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

Abstract

The invention discloses recombinant escherichia coli for producing NMN as well as a preparation method and application of the recombinant escherichia coli, and belongs to the technical field of genetic engineering and bioengineering. According to the invention, nicotinamide phosphoribosyltransferase NAMPT as shown in SEQ ID NO.1, PRPP synthase BaPRS as shown in SEQ ID NO.2 and transporter BMpnuC as shown in SEQ ID NO.3 are expressed in escherichia coli, and a pncC gene, a ushA gene, an nadR gene and a purR gene are knocked out, so that the accumulation amount of NMN in escherichia coli cells is increased. In addition, by optimizing fermentation conditions, NMN can be efficiently accumulated outside cells, 20 g / L or above of NMN can be obtained under a 5L fermentation system, and the method has wide application prospects in the fields of medicines, cosmetics, feed, textiles and the like.

Description

[0001] This invention application is a divisional application based on the Chinese patent application filed on July 4, 2022, with the invention name “A recombinant Escherichia coli for producing NMN and its application” and application number “202210781815.2”. Technical Field

[0002] The present invention relates to a recombinant Escherichia coli for producing NMN and a preparation method and application thereof, belonging to the technical field of genetic engineering and bioengineering. Background Art

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

[0004] The synthesis of NMN is mainly based on chemical synthesis and enzymatic synthesis. The chemical method mainly uses nicotinamide and tetraacetyl ribose as starting materials, condenses and deacetylates with trimethylsilyl trifluoromethanesulfonate (TMSOTf), and then phosphorylates with phosphorus oxychloride / trimethyl phosphate to obtain β-NMN; or β-NMN is prepared by phosphorylating and deprotecting ketal-protected nicotinamide ribose. The chemical synthesis method of NMN technology is relatively mature, but there are many unfavorable factors. On the one hand, some chemical raw materials are highly toxic and cause serious pollution to the environment; on the other hand, some raw materials are expensive, which makes the synthesis cost of NMN high.

[0005] Enzymatic synthesis mainly adopts pure enzyme reaction and whole-cell catalysis, using D-5-phosphoribosyl and nicotinamide as raw materials, using phosphoribosyl pyrophosphate synthetase and nicotinamide phosphoribosyl transferase to catalyze the synthesis of β-NMN; or using nicotinamide and phosphoribosyl pyrophosphate as substrates to catalyze the synthesis of β-NMN. The enzymatic synthesis conversion rate is high, but the substrate cost is high, which is not conducive to scale-up production. Summary of the invention

[0006] This application aims to construct a recombinant strain that is more conducive to NMN accumulation. On the basis of the constructed chassis strain, it expresses the enzyme gene for synthesizing NMN, adjusts the gene expression intensity, and optimizes the fermentation process to increase the yield of NMN.

[0007] The invention provides a genetic engineering strain capable of synthesizing nicotinamide mononucleotide, which expresses nicotinamide phosphoribosyltransferase (NAMPT enzyme), 5-phosphoribosyl-1-pyrophosphate synthetase (PRPP synthase) and transporter BMpnuC.

[0008] In one embodiment, the amino acid sequence of the nicotinamide phosphoribosyltransferase (NAMPT enzyme) 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.

[0009] In one embodiment, the nicotinamide phosphoribosyltransferase is derived from Vibrio alginolyticus phage (Vibrio PhageKVP40) 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.

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

[0011] In one embodiment, the gene BaPRS encoding the PRPP synthase has a nucleotide sequence as shown in SEQ ID NO.5.

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

[0013] In one embodiment, the chassis strain used in 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 one embodiment, the Bacillus subtilis includes but is not limited to B. subtilis 168, B. subtilis WB600, and B. subtilis WB800.

[0015] In one 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 one embodiment, the overexpression vector plasmid used in constructing genetically engineered strains includes but is not limited to pMA5, pWB980, pHT43, pHT01 and pET-22b(+), pET-28a(+), pET-30a(+), pUC57 and other vector plasmids suitable for constructing overexpression genetically engineered strains.

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

[0018] In one embodiment, the nicotinamide phosphoribosyltransferase and PRPP synthase are expressed using pET-28a(+) or pRSFDuet-1.

[0019] In one embodiment, the recombinant Escherichia coli is based on Escherichia coli in which pncC, ushA, nadR, and purR are knocked out.

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

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

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

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

[0024] The invention also provides a starter culture containing the recombinant Escherichia coli.

[0025] The present invention also provides a method for promoting the synthesis of NMN by recombinant Escherichia coli, which is to overexpress the nicotinamide phosphoribosyltransferase gene NAMPT as shown in SEQ ID NO.4, the PRPP synthase gene BaPRS as shown in SEQ ID NO.5 in Escherichia coli, and overexpress the NMN transporter BMpnuC as shown in SEQ ID NO.6.

[0026] In one embodiment, the recombinant Escherichia coli also has pncC, ushA, nadR and purR genes knocked out.

[0027] In one embodiment, the method further comprises inducing IPTG when culturing the recombinant Escherichia coli.

[0028] 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 =0.6-1.0, and then at 25-37°C, use IPTG with a final concentration of ≥0.2mM to induce the synthesis of NMN.

[0029] In one embodiment, the induction time is 24 to 36 hours.

[0030] In one embodiment, the induction is induced by IPTG at a final concentration of 0.5 mM-1 mM.

[0031] In one embodiment, the fermentation uses 500 mg-2000 mg / L nicotinamide as a substrate.

[0032] 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 1.5 to 2 h until the OD 600 =0.6-1.0, then add IPTG to a final concentration of 0.5mM-1mM, and add 500mg-2000mg / L nicotinamide to induce NMN synthesis at 30-37°C.

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

[0034] In one embodiment, nicotinamide is added in batches or continuously during the fermentation process.

[0035] In one embodiment, the batch addition is to add nicotinamide once every 2 to 3 hours after the addition of the inducer.

[0036] In one embodiment, the continuous flow addition is to continuously add the nicotinamide solution after adding the inducer, and control the concentration of nicotinamide in the fermentation system to be less than 3 g / L.

[0037] The present invention also provides the use of the recombinant Escherichia coli, the starter, or the method in the preparation of NMN-containing products in the fields of medicine, cosmetics, feed, and textiles.

[0038] Beneficial effects:

[0039] (1) The present invention enhances the diffusion of NMN outside the cell and promotes the reaction towards NMN synthesis by overexpressing the NMN transporter BMpnuC.

[0040] (2) The present invention optimizes the gene expression intensity by expressing genes related to NMN synthesis on plasmids with different copy numbers, thereby further increasing the biosynthesis of NMN. 1 g / L nicotinamide can be used to synthesize 2.6 g / L NMN.

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

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

[0043] Figure 2 LC-MS images of recombinant E. coli, control and standard products.

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

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

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

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

[0048] Figure 7 The amount of NMN synthesized in a 5L fermenter by horizontal fed-batch feeding.

[0049] Figure 8 The amount of NMN synthesized in a 5L fermenter with constant flow feeding. DETAILED DESCRIPTION

[0050] (I) Culture medium

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

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

[0053] Feed 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] (2) Solution

[0055] 100 g / L nicotinamide solution: 5 g nicotinamide was dissolved in 50 mL ultrapure water and filtered for sterilization.

[0056] 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 / LH3BO3, 10mL / L HCl were used to prepare fermentation medium and feed medium.

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

[0058] (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%.

[0059] (V) Chemical transformation of E. coli: Streak E. coli JM109 on a solid LB plate and culture at 37°C for 12 h. Select a single colony and inoculate it into liquid LB medium and grow it at 37°C, 220 r / min for 10 h. Transfer it to fresh 25 mL liquid LB medium at a 1% inoculum volume and culture it at 37°C for 1.5-2 h. Wait until OD 600 Grow to 0.6-1 and harvest the bacteria to make competent cells.

[0060] The competent cells of E. coli were prepared using TaKaRa's Competent Cell Preparation Kit. The specific operation process is described in the instructions. The competent cells were stored at -80°C and can be transformed with plasmids or fragments.

[0061] (VI) Plasmid assembly method: Gibson reaction system is as follows: add 50ng of DNA fragment, 100ng of vector, 5μL of Gibsonmix, and add sterile ultrapure water to 10μL of the system. Reaction conditions are as follows: 50℃ for 60min, and put on ice immediately after the reaction. Take 10μL and transform it into E. coli competent JM109.

[0062] The seamless cloning reaction system is as follows: 40 ng of target gene, 100 ng of vector, 5 μL of reaction enzyme mixture, and add sterile ultrapure water to make up to 10 μL. The reaction conditions are as follows: 50℃ for 60 min, and immediately put on ice after the reaction. Take 10 μL to transform into E. coli competent JM109.

[0063] (VII) Amino acid and nucleotide sequences

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

[0065] Similarly, nicotinamide phosphoribosyltransferase has an amino acid sequence as shown in SEQ ID NO.1 and an amino acid sequence of the same function obtained by performing one or more amino acid substitutions thereon; PRPP synthase has an amino acid sequence as shown in SEQ ID NO.2 and an amino acid sequence of the same function obtained by performing one or more amino acid substitutions thereon. Genes encoding the proteins shown in SEQ ID NO.1 or SEQ ID NO.2 (e.g., the NAMPT gene of SEQ ID NO.4 or the BaPRS gene shown in SEQ ID NO.5) obtained by cloning, synthesizing or other suitable methods based on existing molecular biology techniques are also included in the scope of the present invention.

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

[0067] (1) Construction of NAMPT expression cassette

[0068] Using the synthetic sequence of NAMPT shown in SEQ ID NO.4 as a template, PCR amplification was performed with primer pair F1 / R1, and Phanta MasterMix (Vazyme) high-fidelity pfu enzyme was selected for the process. The conditions were pre-denaturation at 95°C for 3 min; 30 cycles of amplification at 95°C, 15s, 58°C, 15s, 72°C, 1min; and extension at 72°C for 5min. The PCR product was purified. Using the vector pET-28a(+) as a template, PCR amplification was performed with primer pair F2 / R2, and PhantaMasterMix (Vazyme) high-fidelity pfu enzyme was selected for the process. The conditions were pre-denaturation at 95°C for 3 min; 30 cycles of amplification at 95°C, 15s, 58°C, 15s, 72°C, 3min; and extension at 72°C for 5min. The PCR product was purified. The fragment NAMPT and the vector pET-28a(+) were recombined into the vector pET28a+NAMPT by seamless cloning and assembly. The vector was transformed into E. coli JM109. The obtained vector was sent to Shanghai Bioengineering for sequencing, and the correct recombinant vector pET28a+NAMPT was obtained after correct alignment.

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

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

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

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

[0073] Table 1 Primer information

[0074]

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

[0076] Using chassis strain F004 (E. coli BL21 (DE3), ΔpncC, ΔushA, ΔnadR, ΔpurR) disclosed in the patent application document with publication number CN112795582A) as the host bacteria, 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 bacteria 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 an LB solid plate with appropriate resistance and cultured at 37°C overnight; a single colony was selected and inoculated into LB medium containing the corresponding resistance, cultured at 37°C, 220 r / min, for 10-12 h; the colony was transferred to 30 mL of fermentation medium at a 2% transfer volume and cultured at 37°C until OD 600 When the pH value is 0.6-1.0, cool down to 25°C. Add IPTG with a final concentration of 0.2-0.5 mM, add 1-3 g / L nicotinamide, take samples at regular intervals, centrifuge to obtain the supernatant, and detect the synthesis of NMN by HPLC.

[0078] The results showed that in the HPLC test, an absorption peak was found in the sample with the same peak time as the standard NMN. To further verify whether NMN was synthesized in the sample, the sample was verified by LC-MS. Figure 2 As shown, the synthesis of NMN was confirmed in the sample. When the concentration of substrate nicotinamide was 1 g / L, the extracellular accumulation of NMN reached 980 mg / L within 24 hours.

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

[0080] In order to further increase the amount of NMN synthesis and improve 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, mainly including the induction temperature, IPTG concentration, and substrate addition concentration.

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

[0082] The recombinant strain NMN01 constructed in Example 2 was streaked on an LB solid plate with appropriate resistance and cultured at 37°C overnight; a single colony was selected and inoculated into an LB medium containing kanamycin and chloramphenicol resistance, cultured at 37°C, 220 r / min, for 10 to 12 h; the colony was transferred to 30 mL of fermentation medium at a 2% transfer volume and cultured at 37°C until OD 600 When the concentration of 1g / L nicotinamide was 0.6-1.0, IPTG was added to a final concentration of 0.2-0.5mM, and nicotinamide was added to a final concentration of 1-3g / L, and protein expression was induced at 25°C, 30°C, and 37°C, respectively. Samples were taken at regular intervals, and the supernatant was centrifuged and the NMN content in the supernatant was detected by HPLC. The results are shown in Figure 3 As shown, when the concentration of substrate nicotinamide is 1 g / L, 1560 mg / L of NMN can be synthesized after 24 h of induction at 37°C, which is 35.7% and 24.8% higher than that at induction temperatures of 25°C and 30°C, respectively.

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

[0084] The recombinant strain NMN01 constructed in Example 2 was streaked on an LB solid plate with appropriate resistance and cultured at 37°C overnight; a single colony was selected and inoculated into an LB medium containing kanamycin and chloramphenicol resistance, cultured at 37°C, 220 r / min, for 10 to 12 h; the colony was transferred to 30 mL of fermentation medium at a 2% transfer volume and cultured at 37°C until OD 600 When the pH value was 0.6-1.0, IPTG was added at a final concentration of 0.2, 0.5, and 1.0 mM, and nicotinamide was added at a final concentration of 1-3 g / L to induce protein expression at 37°C. Samples were taken at regular intervals, and the supernatant was obtained by centrifugation. The NMN content in the supernatant was detected by HPLC. Figure 4 As shown, under the conditions of adding substrate nicotinamide 1g / L and induction temperature 30°C, using final concentration 0.5mM IPTG induction for 24h, 1809mg / L of NMN can be synthesized, which is 15% and 8.5% higher than the synthesis capacity when the final IPTG concentration is 0.2 and 1.0mM, respectively.

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

[0086] The recombinant strain NMN01 constructed in Example 2 was streaked on an LB solid plate with appropriate resistance and cultured at 37°C overnight; a single colony was selected and inoculated into an LB medium containing kanamycin and chloramphenicol, cultured at 37°C, 220 r / min, for 10 to 12 h; the colony was transferred to 30 mL of fermentation medium at a 2% transfer volume and cultured at 37°C until OD 600When the concentration of nicotinamide was 0.6-1.0, 0.5 mM IPTG was added to induce protein expression, and nicotinamide was added at final concentrations of 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 g / L, respectively. Samples were taken at regular intervals, and the supernatant was obtained by centrifugation. The NMN content in the supernatant was detected by HPLC. Figure 5 As shown, at nicotinamide concentrations of 0.5 g / L and 2.0 g / L, the NMN production reached more than 1450 mg / L, and at a nicotinamide concentration of 1 g / L, the bacterial NMN production could reach 1871 mg / L.

[0087] Example 4: Using plasmids with different copy numbers to adjust gene expression intensity

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

[0089] The recombinant strain NMN02 was streaked on an LB solid plate with appropriate resistance and cultured at 37°C overnight; a single colony was selected and inoculated into an LB medium containing the corresponding resistance, cultured at 37°C, 220 r / min, for 10-12 h; the colony was transferred to 30 mL of fermentation medium at a 2% transfer volume and cultured at 37°C until OD 600 When the pH value was 0.6-1.0, IPTG was added at a final concentration of 0.5 mM at 37°C to induce protein expression, and nicotinamide was added at a final concentration of 1 g / L. Samples were taken at regular intervals, and the supernatant was collected by centrifugation. The synthesis of NMN was detected by HPLC. Figure 6 As shown, after 24 hours of induction, the recombinant strains NMN01 and NMN02 could accumulate 1.9 g / L and 2.6 g / L, respectively. The recombinant strain NMN02 had stronger NMN synthesis ability, which was 36.8% higher than that of NMN01.

[0090] Example 5: Strain NMN02 expanded to synthesize NMN

[0091] The recombinant bacteria NMN02 was used as the fermentation strain and expanded in a 5L fermenter. The strain NMN02 was streaked from the glycerol tube onto the LB solid medium containing the corresponding resistance and cultured at 37°C for 12h; a single colony was selected and inoculated into the corresponding liquid LB, cultured at 37°C, 220r / min for 10h, and transferred to a 5L fermenter containing 2.5L fermentation medium and 5-10mL metal ion solution according to a transfer volume of 5%-20%. The initial conditions were: pH was controlled at 6.0-7.0 with 35% ammonia water, ventilation volume was 1-2vvm, speed was 300r / min-1000r / min and dissolved oxygen DO was controlled at 30%-50%. When DO rebounded, IPTG with a final concentration of 0.5-1mM was used to continue induction at 37°C, and nicotinamide 2g / L nicotinamide was added at 0h, 2h, 4h, 6h, and 8h of induction (a total of 10g / L was added). Samples were taken regularly to detect the synthesis of NMN. The results showed that after 24 hours of induction, the NMN accumulation was above 12.3 g / L and the OD reached 25-30.

[0092] Example 6: Strain NMN02 expanded to synthesize NMN

[0093] By coupling the amount of intracellular NMN synthesis with growth, a higher NMN concentration can be obtained. The specific implementation method is the same as Example 5, except that the addition method of the substrate nicotinamide is changed to flow addition. Nicotinamide is added at a constant rate at the same time as the inducer is added, and the remaining nicotinamide in the culture medium is detected in real time to control the concentration of nicotinamide to maintain a relatively low concentration (<3g / L). The results showed that the recombinant strain NMN02 can synthesize 20.3g / L of NMN in a 5L fermenter after 25h of fermentation (20h of induction), with 9.6g / L of nicotinamide added in total, and 2.13g / L of nicotinamide remaining. The molar conversion rate of consumed nicotinamide to NMN is 98.97%, and the OD can reach above 30.

[0094] 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. An engineered Escherichia coli strain capable of synthesizing nicotinamide mononucleotide, characterized in that: Nicotinamide phosphoribosyltransferase, 5-phosphoribosyl-1-pyrophosphate synthetase and transporter can be expressed; wherein the transporter contains the amino acid sequence shown in SEQ ID NO.3; the nicotinamide phosphoribosyltransferase contains the amino acid sequence shown in SEQ ID NO.1; and the 5-phosphoribosyl-1-pyrophosphate synthetase contains the amino acid sequence shown in SEQ ID NO.

2.

2. The Escherichia coli engineered strain according to claim 1, characterized in that The E. coli engineered strain also has pncC, ushA, nadR and purR genes knocked out.

3. The engineered Escherichia coli strain according to claim 1, characterized in that The Escherichia coli engineered strain overexpresses genes encoding nicotinamide phosphoribosyltransferase, 5-phosphoribosyl-1-pyrophosphate synthetase and transporter.

4. A method for preparing the Escherichia coli engineered bacteria according to any one of claims 1 to 3, characterized in that: Nicotinamide phosphoribosyltransferase as shown in SEQ ID NO.1, 5-phosphoribosyl-1-pyrophosphate synthetase as shown in SEQ ID NO.2, and NMN transporter as shown in SEQ ID NO.3 were overexpressed in Escherichia coli.

5. The method according to claim 4, characterized in that The method further comprises the step of knocking out the pncC, ushA, nadR and purR genes of Escherichia coli.

6. The method according to claim 4, characterized in that The method further comprises the step of regulating the expression of the transporter protein, optionally by using plasmids with different copy numbers.

7. A method for producing NMN by fermentation, characterized in that: Using the engineered Escherichia coli strain according to any one of claims 1 to 3 as a fermentation microorganism, culturing at 35-40° C. until OD600=0.6-1.0, and inducing the synthesis of NMN with an inducer; Optionally, the inducer may be IPTG at a concentration of 0.5 to 1 mM; Optionally, the induction temperature is 25-37°C; Optionally, the induction time is 24 to 36 hours.

8. A starter culture containing the engineered Escherichia coli strain according to any one of claims 1 to 3.

9. Use of the engineered Escherichia coli strain according to any one of claims 1 to 3, or the method according to any one of claims 4 to 7, or the starter according to claim 8 in the preparation of NMN-containing products in the fields of medicine, cosmetics, feed, and textiles.

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