Recombinant escherichia coli for producing NMN through fermentation and application of recombinant escherichia coli

Abstract

The invention discloses recombinant escherichia coli for producing NMN through fermentation and application of the recombinant escherichia coli, and belongs to the technical field of genetic engineering and biological engineering. According to the invention, nicotinamide phosphoribosyltransferase NAMPT, PRPP synthase BaPRS, transport protein BMpnuC, glucose-6-phosphate 1-dehydrogenase EcZwf, 6-phosphogluconate dehydrogenase EcGnd, fructose-1, 6-diphosphatase II EcGlpX, vitreous hemoglobin enzyme Vhb and NADH transhydrogenase EcSthA are expressed in Escherichia coli, and a pncC gene, a ushA gene, an nadR gene, a purR gene, a pncA gene, an edd gene, an gntR gene, a pfkA gene and a pntAB gene are knocked out, so that the recombinant plasmid is obtained. The accumulation amount of NMN in escherichia coli cells is increased, and fermentation conditions are further optimized, so that the strain can obtain more than 4.9 g / L of NMN at a shake flask level.

Description

Technical Field

[0001] The present invention relates to a recombinant Escherichia coli for producing NMN by fermentation and application thereof, belonging to the technical field of genetic engineering and bioengineering. Background Art

[0002] NAD + It is one of the most popular anti-aging small molecules in recent years, and exogenous intake of β-nicotinamide mononucleotide (β-NMN, hereinafter referred to as NMN) is a supplement of NAD + It is a very direct and effective method. As a complex isomer, the chemical synthesis of NMN is complex and arduous, so NMN is currently mainly synthesized by biological methods. Currently, the biological synthesis of NMN mainly includes two methods: enzyme method and fermentation method.

[0003] The chemical synthesis of NMN mainly uses nicotinamide and tetraacetyl ribose as starting materials, condenses with trimethylsilyl trifluoromethanesulfonate (TMSOTf), deacetylates, and then phosphorylates with phosphorus oxychloride / trimethyl phosphate to obtain β-NMN; or β-NMN is prepared by phosphorylating and deprotecting nicotinamide ribose protected by ketalization. The chemical synthesis method of NMN technology is relatively more complete, but there are many defects. On the one hand, it is not environmentally friendly, and many raw materials are toxic; on the other hand, it is not economically friendly, and some raw materials are expensive, so the cost of chemical synthesis of NMN remains high.

[0004] The enzymatic synthesis of NMN is mainly divided into two types: one is NR as the core substrate, after adding ATP, nicotinamide ribokinase catalyzes the formation of β-NMN; the other is phosphoribosyl-1-pyrophosphate as the core substrate, after introducing ATP and NAM, nicotinamide phosphoribosyltransferase catalyzes the formation of β-NMN. The enzymatic synthesis conversion rate is high, but the substrate cost is still high, which is not conducive to industrial production.

[0005] The fermentation method of NMN production mainly uses NAM and glucose or other sugars that can produce PRPP as substrates, and directly produces NMN through metabolic engineering strains. The fermentation method has a high synthesis conversion rate and low cost. Summary of the invention

[0006] The present application aims to construct a recombinant strain that is more conducive to the accumulation of NMN. On the basis of the constructed chassis strain, the enzyme gene for synthesizing NMN is expressed, the enzyme gene related to PRPP synthesis is expressed, and the enzyme gene that is not conducive to PRPP accumulation is knocked out, so as to optimize the fermentation process and increase the yield of NMN.

[0007] The invention provides a genetically engineered bacterium capable of synthesizing nicotinamide mononucleotide, which expresses nicotinamide phosphoribosyltransferase (NAMPT enzyme), 5-phosphoribosyl-1-pyrophosphate synthetase (PRPP synthase), transporter protein BMpnuC, glucose-6-phosphate 1-dehydrogenase (Zwf), 6-phosphogluconate dehydrogenase (Gnd), fructose-1,6-bisphosphatase II (GlpX), vitreous hemoglobinase (Vhb) and NADH transhydrogenase (SthA).

[0008] In one embodiment, the amino acid sequence of the nicotinamide phosphoribosyltransferase (NAMPT) has at least 95% identity with the sequence SEQ ID NO.1; the amino acid sequence of the 5-phosphoribosyl-1-pyrophosphate synthetase (PRPP synthase) has at least 95% identity with the sequence SEQ ID NO.2; the amino acid sequence of the transporter BMpnuC has at least 95% identity with the sequence SEQ ID NO.3; the amino acid sequence of the glucose-6-phosphate 1-dehydrogenase (Zwf) has at least 95% identity with the sequence SEQ ID NO.4; the amino acid sequence of the 6-phosphogluconate dehydrogenase (Gnd) has at least 95% identity with the sequence SEQ ID NO.5; the amino acid sequence of the fructose-1,6-bisphosphatase II (GlpX) has at least 95% identity with the sequence SEQ ID NO.6; the amino acid sequence of the vitreous hemoglobinase (Vhb) has at least 95% identity with the sequence SEQ ID NO.7; the amino acid sequence of the NADH transhydrogenase (SthA) has at least 95% identity with the sequence SEQ ID NO.8 has at least 95% consistency.

[0009] In one embodiment, the nicotinamide phosphoribosyltransferase is derived from Chitinophaga pinensis and contains the amino acid sequence shown in SEQ ID NO.1; the PRPP synthase is derived from Bacillus amyloliquefaciens and contains the amino acid sequence shown in SEQ ID NO.2; the transporter BMpnuC is derived from Bacillus mycoides and contains the amino acid sequence shown in SEQ ID NO.3; the glucose-6-phosphate 1-dehydrogenase (Zwf) is derived from Escherichia coli and contains the amino acid sequence shown in SEQ ID NO.4; the 6-phosphogluconate dehydrogenase (Gnd) is derived from Escherichia coli and contains the amino acid sequence shown in SEQ ID NO.5; the fructose-1,6-bisphosphatase II (GlpX) is derived from Escherichia coli and contains the amino acid sequence shown in SEQ ID NO. The amino acid sequence shown in NO.6; the vitreous hemoglobinase (Vhb) is derived from Vitreoscilla and contains the amino acid sequence shown in SEQ ID NO.7; the NADH transhydrogenase (SthA) is derived from Escherichia coli and contains the amino acid sequence shown in SEQ ID NO.8.

[0010] In one embodiment, the gene NAMPT encoding the nicotinamide phosphoribosyltransferase has the nucleotide sequence shown in SEQ ID NO.9; the gene prs encoding the PRPP synthase has the nucleotide sequence shown in SEQ ID NO.10; the gene pnuC encoding the transporter has the nucleotide sequence shown in SEQ ID NO.11; the gene zwf encoding the glucose-6-phosphate 1-dehydrogenase has the nucleotide sequence shown in SEQ ID NO.12; the gene gnd encoding the 6-phosphogluconate dehydrogenase has the nucleotide sequence shown in SEQ ID NO.13; the gene glpX encoding the fructose-1,6-bisphosphatase II has the nucleotide sequence shown in SEQ ID NO.14; the gene vhb encoding the vitreous hemoglobinase has the nucleotide sequence shown in SEQ ID NO.15; and the gene sthA encoding the NADH transhydrogenase has the nucleotide sequence shown in SEQ ID NO.16.

[0011] In one embodiment, the chassis strain of the genetically engineered bacteria includes but is not limited to Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and other microbial strains suitable for constructing an expression system.

[0012] In one embodiment, the Bacillus subtilis includes but is not limited to B. subtilis 168 or B. subtilis WS9.

[0013] In one embodiment, the E. coli includes but is not limited to E. coli BL21 (DE3), E. coli BL21star (DE3), E. coli BL21 (DE3) pLysS, E. coli Rosetta (DE3) or E. coli JM109 (DE3).

[0014] In one embodiment, the overexpression vector plasmid used in constructing the genetically engineered strain includes but is not limited to pRSFDuet-1, pETDuet-1, pCDFDuet-1, pMA5, pWB980, pHT43, pHT01 and pET-22b(+), pET-28a(+), pET-30a(+), pUC57 and other vector plasmids suitable for constructing overexpression genetically engineered strains.

[0015] In one embodiment, the transport protein BMpnuC uses plasmid pACYCDuet-1 or pCDFDuet-1 as an expression vector.

[0016] In one embodiment, the nicotinamide phosphoribosyltransferase NAMPT and PRPP synthase are expressed using pRSFDuet-1 or pETDuet-1.

[0017] In one embodiment, the glucose-6-phosphate 1-dehydrogenase, 6-phosphogluconate dehydrogenase, fructose-1,6-bisphosphatase II and vitreous hemoglobinase are expressed using pET-28a(+) or pRSFDuet-1.

[0018] In one embodiment, the chassis strain of the genetically engineered bacteria is Escherichia coli, and the Escherichia coli has knocked out the pncC gene, ushA gene, nadR gene, purR gene, pncA gene, edd gene, gntR gene, pfkA gene and pntAB gene; the Gene ID of the pncC gene in the NCBI database is 947169, the Gene ID of the ushA gene is 947331, the Gene ID of the nadR gene is 948911, the Gene ID of the purR gene is 945226, the Gene ID of the pncA gene is 946276, the Gene ID of the edd gene is 946362, the Gene ID of the gntR gene is 947946, the Gene ID of the pfkA gene is 948412, the Gene ID of the pntA gene is 946628, and the Gene ID of the pntB gene is 946144.

[0019] In one embodiment, the genetically engineered bacteria is based on Escherichia coli BL21 (DE3) ΔpncC ΔushA ΔnadR ΔpurR ΔpncA Δedd ΔpfkA ΔpntAB, using pETDuet-1 as a vector to express the genes BaPrs and NAMPT, using pCDFDuet-1 as a vector to express BMpnuC, and using pRSFDuet-1 as a vector to express the genes Zwf, GlpX, Gnd and Vhb.

[0020] The invention also provides a fermentation agent containing the genetically engineered bacteria.

[0021] The present invention also provides a method for promoting the synthesis of NMN by recombinant Escherichia coli, which is to overexpress nicotinamide phosphoribosyltransferase NAMPT as shown in SEQ ID No.1, express PRPP synthase BaPRS as shown in SEQ ID No.2, express transporter BMpnuC as shown in SEQ ID No.3, express glucose-6-phosphate 1-dehydrogenase EcZwf as shown in SEQ ID No.4, express 6-phosphogluconate dehydrogenase EcGnd as shown in SEQ ID No.5, express fructose-1,6-bisphosphatase II EcGlpX as shown in SEQID No.6, express vitreous hemoglobinase Vhb as shown in SEQ ID No.7 and express NADH transhydrogenase (SthA) as shown in SEQ ID No.8 in Escherichia coli.

[0022] In one embodiment, the recombinant Escherichia coli further has ΔpncC, ΔushA, ΔnadR, ΔpurR, ΔpncA, Δedd, ΔpfkA and ΔpntAB knocked out.

[0023] In one embodiment, the method further comprises inducing IPTG when culturing the recombinant Escherichia coli.

[0024] The present invention also provides a method for producing NMN by fermentation, wherein the recombinant Escherichia coli is used as a fermentation microorganism, and the recombinant Escherichia coli is cultured at 35-40° C. until the OD 600 =3-4, and then at 25-37°C, use IPTG with a final concentration of ≥0.2mM to induce the synthesis of NMN.

[0025] In one embodiment, the fermentation time is 48 to 72 hours.

[0026] In one embodiment, the induction is induced by IPTG at a final concentration of 0.2 mM-1 mM.

[0027] In one embodiment, the fermentation uses 1000 mg-2000 mg / L nicotinamide as a substrate.

[0028] In one embodiment, the method inoculates the recombinant Escherichia coli into a seed culture medium, cultures to obtain a seed solution, and then transfers the seed solution to a fermentation medium at a transfer rate of 1% to 5%, cultures at 35 to 37° C. for 5 to 7 hours until the OD 600 =3-4, then add IPTG to a final concentration of 0.2mM-1mM, and add 500mg-3000mg / L nicotinamide to induce NMN synthesis at 30-37°C.

[0029] In one embodiment, the fermentation medium contains: yeast powder, citric acid, ammonium sulfate, phosphate (K2HPO4, KH2PO4), and glucose.

[0030] The present invention also provides the use of the recombinant Escherichia coli, the starter culture, or the method in the preparation of NMN-containing products in the fields of food, medicine, cosmetics, feed, and textiles.

[0031] Beneficial effects:

[0032] (1) The present invention reduces the degradation of the substrate NAM by knocking out the pncA gene encoding nicotinamide adenine diphosphate (NAM), thereby improving the utilization rate of NAM and making the NMN production of the constructed strain reach 0.35 g / L;

[0033] (2) The present invention improves the protein expression of the strain by optimizing the induction timing, so that the yield after induction is increased to 2.94 g / L when the strain is cultured to an OD of 3 to 4, further increasing the NMN yield of the strain;

[0034] (3) The present invention modified sugar metabolism and enhanced the supply of PRPP by overexpressing glucose-6-phosphate 1-dehydrogenase, 6-phosphogluconate dehydrogenase and fructose-1,6-bisphosphatase II, and knocking out the edd gene and gntR gene, so that the NMN production of the strain reached 4.26 g / L after 48 h of fermentation in a shake flask;

[0035] (3) The present invention improves the balance of energy and reducing power in the strain by overexpressing vitreous hemoglobinase NADH transhydrogenase and knocking out the pntAB gene, so that the NMN production of the strain reaches 4.9 g / L after 48 h of fermentation in a shake flask. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the plasmid map of the recombinant vector pETDuet-1+BaPRS+NAMPT.

[0037] Figure 2 This is the plasmid map of the recombinant vector pRSFDuet-1+Zwf+GlpX+Gnd+Vhb.

[0038] Figure 3 This is the plasmid map of the recombinant vector pCDFDuet-1+BMpunC+SthA.

[0039] Figure 4 This is the effect of pncA gene knockout on NMN synthesis and NAM utilization.

[0040] Figure 5 The effect of different induction times on the synthesis of NMN.

[0041] Figure 6 The effect of glucose metabolism modification on NMN synthesis.

[0042] Figure 7 Effects of energy and reducing power modification on NMN synthesis DETAILED DESCRIPTION

[0043] (I) Culture medium

[0044] LB medium: 5 g / L yeast extract powder, 10 g / L peptone, 10 g / L sodium chloride. Add 15 g / L agar powder to prepare LB solid medium.

[0045] Fermentation medium: KH2PO4 6g-12g / L, K2HPO4 16g-30g / L, ammonium sulfate 1g-5g / L, citric acid monohydrate 1g-5g / L, MgSO4·7H2O 1g-5g / L, yeast powder 10g-30g / L, glucose 10g-30g / L.

[0046] Feed medium: glucose 500 g / L, MgSO4·7H2O 9 g / L, yeast powder 5 g / L, metal ion solution 15 mL, concentrated hydrochloric acid 1 mL.

[0047] (2) Solution

[0048] 100 g / L nicotinamide solution: 5 g nicotinamide was dissolved in 50 mL ultrapure water and filtered to sterilize.

[0049] Metal ion solution: 10g / L FeSO4·7H2O, 1.53g / L CaCl2, 2.2g / L ZnSO4·7H2O, MnSO4·4H2O, 1g / L CuSO4·5H2O, 0.1g / L (NH4)6Mo7O 24 ·4H2O, 0.2g / L Na2B4O7·10H2O, 1g / L NiCl2, 1g / L H3BO3, 10mL / L HCl were used to prepare fermentation medium and feed medium.

[0050] Antibiotic concentration: ampicillin 100 mg / L, kanamycin 30 mg / L, spectinomycin 50 mg / L.

[0051] (III) HPLC detection of NMN: Agilent 1200 HPLC detector equipped with Athena C18-WP (5 μm, 4.6 mm × 250 mm) was used, the UV detector detection wavelength was 260 nm, the column temperature was 25 ° C, the injection volume was 5 μL, the flow rate was 0.6 mL / min, the flowability A was a mixture of water: tetrabutylammonium hydroxide: acetic acid: methanol = 997: 1.5: 1.5: 30, and the flowability B was methanol. The following solvent gradient was applied: 0-10 min, 100% A; 10-15 min, 100% A; 15-20 min, 30% A; 20-30 min, 30% A; 30-37 min, 100% A.

[0052] (IV) Calculation method of NMN conversion rate: molar conversion rate = (NMN concentration / (added niacinamide concentration-remaining niacinamide concentration) × (NMN relative molecular weight / niacinamide relative molecular weight)) × 100%.

[0053] (V) Plasmid assembly method: The one-step cloning reaction system is as follows: add 0.06 pmol of DNA fragment, 0.03 pmol of vector fragment, 5 μL of Exnase II, make up to 10 μL with sterile water, react at 50°C for 5 min, put on ice immediately after the reaction, and take 10 μL to transfer to JM109 competent cell.

[0054] The Megawhop reaction system is as follows: 500 ng of DNA fragment, 50 ng of vector plasmid, and Max Master Mix 25μL, make up to 50μL with sterile water, put into PCR instrument, react at 72℃ for 5min, 98℃ for 2min, then three-step reaction for 30 cycles, 98℃ for 30s, 55℃ for 30min, 55℃ for 1kbp·min-1, 72℃ for 5min. Then take 9.5μL of reaction system, add 0.5μL of Dpn I and digest the template at 37℃ for 2h. Take 10μL and transfer to JM109 competent state.

[0055] Table 1 Primer sequences for knockout genes

[0056]

[0057]

[0058] Table 2 Primer sequences of overexpressed genes

[0059]

[0060] Example 1 Construction of gene knockout strains

[0061] The CRISPR-Cas9 gene knockout system was used to knock out the pncC, ushA, nadR and purR genes in the Escherichia coli BL21 star (DE3) genome. The specific steps are as follows (the primer sequences involved are shown in Table 1):

[0062] (1) Using the genome of Escherichia coli BL21 star (DE3) as a template, the upstream and downstream fragments of pncC, ushA, nadR, purR, edd, pfkA and pntAB were amplified by PCR using primer pairs pncC-up-F / R and pncC-down-F / R, ushA-up-F / R and ushA-down-F / R, nadR-up-F / R and nadR-down-F / R, purR-up-F / R and purR-down-F / R, pncA-up-F / R and pncA-down-F / R, edd-up-F / R and edd-down-F / R, pfkA-up-F / R and pfkA-down-F / R, and pntAB-up-F / R and pntAB-down-F / R, respectively, and then recovered by gel. Then, the upstream and downstream fragments of pncC, ushA, nadR, purR, pncA, edd, pfkA and pntAB were used as templates, and overlapping PCR was performed with primers pncC-up-F / pncC-down-R, ushA-up-F / ushA-down- / R, nadR-up-F / nadR-down-R, purR-up-F / purR-down-R, pncA-up-F / pncA-down-R, edd-up-F / edd-down-R, pfkA-up-F / pfkA-down-R, pntAB-up-F / pntAB-down-R to obtain complete pncC, ushA, nadR, purR, pncA, edd, pfkA and pntAB templates, and the DNA fragments were recovered by gel.

[0063] (2) Using the original pTargetF plasmid (Addgene: #62226) as a template and pncC-sg-F / R, ushA-sg-F / R, nadR-sg-F / R, purR-sg-F / R, pncA-sg-F / R, gntR-sg-F / R, edd-sg-F / R, pfkA-sg-F / R, and pntAB-sg-F / R as primers, PCR amplification was performed to replace the N20 sequence on the original plasmid with N20 sequences complementary to the pncC, ushA, nadR, and purR sequences, respectively, to obtain various pTargetF plasmids targeting pncC, ushA, nadR, purR, pncA, edd, pfkA, and pntAB (i.e., targeting plasmid pTargetF with pncC, ushA, nadR, purR, pncA, edd, pfkA, and pntAB-specific N20 sequences). The competent E. coli JM109 was transformed, coated on LB plates (containing spectinomycin), cultured at 37°C, and the plasmid was extracted and sequenced.

[0064] (3) Take pCas plasmid (Addgene: #60847) and competent E. coli BL21 star (DE3), place on ice for 5 minutes until the competent cells melt, transfer the competent cells to the pre-cooled electrode cup, tap gently to remove bubbles, and cool in ice water for 5-10 minutes; put the electrode cup on the electroporator, adjust the electric shock parameters to 200Ω, 25μF, 2.5kV, hear a buzzer after electric shock, check the time is between 3ms-5ms, take out the electrode cup, and immediately add 900μL of LB liquid culture medium, quickly and gently resuspend the bacterial cells, and then transfer them to a sterilized centrifuge tube; place the centrifuge tube in a 30℃ shaker and gently shake for 60 minutes for recovery, then centrifuge at low speed for 1 minute, discard the supernatant culture medium, keep about 150μL of culture medium to suspend and mix the bacteria, and spread it on LB solid culture medium containing corresponding resistance; culture inverted in a 30℃ constant temperature incubator overnight, pick a single clone colony for colony PCR verification, and add culture medium to culture after verification to obtain Escherichia coli BL21 star(DE3) / pCas competent state.

[0065] (4) 200 ng of the targeting plasmid pTargetF with the pncC-specific N20 sequence constructed in step (2) and 1000 ng of the donor DNA fragment (i.e., the complete pncC template obtained in step (1)) were electroporated into the competent E. coli BL21 star (DE3) / pCas prepared in step (3), spread on LB plates (kanamycin and spectinomycin), and cultured at 30°C for 24 h. The positive colonies on the plates were picked and cultured in LB for 10 h, and then sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing verification.

[0066] (5) The positive clone colonies successfully knocked out by sequencing verification in step (4) were picked into a 4 mL LB liquid test tube, and IPTG and 30 mg / L kanamycin with a final concentration of 1 mM were added, and cultured at 30°C for 8-16 h to remove the pTargetF plasmid, and then cultured at 42°C for 12 h to remove the pCas plasmid to obtain Escherichia coli BL21 star (DE3) ΔpncC with the pncC gene knocked out in the genome, and Escherichia coli BL21 star (DE3) ΔpncC was used as the host bacteria.

[0067] (6) Using the same strategy as above, combined with the targeting plasmid pTargetF carrying the ushA-specific N20 sequence obtained in step (2) and 1000 ng of the donor DNA fragment (i.e., the complete ushA template obtained in step (1)), the ushA gene of the Escherichia coli BL21 star (DE3) ΔNudD genome was knocked out to obtain the gene knockout strain BL21 star (DE3) ΔpncC, ΔushA.

[0068] (7) Using the E. coli engineered bacteria BL21 star (DE3) ΔpncC, ΔushA constructed in step (6) as the starting strain, by referring to steps (2) to (5), the following strains were finally constructed:

[0069] Strain C01: E.coli BL21(DE3)ΔpncC;

[0070] Strain C02: E.coli BL21(DE3)ΔpncCΔushA;

[0071] Strain C03: E. coli BL21(DE3)ΔpncCΔushAΔnadR;

[0072] Strain C04: E. coli BL21(DE3)ΔpncCΔushAΔnadRΔpurR;

[0073] Strain C05: E. coli BL21(DE3)ΔpncCΔushAΔnadRΔpurRΔpncA;

[0074] Strain C06: E. coli BL21(DE3)ΔpncCΔushAΔnadRΔpurRΔpncAΔgntR;

[0075] Strain C07: E. coli BL21(DE3)ΔpncCΔushAΔnadRΔpurRΔpncAΔgntRΔedd;

[0076] Strain C08: E. coli BL21(DE3)ΔpncCΔushAΔnadRΔpurRΔpncAΔgntRΔeddΔpfkA;

[0077] Strain C09: E. coli BL21(DE3)ΔpncCΔushAΔnadRΔpurRΔpncAΔgntRΔeddΔpfkAΔpntAB.

[0078] Example 2 Construction of overexpression plasmid

[0079] The overexpression plasmid in E. coli was constructed using PCR technology, one-step cloning and megawhop technology. The specific steps are as follows (the primer sequences involved are shown in Table 2):

[0080] Obtaining Zwf, Gnd, GlpX, SthA and BaPrs gene fragments: Zwf (shown in SEQ ID NO.12), Gnd (shown in SEQ ID NO.13), GlpX (shown in SEQ ID NO.14), SthA (shown in SEQ ID NO.16) and BaPrs (shown in SEQ ID NO.10) gene fragments were amplified using Zwf-F / Zwf-R, Gnd-F / Gnd-R, GlpX-F / GlpX-R, SthA-F / SthA-R and BaPrs-F / BaPrs-R, respectively, and the DNA fragments were recovered by gel gel. The recovered Zwf, Gnd and GlpX gene fragments were ligated to the vector pRSF-Duet-1 by the megawhop method to obtain plasmids pRSFDuet-1+Zwf, pRSFDuet-1+Zwf+GlpX and pRSFDuet-1+Zwf+GlpX+Gnd. In the same way, SthA was connected to the vector pCDFDuet-1 to obtain the plasmid pCDFDuet-1+SthA. In the same way, BaPrs was connected to the vector pETDuet-1 to obtain the plasmid pETDuet-1+BaPrs.

[0081] Acquisition of NAMPT, BMpnuC, and Vhb gene fragments: Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the nicotinamide phosphoribosyltransferase NAMPT gene sequence from pine chitinophores, the transporter protein BMpunC gene sequence from Bacillus mycoides and the vitreous hemoglobinase Vhb from Vitreoscilla, and the NAMPT (shown in SEQ ID NO.9), BMpnuC (shown in SEQ ID NO.11) and Vhb (shown in SEQ ID NO.15) gene fragments were amplified using NAMPT-F / NAMPT-R, BMpnuC-F / BMpnuC-R, and Vhb-F / Vhb-R, respectively. The DNA fragments were recovered by gel gel, and the recovered NAMPT gene fragment was connected to the vector pETDuet-1+BaPrs by the megawhop method to obtain the plasmid pETDuet-1+BaPrs+NAMPT. In the same way, BMpnuC was connected to the vector pCDFDuet-1 to obtain the plasmid pCDFDuet-1+BMpnuC or BMpnuC was connected to the vector pCDFDuet-1+SthA to obtain the plasmid pCDFDuet-1+BMpnuC+SthA. In the same way, Vhb was connected to the vector pRSFDuet-1+Zwf+GlpX+Gnd to obtain the plasmid pRSFDuet-1+Zwf+GlpX+Gnd+Vhb.

[0082] Example 3 Effect of pncA gene knockout on NMN synthesis and NAM utilization

[0083] Take pETDuet-1+BaPrs+NAMPT plasmid and pCDFDuet-1+BMpnuC plasmid, as well as E. coli BL21star(DE3)ΔpncCΔushAΔnadRΔpurR and E. coli BL21 star(DE3)ΔpncCΔushAΔnadRΔpurRΔpncA competent cells, place them on ice for 5 min until the competent cells melt, transfer the competent cells to the pre-cooled electrode cup, tap gently to remove bubbles, and cool in ice water for 5-10 min; put the electrode cup on the electroporator, adjust the electric shock parameters to 200Ω, 25μF, 2.5kV, hear a buzzer after electric shock, check the time is between 3ms-5ms, take out the electrode cup, and immediately add 900μL in the sterile operating table. LB liquid medium, quickly and gently resuspend the bacterial cells, and then transfer them to a sterilized centrifuge tube; place the centrifuge tube in a 37℃ shaker and shake gently for 60 minutes for recovery, then centrifuge at low speed for 1 minute, discard the supernatant medium, keep about 150μL of medium to suspend and mix the bacteria, and apply it to LB solid medium containing corresponding resistance; incubate inverted overnight in a 37℃ constant temperature incubator, pick a single clone colony for colony PCR verification. The strain Escherichia coli BL21 star (DE3) ΔpncC ΔushA ΔnadR ΔpurR + pETDuet-1 + BaPrs + NAMPT + pCDFDuet-1 + BMpnuC was obtained and named strain F01. The strain Escherichia coli BL21 star (DE3) ΔpncC ΔushA ΔnadR ΔpurR ΔpncA + pETDuet-1 + BaPrs + NAMPT + pCDFDuet-1 + BMpnuC was obtained and named strain F02.

[0084] The strains F01 and F02 were inoculated into LB medium containing the corresponding antibiotics, respectively, and cultured in a shake flask at 37°C, 200 rpm for 12 h to obtain seed solution; the seed solution was then inoculated into 50 mL shake flask MA medium at a 2% inoculum, and cultured in a shake flask at 37°C, 200 rpm until OD 600 3-4; add IPTG with a final concentration of 0.5 mM and nicotinamide (NAM) with a final concentration of 1 g / L, and culture for 48 h at 37°C and 200 rpm. Adjust the pH and add 5 g / L glucose every 12 h.

[0085] After inducing fermentation, samples were taken 48 hours later, and the amount of NMN and NAM produced by the engineered E. coli fermentation of the present invention was detected by HPLC. Figure 4As shown, it was found that the NMN production of strain F02 increased from 0.35 g / L to 0.41 g / L compared with strain F01, and the utilization rate of NAM was increased by 45%.

[0086] Example 3 Effect of different induction times on NMN synthesis

[0087] The strain F02 constructed in Example 2 was inoculated into LB medium containing the corresponding antibiotics, and cultured in a shake flask at 37°C, 200 rpm for 12 h to obtain a seed solution; the seed solution was then inoculated into 50 mL of shake flask MA medium at a 2% inoculum, and cultured in a shake flask at 37°C, 200 rpm until OD 600 are 1, 2, 3, 4, 5, and 6; IPTG was added to a final concentration of 0.5 mM, and NAM was added to a concentration of 1 g / L, and cultured at 37°C and 200 rpm for 48 h.

[0088] After inducing fermentation, samples were taken 48 hours later, and the amount of NMN produced by the engineered E. coli fermentation of the present invention was detected by HPLC. Figure 5 As shown, OD at the time of induction 600 The yield was highest when the concentration was 3-4, reaching 2.94g / L.

[0089] Example 4 Effect of sugar metabolism modification on NMN synthesis

[0090] The pETDuet-1+BaPrs+NAMPT plasmid, pCDFDuet-1+BMpnuC plasmid, pRSFDuet-1+Zwf+GlpX plasmid and pRSFDuet-1+Zwf+GlpX+Gnd were transformed into Escherichia coli BL21 star (DE3) ΔpncCΔushAΔnadRΔpurRΔpncA, BL21 star (DE3) ΔpncCΔushAΔnadRΔpurRΔpncAΔedd and BL21 star (DE3) ΔpncCΔushAΔnadRΔpurRΔpncAΔeddΔpfkA competent cells according to the same method as in Example 3, and single clone colonies were picked for colony PCR verification. The strain Escherichia coli BL21 star (DE3) ΔpncC ΔushA ΔnadR ΔpurR ΔpncA + pETDuet-1 + BaPrs + NAMPT + pCDFDuet-1 + BMpnuC + pRSFDuet-1 + Zwf + GlpX + Gnd was obtained and named as strain F03. The strain Escherichia coli BL21 star (DE3) ΔpncC ΔushA ΔnadR ΔpurR ΔpncA Δedd + pETDuet-1 + BaPrs + NAMPT + pCDFDuet-1 + BMpnuC + pRSFDuet-1 + Zwf + GlpX + Gnd was obtained and named as strain F04. The strain Escherichia coli BL21 star (DE3) ΔpncC ΔushA ΔnadR ΔpurR ΔpncA Δedd ΔpfkA + pETDuet-1 + BaPrs + NAMPT + pCDFDuet-1 + BMpnuC + pRSFDuet-1 + Zwf + GlpX was obtained and named strain F05.

[0091] The strains F03, F04 and F05 were inoculated into LB medium containing the corresponding antibiotics, and cultured in a shake flask at 37°C, 200 rpm for 12 h to obtain seed solution; the seed solution was then inoculated into 50 mL shake flask MA medium at a 2% inoculum, and cultured in a shake flask at 37°C, 200 rpm until OD 600 3-4; add IPTG to a final concentration of .5 mM, and add NAM to a NAM concentration of 2 g / L, and culture for 48 h at 37°C and 200 rpm. Adjust the pH and add 5 g / L glucose every 12 h.

[0092] After inducing fermentation, samples were taken 48 hours later, and the amount of NMN produced by the engineered E. coli fermentation of the present invention was detected by HPLC. Figure 6 As shown, it was found that the NMN production of strain F04 was increased by 1.29 g / L compared with that of strain F03.

[0093] Example 5 Effect of energy and reducing power modification on NMN synthesis

[0094] The pETDuet-1+BaPrs+NAMPT plasmid, pCDFDuet-1+BMpnuC plasmid, pRSFDuet-1+Zwf+GlpX+Gnd plasmid and pRSFDuet-1+Zwf+GlpX+Gnd+Vhb plasmid were transformed into BL21 star(DE3)ΔpncCΔushAΔnadRΔpurRΔpncAΔΔedd and BL21 star(DE3)ΔpncCΔushAΔnadRΔpurRΔpncAΔeddΔpntAB competent cells according to the same method as in Example 3, and single clones were picked for colony PCR verification. The strain Escherichia coli BL21 star (DE3) ΔpncC ΔushA ΔnadR ΔpurR ΔpncA ΔgntR Δedd ΔpntAB + pETDuet-1 + BaPrs + NAMPT + pCDFDuet-1 + BMpnuC + pRSFDuet-1 + Zwf + GlpX + Gnd was obtained and named as strain F06. The strain Escherichia coli BL21 star (DE3) ΔpncC ΔushA ΔnadR ΔpurR ΔpncA Δedd ΔpntAB + pETDuet-1 + BaPrs + NAMPT + pCDFDuet-1 + BMpnuC + pRSFDuet-1 + Zwf + GlpX + Gnd + Vhb was obtained and named as strain F07.

[0095] The strains F04, F05 and F06 were inoculated into LB medium containing the corresponding antibiotics, and cultured in a shake flask at 37°C, 200 rpm for 12 h to obtain seed solution; the seed solution was then inoculated into 50 mL shake flask MA medium at a 2% inoculum, and cultured in a shake flask at 37°C, 200 rpm until OD 600 3-4; add IPTG to a final concentration of 0.5 mM, and add NAM to a NAM concentration of 2 g / L, and culture for 48 h at 37°C and 200 rpm. Adjust the pH and add 5 g / L glucose every 12 h.

[0096] After inducing fermentation, samples were taken 48 hours later, and the amount of NMN produced by the engineered E. coli fermentation of the present invention was detected by HPLC. Figure 7 As shown, it was found that the NMN production of strain F06 was increased by 0.7 g / L compared with that of strain F04.

[0097] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Escherichia coli capable of synthesizing nicotinamide mononucleotide, characterized in that: Nicotinamide phosphoribosyltransferase, 5-phosphoribosyl-1-pyrophosphate synthetase, transporter BMpnuC, glucose-6-phosphate 1-dehydrogenase, 6-phosphogluconate dehydrogenase, fructose-1,6-bisphosphatase II, vitreous hemoglobin, and NADH transhydrogenase were expressed.

2. The recombinant Escherichia coli according to claim 1, characterized in that One or more genes among pncC, ushA, nadR, purR, pncA, edd, pfkA, and pntAB were also knocked out.

3. The recombinant Escherichia coli according to claim 1 or 2, characterized in that pETDuet-1 was used as a vector to express PRPP synthase and the gene nicotinamide phosphoribosyltransferase, and / or pCDFDuet-1 was used as a vector to express the transporter BMpnuC, and / or pRSFDuet-1 was used as a vector to express glucose-6-phosphate 1-dehydrogenase, fructose-1,6-bisphosphatase II, 6-phosphogluconate dehydrogenase, and vitreous hemoglobin.

4. The recombinant Escherichia coli according to any one of claims 1 to 3, characterized in that The nicotinamide phosphoribosyltransferase contains the amino acid sequence shown in SEQ ID NO.1; the 5-phosphoribosyl-1-pyrophosphate synthetase contains the amino acid sequence shown in SEQ ID NO.2; the transporter contains the amino acid sequence shown in SEQ ID NO.3; the glucose-6-phosphate 1-dehydrogenase contains the amino acid sequence shown in SEQ ID NO.4; the 6-phosphogluconate dehydrogenase contains the amino acid sequence shown in SEQ ID NO.5; the fructose-1,6-bisphosphatase II contains the amino acid sequence shown in SEQ ID NO.6; the vitreous hemoglobinase contains the amino acid sequence shown in SEQ ID NO.7; and the NADH transhydrogenase contains the amino acid sequence shown in SEQ ID NO.

8.

5. A starter culture containing the recombinant Escherichia coli according to any one of claims 1 to 4.

6. A method for promoting the synthesis of NMN by recombinant Escherichia coli, characterized in that: Nicotinamide phosphoribosyltransferase as shown in SEQ ID NO.1, 5-phosphoribosyl-1-pyrophosphate synthetase as shown in SEQ ID NO.2, NMN transporter as shown in SEQ ID NO.3, glucose-6-phosphate 1-dehydrogenase as shown in SEQ ID NO.4, 6-phosphogluconate dehydrogenase as shown in SEQ ID NO.5, fructose-1,6-bisphosphatase II as shown in SEQ ID NO.6, vitreous hemoglobinase Vhb as shown in SEQ ID No.7 and NADH transhydrogenase EcSthA as shown in SEQ ID No.8 were overexpressed in Escherichia coli.

7. The method according to claim 6, characterized in that The pncC, ushA, nadR, purR, pncA, edd, pfkA, and pntAB genes of Escherichia coli were also knocked out.

8. A method for producing NMN by fermentation, characterized in that: The recombinant Escherichia coli according to any one of claims 1 to 4 is cultured at 35 to 42°C until OD 600 =3-4, use inducer to induce NMN synthesis; Optionally, the inducer is IPTG, with a concentration of 0.5 to 1 mM; Optionally, the induction temperature is 25-37°C.

9. The method according to claim 8, characterized in that Glucose is also added during the fermentation process.

10. Use of the recombinant Escherichia coli engineering described in any one of claims 1 to 4, or the starter culture described in claim 5, or the method described in any one of claims 6 to 9 in the preparation of NMN-containing products in the fields of food, medicine, cosmetics, feed, and textiles.

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