A strain and its use in the production of beta-nicotinamide mononucleotide

Abstract

The application discloses a strain and application thereof in production of beta-nicotinamide mononucleotide. The strain is Escherichia coli, and the preservation number of the strain is CCTCC M2022922. The application provides a new strain for producing beta-nicotinamide mononucleotide (NMN), and the strain has the advantages of high yield and simple method in preparation of NMN.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation, specifically relating to a strain and its application in the production of β-nicotinamide mononucleotide. Background Technology

[0002] β-Nicotinamide mononucleotide (NMN), also known as nicotinamide mononucleotide, is a naturally occurring substance in the human body and an important source of cellular energy. NMN acts as a coenzyme I in mammals—nicotinamide adenine dinucleotide (NAD) + NMN is one of the key precursors in NAD+ synthesis and is gradually being understood and studied by researchers. NMN works by increasing NAD+ levels. + It exerts its anti-aging function. With the deepening of research on NMN, researchers believe that NMN supplementation can repair brain damage, improve pancreatic function, protect the heart from ischemia-reperfusion injury, and repair mitochondrial respiratory defects in the brain. It also has certain therapeutic effects on age-related degenerative diseases and retinal degenerative diseases. Currently, due to limitations in synthesis technology, NMN is relatively expensive.

[0003]

[0004] Currently, the technology for preparing NMN by chemical synthesis is relatively mature, such as the bromoacetylribose method, the TMSOTF catalytic condensation method, the AMP acid hydrolysis catalytic method, and the ketalization protected synthesis method. However, these methods have drawbacks such as harsh preparation conditions, low safety, and high production costs.

[0005] The biosynthesis of NMN includes enzymatic and fermentation methods. Enzymatic route one uses phosphoribosyl pyrophosphate (PRPP) and nicotinamide (NAM) as substrates to generate NMN under the catalysis of nicotinamide phosphoribosyltransferase (NAMPT). Route two uses nicotinamide nucleoside (NR) as a substrate and ATP as a phosphate donor, undergoing phosphorylation under the catalysis of nicotinamide nucleoside kinase (NRK) to generate NMN. Route three uses D-5-phosphoribose and nicotinamide as raw materials, and in the presence of ATP, utilizes a combination of phosphoribosyl pyrophosphate synthase (Prs) and NAMPT enzymes to synthesize NMN.

[0006]

[0007] Fermentation methods, such as those described by 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.), involve modifying the genes of enzymes involved in NMN production along metabolic pathways to obtain E. coli strains that produce NMN. These strains use glucose and nicotinamide as substrates to ferment and produce NMN, achieving a yield of up to 6.79 g / L. However, this method has relatively low yields, making it unsuitable for large-scale industrial production. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a strain and its application in the production of β-nicotinamide mononucleotide (NMN). This invention provides a novel strain for producing β-nicotinamide mononucleotide (NMN). Using this strain to prepare NMN offers advantages such as high yield and simple method. For example, the NMIS208 strain of this invention can achieve a high yield of up to 14.50 g / L in NMN production.

[0009] The first aspect of the present invention provides a strain of Escherichia coli, the strain having the accession number CCTCC M2022922.

[0010] The Escherichia coli strain described in this invention is the NMIS208 strain.

[0011] A second aspect of the present invention provides a microbial cell obtained by culturing the strain described in the first aspect of the present invention.

[0012] The culture medium used for the culture can be a conventional culture medium in the art suitable for the growth of Escherichia coli.

[0013] Preferably, the culture medium used is TB medium or M9 medium.

[0014] The TB medium or the M9 medium can be a conventional culture medium in the art. Preferably, the M9 medium formula (concentration g / L) is: disodium hydrogen phosphate dodecahydrate 17.9; potassium dihydrogen phosphate 6.8; glycerol 5 mL; tryptone 5; yeast extract 5; ammonium chloride 2.7; anhydrous sodium sulfate 0.71; anhydrous magnesium sulfate 0.24; manganese chloride tetrahydrate 0.02; ferric chloride 0.016; calcium chloride 0.01.

[0015] Preferably, the TB culture medium formulation (g / L) is: tryptone 10; yeast extract 18; glycerol 4 mL; dipotassium hydrogen phosphate 16.43; potassium dihydrogen phosphate 2.31.

[0016] More preferably, the culture medium for culturing the NMIS208 strain contains 40 μg / mL Km, 40 μg / mL Spec, and 20 μg / mL Cm.

[0017] Km stands for kanamycin, Spec for streptomycin, and Cm for chloramphenicol.

[0018] In a preferred embodiment, the cultivation includes the following steps:

[0019] a) Use TB medium or M9 medium for culture; the culture conditions are 25-40℃ and 100-300rpm.

[0020] b) When OD is cultured in a) 600 When the concentration reaches 0.6-1, add 0.1mM of an inducer for induction culture, wherein the inducer is IPTG, and the induction culture conditions are 25-40℃, 100-300rpm, 12-24h.

[0021] Preferably, the culture medium also contains 40 μg / mL kanamycin, 40 μg / mL streptomycin, and 20 μg / mL chloramphenicol.

[0022] In a preferred embodiment, the microbial cells are obtained by the following method:

[0023] The glycerol strain was inoculated at a 4% inoculum into 5 mL of LB medium (containing 50 μg / mL Km, 50 μg / mL Spec, and 25 μg / mL Cm) and cultured at 37℃ and 250 rpm for 4 h. Then, it was transferred at a 1% inoculum to TB medium or M9 medium (containing 40 μg / mL Km, 40 μg / mL Spec, and 20 μg / mL Cm) and cultured at 37℃ and 250 rpm. When OD... 600 When the concentration reaches approximately 0.8, add IPTG to a final concentration of 0.1 mM and incubate at 25°C and 250 rpm for 16 h.

[0024] A third aspect of the present invention provides a method for producing NMN, which uses the microbial cells described in the second aspect of the present invention to react with a substrate in a reaction system to produce the NMN, wherein the substrate is glucose and nicotinamide.

[0025] In the method, preferably, the reaction system contains phosphate buffer and / or culture medium.

[0026] The culture medium may be a conventional culture medium in the art suitable for the growth of Escherichia coli.

[0027] Preferably, the culture medium is TB medium or M9 medium.

[0028] Preferably, the phosphate buffer solution is PBS buffer and / or MB buffer, wherein the MB buffer solution comprises Na2HPO4, K2HPO4, NH4Cl and NaCl.

[0029] In a preferred embodiment, the MB buffer solution comprises 6.8 g / L Na2HPO4, 3.0 g / L K2HPO4, 1.0 g / L NH4Cl, and 0.5 g / L NaCl.

[0030] The PBS buffer is standard in the art.

[0031] In a preferred embodiment, the phosphate buffer is a mixed buffer of PBS buffer and MB buffer.

[0032] Preferably, the mixing buffer comprises 300 mM PBS buffer and 2 MB buffer.

[0033] In a preferred embodiment, the microbial cells are prepared into a suspension and then mixed with the substrate to obtain the reaction system.

[0034] The preparation of the bacterial suspension includes any one of the following:

[0035] i: The microbial cells are suspended in phosphate buffer to obtain the cell suspension; preferably, the mass-to-volume ratio of the microbial cells to the phosphate buffer is 1:(5-20), for example 1:10;

[0036] ii: The microbial cells are mixed with the supernatant of the culture medium from which they are cultured to obtain the microbial cell suspension; preferably, the mass-to-volume ratio of the microbial cells to the supernatant of the culture medium is 1:(5-20), for example, 1:10.

[0037] Preferably, when the bacterial suspension is prepared by method i, the substrate is dissolved in the PBS phosphate buffer and mixed with the bacterial suspension; when the bacterial suspension is prepared by method ii, the substrate is dissolved in the mixing buffer and mixed with the bacterial suspension.

[0038] In the method, preferably, in the reaction system:

[0039] The concentration of nicotinamide in the reaction system is 5-10 g / L, for example, 8 g / L.

[0040] The mass ratio of nicotinamide to glucose is 1:(1.5-5), for example 1:2.625.

[0041] The mass-to-volume ratio of the microbial cells to the reaction system is 1:(10-30), for example, 1:20.

[0042] The mass-to-volume ratio refers to g / mL.

[0043] The mass ratio of the microbial cells to the nicotinamide is 1:(0.1 to 0.3), for example, 1:0.16.

[0044] In a preferred embodiment, the microbial cells are wet cells obtained by centrifuging the bacterial culture solution of the strain described in the first aspect. Specifically, they can be obtained by centrifuging the bacterial culture solution at 4°C and 4000 rpm for 15 min, collecting the supernatant and wet cells separately, and storing them at 4°C for later use.

[0045] In a preferred embodiment, the reaction is carried out by stirring or shaking.

[0046] Preferably, the reaction conditions are 25-35℃, 200-300rpm and 6-24h, more preferably 30℃, 220rpm and 8-24h.

[0047] In a preferred embodiment, when the reaction time reaches 4-6 hours, the substrate is supplemented so that the concentration of glucose in the reaction system is 7-15 g / L, for example 10.5 g / L, and the concentration of nicotinamide in the reaction system is 3-5 g / L, for example 4 g / L.

[0048] The fourth aspect of this invention provides the application of the Escherichia coli strain described in the first aspect of this invention or the microbial cells described in the second aspect of this invention in the preparation of NMN.

[0049] The NMN mentioned in this invention refers to β-nicotinamide mononucleotide (also known as nicotinamide mononucleotide, NMN).

[0050] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0051] The reagents and raw materials used in this invention are all commercially available.

[0052] The positive and progressive effects of this invention are as follows: This invention provides a new strain for producing NMN. The preparation of NMN using this strain has the advantages of high yield and simple method. For example, the NMIS208 strain of this invention can bring a high yield of up to 14.50 g / L when preparing NMN.

[0053] Information on the preservation of biological materials

[0054] The Escherichia coli NMIS208 strain of the present invention was deposited on June 20, 2022, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Luojia Mountain, Wuchang District, Wuhan, Hubei Province, China, 430072, with accession number CCTCC M2022922 and culture name Escherichia coli NMIS208 strain. Detailed Implementation

[0055] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0056] In the examples, the M9 culture medium formulation (concentration g / L) used was as follows: disodium hydrogen phosphate dodecahydrate 17.9; potassium dihydrogen phosphate 6.8; glycerol 5 mL; tryptone 5; yeast extract 5; ammonium chloride 2.7; anhydrous sodium sulfate 0.71; anhydrous magnesium sulfate 0.24; manganese chloride tetrahydrate 0.02; ferric chloride 0.016; calcium chloride 0.01.

[0057] TB medium formula (g / L): tryptic peptone 10; yeast extract 18; glycerol 4 mL; dipotassium hydrogen phosphate 16.43; potassium dihydrogen phosphate 2.31.

[0058] MB buffer (g / L): Na2HPO4 6.8, K2HPO4 3.0, NH4Cl 1.0, NaCl 0.5.

[0059] In the examples, the experimental results were detected using high performance liquid chromatography (HPLC). The chromatographic column was Welch Ultimate AQ-C18 (5 μm, 250 × 4.6 mm), the flow rate was 1 mL / min, the column temperature was 35 °C, the detection wavelength was 260 nm, and the injection volume was 5 μL.

[0060] Mobile phase: A: 10mM ammonium acetate + 0.1% formic acid, B: acetonitrile.

[0061] The gradient elution procedure is shown in Table 1 below:

[0062] Table 1

[0063]

[0064] Example 1: Culture of NMIS208 strain

[0065] Inoculate 4% of NMIS208 glycerol bacteria into 5 mL of LB medium (containing 50 μg / mL Km, 50 μg / mL Spec, and 25 μg / mL Cm) and incubate at 37℃ and 250 rpm for 4 h. Then, transfer 1% of the inoculum to TB or M9 medium (containing 40 μg / mL Km, 40 μg / mL Spec, and 20 μg / mL Cm) and incubate at 37℃ and 250 rpm. When OD... 600 When the concentration reaches approximately 0.8, add IPTG to a final concentration of 0.1 mM and incubate at 25°C and 250 rpm for 16 h.

[0066] Centrifuge the above bacterial culture at 4℃ and 4000rpm for 15min, collect the supernatant and wet bacterial cells separately, and store them at 4℃ for later use.

[0067] Example 2: Preparation of NMN by Fermentation TB-Catalysis

[0068] (1) Preparation of reaction substrate solution: Add glucose and nicotinamide to 300mM PBS buffer to obtain reaction substrate solution, wherein the glucose concentration is 42g / L and the nicotinamide concentration is 16g / L.

[0069] (2) Preparation of bacterial suspension:

[0070] Dilute the 10*MB buffer solution 5 times to obtain a 2*MB buffer solution. Take the NMIS208 wet cells described in Example 1 and add them to the 2*MB buffer solution at a ratio of 1:10 (g / mL) to suspend them, thus obtaining a cell suspension.

[0071] (3) Transformation reaction and result detection: The prepared reaction substrate solution and bacterial suspension were mixed at a volume ratio of 1:1 to obtain a 20 mL reaction system, and reacted at 30℃ and 220 rpm. Samples were taken for detection after 4 h, and the HPLC detection results are shown in Table 2.

[0072] The detection method is as follows: Take 1 mL of reaction solution, centrifuge at 12000 rpm for 10 min, take 100 μL of supernatant, add 880 μL of deionized water and 20 μL of 20% hydrochloric acid solution, mix, centrifuge at 12000 rpm for 10 min, filter the obtained supernatant and perform HPLC determination.

[0073] (4) Feeding: After 6 hours of reaction conversion, glucose and nicotinamide were added to the reaction solution until their concentrations reached 10.5 g / L and 4 g / L, respectively. Samples were taken again after 24 hours, and the detection method was the same as above. The detection results are shown in Table 2.

[0074] Table 2. Experimental results of NMN preparation by fermentation TB-catalysis method

[0075]

[0076] The results show that, under the conditions of this embodiment, when the reaction conversion lasts for 24 hours, the yield of NMN can reach 12.96 g / L.

[0077] Example 3: Preparation of NMN by Two-Stage Fermentation Method

[0078] (1) Preparation of reaction substrate solution: Mix and dilute 1.5M PBS buffer and 10*MB buffer to obtain 300mMPBS+2*MB mixed buffer. Add glucose and nicotinamide to prepare reaction substrate solution, wherein the glucose concentration is 42g / L and the nicotinamide concentration is 16g / L.

[0079] (2) Preparation of bacterial suspension: The NMIS208 wet bacterial cells described in Example 1 and the supernatant of culture medium M9 were mixed at a mass-volume ratio of 1:10 (g / mL) to obtain the bacterial suspension.

[0080] (3) Transformation reaction and result detection: The prepared reaction substrate solution and bacterial suspension were mixed at a volume ratio of 1:1 to obtain a 20 mL reaction system, and the reaction was carried out at 30℃ and 220 rpm. After 4 h of reaction, samples were taken for detection. The detection method was the same as described in Example 2. The HPLC detection results are shown in Table 3.

[0081] (4) Feeding: After 6 hours of reaction conversion, glucose and nicotinamide were added to the reaction solution until their concentrations reached 10.5 g / L and 4 g / L, respectively. Samples were taken again after 24 hours, and the detection method was the same as above. The HPLC detection results are shown in Table 3.

[0082] Table 3. Experimental results of NMN preparation by the two-stage fermentation method.

[0083]

[0084] The results show that when the bacterial cells are resuspended in the original culture medium and the reaction is carried out for 24 hours, the yield of NMN can reach 14.50 g / L.

Claims

1. A type of Escherichia coli ( Escherichia coli ), characterized in that, The preservation number of the *Escherichia coli* is CCTCCM2022922.

2. A method for culturing Escherichia coli as described in claim 1, characterized in that... The cultivation process includes the following steps: a) Use TB medium or M9 medium for culture; the culture conditions are 25-40℃ and 100-300 rpm. b) When OD is cultured in a) 600 When the concentration reaches 0.6-1, add 0.1 mM of an inducer, namely IPTG, for induction culture. The induction culture conditions are 25-40℃, 100-300 rpm, and 12-24 h.

3. The cultivation method as described in claim 2, characterized in that, The culture medium described in step a) also contains 40 μg / mL kanamycin, 40 μg / mL streptomycin, and 20 μg / mL chloramphenicol.

4. A method for producing NMN, characterized in that, The NMN is produced by reacting Escherichia coli as described in claim 1 with a substrate in a reaction system, wherein the substrate is glucose and nicotinamide.

5. The method as described in claim 4, characterized in that, The reaction system contains phosphate buffer and / or culture medium.

6. The method as described in claim 5, characterized in that, The culture medium is TB medium or M9 medium, the phosphate buffer is PBS buffer and / or MB buffer, and the MB buffer solution includes Na2HPO4, K2HPO4, NH4Cl and NaCl.

7. The method as described in claim 6, characterized in that, The MB buffer solution comprises 6.8 g / L Na2HPO4, 3.0 g / L K2HPO4, 1.0 g / L NH4Cl, and 0.5 g / L NaCl.

8. The method as described in claim 5, characterized in that, The phosphate buffer is a mixture of PBS buffer and MB buffer.

9. The method as described in claim 8, characterized in that, The mixing buffer contains 300 mM PBS buffer and 2 MB buffer.

10. The method as described in claim 4, characterized in that, In the reaction system: The concentration of nicotinamide in the reaction system is 5-10 g / L; and / or, The mass ratio of nicotinamide to glucose is 1:(1.5~5); and / or, The mass-to-volume ratio of *E. coli* in the reaction system is 1:(10~30); and / or, The mass ratio of Escherichia coli to nicotinamide is 1:(0.1~0.3).

11. The method as described in claim 10, characterized in that, In the reaction system: The concentration of 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 Escherichia coli accounts for a mass-to-volume ratio of 1:20 in the reaction system; and / or, The mass ratio of Escherichia coli to nicotinamide is 1:0.

16.

12. The method according to any one of claims 4-11, characterized in that, The reaction is carried out by stirring or shaking.

13. The method as described in claim 12, characterized in that, The reaction conditions are 25-35 °C, 200-300 rpm, and 6-24 h.

14. The method as described in claim 13, characterized in that, The reaction conditions were 30 °C, 220 rpm, and 8–24 h.

15. The method as described in claim 12, characterized in that, When the reaction time reaches 4-6 h, the substrate is replenished so 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. The method as described in claim 15, characterized in that, When the reaction time reaches 6 h, the substrate is replenished so that the concentration of glucose in the reaction system is 10.5 g / L and the concentration of nicotinamide in the reaction system is 4 g / L.

17. The *Escherichia coli* as described in claim 1 (… Escherichia coli Application of NMN in preparation.

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