Recombinant escherichia coli for producing nmn and preparation method and application thereof

Abstract

The application discloses a kind of recombinant escherichia coli for producing NMN and its preparation method and application, belong to genetic engineering and bioengineering technical field.The application expresses nicotinamide phosphoric acid ribosyltransferase NAMPT as shown in SEQ ID NO.1 in escherichia coli, expresses PRPP synthetase BaPRS as shown in SEQ ID NO.2, expresses transporter BMpnuC as shown in SEQ ID NO.3, and knocks out pncC gene, ushA gene, nadR gene and purR gene, improves the accumulation amount of NMN in escherichia coli cell.The application also optimizes fermentation condition, so that NMN can be accumulated efficiently outside cell, and more than 20g / L NMN can be obtained in 5L fermentation system, and has wide application prospect in the field of medicine, cosmetics, feed, textile and the like.

Description

[0001] The present application is a divisional application of the Chinese Patent Application No. 202210781815.2, filed on July 4, 2022, entitled "Recombinant Escherichia coli for producing NMN and application thereof". TECHNICAL FIELD

[0002] The present application relates to a recombinant Escherichia coli for producing NMN, a preparation method and application thereof, and belongs to the field of genetic engineering and bioengineering technology. BACKGROUND

[0003] In recent years, NAD + and its derivatives have attracted much attention as potential anti-aging drugs, and great efforts have been made to produce them effectively. Since β-nicotinamide mononucleotide (NMN) is a complex structural isomer, its chemical synthesis is a challenging task. Currently, NMN is mainly produced by biological synthesis. In different organisms, the biosynthesis of NAD + slightly differs, but both mainly include two pathways: de novo synthesis pathway and salvage synthesis pathway. The salvage synthesis pathway plays a major role and is the main path for NMN synthesis.

[0004] The main methods for synthesizing NMN are chemical synthesis and enzymatic synthesis. The chemical method mainly uses nicotinamide and tetraacetyl ribose as starting materials, which are condensed by trifluoromethylsilicane trimethylsilicane (TMSOTf), deacetylated, and then phosphorylated by phosphorus oxychloride / trimethyl phosphate to obtain β-NMN; or β-NMN is prepared by phosphorylation of ketal-protected nicotinamide ribose and deprotection. The chemical synthesis method of NMN is relatively mature, but there are many unfavorable factors. On the one hand, some chemical raw materials are highly toxic and pollute the environment; on the other hand, some raw materials are expensive, making the synthesis cost of NMN high.

[0005] Enzymatic synthesis mainly uses pure enzyme reaction and whole-cell catalysis to synthesize β-NMN from D-5-phosphoribose and nicotinamide using phosphoribosyl pyrophosphate synthase and nicotinamide phosphoribosyltransferase; or β-NMN is synthesized from nicotinamide and phosphoribosyl pyrophosphate. The conversion rate of enzymatic synthesis is high, but the substrate cost is high, which is not conducive to scale-up production. SUMMARY

[0006] The present application aims to construct a recombinant strain that is more conducive to NMN accumulation. On the basis of the constructed chassis strain, the enzyme genes for synthesizing NMN are expressed, the gene expression strength is adjusted, and the fermentation process is optimized to improve the yield of NMN.

[0007] The application provides a genetically engineered strain with nicotinamide mononucleotide synthesis capacity, which expresses nicotinamide phosphoribosyltransferase (NAMPT enzyme), 5-phosphoribosyl-1-pyrophosphate synthetase (PRPP synthetase) and transporter protein BMpnuC.

[0008] In an embodiment, the amino acid sequence of the nicotinamide phosphoribosyltransferase (NAMPT enzyme) has at least 95% identity with the sequence of SEQ ID NO. 1; the amino acid sequence of the 5-phosphoribosyl-1-pyrophosphate synthetase (PRPP synthetase) has at least 95% identity with the sequence of SEQ ID NO. 2; and the amino acid sequence of the transporter protein BMpnuC has at least 95% identity with the sequence of SEQ ID NO. 3.

[0009] In an embodiment, the nicotinamide phosphoribosyltransferase is derived from Vibrio Phage KVP40 and contains the amino acid sequence shown in SEQ ID NO. 1; the PRPP synthetase is derived from Bacillus amyloliquefaciens and contains the amino acid sequence shown in SEQ ID NO. 2; and the transporter protein BMpnuC is derived from Bacillus mycoides and contains the amino acid sequence shown in SEQ ID NO. 3.

[0010] In an embodiment, the gene NAMPT encoding the nicotinamide phosphoribosyltransferase has the nucleotide sequence shown in SEQ ID NO. 4.

[0011] In an embodiment, the gene BaPRS encoding the PRPP synthetase has the nucleotide sequence shown in SEQ ID NO. 5.

[0012] In an embodiment, the gene BMpnuC encoding the transporter protein has the nucleotide sequence shown in SEQ ID NO. 6.

[0013] In an embodiment, the chassis strain used by the genetically engineered strain includes but is not limited to Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and other microorganisms suitable for constructing an expression system.

[0014] In an embodiment, the Bacillus subtilis includes but is not limited to B. subtilis 168, B. subtilis WB600, B. subtilis WB800.

[0015] In an embodiment, the E. coli includes, but is not limited to, E. coli BL21(DE3), E. coli BL21(DE3)pLysS, E. coli Rosetta(DE3) or E. coli JM109(DE3).

[0016] In an embodiment, the overexpression vector plasmid used in the construction of the genetically engineered strain includes, but is not limited to, pMA5, pWB980, pHT43, pHT01 and pET-22b(+), pET-28a(+), pET-30a(+), pUC57 and other suitable vector plasmids for the construction of overexpression genetically engineered strains.

[0017] In an embodiment, the transporter protein BMpnuC uses plasmid pACYCDuet-1 or pCDFDuet-1 as an expression vector.

[0018] In an embodiment, the nicotinamide phosphoribosyltransferase and PRPP synthetase use pET-28a(+) or pRSFDuet-1 as an expression vector.

[0019] In an embodiment, the recombinant E. coli uses E. coli with pncC, ushA, nadR, purR knocked out as a starting strain.

[0020] In an embodiment, the starting strain is E. coli F004 (E. coli BL21(DE3), ΔpncC, ΔushA, ΔnadR, ΔpurR), which has been disclosed in the patent application file with publication number CN112795582A.

[0021] The present application also provides a recombinant plasmid carrying the nicotinamide phosphoribosyltransferase gene, 5-phosphoribosyl-1-pyrophosphate synthetase gene and / or transporter gene.

[0022] In an embodiment, the backbone of the recombinant vector carrying the transporter gene is any one of the pACYCDuet-1 or pCDFDuet-1 series of vectors.

[0023] In an embodiment, the backbone of the recombinant vector carrying the nicotinamide phosphoribosyltransferase gene and 5-phosphoribosyl-1-pyrophosphate synthetase gene is any one of the pET-28a(+) or pRSFDuet-1 series of vectors.

[0024] The present application also provides a fermenting agent containing the recombinant E. coli.

[0025] The application also provides a method for promoting NMN synthesis by recombinant E. coli, which comprises overexpressing nicotinamide phosphoribosyltransferase gene NAMPT as shown in SEQ ID NO. 4, PRPP synthase gene BaPRS as shown in SEQ ID NO. 5, and NMN transporter protein BMpnuC as shown in SEQ ID NO. 6 in E. coli.

[0026] In an embodiment, the recombinant E. coli further knocks out pncC, ushA, nadR and purR genes.

[0027] In an embodiment, the method further comprises using IPTG for induction when culturing the recombinant E. coli.

[0028] The application also provides a method for fermentative production of NMN, which uses the recombinant E. coli as fermentative microorganism, and the fermentation is carried out at 35-40℃ until OD 600 = 0.6-1.0, and then adding IPTG with a final concentration of ≥0.2 mM and nicotinamide with a final concentration of 500-2000 mg / L to induce NMN synthesis at 30-37℃.

[0029] In an embodiment, the induction time is 24-36 h.

[0030] In an embodiment, the induction uses IPTG with a final concentration of 0.5-1 mM.

[0031] In an embodiment, the fermentation uses nicotinamide with a final concentration of 500-2000 mg / L as substrate.

[0032] In an embodiment, the method inoculates the recombinant E. coli into seed culture medium, cultures to obtain seed liquid, and then inoculates the seed liquid into fermentation culture medium according to a transfer amount of 1-5%, and cultures at 35-37℃ for 1.5-2 h until OD 600 = 0.6-1.0, and then adding IPTG with a final concentration of 0.5-1 mM and nicotinamide with a final concentration of 500-2000 mg / L to induce NMN synthesis at 30-37℃.

[0033] In an embodiment, the fermentation culture medium contains yeast powder, citric acid, ammonium sulfate, phosphate (K2HPO4, KH2PO4) and glucose.

[0034] In an embodiment, the fermentation process further comprises batchwise or continuous flow addition of nicotinamide.

[0035] In an embodiment, the batchwise addition is adding nicotinamide every 2-3 h after adding the inducer.

[0036] In an embodiment, the continuous flow addition is continuous flow addition of nicotinamide solution after self-addition of an inducing agent, and the concentration of nicotinamide in the fermentation system is controlled to be less than 3 g / L.

[0037] The application also provides use of the recombinant E. coli, the starter, or the method in the field of pharmaceuticals, cosmetics, feed, and textiles to prepare a product containing NMN.

[0038] Advantages:

[0039] (1) The application strengthens diffusion of NMN to the outside of the cell by overexpressing NMN transporter protein BMpnuC, and promotes the reaction to proceed in the direction of NMN synthesis.

[0040] (2) The application optimizes the expression strength of genes, further improves the amount of NMN biosynthesis by expressing NMN synthesis-related genes on plasmids with different copy numbers, and can synthesize 2.6 g / L NMN from 1 g / L nicotinamide.

[0041] (3) The application also optimizes the fermentation conditions, controls the fermentation process by expanding the culture, and can obtain more than 20 g / L NMN in a fermentation system containing 10 g / L nicotinamide for 25 h, with a conversion rate of more than 90%, which has potential for industrial application. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 The figure is a plasmid map of the recombinant vector pET28a+BaPRS+NAMPT.

[0043] Figure 2 The figure is an LC-MS chart of the recombinant E. coli, the control, and the standard.

[0044] Figure 3 The figure is the effect of different induction temperatures on the amount of NMN synthesis.

[0045] Figure 4 The figure is the effect of different IPTG concentrations on the amount of NMN synthesis.

[0046] Figure 5 The figure is the effect of different substrate nicotinamide concentrations on the amount of NMN synthesis.

[0047] Figure 6 The figure is the effect of different recombinant strains on the amount of NMN synthesis.

[0048] Figure 7 The figure is the amount of NMN synthesis in a 5L fermenter with horizontal batch feeding.

[0049] Figure 8 The figure is the amount of NMN synthesis in a 5L fermenter with horizontal constant-speed flow feeding. DETAILED DESCRIPTION

[0050] (I) Culture medium

[0051] LB medium: Yeast extract powder 5 g / L, peptone 10 g / L, sodium chloride 10 g / L. Add 15 g / L agar strip to prepare LB solid medium.

[0052] Fermentation medium: KH2PO4 6-12 g / L, K2HPO4 16-30 g / L, ammonium sulfate 5-10 g / L, citric acid monohydrate 1-5 g / L, MgSO4·7H2O 1-5 g / L, yeast powder 10-30 g / L, glucose 15-50 g / L.

[0053] Fed-batch medium: glucose 700 g / L, ammonium sulfate 73 g / L, MgSO4·7H2O 9 g / L, yeast powder 5 g / L, metal ion solution 15 mL, concentrated hydrochloric acid 1 mL.

[0054] (II) Solution

[0055] 100 g / L nicotinamide solution: 5 g nicotinamide is dissolved in 50 mL ultrapure water, and filtered to remove bacteria.

[0056] Metal ion solution: 10 g / L FeSO4·7H2O, 1.53 g / L CaCl2, 2.2 g / L ZnSO4·7H2O, MnSO4·4H2O, 1 g / L CuSO4·5H2O, 0.1 g / L (NH4)6Mo7O 24 ·4H2O, 0.2 g / L Na2B4O7·10H2O, 1 g / L NiCl2, 1 g / L H3BO3, 10 mL / L HCl for preparing fermentation medium and fed-batch medium.

[0057] (III) HPLC detection of NMN: Use a chromatographic column (250 x 4.6 mm, 5 μm, Thermo-Fisher, MA, USA), and use a SPD-20A detector of Japan Shimadzu under the detection condition of 30°C, the mobile phase is 20 mM ammonium acetate containing 5% acetonitrile; flow rate: 1.0 mL / min; detection wavelength: 259 nm; column oven temperature: 30°C.

[0058] (IV) NMN conversion rate calculation method: Molar conversion rate = (NMN concentration / (supplemented nicotinamide concentration - residual nicotinamide concentration) x (relative molecular mass of NMN / relative molecular mass of nicotinamide)) x 100%.

[0059] (five) E. coli chemical transformation method: E. coli JM109 was streaked on solid LB plate, 37°C incubated for 12h, single colony was picked and inoculated in liquid LB medium, 37°C, 220r / min grown for 10h, 1% inoculation was transferred to fresh 25mL liquid LB medium, 37°C incubated for 1.5-2h, when OD 600 up to 0.6-1, the bacteria were collected to make competent cells.

[0060] The E. coli competence was prepared using TaKaRa Competent Cell Preparation Kit, and the specific operation process was referred to the instruction. The prepared competent cells were stored at -80°C, and then the plasmid or fragment could be transformed.

[0061] (six) plasmid assembly method: Gibson reaction system was as follows, 50ng of DNA fragment was added, 100ng of vector was added, 5μL of Gibson mix was added, and sterile ultrapure water was added to 10μL of system. The reaction conditions were as follows, 50°C reaction for 60min, and immediately placed on ice after reaction. 10μL was taken and transformed into E. coli competent JM109.

[0062] The reaction system of seamless cloning was as follows, 40ng of target gene was added, 100ng of vector was added, 5μL of reaction enzyme mixture was added, and sterile ultrapure water was added to 10μL. The reaction conditions were as follows, 50°C reaction for 60min, and immediately placed on ice after reaction. 10μL was taken and transformed into E. coli competent JM109.

[0063] (seven) amino acid and nucleotide sequence

[0064] In the following specific embodiments, the transport protein PnuC includes the amino acid sequence as shown in SEQ ID NO. 3 and the same functional amino acid sequence obtained by substituting one or more amino acids based thereon. Based on the amino acid sequence of PnuC disclosed in the present application, the skilled in the art can obtain the BMpnuC gene of the present application by using cloning or synthetic method or other suitable method based on existing molecular biology technology. In addition, the pnuC gene from different strains, species or other species has similar function, therefore, the nucleotide sequence encoding the above-mentioned pnuC gene is not limited to the nucleotide sequence as shown in SEQ ID NO. 6. If the obtained protein has no obvious functional difference with the protein as shown in SEQ ID NO. 3, it is also included in the scope of the present application.

[0065] Similarly, nicotinamide phosphoribosyltransferase has an amino acid sequence as shown in SEQ ID NO. 1 and a same functional amino acid sequence obtained by substituting one or more amino acids on this basis; PRPP synthase has an amino acid sequence as shown in SEQ ID NO. 2 and a same functional amino acid sequence obtained by substituting one or more amino acids on this basis, and a gene (for example, the NAMPT gene of SEQ ID NO. 4 or the BaPRS gene of SEQ ID NO. 5) encoding the protein shown in SEQ ID NO. 1 or SEQ ID NO. 2 obtained by cloning or synthesis method or other suitable method based on the existing molecular biology technology is also included in the scope of the present application.

[0066] Example 1: Construction of NMN biosynthesis-related expression plasmid

[0067] (1) Construction of NAMPT expression frame

[0068] The NAMPT synthetic sequence shown in SEQ ID NO. 4 was used as a template, and primer pair F1 / R1 was used for PCR amplification. Phanta MasterMix (Vazyme) high-fidelity pfu enzyme was selected, and the conditions were pre-denaturation at 95°C for 3 min; amplification stage for 30 cycles, according to 95°C, 15s, 58°C, 15s, 72°C, 1 min; extension at 72°C for 5 min. The PCR product was purified, and the vector pET-28a(+) was used as a template, and primer pair F2 / R2 was used for PCR amplification. Phanta MasterMix (Vazyme) high-fidelity pfu enzyme was selected, and the conditions were pre-denaturation at 95°C for 3 min; amplification stage for 30 cycles, according to 95°C, 15s, 58°C, 15s, 72°C, 3 min; extension at 72°C for 5 min. The PCR product was purified. The fragments NAMPT and the vector pET-28a(+) were recombined into the vector pET28a+NAMPT by the method of seamless cloning. The obtained vector was sent to Shanghai Biosciences for sequencing, and the correct recombination vector pET28a+NAMPT was obtained after correct comparison.

[0069] (2) Construction of BaPRS+NAMPT expression frame

[0070] The pET28a+NAMPT constructed in step (1) was used as a template, and primer pair F3 / R3 was used for PCR amplification, Phanta MasterMix (Vazyme) high-fidelity pfu enzyme was selected, and the conditions were pre-denaturation at 95 °C for 3 min; 30 cycles of amplification, according to 95 °C, 15 s, 58 °C, 15 s, 72 °C, 3 min; extension at 72 °C for 5 min. The PCR product was purified to obtain the pET28a+NAMPT fragment; BaPRS represented by SEQ ID NO. 5 was used as a template, and primer pair F4 / R4 was used for PCR amplification, Phanta MasterMix (Vazyme) high-fidelity pfu enzyme was selected, and the conditions were pre-denaturation at 95 °C for 3 min; 30 cycles of amplification, according to 95 °C, 15 s, 58 °C, 15 s, 72 °C, 30 s; extension at 72 °C for 5 min. The PCR product was purified to obtain the BaPRS fragment. The pET28a+NAMPT fragment and the BaPRS fragment were recombined into the vector by the method of seamless cloning, and E. coli JM109 was transformed. The obtained vector was sent to Shanghai Biosciences for sequencing, and the correct recombinant vector pET28a+BaPRS+NAMPT was obtained after comparison. Figure 1

[0071] (3) Construction of NMN transporter expression frame

[0072] The BMpnuC gene sequence represented by SEQ ID NO. 6 was used as a template, and primer pair F5 / R5 was used for PCR amplification, Phanta MasterMix (Vazyme) high-fidelity pfu enzyme was selected, and the conditions were pre-denaturation at 95 °C for 3 min; 30 cycles of amplification, according to 95 °C, 15 s, 58 °C, 15 s, 72 °C, 1 min; extension at 72 °C for 5 min. The PCR product was purified, and the vector pACYCDuet-1 was used as a template, and primer pair F6 / R7 was used for PCR amplification, Phanta MasterMix (Vazyme) high-fidelity pfu enzyme was selected, and the conditions were pre-denaturation at 95 °C for 3 min; 30 cycles of amplification, according to 95 °C, 15 s, 58 °C, 15 s, 72 °C, 3 min; extension at 72 °C for 5 min. The PCR product was purified. The fragment BMpnuC and the vector pACYCDuet-1 were recombined into the vector pACYCDuet+BMpnuC by the method of seamless cloning. And E. coli JM109 was transformed. The obtained vector was sent to Shanghai Biosciences for sequencing, and the correct recombinant vector pACYCDuet+BMpnuC was obtained after comparison.

[0073] Table 1 primer information ​

[0074]

[0075] Example 2: Construction and fermentation of recombinant strain for biosynthesis of NMN

[0076] The bottom strain F004 (E. coli BL21 (DE3), ΔpncC, ΔushA, ΔnadR, ΔpurR) disclosed in the patent application document with publication number CN112795582A) was used as the host strain. The NMN synthesis plasmid pET28a+BaPRS+NAMPT and the NMN transport plasmid pACYCDuet+BMpnuC constructed according to the method of Example 1 were transformed into the host strain to obtain the recombinant strain NMN01.

[0077] To verify the ability of the recombinant strain NMN01 to synthesize NMN, the recombinant strain NMN01 was streaked on a LB solid plate with appropriate resistance and incubated at 37°C overnight. A single colony was inoculated in a LB medium containing the corresponding resistance and incubated at 37°C, 220 r / min for 10-12 h. The culture was transferred into 30 mL of fermentation medium at a ratio of 2%, and incubated at 37°C until the OD 600 When the OD was 0.6-1.0, the temperature was lowered to 25°C. The final concentration of IPTG was 0.2-0.5 mM, and 1-3 g / L nicotinamide was added. Samples were taken at regular intervals, centrifuged to obtain the supernatant, and HPLC was used to detect the synthesis of NMN.

[0078] The results showed that in the HPLC detection, an absorption peak consistent with the peak time of the standard NMN was found in the sample. To further verify whether NMN was synthesized in the sample, LC-MS was used to verify the sample, as shown in Figure 2 The results showed that NMN was synthesized in the sample. When the concentration of the substrate nicotinamide was 1 g / L, the extracellular accumulation of NMN reached 980 mg / L after 24 h of induction.

[0079] Example 3: Optimization of NMN synthesis conditions at the shake flask level

[0080] To further improve the synthesis of NMN and the conversion rate, the recombinant strain NMN01 with the ability to synthesize NMN constructed in Example 2 was used as the fermentation microorganism, and the NMN fermentation process conditions were optimized at the shake flask level, including induction temperature, IPTG concentration, and substrate addition concentration.

[0081] (1) Effect of different induction temperatures on NMN synthesis

[0082] The recombinant strain NMN01 constructed in Example 2 was streaked on LB solid plates with appropriate resistance, and incubated at 37°C overnight; a single colony was inoculated in LB medium containing kanamycin and chloramphenicol, and incubated at 37°C, 220 r / min for 10-12 h; 2% of the inoculum was transferred into 30 mL fermentation medium, and incubated at 37°C until OD 600 was 0.6-1.0, 0.2, 0.5, and 1.0 mM IPTG was added respectively, and 1-3 g / L nicotinamide was added, and protein expression was induced at 37°C. Timely sampling, centrifugation to obtain supernatant, and HPLC detection of NMN content in the supernatant were performed. As shown in Table 2, under the conditions of 1 g / L nicotinamide added as substrate and 30°C induction temperature, 1809 mg / L NMN was synthesized by induction for 24 h using 0.5 mM IPTG, which was 15% and 8.5% higher than the synthesis capacity of 0.2 mM and 1.0 mM IPTG respectively. Figure 3

[0083] (2) Effect of IPTG concentration on NMN synthesis

[0084] The recombinant strain NMN01 constructed in Example 2 was streaked on LB solid plates with appropriate resistance, and incubated at 37°C overnight; a single colony was inoculated in LB medium containing kanamycin and chloramphenicol, and incubated at 37°C, 220 r / min for 10-12 h; 2% of the inoculum was transferred into 30 mL fermentation medium, and incubated at 37°C until OD 600 was 0.6-1.0, 0.2, 0.5, and 1.0 mM IPTG was added respectively, and 1-3 g / L nicotinamide was added, and protein expression was induced at 37°C. Timely sampling, centrifugation to obtain supernatant, and HPLC detection of NMN content in the supernatant were performed. As shown in Table 2, under the conditions of 1 g / L nicotinamide added as substrate and 30°C induction temperature, 1809 mg / L NMN was synthesized by induction for 24 h using 0.5 mM IPTG, which was 15% and 8.5% higher than the synthesis capacity of 0.2 mM and 1.0 mM IPTG respectively. Figure 4

[0085] (3) Effect of substrate addition amount on NMN synthesis amount

[0086] The recombinant strain NMN01 constructed in Example 2 was streaked on LB solid plates with appropriate resistance, and incubated at 37°C overnight; a single colony was inoculated in LB medium containing kanamycin and chloramphenicol, and incubated at 37°C, 220 r / min for 10-12 h; 2% of the inoculum was transferred into 30 mL fermentation medium, and incubated at 37°C until OD 600 ​​When the value of OD600 is 0.6-1.0, add 0.5 mM IPTG to induce protein expression, and add nicotinamide with a final concentration of 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 g / L, respectively. Take samples at regular intervals, centrifuge to obtain supernatant, and detect the NMN content in the supernatant by HPLC. The results are shown in Table 1. Figure 5 As shown in Table 1, the NMN yield is more than 1450 mg / L at a nicotinamide concentration of 0.5 g / L and 2.0 g / L, and the yield of NMN in the cells is 1871 mg / L at a nicotinamide concentration of 1 g / L.

[0087] Example 4: Adjustment of gene expression intensity by using plasmids with different copy numbers

[0088] The recombinant strain was constructed according to the strategy of Examples 1-2, except that the NMN transporter plasmid of strain NMN01 was replaced by pCDFDuet-1, and the obtained recombinant strain was named NMN02.

[0089] The recombinant strain NMN02 was streaked on a LB solid plate with appropriate resistance and incubated at 37°C overnight. A single colony was inoculated in a LB medium with appropriate resistance and incubated at 37°C, 220 r / min for 10-12 h. The culture was transferred into 30 mL fermentation medium at a ratio of 2%, and incubated at 37°C until the OD600 value was 0.6-1.0. Then, 0.5 mM IPTG was added to induce protein expression, and 1 g / L nicotinamide was added. Samples were taken at regular intervals, centrifuged to obtain supernatant, and the synthesis of NMN was detected by HPLC. 600 When the value of OD600 is 0.6-1.0, add 0.5 mM IPTG to induce protein expression, and add nicotinamide with a final concentration of 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 g / L, respectively. Take samples at regular intervals, centrifuge to obtain supernatant, and detect the NMN content in the supernatant by HPLC. The results are shown in Table 1. Figure 6 As shown in Table 1, the NMN yield is more than 1450 mg / L at a nicotinamide concentration of 0.5 g / L and 2.0 g / L, and the yield of NMN in the cells is 1871 mg / L at a nicotinamide concentration of 1 g / L.

[0090] Example 5: Synthesis of NMN by scale-up culture of strain NMN02

[0091] The recombinant strain NMN02 is used as the fermentation strain and expanded in a 5L fermenter. The strain NMN02 is streaked from a glycerol tube to LB solid medium containing the corresponding resistance and incubated at 37°C for 12h. A single colony is selected and inoculated into liquid LB containing the corresponding resistance and incubated at 37°C and 220r / min for 10h. The culture is transferred into a 5L fermenter containing 2.5L fermentation medium and 5-10mL metal ion solution at a transfer amount of 5%-20%. The initial conditions are as follows: pH is controlled at 6.0-7.0 by using 35% ammonia water, the aeration amount is 1-2vvm, the rotation speed is 300r / min-1000r / min, and the dissolved oxygen DO is controlled at 30%-50%. When the DO rebound occurs, IPTG is added at a final concentration of 0.5-1mM for further induction at 37°C. Nicotinamide is added at 2g / L at 0h, 2h, 4h, 6h, and 8h, respectively, and the total amount of nicotinamide added is 10g / L. The synthesis of NMN is detected by sampling at regular time intervals. The results show that the accumulation of NMN is more than 12.3g / L and the OD is 25-30 after 24h of induction.

[0092] Example 6: Expansion of the strain NMN02 for NMN synthesis

[0093] The synthesis of intracellular NMN is coupled with the growth to obtain higher NMN concentration. The specific implementation is the same as that in Example 5, except that the nicotinamide is added in a flow addition mode. The flow addition of nicotinamide is started at the same time when the inducer is added. The residual nicotinamide in the medium is detected in real time, and the concentration of nicotinamide is controlled to be relatively low (<3g / L). The results show that the recombinant strain NMN02 can synthesize 20.3g / L of NMN in a 5L fermenter after 25h of fermentation (20h of induction). The total amount of added nicotinamide is 9.6g / L, the residual nicotinamide is 2.13g / L, the molar conversion rate of consumed nicotinamide to NMN is 98.97%, and the OD is more than 30.

[0094] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application. Therefore, the protection scope of the present application should be defined by the claims.

Claims

1. An engineered Escherichia coli strain having the ability of nicotinamide mononucleotide synthesis, characterized in that, The nicotinamide phosphoribosyltransferase, 5-phosphoribosyl-1-pyrophosphate synthetase and transporter protein can be expressed; wherein the transporter protein is an amino acid sequence shown as SEQ ID NO. 3; the nicotinamide phosphoribosyltransferase is an amino acid sequence shown as SEQ ID NO. 1; and the 5-phosphoribosyl-1-pyrophosphate synthetase is an amino acid sequence shown as SEQ ID NO. 2; and the engineered E. coli strain further knocks out pncC, ushA, nadR and purR genes.

2. The E. coli engineered strain of claim 1, characterized in that, The engineered E. coli strain overexpresses genes encoding nicotinamide phosphoribosyltransferase, 5-phosphoribosyl-1-pyrophosphate synthetase and transporter protein.

3. A method of preparing the engineered E. coli bacterium of any one of claims 1-2, comprising, The method further comprises the step of knocking out pncC, ushA, nadR and purR genes of the E. coli.

4. The method of claim 3, wherein, The method further comprises the step of adjusting the expression of the transporter protein.

5. The method of claim 3, wherein, The expression of the transporter protein is adjusted by using plasmids with different copy numbers.

6. A method for fermentative production of NMN, characterized in that, The engineered E. coli strain according to any one of claims 1-2 is used as the fermentation microorganism, and is cultured at 35-40°C to OD 600 =0.6-1.0, and NMN synthesis is induced using an inducer.

7. The method of claim 6, wherein: The inducer can be IPTG, and the concentration is 0.5 ~1 mM.

8. The method of claim 6, wherein: The induction temperature is 25~37℃.

9. The method of claim 6, wherein: The induction time is 24~36 h.

10. A fermenting agent containing the engineered E. coli strain of any one of claims 1-2.

11. Use of the engineered E. coli strain of any one of claims 1-2, or the method of any one of claims 3-9, or the fermenting agent of claim 10 in the preparation of products containing NMN in the fields of pharmaceuticals, cosmetics, feed, and textiles.

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