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CN-117402766-B - Strain and application thereof in production of beta-nicotinamide mononucleotide

CN117402766BCN 117402766 BCN117402766 BCN 117402766BCN-117402766-B

Abstract

The invention discloses a strain and application thereof in the production of beta-nicotinamide mononucleotide. A strain of escherichia coli (ESCHERICHIA COLI) having a accession number cctccc M2022922. The invention provides a novel strain for producing beta-Nicotinamide Mononucleotide (NMN), and the strain is used for preparing NMN, and has the advantages of high yield and simple method.

Inventors

  • WU YAN
  • WANG SHU
  • ZHENG XIAOFU
  • TIAN ZHENHUA

Assignees

  • 弈柯莱生物科技(集团)股份有限公司

Dates

Publication Date
20260512
Application Date
20220706

Claims (17)

  1. 1. An escherichia coli (ESCHERICHIA COLI) is characterized by having a preservation number of CCTCC M2022922.
  2. 2. A method of culturing escherichia coli as set forth in claim 1, characterized in that the culturing comprises the steps of: a) Culturing in TB medium or M9 medium at 25-40deg.C and 100-300 rpm; b) When the OD 600 in the culture in the a) reaches 0.6-1, adding an inducer of 0.1 mM for induction culture, wherein the inducer is IPTG, and the induction culture conditions are 25-40 ℃,100-300 rpm and 12-24 h.
  3. 3. The method according to claim 2, wherein 40. Mu.g/mL kanamycin, 40. Mu.g/mL streptomycin, 20. Mu.g/mL chloramphenicol are further added to the medium in step a).
  4. 4. A method for producing NMN, characterized in that the NMN is produced by reacting escherichia coli according to claim 1 with a substrate in a reaction system, wherein the substrate is glucose and nicotinamide.
  5. 5. The method of claim 4, wherein the reaction system comprises phosphate buffer and/or culture medium.
  6. 6. The method of claim 5, wherein the medium is TB medium or M9 medium, the phosphate buffer is PBS buffer and/or MB buffer, and the MB buffer solution comprises Na 2 HPO 4 、K 2 HPO 4 、NH 4 Cl and NaCl.
  7. 7. The method of claim 6, wherein the MB buffer solution comprises Na 2 HPO 4 6.8 g/L、K 2 HPO 4 3.0 g/L、NH 4 Cl 1.0 g/L, naCl 0.5 g/L.
  8. 8. The method of claim 5, wherein the phosphate buffer is a mixed buffer of PBS buffer and MB buffer.
  9. 9. The method of claim 8, wherein the mixed buffer comprises 300 mM PBS buffer and 2 MB buffer.
  10. 10. The method according to claim 4, wherein in the reaction system: the concentration of the nicotinamide in the reaction system is 5-10 g/L, and/or, The mass ratio of the nicotinamide to the glucose is 1 (1.5-5), and/or, The mass volume ratio of the escherichia coli to the reaction system is 1 (10-30), and/or, The mass ratio of the escherichia coli to the nicotinamide is 1 (0.1-0.3).
  11. 11. The method of claim 10, wherein in the reaction system: the concentration of the nicotinamide in the reaction system is 8 g/L, and/or, The mass ratio of nicotinamide to glucose is 1:2.625, and/or, The mass volume ratio of the escherichia coli to the reaction system is 1:20, and/or, The mass ratio of the escherichia coli to the nicotinamide is 1:0.16.
  12. 12. The method according to any one of claims 4 to 11, wherein the reaction is carried out by stirring or shaking.
  13. 13. The method of claim 12, wherein the reaction conditions are 25-35 ℃, 200-300rpm, and 6-24 h.
  14. 14. The method of claim 13, wherein the reaction conditions are 30 ℃, 220 rpm, and 8-24 hours.
  15. 15. The method of claim 12, wherein the substrate is replenished when the reaction time reaches 4-6 h, such that the concentration of glucose in the reaction system is 7-15 g/L and the concentration of nicotinamide in the reaction system is 3-5 g/L.
  16. 16. The method of claim 15, wherein the substrate is replenished when the reaction time reaches 6h, such that the glucose is at a concentration of 10.5g/L and the nicotinamide is at a concentration of 4.4 g/L.
  17. 17. Use of escherichia coli (ESCHERICHIA COLI) according to claim 1 for the preparation of NMN.

Description

Strain and application thereof in production of beta-nicotinamide mononucleotide Technical Field The invention belongs to the field of biological fermentation, and particularly relates to a strain and application thereof in the production of beta-nicotinamide mononucleotide. Background Beta-nicotinamide mononucleotide, also known as nicotinamide mononucleotide (nicotinamide mononucleotide, NMN), is a naturally occurring substance in the human body and is one of the important sources of cellular energy. NMN is becoming known and studied by researchers as one of the key precursors for the synthesis of nicotinamide adenine nucleotide (nicotinamide adenine dinucleotide, NAD +) which is coenzyme I in mammals. NMN exerts anti-aging functions by increasing NAD + levels. With the gradual deep research on NMN, researchers consider that the NMN can repair brain injury, improve islet function, protect heart from ischemia reperfusion injury and repair brain mitochondrial respiratory defects, and has certain treatment effects on senile degenerative diseases, retinal degenerative diseases and the like. At present, NMN is expensive due to the limitation of the synthesis process. The existing technology for preparing NMN by chemical synthesis is mature, such as bromoacetyl ribose method, TMSOTF catalytic condensation method, AMP acid hydrolysis catalytic method, ketalization protection synthesis method and the like, but has the defects of harsh preparation conditions, low safety, high production cost and the like. Biosynthesis of NMN includes enzymatic and fermentation processes. The first enzymatic method uses phosphoribosyl pyrophosphate (PRPP) and Nicotinamide (NAM) as substrates to generate NMN under the catalysis of nicotinamide phosphoribosyl transferase (NAMPT), the second enzymatic method uses Nicotinamide Riboside (NR) as substrates and ATP as a phosphate donor to generate NMN by phosphorylation under the catalysis of Nicotinamide Riboside Kinase (NRK), and the third enzymatic method uses D-5-phosphoribosyl and nicotinamide as raw materials to synthesize NMN by catalyzing with the combination of phosphoribosyl pyrophosphate synthetase (Prs) and NAMPT enzyme in the presence of ATP. Fermentation processes such as Ss A,Ty A,Hm A,et al.(Metabolic design for selective production of nicotinamide mononucleotide from glucose and nicotinamide-ScienceDirect[J].Metabolic Engineering,2020.) produce NMN-producing E.coli strains by genetic engineering of enzymes in the metabolic pathway that are involved in NMN production. The strain takes glucose and nicotinamide as substrates, NMN is prepared by fermentation, and the yield can reach 6.79g/L. The method has relatively low yield and is unfavorable for large-scale industrialized production. Disclosure of Invention In order to solve the technical problems, the invention provides a strain and application thereof in the production of beta-nicotinamide mononucleotide. The invention provides a novel strain for producing beta-nicotinamide mononucleotide (also called nicotinamide mononucleotide, nicotinamide mononucleotide, NMN), and the preparation of NMN by using the strain has the advantages of high yield and simple method, for example, the NMIS208 strain can produce NMN with high yield of up to 14.50 g/L. The first aspect of the invention provides a strain of escherichia coli (ESCHERICHIA COLI), wherein the preservation number of the strain is CCTCC M2022922. The strain of the escherichia coli (ESCHERICHIA COLI) is NMIS208 strain. In a second aspect, the present invention provides a microbial cell obtained by culturing the strain according to the first aspect of the present invention. The medium used for the culture may be a medium conventional in the art suitable for E.coli growth. Preferably, the culture medium used for the culture is a TB medium or an M9 medium. The TB medium or the M9 medium can be a conventional medium in the art, and preferably, the formula (concentration g/L) of the M9 medium comprises 17.9 parts of disodium hydrogen phosphate dodecahydrate, 6.8 parts of potassium dihydrogen phosphate, 5mL of glycerol, 5 parts of tryptone, 5 parts of yeast extract, 2.7 parts of ammonium chloride, 0.71 parts of anhydrous sodium sulfate, 0.24 parts of anhydrous magnesium sulfate, 0.02 parts of manganese chloride tetrahydrate, 0.016 parts of ferric chloride and 0.01 parts of calcium chloride. Preferably, the TB medium formulation (g/L) is tryptone 10, yeast extract 18, glycerol 4mL, dipotassium hydrogen phosphate 16.43, potassium dihydrogen phosphate 2.31. More preferably, the medium contains 40. Mu.g/mL Km, 40. Mu.g/mL Spec, 20. Mu.g/mL Cm when the NMIS208 strain is cultured. Km is kanamycin, spec is streptomycin, cm is chloramphenicol. In a preferred embodiment, the culturing comprises the steps of: a) Culturing in TB medium or M9 medium at 25-40deg.C and 100-300rpm. B) When the OD 600 in the a) is 0.6-1, adding 0.1mM inducer for induction culture, wherein the inducer is IPTG