CN116926033A - Mutant for improving yield of beta-nicotinamide mononucleotide, coding gene, amino acid sequence and application thereof - Google Patents

Mutant for improving yield of beta-nicotinamide mononucleotide, coding gene, amino acid sequence and application thereof Download PDF

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CN116926033A
CN116926033A CN202310968235.9A CN202310968235A CN116926033A CN 116926033 A CN116926033 A CN 116926033A CN 202310968235 A CN202310968235 A CN 202310968235A CN 116926033 A CN116926033 A CN 116926033A
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范超
李倩
齐佳琨
程杨好
洪皓
吴文忠
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Innobio Corp ltd
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Abstract

The application discloses a mutant for improving the yield of beta-nicotinamide mononucleotide, a coding gene, an amino acid sequence and application thereof, wherein the mutant comprises mutants Nampt1, pnuC2 and/or 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; the amino acid sequence of the mutant NiaP1 is shown as SEQ ID NO. 6; the recombinant bacteria obtained by the method can improve the NMN production capacity, the production process is easy to amplify, and the NMN content obtained by the method reaches 26.25g/L; the combination of the mutants designed by the application can make each gene exert the respective capacity to the maximum extent, the optimal combination is improved by 5.7 times, and the production time is shortened by 35%. The recombinant thallus is applied to the fields of food, medicine and cosmetics.

Description

Mutant for improving yield of beta-nicotinamide mononucleotide, coding gene, amino acid sequence and application thereof
Technical Field
The application relates to the technical field of molecular biology, in particular to a mutant for improving the yield of beta-nicotinamide mononucleotide, and a coding gene, an amino acid sequence and application thereof.
Background
Beta-nicotinamide mononucleotide (Nicotinamide mononucleotide, NMN) is a naturally occurring active nucleotide that is widely found in a variety of organisms. NMN is converted into NAD+ in human body, so that the NMN has a plurality of physiological functions, such as treatment effects on neurodegenerative diseases, senile degenerative diseases, heart and brain diseases, diabetes and the like, and particularly NMN has remarkable correlation with aging-related diseases, so that NMN has great application potential in the fields of medical cosmetology, health care products and daily chemicals, and the demand of NMN in the future is great, so that the scientific community and industry are urgent to find NMN synthesis methods with cost effectiveness, environmental friendliness and high efficiency.
NMN can be synthesized by chemical, enzymatic and biological fermentation methods. Prior to 2010, large-scale NMN production was mostly achieved by chemical synthesis, which is extremely environmentally polluting, low in yield and high in production cost, and only 2/3 of the synthesized NMN is a biologically active isomer. The biological enzyme method of NMN is to take nicotinamide riboside (nicotinamide riboside, NR) as a starting material, and to react with nicotinamide riboside kinase (NR kinase, NRK) and ATP to obtain NMN in one step. The disadvantages of the enzyme catalysis method are that the addition of ATP is required, and the overall efficiency is low in a multi-enzyme system, 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 condition, simpler separation process and the like, but the current biological method for synthesizing NMN has lower production intensity, cannot meet the requirement of industrialized mass production, and leads to higher NMN price. The biological fermentation method uses sugar as raw material to produce nucleotide substances, overcomes the limiting factor in the production of enzyme method, but the optimization of metabolic pathway, the limitation of key enzyme and the entry and exit of substrate and product still have key points of breakthrough space. As an innovative method for improving the enzymatic activity of NMN biosynthetic enzyme Nampt in CN202110468908, 10 mutant strains are constructed by designing primers, and the activity of 8 mutants is higher than that of a wild type strain, wherein the activity of Nampt V365L mutants is improved by 62%, and the NMN yield of Nampt S248A, namptN L, nampt S382M, namptA T and NamptA208G is respectively improved by 34%,27%,27%,22% and 17%, but the highest yield of the optimal mutants is only 45.4mg/L. The patent HK40077691A utilizes the frame of the genetically engineered bacteria of the fermenting NMN by utilizing the amide, and introduces exogenous nicotinamide phosphoribosyl transferase NAMPT into the technical scheme of the patent, expresses substrate intake protein NiaP and product efflux protein PnuC, and finally improves the yield of NMN by less than 0.08mg/L and less than 5 times even compared with the original strain, thereby indicating that the catalytic capability and proper strength of key enzyme are important to the influence of the production level. Zhou Jingwen group in ACS Synth Biol,2022,11:2979-2988, article Systematic engineering ofEscherichia coli for efficientproduction ofnicotinamide mononucleotide from nicotinamide, teaches that the introduction of the nicotinamide transporter BcniaP does not significantly increase the shake flask level NMN yield, but rather reduces NMN yield.
In view of the foregoing, there is a need in the art for a more cost-effective, environmentally friendly and efficient method of synthesizing NMN.
Disclosure of Invention
Aiming at the defects in the prior art, the application aims to greatly improve the production efficiency of NMN by mutating key enzymes in the NMN synthesis path in the strain capable of producing NMN, and brings great potential for industrial production of NMN.
The first aspect of the application provides mutants for improving the yield of beta-nicotinamide mononucleotide, comprising mutants 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; the amino acid sequence of the mutant NiaP1 is shown as SEQ ID NO. 6.
The second aspect of the present application consists in protecting the nucleotide sequence of the gene encoding the mutant described above.
For the technical scheme, 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; the nucleotide sequence of the mutant NiaP1 is shown as SEQ ID NO. 3.
A third aspect of the present application resides in protecting a recombinant vector comprising a nucleotide sequence as described above.
A fourth aspect of the application resides in protecting recombinant cells expressing the mutants described above.
For the above technical scheme, further, the host cell of the recombinant bacterial body is selected from one of escherichia coli BL21 (DE 3), BL21 (AI), rosetta, origamiB (DE 3), JM109 (DE 3), and W3110 (DE 3). Specifically, in the examples, the recombinant vectors containing the coding genes of the mutants are pETDuet-zwf-pgl-pgi and pRSFDuet-Nampt1-PnuC2/NiaP1;
the construction process of the recombinant vector is as follows:
endogenous pgi (np_ 418449), pgl (np_ 415288), zwf (np_ 416366) were overexpressed by the plasmid vector petguet.
The Nampt1 (WP_ 012788281.1) mutant from Chitinophagapiensis, the PnuC2 (AJH 20194.1) mutant from Bacillus mycoides and the NiaP1 (CDN 64082.1) mutant from Burkholderia cenocepacia are over-expressed by another plasmid vector.
In a fifth aspect, the application resides in a method for increasing the yield of β -nicotinamide mononucleotide by converting a substrate to β -nicotinamide mononucleotide using recombinant cells expressing the mutants described above.
For the technical scheme, in the method, E.coli BL21 (DE 3) - [ delta ] nadR [ delta ] amp:: prs-Deltapncc:: lacI-NadE strain is used as a starting strain;
for the technical scheme, in the method, recombinant thalli expressing the mutant is BL-G17 delta nadR:: P T7 Nampt1, designated BLN-G17. Namely: the Nampt1 gene was integrated into the E.coli genome at the nadR sequence position, replaced with the gene, and started with the T7 promoter.
For the technical scheme, the method further comprises the step of culturing the recombinant bacterial body to OD in a culture system 600 0.5-1mM IPTG with inducer 0.5-1mM is added, induction is carried out for 12-20h at 30-37 ℃, and substrate is added at the same time, and reaction is carried out for 12-20h.
For the above-described technical scheme, further, the substrate concentration in the reaction system of the method is 0.5-10g/L nicotinamide mM, and even more preferably, in the reaction system of the method, the strain OD 600 Is reacted at pH6.0-7.0 and 30-37 deg.c in 0.5-52.
The sixth aspect of the present application is to protect the mutant, the vector or the recombinant bacterial cells from being used in the fields of foods, medicines and cosmetics.
Compared with the prior art, the application has the following beneficial effects:
(1) The recombinant obtained by the application can improve the NMN production capacity, and the production process can be amplified to obtain the NMN content reaching 26.25g/L;
(2) The combination of the mutants designed by the application can make each gene exert the respective capacity to the maximum extent, and the optimal combination is 5.7 times of the worst combination;
(3) The substrate is added to start the reaction at the same time of induction, and the production time is shortened by 35%.
Drawings
FIG. 1 shows NMN content detection in supernatants of recombinant strains BL00, BL00/1, BL 00/2.
FIG. 2 shows the NMN content detection results in recombinant strain BL00/2 and its nicotinamide riboside transferase (Nampt) nicotinamide riboside transporter (PnuC) and nicotinic acid transporter (NiaP).
FIG. 3 shows NMN content detection results in supernatants of recombinant strains BLG 1-G18.
FIG. 4 shows the effect of induction temperature on NMN content and strain growth.
FIG. 5 shows the effect of IPTG concentration on NMN content and strain growth.
Figure 6 shows the effect of induction timing on NMN content and strain growth.
Detailed Description
The present application will be further described with reference to examples, but it should be understood that the scope of the present application is not limited by the examples.
In the present application, percentages and percentages are by mass unless explicitly stated otherwise. Unless otherwise specified, all experimental procedures used are conventional and all materials, reagents, etc. used are commercially available.
1. Culture medium and reagent
LB medium (1L): 10g of tryptone, 5g of yeast powder, 10g of NaCl, and 20g of agar powder are added into the solid;
MA medium (1L): 6g of monopotassium phosphate, 16.4g of dipotassium phosphate, 5g of ammonium sulfate, 1.1g of citric acid monohydrate, 1g of magnesium sulfate and 10g of yeast powder.
2. High Performance Liquid Chromatograph (HPLC) detection
HPLC detection HPLC analysis was performed using an LC-20AD system (Shimadzu, tokyo, japan) equipped with SPD-M20A UV/Vis detector and a C18-H column (Alphasil XD-C18CH, 4.6X108 mm,5 μm). The sample injection amount is 20 mu L, the mobile phase A is 0.01mol/L potassium dihydrogen phosphate, the mobile phase B is 100% acetonitrile, and the flow rate is 1.0mL/min. At 0-8min, mobile phase A: b=100:0, mobile phase a at 8-20 min: b=30:70, mobile phase a at 20-30 min: b=30:70, column temperature 25 ℃, signal of NMN and nicotinamide was detected at 210 nm.
Preparing a standard solution related to NMN, gradually diluting the standard solution into various concentrations, establishing a standard curve graph, and calculating the NMN content of the sample to be detected according to the standard curve and the peak area result of the sample to be detected. Preparing a standard solution of nicotinamide, gradually diluting the standard solution into various concentrations, establishing a standard curve graph, and calculating the nicotinamide content of the sample to be detected according to the standard curve and the peak area result of the sample to be detected.
TABLE 1 plasmid
TABLE 2 strains
Example 1
Construction of NMN-producing strains
Expression of endogenous prs (NP-415725), pgi (NP-418449), pgl (NP-415288) gnd (np_ 416533), zwf (np_ 416366), rpiA (np_ 417389), rpiB (np_ 418514). Specific: gene prs, pgi, pgl, gnd, zwf, rpiA, rpiB, which is sequence optimized for E.coli, is sent to a manufacturer for total gene synthesis, and the synthesized products are respectively connected with pETDuet plasmid skeletons to respectively obtain E1 and E2 plasmids (Table 1).
The E1 and E2 plasmids were respectively electrotransformed into E.coli BL00 according to different combinations to obtain recombinant strains BL00/1 and BL00/2 (Table 2).
After streaking and activating the obtained recombinant strain, picking up a single clone to culture in LB liquid medium (100 mg/L containing ampicillin) at 37 ℃ and 180rpm for 16 hours, transferring 2% (v/v) seed solution to 20mL MA medium, and culturing OD 600 To 0.6, IPTG and nicotinamide were added, induced culture at 30℃and 180rpm, low-temperature centrifugation, and supernatants of the recombinant strains were collected, respectively.
The NMN content of the fermentation supernatant was measured by HPLC.
As shown in FIG. 1, the BL00/2 NMN content was highest in the fermentation supernatant. BL00/2 was therefore selected as the starting strain for subsequent studies.
Example 2
Mutant construction
Gene Nampt, pnuC, niaP, which was sequence-optimized for E.coli, was sent to a manufacturer for total gene synthesis, and the synthesized products were ligated to pRSFDuet plasmid backbones, respectively, to obtain R1, R2, R3 plasmids (Table 1).
The mutant takes R1, R2 and R3 as templates respectively for random mutation:
(1) according to QuickMutation TM The gene random mutation kit designs a primer and random mutation, wherein the primer sequence is as follows:
Random-Nampt-F：gtttaactttaataaggaggaattcATGACCAAAGAAAATCTGATTC；SEQ IDNO:7
Random-Nampt-R：atggtatatctccttgagctcTTAGATGGTGGCATTTTTAC；SEQ IDNO:8
Random-PnuC-F：aagagctcaaggagatataccatggTTCGTAGTCCACTGTTTC；SEQ IDNO:9
Random-PnuC-R：ttaagcattatgcggccgcaagcttTCAAATATAATTATTCACCCTTTC；SEQ IDNO:10
Random-NiaP-F：atgtttgaacattaaggtaccAAGGAGATATACCATGCC；SEQ IDNO:11
Random-NiaP-R：cagcggtttctttaccagactcgagTTAACTAGCTTTATCCGC；SEQ IDNO:12
(2) random mutation PCR reaction
Random mutation PCR reaction a random mutation PCR reaction system was set up with reference to table 3 below:
TABLE 3 Table 3
The PCR instrument was set up according to the following parameters:
TABLE 4 Table 4
(3) Transforming competent cells
The PCR product was taken in 4. Mu.L, and the band concentration and specificity were detected by agarose electrophoresis at 1%. And (5) electrophoresis of the rest PCR products, and cutting gel to recover the target DNA fragment. The mutated fragment is connected with a carrier skeleton and is transformed into competent cells of the escherichia coli strain, and after being uniformly mixed, the fragment is subjected to ice bath for 30min, and then is subjected to water bath heat shock for 45s at 42 ℃, and is immediately placed on ice for 2-5min. Then 250. Mu.L of LB medium was added thereto, and the mixture was incubated at 200rpm and 37℃for 1 hour, and 100 to 200. Mu.L of the bacterial liquid was allowed to incubate on a kanamycin-and ampicillin-resistant plate overnight. Obtaining a plurality of mutants, respectively transferring plasmids into BL00/2 competent cells after extracting, streaking and activating the obtained recombinant strain, picking up a single clone in LB liquid medium (100 mg/L containing ampicillin and 50mg/L containing kanamycin), culturing at 37 ℃ and 180rpm for 16h, transferring 2% (v/v) seed solution into 20mL MA medium (100 mg/L containing ampicillin and 50mg/L containing kanamycin), and culturing OD 600 To 0.6
In addition, OD will be cultivated 600 Cells overexpressing nicotinamide riboside transporter (PnuC) and mutants thereof to 0.6 were added with 0.5mM IPTG and 1g/LNMN, induced at 30℃and 180rpm, centrifuged at low temperature, and assayed for intracellular and extracellular NMN content, respectively.
Will culture OD 600 Cells overexpressing nicotinic acid transporter (NiaP) and mutants thereof to 0.6 were added with 0.5mM IPTG and 1g/L nicotinamide, induced culture at 30℃and 180rpm, and centrifuged at low temperature to detect intracellular and extracellular nicotinamide content, respectively.
A mutant of nicotinamide phosphoribosyl transferase (Nampt) results in a significant increase in NMN production, designated R11, and a strain designated BL-R11.HPLC detection of intracellular and extracellular NMN content by nicotinamide riboside transporter (PnuC) and mutants thereof, the mutants with improved properties were named R21 and R22, and the ratio of intracellular to extracellular NMN content of R2, R21 and R22 was 0.45, respectively: 0.55,0.31:0.69,0.22:0.78.
mutants of nicotinic acid transporter (NiaP) resulted in significantly lower extracellular nicotinamide concentrations were designated R31, R32. The ratio of the intracellular and extracellular nicotinamide contents of R3, R31 and R32 is 0.65:0.35,0.72:0.28,0.89:0.11.
will culture OD 600 The strain was subjected to induction culture at 30℃and 180rpm with the addition of 0.5mM IPTG and 0.5g/L nicotinamide to 0.6, and the supernatant of the recombinant strain was collected by low-temperature centrifugation, and the NMN content in the supernatant was measured.
The results of the assay are shown in FIG. 2, and it can be seen that the property enhancement of the key gene nicotinamide riboside transferase (Nampt) nicotinamide riboside transporter (PnuC) nicotinic acid transporter (NiaP) is helpful for the accumulation of NMN concentration.
Example 3
Construction of combinatorial optimized strains
According to the conventional technical means of molecular biology, the G1-G18 plasmid is expressed in BL00/2 to obtain recombinant bacterium BL00/2-G1-G18. After streaking and activating the obtained recombinant strain, picking up a single clone in LB liquid medium (100 mg/L containing ampicillin and 50mg/L containing kanamycin), culturing at 37 ℃ and 180rpm for 16h, transferring 2% (v/v) seed solution into 20mL MA medium, and culturing OD 600 To 0.6, 0.5mM IPTG and 1g/L nicotinamide were added, induced culture was performed at 30℃and 180rpm, and the supernatants of the recombinant strains were collected separately by low-temperature centrifugation, and the NMN content of the fermentation supernatants was detected by HPLC. The detection results are shown in FIG. 3. As can be seen from the results of the yields of the relevant vectors, the activity of the nicotinic acid transporter (NiaP) is not as high as possible without enhancing nicotinamide riboside transferase (Nampt), and the enhancement of nicotinic acid transport requires the cooperation of the subsequent synthesis steps and the enhancement of product excretion. As can be seen from the results of the yields of BL-G1, BL-G7, BL-G13 and related vectors, enhancement of nicotinamide riboside transporter (PnuC) can release feedback inhibition of the product, and the effect is evident with the enhancement of the overall productivity. BL-G1, BL-G4; as a result of the production of the expression strain having only nicotinamide riboside transferase as a variant, such as BL-G2 or BL-G5, it was found that enhancement of nicotinamide riboside transferase (Nampt) can improve productivity, and theoretically, the activity of the enzymeThe higher the property, the better.
Example 4
Further enhancement of nicotinamide riboside phosphate transferase (Nampt)
From the conclusion in example 3, it was found that the enhancement of nicotinamide riboside transferase did not reach the upper limit of metabolic flux, and that the gene of mutant Nampt1 was integrated into the genome in order to further increase the productivity. The integration fragment was ligated with the upstream and downstream homology arms of the integration site ΔnadR, and was then seamlessly cloned into the PstI and EcoRI sites of plasmid pUC19 to obtain pUC19-Nampt1 plasmid (Table 1). After sequencing to verify the sequence correctness, the donor fragments are integrated into the BL-G17 genome according to the escherichia coli gene replacement operation to obtain the recombinant strain BL-G17 delta nadR:: P T7 Nampt1, designated BLN-G17.
After streaking and activating the obtained recombinant strain, picking up a single clone in LB liquid medium (ampicillin 100mg/L, kanamycin 50 mg/L), culturing at 37 ℃ and 180rpm for 16 hours, transferring 2% (v/v) seed solution into 20mL MA medium (ampicillin 100mg/L, kanamycin 50 mg/L), and culturing OD 600 0.6 m MIPTG and 1G/L nicotinamide are added, induced culture is carried out at 30 ℃ and 180rpm, supernatant of the recombinant strain is collected respectively, HPLC is adopted to detect the NMN content in the fermentation supernatant, 563.6 +/-9.8 mg/L is achieved, and the ratio is improved by 1.14 times compared with BL-G17.
Comparative example 1
Further enhancement of nicotinamide riboside transporter proteins
The gene of mutant PnuC2 was integrated into the genome. The integration fragment was ligated with the upstream and downstream homology arms of the integration site ΔnadR, and was then seamlessly cloned into the PstI and EcoRI sites of plasmid pUC19 to obtain pUC19-PnuC2 plasmid (Table 2). After verifying the correct sequence by sequencing, integrating the donor fragments into the BL-G16 genome according to the escherichia coli gene replacement operation to obtain BL-G17 delta nadR: PT7-PnuC2 recombinant strain, streaking and activating the obtained recombinant strain, picking up a single clone in LB liquid medium (containing ampicillin 100mg/L and kanamycin 50 mg/L), culturing at 37 ℃ for 16 hours at 180rpm, transferring 2% (v/v) seed solution into 20mL MA medium(ampicillin 100mg/L, kanamycin 50 mg/L), OD was cultured 600 To 0.6, 0.5mM IPTG and 1G/L nicotinamide were added, induced culture was performed at 30℃and 180rpm, the supernatants of the recombinant strains were collected separately, and the NMN content in the fermentation supernatants was detected by HPLC, as detected, the content was only 463.1.+ -. 6.7mg/L, which was relatively low compared to BL-G17 production. But its growth rate becomes slow after induction, resulting in an extended fermentation time. The method shows that the outward transfer capability of the thalli is almost saturated, and the enhanced nicotinamide riboside transporter cannot further improve the yield, but has a certain influence on the growth.
Comparative example 2
Further enhancement of nicotinic acid transporter
The NiaP1 gene was integrated into the genome. The integration fragment was ligated with the upstream and downstream homology arms of the integration site ΔnadR, and then seamlessly cloned into the PstI and EcoRI sites of plasmid pUC19 to obtain pUC19-NiaP1 plasmid. After verifying the correct sequence by sequencing, integrating the donor fragment into the BL-G17 genome according to the escherichia coli gene replacement operation to obtain a recombinant strain BL-G17 delta nadR: PT7-NiaP1, streaking and activating the obtained recombinant strain, picking up a single clone in LB liquid medium (100 mg/L ampicillin and 50mg/L kanamycin) for 16h at 37 ℃, transferring 2% (v/v) seed solution into 20mL MA medium (100 mg/L ampicillin and 50mg/L kanamycin) for OD culture 600 To 0.6, 0.5mM IPTG and 1G/L nicotinamide are added, induced culture is carried out at 30 ℃ and 180rpm, low-temperature centrifugation is carried out, supernatant of the recombinant strain is collected, HPLC is adopted to detect the NMN content in the fermentation supernatant, the content only reaches 355.1 +/-6.7 mg/L, the production capacity of the recombinant strain is obviously reduced compared with BL-G17, and 16 hours are needed to reach the production capacity. It can be seen that although the copy number of the nicotinic acid transporter was increased, the productivity was not increased but decreased.
From the experimental results of the above comparative examples 1 and 2, it is apparent that the best production efficiency of NMN synthesis can be achieved without further strengthening of nicotinamide riboside transporter and nicotinic acid transporter.
Example 5
Optimization of induced fermentation conditions
Important factors influencing bacterial growth and fermentation ability when optimizing the induction conditions. By optimizing the induction process of the strain, higher product concentration, stronger production strength and lower production cost can be obtained. The induction temperature, the inducer concentration and the induction timing of BL-G17 were each optimized for this purpose.
The following experiments were optimized by a one-factor controlled variable method, cultured at 37℃and 180rpm after induction, and biomass was measured by a spectrophotometer.
Other conditions were unchanged, and the induction temperature was set at 18 ℃, 25 ℃,30 ℃ and 37 ℃. Each combination was repeated 3 times. As shown in FIG. 4, NMN expression was maximized when the induction temperature was 37 ℃.
Other conditions were unchanged, and the inducer IPTG concentrations were set at 0mM, 0.1mM, 0.25mM, 0.5mM, 0.75mM and 1.0mM. As shown in FIG. 5, NMN expression was highest at an inducer concentration of 0.5 mM.
Other conditions were unchanged, the induction timing (OD was set 600 ) 0.3, 0.6, 0.9, 1.2 and 1.5. Each combination was repeated 3 times. As shown in FIG. 6, when OD is induced 600 The NMN expression level reached the highest at 0.9.
The optimal fermentation conditions obtained by combining the results are as follows: the optimal induction temperature is 37 ℃, the optimal inducer concentration is 0.5mM, and the optimal induction time is OD 600 The NMN content reaches 586.24+ -4.2 mg/L, which is 18.97 times that of the starting strain BL00 (30.9+ -3.8 mg/L). And through the detection of sampling points at other moments, the moment when the yield reaches the highest is found to be 13h after induction, and the time is shortened by 35% compared with 20h required by the original strain.
Recombinant strain BLN-G17 glycerol seed solution was streaked on LB solid plates (ampicillin 100mg/L, kanamycin 50 mg/L) and incubated for activation at 37 ℃. After activation, the cells were inoculated into fresh liquid LB medium (ampicillin: 100 mg/L; kanamycin: 50 mg/L) and cultured with shaking at 37℃and 180rpm for 16 hours. Inoculating 2% (v/v) of the seed solution into MA fermenter (containing ampicillin 100mg/L and kanamycin 50 mg/L), maintaining the temperature at 37deg.C+ -0.5deg.C and pH at 6.7+ -0.1, and regulating ventilation (2)0-100 L.h-1) and stirring speed (100-600 rpm) to control dissolved oxygen to 10% -40%, when OD 600 At a value of 0.9, IPTG was additionally added to the medium at a final concentration of 0.5mM to induce protein expression, and cultivation was continued until a total of 10g/L nicotinamide solution was added to the medium at the late logarithmic phase to promote NMN synthesis. After fermentation for 12-13h, the HPLC assay was sampled. Determination of NMN content to 26.25g/L, biomass OD 600 =52.42, the conversion to nicotinamide reached 89%.
In summary, the comparative analysis of the data from the examples of the present application shows that: the synthesis of NMN is greatly affected by the key enzymatic activity in its pathway, the content of substrates and products in the reaction system. The nicotinamide riboside transferase (Nampt) mutant with high activity has great effect on the synthesis efficiency of NMN, and the improvement of the functions of the efflux proteins nicotinamide riboside transporter (PnuC) and nicotinic acid transporter (NiaP) is also beneficial to the synthesis of NMN, but the two are combined under reasonable relative strength to achieve the optimal effect.
The foregoing is only a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and those skilled in the art can easily understand the changes and substitutions within the scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims (10)

1. The mutant for improving the yield of the beta-nicotinamide mononucleotide is characterized by comprising mutants 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; the amino acid sequence of the mutant NiaP1 is shown as SEQ ID NO. 6.
2. A nucleotide sequence of a gene encoding the mutant of claim 1.
3. The nucleotide sequence of a gene encoding the mutant according to claim 1, wherein the nucleotide sequence of the mutant Nampt1 is shown in SEQ ID NO. 1; the nucleotide sequence of the mutant PnuC2 is shown as SEQ ID NO. 2; the nucleotide sequence of the mutant NiaP1 is shown as SEQ ID NO. 3.
4. A recombinant vector comprising the nucleotide sequence of claim 2.
5. A recombinant strain expressing the mutant of claim 1.
6. The recombinant bacterium according to claim 5, wherein said recombinant bacterium is selected from one of E.coli BL21 (DE 3), BL21 (AI), rosetta, origamiB (DE 3), JM109 (DE 3) and W3110 (DE 3).
7. A method for increasing the yield of beta-nicotinamide mononucleotide, which uses the recombinant bacteria of claim 5 to transform a substrate to generate beta-nicotinamide mononucleotide.
8. The method according to claim 7, wherein the recombinant bacterial cells are cultured to OD in a culture system 600 0.5-1mM of inducer IPTG is added, the induction is carried out for 12-20 hours at the temperature of 30-37 ℃, and simultaneously, substrate is added for reaction for 12-20 hours; the recombinant bacterial body is E.coli BL21 (DE 3) - [ delta ] nadR [ delta ] aman:: prs-delta ] pncC:: lacI-NadE.
9. The method according to claim 7, wherein the substrate concentration in the reaction system of the method is 0.5-10g/L nicotinamide.
10. The mutant according to claim 1, the vector according to claim 4 or the recombinant bacterial strain according to claim 5 for use in the fields of food, medicine and cosmetics.
CN202310968235.9A 2023-08-03 2023-08-03 Mutant for improving yield of beta-nicotinamide mononucleotide, coding gene, amino acid sequence and application thereof Pending CN116926033A (en)

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