogenases GDH1 and GDH2 into the engineered bacteria constructed in (2), and using the EsaI-EsaR quorum response system to dynamically control their expression intensity to balance the reducing power level in the cell.

[0011] (4) Perform shake flask fermentation on the engineered bacteria constructed in step (3) to test the NMN synthesis performance of the engineered bacteria.

[0012] The strain involved in this scheme is Escherichia coli, specifically Escherichia coli BL21. By introducing the nadV gene and prs D128A The introduction of the pnuC gene effectively improves the synthesis efficiency of NMN with NAM as the substrate; the introduction of the GDH1 gene and the GDH2 gene can transport the NADPH / NADH imbalance problem generated during the production process; the introduction of the esaR gene and the esaI gene can finely regulate the expression of the proteins encoded by the GDH1 gene and the GDH2 gene, optimize the reducing power level and the balance between strain growth and production, among which:

[0013] nadV is the gene with the DNA sequence shown in SEQ ID NO: 1 in the sequence listing;

[0014] prs D128A A gene having a DNA sequence as shown in SEQ ID NO: 2 in the sequence listing;

[0015] pnuC is the gene with the DNA sequence shown in SEQ ID NO: 3 in the sequence listing;

[0016] GDH1 is the gene with the DNA sequence shown in SEQ ID NO: 4 in the sequence listing;

[0017] GDH2 is the gene with the DNA sequence shown in SEQ ID NO: 5 in the sequence listing;

[0018] esaR is the gene with the DNA sequence shown in SEQ ID NO: 6 in the sequence listing;

[0019] esaI is a gene having a DNA sequence shown in SEQ ID NO: 7 in the sequence listing.

[0020] The strain involved in this technical solution is E. coli BL21, but the scope of strain protection in this patent application is Escherichia coli, and there is no special limitation on the specific bacterial species.

[0021] The specific implementation method of this scheme is to use pET-28a plasmid as a vector to overexpress the nicotinamide phosphoribosyltransferase gene nadV and the transport vector pnuC ( Figure 1); introducing the population response gene elements esaR and esaI into E. coli BL21, and making esaI located at PL 13 promoter; using pSTV28 plasmid as a vector to introduce transhydrogenase genes GDH1 and GDH2 into E. coli BL21, and making GDH1 and GDH2 in P esaR Promoter, BCD 18 and BCD 13 Under control Figure 2 ). CRISPR / Cas9 was used to knock out the pncC, ushA, nudC, mazG, pfkA, and talA genes. D128A Perform genome integration.

[0022] The second technical solution provided by the present invention is a method for detecting the intracellular NADPH content during the fermentation production of NMN by the above-mentioned genetically engineered bacteria, which is specifically as follows:

[0023] (1) This scheme introduces mBFP, a fluorescent protein that can specifically bind to NADPH, to monitor the changes in intracellular NADPH in the strain during the fermentation process, as well as the changes in intracellular NADPH in the strain before and after the introduction of the reducing power regulation system. The fluorescence intensity of this protein will increase after binding to NADPH, and this fluorescence change can be detected by a microplate reader under the conditions of excitation wavelength and emission wavelength of 350nm and 451nm respectively.

[0024] (2) mBFP was introduced into the genetically engineered bacteria. During the shake flask fermentation, 1 mL of bacterial solution was taken every 4 h, washed with PBS, and the OD was measured in a black ELISA plate. 600 Finally, the fluorescence changes of the engineering strain per unit cell during fermentation were calculated.

[0025] The third technical solution provided by the present invention is a method for producing NMN by shake flask fermentation of the genetically engineered bacteria, which is specifically as follows:

[0026] (1) Slant culture: Take the -80℃ preserved bacteria and inoculate them on the activated slant, culture them at 37℃ for 12h, and subculture them once;

[0027] (2) Shake flask seed culture: Use an inoculation loop to scrape a ring of slant seeds and inoculate it into a shake tube containing 5 mL of seed culture medium, and culture at 37°C and 220 rpm for 8-10 hours;

[0028] (3) Shake flask fermentation: Take 1 mL of seed solution and inoculate it into a 250 mL Erlenmeyer flask filled with fermentation medium (final volume is 30 mL), seal it with sealing film, shake and culture at 37°C and 220 rpm. During the fermentation process, add 30 μL of 1 M IPTG inducer and 1 mL of 30 g / L NAM solution. The fermentation period is 24 h.

[0029] Beneficial effects: The present invention constructs an engineered bacterium that efficiently synthesizes NMN using NAM as a substrate, and introduces the transhydrogenase encoding genes GDH1 and GDH2 from Saccharomyces cerevisiae into the NMN engineered strain, effectively solving the NADPH / NADH imbalance problem caused by the NMN production process; the esaR-esaI population response system is introduced to achieve joint control of transhydrogenase expression and strain growth status, and inhibits hydrogenase expression in the early stage to avoid strain growth inhibition. Figure 3 The fluorescence intensity of strain S2 containing the reducing power balance system was always lower than that of S1 during the fermentation process, and reached the fluorescence peak at around 16h, and the maximum fluorescence intensity decreased by 27% compared with the S1 strain. This proves that the system can solve the problem of reducing power imbalance caused by NMN accumulation during the fermentation process. Figure 5 As shown, based on the above strategy, the production capacity of the NMN engineered strain S2 was significantly improved. After 24 hours of shake flask fermentation, 3.0 g / L of NMN could be accumulated, which was 67% higher than the NMN production of the S1 strain (1.8 g / L), and had industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the pET-28a-PT7-nadV-pnuC plasmid map;

[0031] Figure 2 pSTV28-P esaR -GDH1-GDH2 plasmid map;

[0032] Figure 3 The changes in NADPH content of the strain during the NMN shake flask fermentation process;

[0033] Figure 4 This is the standard curve for liquid phase detection of NMN;

[0034] Figure 5 This is the NMN shake flask fermentation yield. DETAILED DESCRIPTION

[0035] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] Example 1: Method for editing the genome of engineered strains and enhancing the NMN synthesis pathway

[0037] 1. Genome Editing Methods

[0038] This example uses the CRISPR / Cas9 system for gene editing. The gRNA sequence for gene knockout (PAM: 5'-NGG-3') was designed online using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ) to knock out the NMN degradation pathway genes pncC, ushA, nudC, mazG, the pyrimidine nucleoside metabolism repressor protein encoding gene purR, and the key genes pfkA and talA in the central carbon metabolism pathway. In addition, while knocking out related genes, primers were designed to knock out multiple genes esaI, esaR, and prs D128A The heterologous genes used were synthesized by Suzhou Genewise Co., Ltd. The amplification primers required for integration and knockout fragments are shown in the sequence table in "4."

[0039] 2. pET-28a-PT7-nadV-pnuC and pSTV28-P esaR -GDH1-GDH2 plasmid construction method

[0040] 2.1 Gene synthesis

[0041] Codon optimization (for Escherichia coli ) and synthesis of genes nadV, pnuC, GDH1, GDH2, esaR, and esaI were performed at Suzhou Genewise Biotechnology Co., Ltd.;

[0042] 2.2 Gene amplification

[0043] (1) Using the gene synthesized in "2.1" as a template, nadV was amplified by PCR using nadV-F and nadV-R as primers, pnuC was amplified by PCR using pnuC-F and pnuC-R as primers, GDH1-GDH2 was amplified by PCR using GDH-F and GDH-R as primers, esaR was amplified by PCR using esaR-F and esaR-R as primers, and esaI was amplified by PCR using esaI-F and esaI-R as primers. The DNA polymerase used for PCR was Phanta enzyme, and the PCR system used was as shown in the following table;

[0044] 50μL Phanta enzyme PCR amplification system: 2μL DNA, 2μL upstream primer (10μmol / L), 2μL downstream primer (10μmol / L), 25μL 2×PhantaMix and 19μL ddH2O.

[0045] (2) Use NcoI and BamHI restriction endonucleases to digest the vector plasmid pET-28a stored in the laboratory of Tianjin University, and use KpnI restriction endonuclease to digest pSTV-28a. 50μL restriction digestion system: pET-28a plasmid 5000ng, endonuclease 12.5μL, endonuclease 22.5μL, 10× Buffer 5μL and the remaining ddH2O. The reaction conditions are 37℃, 30min.

[0046] (3) Using the amplified fragments obtained in steps (1) and (2) and the linearized vector as templates, a seamless cloning recombination reaction was performed to construct pET-28a-PT7-nadV-pnuC and pSTV28-P esaR -GDH1-GDH2 plasmid. Recombination reaction conditions: 50°C, 10 min;

[0047] 10μL recombinant system: 2×CE II Buffer 5μL, linearized cloning vector 1μL, insert fragment cloning vector 2μL and ddH2O 2μL.

[0048] 2.3 Plasmid verification

[0049] After the reaction in "2.2(3)", add the recombinant system to 100 μL DH5α competent cells, mix gently, place on ice for 20 minutes, heat shock at 42℃ for 90 seconds, place on ice for 2-3 minutes, add 900 μL SOC, and resuscitate at 37℃ for 1 hour. Centrifuge at 8000rpm for 2 minutes, discard part of the supernatant, and keep about 200 μL to resuspend the bacteria and spread them on LB plates containing 100 mg / L kanamycin (pET-28a-PT7-nadV-pnuC) and 100 mg / L chloramphenicol (pSTV28-PesaR-GDH1-GDH2). Invert the plates and culture at 37℃ overnight. After a single colony grows on the plate, identify it by colony PCR and select positive recombinants. Inoculate the PCR-positive colony into LB medium containing 100 mg / L kanamycin, culture overnight, and preserve the bacteria to extract the plasmid.

[0050] The colony PCR system is as follows:

[0051] 15 μL Taq enzyme colony PCR system: 0.5 μL upstream primer (10 μmol / L), 0.5 μL downstream primer (10 μmol / L), 7.5 μL 2x RapidTaq MasterMix and 6.5 μL ddH2O.

[0052] 3. Methods for constructing engineered strains

[0053] 3.1 Plasmid transformation

[0054] The E. coli S1 strain with enhanced NMN pathway and central metabolism was cultured in LB medium at 37°C until OD 600 = 0.5-0.6, prepare the competent cells for transformation. esaR -GDH1-GDH2 plasmid, take 100ng and add it to 100μLE.coli S1 transfection competent cells, mix gently and place on ice for 20min, heat shock at 42℃ for 45-90s, immediately place on ice for 2-3min, add 900μL SOC, and recover at 37℃ for 1h. Centrifuge at 8000rpm for 2min, discard part of the supernatant, keep about 200μL to resuspend the bacteria and spread it on a plate containing 100mg / L kanamycin, turn the plate upside down, and culture it at 37℃ overnight. After a single colony grows on the plate, identify it by colony PCR and select positive recombinants. Using the same method, prepare the transfection competent strain containing pET-28a-PT7-nadV-pnuC, and transform the plasmid pSTV28-P according to the above method. esaR -GDH1-GDH2. After recovery, the bacterial solution was spread on a double-resistance plate containing kanamycin and chloramphenicol, the plate was inverted, and cultured at 37°C overnight. Finally, the strains containing pET-28a-PT7-nadV-pnuC and pSTV28-P esaR -NMN synthesis strain S2 of GDH1-GDH2.

[0055] 4. The primers involved in the strain transformation process are shown in the following table:

[0056]

[0057]

[0058]

[0059] Example 2: Production of NMN by shake flask fermentation using NMN engineered bacteria

[0060] A specific operation of producing NMN by using NMN engineered bacteria shake flask fermentation is as follows:

[0061] Slant culture: Take the -80℃ stored strain and inoculate it on the activated slant, culture it at 37℃ for 12h, and subculture it once;

[0062] Seed culture: scrape a ring of slant seeds with an inoculation loop and inoculate it into a shaking tube containing 5 mL of seed culture medium, and culture at 37°C and 220 rpm for 8-10 hours;

[0063] Shake flask fermentation: 1 mL of seed solution was inoculated into a 250 mL Erlenmeyer flask containing fermentation medium (final volume was 30 mL), sealed with sealing film, cultured at 37°C, 220 rpm, and added 30 μL of 1 MIPTG inducer and 1 mL of 30 g / L NAM mother solution during the fermentation process. The fermentation cycle was 24 h.

[0064] Detection of NMN content in fermentation supernatant: Take 1mL of fermentation broth and centrifuge at 12000rpm for 2min. Aspirate the supernatant with a syringe, filter through a 0.22μm filter membrane, and add it to the liquid phase tube for testing. The chromatographic conditions are: chromatographic column Kromasil C18 column (250mm×460mm, 5μm), mobile phase is 95% 20mM ammonium acetate solution and 5% acetonitrile, flow rate 1.0mL / min, column temperature 30℃, detection wavelength 259nm, injection volume 20μL, peak time is about 1.5min, and the concentration of NMN in the fermentation broth is calculated according to the peak area according to the drawn standard curve;

[0065] Drawing of NMN standard curve: Prepare solutions with NMN concentrations of 0 mg / L, 2.5 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L, respectively, and use liquid chromatography to determine the peak areas of NMN at the corresponding concentrations according to the above chromatographic conditions, and draw a standard curve related to NMN concentration and its peak area as shown in the attached figure. Figure 4 shown.

[0066] The activation medium components are: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 15g / L, the rest is water, pH 7.0-7.2, sterilized in a high pressure steam cooker at 121℃ for 20min;

[0067] The seed culture medium consists of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and the rest is water, pH 7.0-7.2, sterilized in a high pressure steam cooker at 121°C for 20 min;

[0068] The components of the fermentation medium are: glucose 20 g / L, (NH4)2SO4 5 g / L, KH2PO4 12 g / L, MgSO4 1 g / L, yeast extract 5 g / L, NaCl 1 g / L, citric acid 1.1 g / L, MOPS 10 g / L, and the rest is water, pH 7.0-7.2, sterilized in a high-pressure steam cooker at 115°C for 15 min.

[0069] The NMN engineered strain constructed above was used for fermentation, and the strain S1 preserved in the laboratory of Tianjin University without central metabolic reconstruction and without the introduction of the reducing power system was fermented under the same conditions as a control. The experimental results are as follows Figure 5As shown, the NMN engineered strain S2 accumulated NMN up to 3.0 g / L, an increase of 67% compared to the control strain S1.

[0070] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing an engineered strain for efficient synthesis of β-nicotinamide mononucleotide, characterized in that: The following steps are involved: (1) Using Escherichia coli as the base bacterium, the coding genes of nicotinamide phosphoribosyltransferase, phosphoribosyl pyrophosphokinase and nicotinamide mononucleotide transporter were introduced into the genome of the Escherichia coli to construct an Escherichia coli NMN efficient production bacterium; (2) Knock out the genes encoding PncC, NadR, UshA, NudC, MazG, PfkA, and TalA enzymes in the Escherichia coli NMN efficient production bacteria to obtain engineered bacteria with enhanced NMN synthesis; (3) Introducing the coding genes of yeast transhydrogenase GDH1 and GDH2 into the engineered bacteria in step (2), and dynamically controlling their expression intensity using the EsaI-EsaR population response system to obtain the engineered strain for efficient synthesis of β-nicotinamide mononucleotide.

2. The construction method according to claim 1, characterized in that: The gene encoding the nicotinamide phosphoribosyltransferase in step (1) is nadV, and its DNA sequence is shown in SEQ ID NO: 1; The coding gene of the phosphoribosyl pyrophosphate kinase is prs D128A , whose DNA sequence is shown in SEQ ID NO: 2; The coding gene of the nicotinamide mononucleotide transporter is pnuC, and its DNA sequence is shown in SEQ ID NO:

3.

3. The construction method according to claim 1, characterized in that: The gRNA sequences used for the knockout in step (2) are shown as SEQ ID NO: 56-SEQ ID NO:

69.

4. The construction method according to claim 1, characterized in that: In step (3), the population response gene elements esaR and esaI are introduced, and esaI is located at PL 13 under the control of the promoter; The sequence of esaR is shown in SEQ ID NO: 6; The esaI sequence is shown in SEQ ID NO:

7.

5. The construction method according to claim 4, characterized in that: The PL 13 The sequence of the promoter is shown in SEQ ID NO:

11.

6. The construction method according to claim 4, characterized in that: In step (3), pSTV28 plasmid is used as a vector to introduce transhydrogenase genes GDH1 and GDH2, and GDH1 and GDH2 are expressed in P esaR Promoter, BCD 18 and BCD 13 under control; The sequence of GDH1 is shown in SEQ ID NO: 4; The sequence of GDH2 is shown in SEQ ID NO:

5.

7. The construction method according to claim 6, characterized in that: The P esaR Promoter, BCD 18 and BCD 13 The sequences are shown in SEQ ID NO: 8 to SEQ ID NO:

10.

8. An engineered strain for efficiently synthesizing β-nicotinamide mononucleotide obtained by the construction method according to any one of claims 1 to 7.

9. Use of the engineered strain according to claim 9 in the production of NMN.

10. A method for producing NMN using the engineered strain according to claim 9, characterized in that: The method comprises using the engineered strain to perform shake flask fermentation to obtain NMN.

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