VniNatt enzyme variant and nicotinamide mononucleotide biosynthesis method

Abstract

The invention provides a VniNatt enzyme mutant, nucleic acid, a carrier or a host cell, and application of the VniNatt enzyme mutant, nucleic acid, a carrier or a host cell in fermentation production of NMN. Compared with a wild type, the mutant can catalytically produce NMN with higher efficiency, and has good thermal stability. BL21 engineering bacteria are constructed on the basis of the VniNamt enzyme mutant and are used for producing NMN through fermentation, and the yield of the NMN can reach 54 g / L by adopting a fed-batch fermentation process.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of synthetic biology, and particularly relates to a VniNampt enzyme variant and a nicotinamide mononucleotide biosynthesis method. BACKGROUND

[0002] β-nicotinamide mononucleotide (NMN) as a key precursor of NAD + , has broad application prospects in the fields of anti-aging and metabolic regulation. In the prior art, NMN biosynthesis has been achieved through gene knockout (such as knocking out pncA, nadR and other genes in the NMN degradation pathway of E. coli) and expressing exogenous synthetic genes (such as NAMPT, PRS and PnuC).

[0003] CN120290510A provides a nicotinamide phosphoribosyltransferase mutant and its application in preparing β-nicotinamide mononucleotide. The nicotinamide phosphoribosyltransferase mutant mutates the 259 lysine (K) in the amino acid sequence shown in SEQ ID NO: 2 to 259 valine (V), which is used in the fermentation production of nicotinamide mononucleotide, and slows down the feedback inhibition. After optimization of the fermentation conditions, the yield can reach 12.6 mg / L.

[0004] CN120173906A provides a nicotinamide phosphoribosyltransferase mutant, a genetically engineered bacterium and its application. The mutant enzyme strain has significantly improved NMN production capacity compared to the wild enzyme strain, and the NMN yield reaches 108.66 mg / L.

[0005] CN119776389A improves the accumulation of NMN through metabolic flow redirection and gene knockout, successfully establishes the synthesis path of NMN in E. coli, further optimizes the NAM-NMN transport system and PRPP synthesis pathway, effectively improves the yield of NMN, and further optimizes the production process of NMN, achieving a maximum NMN yield of 28.5 g / L in the fed-batch fermentation system.

[0006] CN119736324A improves the NMN synthesis flux by introducing NadV and PrsD128A in E. coli; introduces PnuC to promote NMN efflux; knocks out PncC, NadR, UshA, NudC, MazG; knocks out PfkA, TalA to improve the carbon flux of the pentose phosphate pathway. Heterologous expression of GDH1 and GDH2 in the engineering bacteria; a reducing power balance system is established to convert the excess NADPH accumulated in the NMN synthesis pathway into NADH, and the esaR-esaI population response element is introduced to dynamically regulate the system. The engineering strain constructed by using this method can accumulate 3.0g / L NMN in 24h shake flask fermentation.

[0007] CN119193443A expresses nicotinamide phosphoribosyltransferase NAMPT, PRPP synthase BaPRS, transporter BMpnuC, glucose-6-phosphate 1-dehydrogenase EcZwf, 6-phosphogluconate dehydrogenase EcGnd, fructose-1,6-bisphosphatase II EcGlpX, vitreous hemoglobinase Vhb, NADH transhydrogenase EcSthA in E. coli, and knocks out pncC gene, ushA gene, nadR gene, purR gene, pncA gene, edd gene, gntR gene, pfkA gene and pntAB gene, which improves the accumulation of NMN in E. coli cells, and further optimizes the fermentation conditions, so that the strain can obtain more than 4.9g / L NMN at the shake flask level.

[0008] CN118440880A knocks out the regulatory genes and product degradation enzyme genes in the E. coli host strain that affect NMN synthesis, and then introduces a recombinant plasmid overexpressing key enzymes in the NMN synthesis pathway, to construct a genetically engineered E. coli strain that efficiently synthesizes NMN. Then, using ribose and nicotinamide as precursors, high-level microbial metabolism is used to prepare NMN. The yield of NMN produced by 5L tank genetic engineering bacteria YWX001 fermentation reaches 45.2mM (about 15.1g / L).

[0009] However, the catalytic efficiency and solubility of the rate-limiting enzyme VniNampt still need to be improved, and the metabolic burden and product secretion capacity of the chassis strain still limit further improvement of the yield. SUMMARY

[0010] To solve the above problems in the prior art, the present application provides, in a first aspect, a VniNampt enzyme mutant, the amino acid sequence of which is obtained by mutating S at position 143 of SEQ ID NO: 10 to T.

[0011] The present application provides, in a second aspect, a nucleic acid encoding the VniNampt enzyme mutant as described above.

[0012] The present application provides, in a third aspect, a vector comprising the nucleic acid as described above.

[0013] The present application provides, in a fourth aspect, a host cell comprising the nucleic acid or the vector as described above.

[0014] Preferably, it is E. coli BL21.

[0015] Preferably, the E. coli BL21 further expresses BsPrs and BmPnuC.

[0016] Preferably, the E. coli BL21 is knocked out of pncA gene, nadR gene and ushA gene.

[0017] The present application provides, in a fifth aspect, use of the VniNampt enzyme mutant, the nucleic acid, the vector or the host cell as described above in fermentation production of NMN.

[0018] The present application provides, in a sixth aspect, a method for fermentation production of NMN, the method comprising culturing the host cell as described above in a culture medium.

[0019] Through the above technical solutions, the present application can achieve the following beneficial effects:

[0020] 1. A VniNampt enzyme mutant is provided, which can catalyze production of NMN with higher efficiency compared with wild type, and has good thermal stability.

[0021] 2. Based on the VniNampt enzyme mutant, an E. coli BL21 engineering strain for production of NMN is obtained.

[0022] 3. Based on the engineering strain, NMN is produced by fermentation, and a fed-batch fermentation process is adopted, and the yield of NMN can reach 54 g / L. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 M1~M5 72h flask yield of the present application;

[0024] Figure 2 L1~L4 72h flask yield of the present application;

[0025] Figure 3 M3-L2 fermentation condition chart of the present application. DETAILED DESCRIPTION

[0026] The present application will be further described below in combination with the drawings and examples of the specification.

[0027] Example 1 Preparation of chassis bacteria

[0028] Knock out pncA (encoding nicotinamide), nadR (encoding NAD + regulatory protein) and ushA (encoding UDP-sugar hydrolase) in E. coli BL21 (purchased from Shengong Bioengineering (Shanghai) Co., Ltd.) in turn, the above-mentioned genes are NMN degradation pathway related genes known in the art, KEGG database number: pncA: ECD_01737; nadR: ECD_04266; ushA: ECD_00431, obtain the chassis BL21 (ΔpncAΔnadRΔushA). Any existing gene knockout technology can be used for gene knockout of E. coli, which does not constitute the focus of the present application, and thus is not described here.

[0029] Example 2 VniNampt mutant design

[0030] With the help of computer services (analysis method tools include aMIc, BLAST, ClustalΩ, FoldX, Rosetta, SCA, USEARCH, etc.), the conserved sequences of NAMPT of different species are aligned, and the potential residue sites in VniNampt (gene sequence as shown in SEQ ID NO: 3, amino acid sequence as shown in SEQ ID NO: 10) that may enhance enzyme activity are screened. The substrate binding capacity is enhanced by site-directed mutation, and the catalytic efficiency is improved.

[0031] Free energy calculation verification:

[0032] The folding free energy (ΔΔG) of the mutant is calculated by molecular dynamics simulation, and the mutant with ΔΔG reduction ≥0.5 kcal / mol is screened to enhance the protein structure stability and expand the temperature adaptation range.

[0033] Combined with two kinds of prediction theory, five potential mutation points are screened (see Table 1).

[0034] Table 1: Candidate VniNampt mutants Serial number Mutant site Wild-type amino acid Mutated amino acid Abbreviation AG Fold (kcal / mol) M1 41 Y F Y41F -1.15 M2 49 D K D49K -2.06 M3 143 S T S143T -1.06 M4 288 V I V288I -0.68 M5 437 D N D437N -1.04

[0035] Example 3 Expression of VniNampt mutants in chassis

[0036] The single-point mutants to be constructed include: Y41F, D49K, S143T, V288I, D437N (named M1~M5 in turn). The mutation is introduced by primer PCR, and is connected to pET-28a (+) vector by enzyme digestion (NcoⅠ / XhoⅠ), which is placed downstream of T7 promoter to realize high-efficiency expression induced by IPTG (kanamycin resistance screening). The sequencing verification result shows that no random mutation occurs except the required mutation site, so the mutant plasmid is successfully constructed.

[0037] BsPrs (gene sequence as shown in SEQ ID NO: 1, amino acid sequence as shown in SEQ ID NO: 8) and NMN transporter (BmPnuC, gene sequence as shown in SEQ ID NO: 2, amino acid sequence as shown in SEQ ID NO: 9) were cloned in series into pCDF-1b vector downstream of tac promoter (streptomycin resistance screening). The constructed vector is written as pCDF- BsPrs- BmPnuC.

[0038] The constructed mutant plasmid and pCDF- BsPrs- BmPnuC were transformed into the chassis strain, and positive clones were screened by double antibiotics (kanamycin + spectinomycin). For ease of understanding, the corresponding bacteria are also recorded as M1~M5.

[0039] Example 4 Comparison of yields in flask fermentation

[0040] The modified BL21 E. coli was subjected to flask culture, and the composition of the culture medium in the fermentation flask was Na2HPO4(5.8 g / L), KH2PO4(3 g / L), NaCl (0.5 g / L), NH4Cl (1 g / L), and MgSO4(1 mM), and the carbon source was glucose (10 g / L). The fed-batch culture medium contained 10 g glucose, 10 g xylose, 0.5 g NAM, 4 g (NH4)2HPO4, 13.3 g KH2PO4, 1.2 g MgSO 4· 7H2O, 1.7 g citric acid, and 10 mL trace element solution (containing 10 g FeSO 4· 7H2O, 2.25 g ZnSO 4· 7H2O, 1 g CuSO 4· 5H2O, 0.5 g MnSO 4· 5H2O, 0.23 g Na2B4O 7· 10H2O, 2 g CaCl 2· 2H2O, and 0.1 g (NH4)6MoO 24 The culture temperature was 37℃, the shaking speed was 220 rpm, and the yield of NMN was determined after 72 h of culture, with the initial engineering strain into which wild-type VniNampt was taken as a control. The determination results are shown in Table 1. Figure 1 Among them, the yield of E. coli expressing mutant M3 in the flask was the highest, reaching 12.4 g / L.

[0041] NMN and its analogs were detected using a high-performance liquid chromatography (HPLC) system equipped with a ZORBAX SB-AQ stable-bonded analytical column (5 μm, 4.6 × 250 mm; Agilent Technologies, Santa Clara, USA) at a flow rate of 1.0 mL / min, a column temperature of 30 °C, an injection volume of 10 μL, and a detection wavelength of 263 nm. The mobile phase A was 0.1% (v / v) trifluoroacetic acid aqueous solution, and the mobile phase B was acetonitrile. The elution conditions were as follows: 0-5 min, acetonitrile concentration gradient from 0% to 5%; 5-10 min, acetonitrile concentration gradient from 5% to 20%; 10-17 min, acetonitrile concentration gradient from 20% to 100%; 17-20 min, acetonitrile concentration maintained at 100%; 20-25 min, acetonitrile concentration gradient from 100% to 0%; and 25-30 min, acetonitrile concentration maintained at 0%. Glucose and xylose were detected using an HPLC system equipped with a BioRad Aminex HPX-87H chromatographic column (7.8 mm × 300 mm; Hercules, USA) and a differential refractive index detector. The column temperature was set to 30 °C, and the mobile phase was 5 mM sulfuric acid solution at a flow rate of 0.6 mL / min.

[0042] Example 5 Screening and optimization of solubility tags

[0043] For the optimal mutant M3, different solubility tags were introduced at its N-terminus. The tags were connected to the enzyme through a flexible linker ((GGGGS)2) to avoid steric hindrance affecting enzyme activity. The VniNampt M3 mutant genes containing different tags and flexible linkers (GGGGS)2 were synthesized and ligated to the pET-28a(+) vector through enzyme digestion (Nco I / Xho I). The resulting plasmids were co-transformed with pCDF-BsPrs-BmPnuC into the chassis strain BL21 (ΔpncA ΔnadR ΔushA). The different tags were SUMO (gene sequence as shown in SEQ ID NO: 5, and amino acid sequence as shown in SEQ ID NO: 12), CBM66 (gene sequence as shown in SEQ ID NO: 7, and amino acid sequence as shown in SEQ ID NO: 14), NT11 (gene sequence as shown in SEQ ID NO: 4, and amino acid sequence as shown in SEQ ID NO: 11), and GST (gene sequence as shown in SEQ ID NO: 6, and amino acid sequence as shown in SEQ ID NO: 13), which were denoted as L1-L4, respectively. For ease of understanding, the corresponding strains were also denoted as L1-L4.

[0044] Shake flask fermentation was performed according to the method of Example 4. M3 without the addition of water-soluble tags was used as a control, and the NMN yield was determined after 72 h. The determination results are shown in Table 2. Figure 2Among them, the VniNampt mutant (L2) modified by CBM66 tag + flexible linker has the highest solubility, and the NMN yield is more than 15 g / L, which is significantly better than SUMO and NT11.

[0045] Example 6 Fed-batch fermentation

[0046] Using L2 as the active bacteria, the medium in Example 4 was used in a 3L bioreactor, and the OD 600 was controlled at 0.8, 0.5 mM IPTG was added for induction, the pH was controlled at 7.0, the dissolved oxygen was controlled at 20%, and the feed was added (see Example 4). The high-density fermentation culture was carried out for 96 h, and the changes of each component were monitored every 12 h during the fermentation process (see Example 4). Figure 3 The yield of NMN can reach 54 g / L.

[0047] The specific description of the above examples cannot be understood as a limitation on the protection scope of the present application, and some non-essential improvements and adjustments made by those skilled in the art based on the content of the above application fall within the protection scope of the present application.

Claims

1. A mutant VniNampt enzyme, whose amino acid sequence is obtained after mutating the amino acid S at position 143 of SEQ ID NO: 10 to T.

2. A nucleic acid encoding the mutant VniNampt enzyme of claim 1.

3. A vector comprising the nucleic acid of claim 2.

4. A host cell comprising the nucleic acid of claim 2 or the vector of claim 3.

5. The host cell of claim 4, which is E. coli BL21.

6. The host cell of claim 5, wherein the E. coli BL21 further expresses BsPrs and BmPnuC.

7. The host cell of claim 5 or 6, wherein the E. coli BL21 is knocked out of pncA gene, nadR gene and ushA gene.

8. Use of the mutant VniNampt enzyme of claim 1, the nucleic acid of claim 2, the vector of claim 3, or the host cell of any one of claims 4-7 in fermentation production of NMN.

9. A method of fermentation production of NMN, comprising culturing the host cell of any one of claims 4-7 in a culture medium.

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