Mutants for improving the yield of beta-nicotinamide mononucleotide, encoding genes, amino acid sequences and applications thereof

Abstract

The application discloses a mutant for improving the yield of beta-nicotinamide mononucleotide, a coding gene, an amino acid sequence and application thereof, and the mutant comprises a mutant Nampt1, a mutant PnuC2 and / or a mutant NiaP1; the amino acid sequence of the mutant Nampt1 is shown as SEQ ID NO: 4; the amino acid sequence of the mutant PnuC2 is shown as SEQ ID NO: 5; and the amino acid sequence of the mutant NiaP1 is shown as SEQ ID NO: 6; the recombinant bacteria obtained by the application can improve the production capacity of NMN, the production process is easy to scale up, the content of NMN obtained by the method reaches 26.25 g / L; the combination of the mutants designed by the application can maximize the respective abilities of the genes, the optimal combination is improved by 5.7 times, and the production time is shortened by 35%. The application also discloses the application of the recombinant bacteria in the fields of food, medicine and cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, in particular to a mutant for improving the yield of β-nicotinamide mononucleotide, a coding gene thereof, an amino acid sequence and application thereof. BACKGROUND

[0002] Nicotinamide mononucleotide (NMN) is a naturally occurring active nucleotide, which exists widely in various organisms. NMN is converted into NAD+ in the human body, and has many physiological functions, such as treatment of neurodegenerative diseases, senile degenerative diseases, heart and brain diseases, diabetes and the like. In particular, NMN has a significant correlation with aging-related diseases, which has attracted widespread attention, and thus NMN shows great application potential in the fields of medicine and beauty, health products and daily chemicals. The demand for NMN will be huge in the future, so the scientific and industrial communities are urgently seeking a cost-effective, environmentally friendly and efficient method for synthesizing NMN.

[0003] NMN can be synthesized by chemical method, enzyme catalysis method and biological fermentation method. Before 2010, most of the large-scale NMN production was achieved by chemical synthesis, which caused great environmental pollution, low yield, high production cost, and only 2 / 3 of the synthetic NMN was the bioactive isomer. The biological enzyme method for preparing NMN takes nicotinamide riboside (NR) as the starting material, and under the action of nicotinamide riboside kinase (NRK) and ATP, NMN is obtained by one-step reaction. The disadvantage of enzyme catalysis method is the need for ATP addition, and in the multi-enzyme system, the overall efficiency is low, resulting in high production cost. The biological method for synthesizing NMN has the advantages of high stereoselectivity, high conversion rate, no organic solvent residue, mild conditions and simple separation process, but the current biological method for synthesizing NMN has low production intensity, which cannot meet the requirements of large-scale industrial production, resulting in high price of NMN. The biological fermentation method takes sugar as the raw material to produce nucleotides, which overcomes the limitations of enzyme production, but the optimization of metabolic pathway, the limitation of key enzymes and the import and export of substrates and products are still key points for breakthrough. For example, CN202110468908, an innovative method for improving the enzyme activity of NMN biosynthesis enzyme Nampt, designed primers to construct 10 mutant strains, and the activity of 8 mutants was higher than that of the wild-type strain. Among them, the Nampt V365L mutant increased by 62%, and the NMN yield of Nampt S248A, Nampt N164L, Nampt S382M, Nampt A245T and Nampt A208G increased by 34%, 27%, 27%, 22% and 17% respectively, but the highest yield of the best mutant was only 45.4 mg / L. Patent HK40077691A utilizes the construction and application of genetically engineered bacteria for NMN fermentation, which introduces exogenous nicotinamide phosphoribosyltransferase NAMPT and expresses substrate uptake protein NiaP and product efflux protein PnuC. The final NMN yield is less than 0.08 mg / L, and even compared with the starting strain, the increase is less than 5 times, which shows that the catalytic ability of key enzymes and the appropriate intensity are crucial for the production level. In the article "Systematic engineering of Escherichia coli for efficient production of nicotinamide mononucleotide from nicotinamide" published by Zhou Jingwen's research group in ACS Synth Biol, 2022, 11:2979-2988, it is considered that the introduction of nicotinamide transporter BcniaP cannot significantly improve the NMN yield at the level of shake flask, but on the contrary, it reduces the NMN yield.

[0004] In view of the above, there is an urgent need in the art for a more cost-effective, environmentally friendly and efficient method for synthesizing NMN. SUMMARY

[0005] In view of the deficiencies in the prior art, the purpose of the present application is to, in a strain capable of producing NMN, by mutating key enzymes in the NMN synthesis pathway, the optimal combination of the mutants can greatly improve the production efficiency of NMN, bringing great potential for industrial production of NMN.

[0006] The first aspect of the present application provides a mutant for improving the yield of beta-nicotinamide mononucleotide, comprising a mutant Nampt1, PnuC2 and / or NiaP1; wherein the amino acid sequence of the mutant Nampt1 is shown as SEQ ID NO: 4; the amino acid sequence of the mutant PnuC2 is shown as SEQ ID NO: 5; and the amino acid sequence of the mutant NiaP1 is shown as SEQ ID NO: 6.

[0007] The second aspect of the present application is to protect the nucleotide sequence of the gene encoding the mutant described above.

[0008] For the technical solution described above, further, the nucleotide sequence of the mutant Nampt1 is shown as SEQ ID NO: 1; the nucleotide sequence of the mutant PnuC2 is shown as SEQ ID NO: 2; and the nucleotide sequence of the mutant NiaP1 is shown as SEQ ID NO: 3.

[0009] The third aspect of the present application is to protect the recombinant vector containing the nucleotide sequence described above.

[0010] The fourth aspect of the present application is to protect the recombinant bacteria expressing the mutant described above.

[0011] For the technical solution described above, further, the host cell of the recombinant bacteria is selected from one of Escherichia coli BL21(DE3), BL21(AI), Rosetta, OrigamiB(DE3), JM109(DE3), W3110(DE3). Specifically, in the examples, the recombinant vector containing the coding gene of the mutant is pETDuet-zwf-pgl-pgi and pRSFDuet-Nampt1-PnuC2 / NiaP1;

[0012] The construction process of the above-mentioned recombinant vector is as follows:

[0013] Overexpress endogenous pgi (NP_418449), pgl (NP_415288), zwf (NP_416366) through plasmid vector pETDuet.

[0014] By another plasmid vector, overexpressing Nampt 1 (WP_012788281.1) mutant from Chitinophaga pinensis, PnuC2 (AJH20194.1) mutant from Bacillus mycoides, NiaP1 (CDN64082.1) mutant from Burkholderia cenocepacia.

[0015] The fifth aspect of the present application is to protect a method for improving the yield of beta-nicotinamide mononucleotide, using a recombinant bacterium expressing the mutant described above to convert the substrate to beta-nicotinamide mononucleotide.

[0016] For the technical solutions described above, further, in the method, the E. coli BL21 (DE3)-△nadR△amn::prs-△pncC::lacI-NadE strain is used as the starting strain.

[0017] For the technical solutions described above, further, in the method, the recombinant bacterium expressing the mutant described above is BL-G17△nadR::P T7 -Nampt1, denoted as BLN-G17. That is, the Nampt1 gene is integrated into the nadR sequence position of the E. coli genome, replacing the gene, and is activated by the T7 promoter.

[0018] For the technical solutions described above, further, the method is to culture the recombinant bacterium in a culture system to an OD 600 0.5-1, 0.5-1 mM IPTG is added as an inducer, and the reaction is induced at 30-37°C for 12-20 h, while the substrate is added, and the reaction is carried out for 12-20 h.

[0019] For the technical solutions described above, further, in the reaction system of the method, the substrate concentration is 0.5-10 g / L nicotinamide mM, and more preferably, in the reaction system of the above method, the strain OD 600 0.5-52, and the reaction is carried out at 30-37°C and pH 6.0-7.0.

[0020] The sixth aspect of the present application is to protect the use of the mutant, the vector or the recombinant bacterium in the field of food, medicine and cosmetics.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] (1) The recombinant obtained by the present application can improve the production capacity of NMN, and the production process can be scaled up to obtain NMN content of 26.25 g / L;

[0023] (2) The combination of the mutants designed in the application can maximize the ability of each gene, and the best combination is 5.7 times the worst combination;

[0024] (3) The substrate is added at the same time as induction to start the reaction, and the production time is shortened by 35%. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 NMN content detection in supernatant of recombinant strains BL00, BL00 / 1, and BL00 / 2.

[0026] Figure 2 NMN content detection results in recombinant strain BL00 / 2 and its nicotinamide phosphoribosyltransferase (Nampt), nicotinamide nucleoside transporter (PnuC), and nicotinic acid transporter (NiaP).

[0027] Figure 3 NMN content detection results in supernatant of recombinant strains BLG1-G18.

[0028] Figure 4 Effect of induction temperature on NMN content and strain growth.

[0029] Figure 5 Effect of IPTG concentration on NMN content and strain growth.

[0030] Figure 6 Effect of induction timing on NMN content and strain growth. DETAILED DESCRIPTION

[0031] The application will be further described below in conjunction with examples, but it should be understood that the protection scope of the application is not limited by the examples.

[0032] In the application, unless otherwise explicitly stated, percentages and percentage contents are by mass. Unless otherwise specified, the experimental methods used are conventional methods, and the materials, reagents, etc. used can be purchased through commercial channels.

[0033] 1. Culture medium and reagents

[0034] LB medium (1 L): 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, and 20 g of agar powder for solidification;

[0035] MA medium (1 L): 6 g of potassium dihydrogen phosphate, 16.4 g of dipotassium hydrogen phosphate, 5 g of ammonium sulfate, 1.1 g of citric acid monohydrate, 1 g of magnesium sulfate, and 10 g of yeast powder.

[0036] 2. High-performance liquid chromatography (HPLC) detection

[0037] HPLC detection HPLC analysis was performed using an LC-20AD system (Shimadzu, Tokyo, Japan) equipped with an SPD-M20A UV / Vis detector and a C18-H column (Alphasil XD-C18CH, 4.6 x 250 mm, 5 μm). The injection volume was 20 μL, the mobile phase A was 0.01 mol / L potassium dihydrogen phosphate, the mobile phase B was 100% acetonitrile, and the flow rate was 1.0 mL / min. The mobile phase A:B = 100:0 at 0-8 min, the mobile phase A:B = 30:70 at 8-20 min, the mobile phase A:B = 30:70 at 20-30 min, the column temperature was 25 °C, and the signal of NMN and nicotinamide was detected at 210 nm.

[0038] A standard solution of NMN was prepared and then diluted step by step to various concentrations to establish a standard curve. The NMN content of the sample to be tested was calculated according to the standard curve and the peak area of the sample to be tested. A standard solution of nicotinamide was prepared and then diluted step by step to various concentrations to establish a standard curve. The nicotinamide content of the sample to be tested was calculated according to the standard curve and the peak area of the sample to be tested.

[0039] Table 1 Plasmids

[0040]

[0041]

[0042] Table 2 Strains

[0043]

[0044] Example 1

[0045] Construction of NMN production strain

[0046] The endogenous prs (NP_415725), pgi (NP_418449), pgl (NP_415288), gnd (NP_416533), zwf (NP_416366), rpiA (NP_417389), and rpiB (NP_418514) were expressed. Specifically, the genes prs, pgi, pgl, gnd, zwf, rpiA, and rpiB, which were respectively sequence-optimized for E. coli, were sent to Genscript for full synthesis, and the synthesized products were respectively connected to a pETDuet plasmid backbone to obtain plasmids E1 and E2 (Table 1).

[0047] The plasmids E1 and E2 were respectively electroporated into E. coli BL00 in different combinations to obtain recombinant strains BL00 / 1 and BL00 / 2 (Table 2).

[0048] After the obtained recombinant strains were activated by streaking, single colonies were picked and inoculated in LB liquid medium (containing ampicillin 100 mg / L) and cultured at 37°C, 180 rpm for 16 h. Then, 2% (v / v) seed liquid was transferred into 20 mL MA medium, and the OD 600 was adjusted to 0.6. IPTG and nicotinamide were added, and the culture was induced at 30°C, 180 rpm. The supernatant of the recombinant strain was collected by low-temperature centrifugation.

[0049] The content of NMN in the fermentation supernatant was detected by HPLC.

[0050] The detection results are shown in Table 1. The NMN content of BL00 / 2 was the highest in the fermentation supernatant. Therefore, BL00 / 2 was selected as the starting strain for subsequent research. Figure 1

[0051] Example 2

[0052] Construction of mutants

[0053] The sequence-optimized genes Nampt, PnuC and NiaP for E. coli were synthesized by Shengong, and the synthesized products were connected to the pRSFDuet plasmid backbone, respectively, to obtain R1, R2 and R3 plasmids (Table 1).

[0054] The mutants were designed and randomly mutated using R1, R2 and R3 as templates, respectively.

[0055] ①According to the QuickMutation TM kit, the primers and random mutations were designed. The primer sequences were as follows:

[0056] Random-Nampt-F: gtttaactttaataaggaggaattcATGACCAAAGAAAATCTGATTC; SEQ ID NO: 7

[0057] Random-Nampt-R: atggtatatctccttgagctcTTAGATGGTGGCATTTTTAC; SEQ ID NO: 8

[0058] Random-PnuC-F: aagagctcaaggagatataccatggTTCGTAGTCCACTGTTTC; SEQ ID NO: 9

[0059] ​Random-PnuC-R: ttaagcattatgcggccgcaagcttTCAAATATAATTATTCACCCTTTC; SEQ ID NO: 10

[0060] Random-NiaP-F: atgtttgaacattaaggtaccAAGGAGATATACCATGCC; SEQ ID NO: 11

[0061] Random-NiaP-R: cagcggtttctttaccagactcgagTTAACTAGCTTTATCCGC; SEQ ID NO: 12

[0062] 2. Random mutation PCR reaction

[0063] The random mutation PCR reaction system was set up according to the following Table 3:

[0064] Table 3

[0065]

[0066]

[0067] The PCR instrument was set according to the following parameters:

[0068] Table 4

[0069]

[0070] 3. Transformation of competent cells

[0071] 4 μL of the PCR product was taken and 1% agarose electrophoresis was performed to detect the band concentration and specificity. The remaining PCR product was electrophoresed and the target DNA fragment was recovered by cutting the gel. The mutated fragment was ligated with the vector skeleton, transformed into the competent cells of the E. coli strain, mixed, and then placed in ice bath for 30 min. Then, the cells were subjected to 42°C water bath heat shock for 45 s and immediately placed on ice for 2-5 min. Then, 250 μL of LB medium was added, and the cells were cultured at 200 rpm and 37°C for 1 h. 100-200 μL of the bacterial solution was inoculated on a plate resistant to kanamycin and ampicillin and cultured overnight. Multiple mutants were obtained, and the plasmids were extracted and then transferred into the competent cells of BL00 / 2. The obtained recombinant strains were streaked and activated, and single colonies were inoculated in LB liquid medium (containing ampicillin 100 mg / L and kanamycin 50 mg / L) at 37°C and 180 rpm for 16 h. Then, 2% (v / v) of the seed liquid was inoculated in 20 mL of MA medium (containing ampicillin 100 mg / L and kanamycin 50 mg / L), and the culture was incubated at 37°C and 180 rpm until the OD 600 0.6

[0072] In addition, the cells with OD 600 The cells with OD 600 to 0.6 were added with 0.5 mM IPTG and 1 g / L NMN, and induced to culture at 30°C, 180 rpm, and centrifuged at low temperature, and the intracellular and extracellular NMN contents were detected, respectively.

[0073] The cells with OD 600 to 0.6 were added with 0.5 mM IPTG and 1 g / L NMN, and induced to culture at 30°C, 180 rpm, and centrifuged at low temperature, and the intracellular and extracellular NMN contents were detected, respectively.

[0074] The mutant of nicotinamide phosphoribosyltransferase (Nampt) that significantly improved the NMN yield was named R11, and the strain was named BL-R11. The intracellular and extracellular NMN contents caused by nicotinamide riboside transporter (PnuC) and its mutants were detected by HPLC, and the mutants with improved properties were named R21 and R22. The intracellular and extracellular NMN contents of R2, R21 and R22 were 0.45:0.55, 0.31:0.69 and 0.22:0.78, respectively.

[0075] The mutants of nicotinic acid transporter (NiaP) that significantly reduced the extracellular nicotinamide concentration were named R31 and R32. The intracellular and extracellular nicotinamide contents of R3, R31 and R32 were 0.65:0.35, 0.72:0.28 and 0.89:0.11, respectively.

[0076] The cells with OD 600 to 0.6 were added with 0.5 mM IPTG and 0.5 g / L nicotinamide, and induced to culture at 30°C, 180 rpm, and centrifuged at low temperature, and the supernatant of the recombinant strain was collected, and the NMN content in the supernatant was detected.

[0077] The detection results are shown in Table 1. Figure 2 Therefore, it can be known that the property improvement of the key genes nicotinamide phosphoribosyltransferase (Nampt), nicotinamide riboside transporter (PnuC) and nicotinic acid transporter (NiaP) is helpful for the accumulation of NMN concentration.

[0078] Example 3

[0079] Construction of a combination-optimized strain

[0080] The G1-G18 plasmid was expressed in BL00 / 2 according to the conventional technical means of molecular biology to obtain recombinant strain BL00 / 2-G1-G18. After the obtained recombinant strain was activated by streaking, a single colony was picked in LB liquid medium (containing ampicillin 100 mg / L, kanamycin 50 mg / L) and cultured at 37°C, 180 rpm for 16 h, then 2% (v / v) seed liquid was transferred into 20 mL MA medium, and the culture was induced at 30°C, 180 rpm until OD 600 0.6, 0.5 mM IPTG and 1 g / L nicotinamide were added, and the culture was induced at 30°C, 180 rpm, then low-temperature centrifugation was performed, and the supernatant of the recombinant strain was collected, and the content of NMN in the fermentation supernatant was detected by HPLC. The detection results are shown in Table 2. Figure 3 As can be seen from the yield results of the related vectors, under the condition that nicotinamide phosphoribosyltransferase (Nampt) is not strengthened, the activity of nicotinic acid transporter (NiaP) is not the higher the better, and the strengthening of nicotinic acid transport needs to be matched with the strengthening of the subsequent synthesis step and product efflux. As can be seen from the yield results of BL-G1, BL-G7, BL-G13 and their related vectors, the strengthening of nicotinamide nucleoside transporter (PnuC) can relieve the feedback inhibition of the product, and the effect is obvious with the increase of the overall production capacity. As can be seen from the yield results of the expression strains of BL-G1, BL-G4; BL-G2, BL-G5 and the like with only nicotinamide phosphoribosyltransferase as different mutants, the strengthening of nicotinamide phosphoribosyltransferase (Nampt) can improve the production capacity, and theoretically the higher the activity of the enzyme is, the better.

[0081] Example 4

[0082] Further strengthening of nicotinamide phosphoribosyltransferase (Nampt)

[0083] As can be seen from the conclusion in Example 3, the strengthening of nicotinamide phosphoribosyltransferase does not reach the upper limit of metabolic flow, in order to further improve the production capacity, the gene of mutant Nampt1 was integrated into the genome. The integration fragment was connected with the homologous arms upstream and downstream of the integration site ΔnadR, and then was connected into the Pst I and EcoR I sites of plasmid pUC19 by seamless cloning to obtain pUC19-Nampt1 plasmid (Table 1). After the sequence was verified by sequencing, the donor fragments were integrated into the BL-G17 genome according to the gene replacement operation of Escherichia coli to obtain recombinant strain BL-G17 ΔnadR::P T7 -Nampt1, which is denoted as BLN-G17.

[0084] The obtained recombinant strain was activated by streaking, and a single colony was picked and inoculated in LB liquid medium (containing ampicillin 100 mg / L, kanamycin 50 mg / L) and cultured at 37°C, 180 rpm for 16 h. Then, 2% (v / v) seed liquid was transferred into 20 mL MA medium (containing ampicillin 100 mg / L, kanamycin 50 mg / L) and cultured at 30°C, 180 rpm until OD 600 0.6, 0.5 mM IPTG and 1 g / L nicotinamide were added, and induction culture was carried out at 30°C, 180 rpm. Low-temperature centrifugation was performed, and the supernatant of the recombinant strain was collected. HPLC was used to detect the content of NMN in the fermentation supernatant. The content reached 563.6 ± 9.8 mg / L, which was 1.14 times higher than that of BL-G17.

[0085] Comparative Example 1

[0086] Further strengthening of nicotinamide nucleoside transporters is intended

[0087] The gene of mutant PnuC2 was integrated into the genome. The integration fragment was connected with the homologous arms upstream and downstream of the integration site ΔnadR, and then was connected into the Pst I and EcoR I sites of plasmid pUC19 by seamless cloning to obtain pUC19-PnuC2 plasmid (Table 2). After sequence verification to confirm the correct sequence, these donor fragments were integrated into the BL-G16 genome according to the Escherichia coli gene replacement operation to obtain BL-G17 ΔnadR::PT7-PnuC2 recombinant strain. The obtained recombinant strain was activated by streaking, and a single colony was picked and inoculated in LB liquid medium (containing ampicillin 100 mg / L, kanamycin 50 mg / L) and cultured at 37°C, 180 rpm for 16 h. Then, 2% (v / v) seed liquid was transferred into 20 mL MA medium (containing ampicillin 100 mg / L, kanamycin 50 mg / L) and cultured at 30°C, 180 rpm until OD 600 0.6, 0.5 mM IPTG and 1 g / L nicotinamide were added, and induction culture was carried out at 30°C, 180 rpm. Low-temperature centrifugation was performed, and the supernatant of the recombinant strain was collected. HPLC was used to detect the content of NMN in the fermentation supernatant. The content reached 563.6 ± 9.8 mg / L, which was 1.14 times higher than that of BL-G17.

[0088] Comparative Example 2

[0089] Further strengthening of nicotinic acid transporters is intended

[0090] The NiaP1 gene was integrated into the genome. The integration fragment was ligated with the upstream and downstream homologous arms of the integration site ΔnadR, and then connected into the Pst I and EcoR I sites of plasmid pUC19 by seamless cloning to obtain pUC19-NiaP1 plasmid. After the sequence was verified by sequencing, the donor fragment was integrated into the genome of BL-G17 according to the gene replacement operation of E. coli to obtain the recombinant strain BL-G17 ΔnadR::PT7-NiaP1. After the obtained recombinant strain was streaked and activated, a single colony was picked in LB liquid medium (containing ampicillin 100 mg / L, kanamycin 50 mg / L) and cultured at 37°C, 180 rpm for 16 h, and then 2% (v / v) seed liquid was transferred into 20 mL of MA medium (containing ampicillin 100 mg / L, kanamycin 50 mg / L) and cultured at 30°C, 180 rpm until the OD600 of the culture reached 0.6, 0.5 mM IPTG and 1 g / L nicotinamide were added, and the culture was induced at 30°C, 180 rpm. After low-temperature centrifugation, the supernatant of the recombinant strain was collected, and the content of NMN in the fermentation supernatant was detected by HPLC. The detection showed that the content reached only 355.1 ± 6.7 mg / L, which was significantly lower than the production capacity of BL-G17, and it took 16 h to reach the production capacity. It can be seen that although the copy number of nicotinamide transporter was increased, the production capacity was not increased but decreased. 600 600 600 The experimental results of the above Comparative Examples 1 and 2 show that the optimal production efficiency of NMN cannot be achieved by further strengthening the nicotinamide transporter and further strengthening the nicotinamide transporter.

[0092] Example 5

[0093] Optimization of induction fermentation conditions

[0094] The induction conditions are important factors affecting bacterial growth and fermentation capacity. By optimizing the induction process of the strain, higher product concentration, stronger production intensity, and lower production cost can be obtained. Therefore, the induction temperature, inducer concentration, and induction timing of BL-G17 were optimized respectively.

[0095] In the following experiments, single-factor control variable method was used for optimization. After induction, the culture was incubated at 37°C, 180 rpm, and the biomass was determined by spectrophotometer.

[0096] Other conditions were unchanged, and the induction temperature was set to 18°C, 25°C, 30°C and 37°C. Each combination was repeated 3 times. As shown in Figure 4 induction temperature was 37°C, the expression of NMN reached the highest.

[0097] ​The other conditions remain unchanged, set the inducer IPTG concentration of 0 mM, 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM and 1.0 mM. As shown in Fig. 1, when the inducer concentration is 0.5 mM, the expression of NMN is the highest. Figure 5

[0098] The other conditions remain unchanged, set the induction opportunity (OD 600 ) of 0.3, 0.6, 0.9, 1.2 and 1.5. Each group is repeated 3 times. As shown in Fig. 3, when the induction OD 600 is 0.9, the expression of NMN reaches the highest. Figure 6

[0099] Based on the above results, the optimal fermentation conditions are obtained: the optimum induction temperature is 37℃, the optimum inducer concentration is 0.5 mM, the optimum induction opportunity is OD 600 = 0.9, and the NMN content reaches 586.24 ± 4.2 mg / L, which is 18.97 times of the starting strain BL00 (30.9 ± 3.8 mg / L). Through the detection of other sampling points, it is found that the highest yield is reached at 13 h after induction, which is 35% shorter than the 20 h required by the starting strain.

[0100] The recombinant strain BLN-G17 glycerol seed liquid was streaked on LB solid plate (containing ampicillin 100 mg / L, kanamycin 50 mg / L) for 37℃ incubation and activation. After activation, it was inoculated into fresh liquid LB medium (containing ampicillin 100 mg / L, kanamycin 50 mg / L) and cultured at 37℃, 180 rpm for 16 h. The seed liquid was inoculated into the MA fermenter (containing ampicillin 100 mg / L, kanamycin 50 mg / L) at 2% (v / v), and the temperature was maintained at 37℃ ± 0.5℃, the pH was 6.7 ± 0.1, the dissolved oxygen was controlled at 10%-40% by adjusting the aeration rate (20-100 L·h-1) and stirring speed (100-600 rpm), and when the OD 600 value was 0.9, 0.5 mM IPTG was added to the medium to induce protein expression, and 10 g / L nicotinamide solution was continuously added to the medium to promote the synthesis of NMN. After 12-13 h of fermentation, sampling was performed for HPLC determination. The NMN content reached 26.25 g / L, the biomass OD 600 = 52.42, and the conversion rate of nicotinamide reached 89%.

[0101] ​​In summary, by comparative analysis of the example data of the present application, it can be seen that the synthesis of NMN is greatly affected by the key enzyme activity in the pathway, the content of substrates and products in the reaction system. The mutant of nicotinamide phosphoribosyltransferase (Nampt) with high activity has a great effect on the synthesis efficiency of NMN, and the improvement of the functions of efflux proteins nicotinamide nucleoside transporter (PnuC) and nicotinic acid transporter (NiaP) is also beneficial to the synthesis of NMN, but the three need to be combined in a reasonable relative intensity to achieve the best effect.

[0102] The above merely provides the preferred, specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any changes or substitutions easily thought of by those skilled in the art within the technical range disclosed by the present application should be encompassed within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A mutant that improves the production of β-nicotinamide mononucleotide, characterized in that, The mutants were selected from: Mutant Nampt1; Or a combination of mutant Nampt1 and mutant PnuC2; Or a combination of mutant Nampt1 and mutant NiaP1; Or a combination of mutants Nampt1, PnuC2 and NiaP1; The amino acid sequence of the mutant Nampt1 is shown in SEQ ID NO:4; the amino acid sequence of the mutant PnuC2 is shown in SEQ ID NO:5; and the amino acid sequence of the mutant NiaP1 is shown in SEQ ID NO:

6.

2. A class of nucleotides encoding a gene of the mutant of claim 1, characterized in that, The nucleotides are selected from: Nucleotides encoding the mutant Nampt1; Or a combination of nucleotides encoding mutant Nampt1 and nucleotides encoding mutant PnuC2; Or a combination of nucleotides encoding mutant Nampt1 and nucleotides encoding mutant NiaP1; Or a combination of nucleotides encoding mutants Nampt1 and PnuC2 and nucleotides encoding mutant NiaP1.

3. A nucleotide of the class of genes encoding the mutant of claim 1 according to claim 2, characterized in that, The nucleotide sequence of the mutant Nampt1 is shown in SEQ ID NO:1; the nucleotide sequence of the mutant PnuC2 is shown in SEQ ID NO:2; and the nucleotide sequence of the mutant NiaP1 is shown in SEQ ID NO:

3.

4. A type of recombinant vector containing the nucleotide sequence of claim 2.

5. A class of recombinant bacterial cells expressing the mutant of claim 1.

6. The recombinant bacterial cell of claim 5, wherein, The recombinant bacterial strain is selected from one of Escherichia coli BL21(DE3), BL21(AI), Rosetta, OrigamiB(DE3), JM109(DE3), and W3110(DE3).

7. A method for increasing the yield of β-nicotinamide mononucleotide, comprising transforming a substrate to generate β-nicotinamide mononucleotide using the recombinant bacterial cells of claim 5.

8. The method according to claim 7, characterized in that, The recombinant bacteria are cultured in a culture system to OD 600 0.5-1, 0.5-1 mM of inducer IPTG is added, and the bacteria are induced at 30-37℃ for 12-20 h, while a substrate is added, and the reaction is carried out for 12-20 h; the recombinant bacteria are E. coli BL21(DE3)-△nadR△amn::prs-△pncC::lacI-NadE.

9. The method according to claim 7, characterized in that, The substrate concentration in the reaction system of the method is 0.5-10 g / L nicotinamide.

10. The use of the mutant of claim 1, the vector of claim 4, or the recombinant bacterial cell of claim 5 in the production of β-nicotinamide mononucleotide.

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