WO2024008046A1 - STRAIN AND USE THEREOF IN PRODUCTION OF β-NICOTINAMIDE MONONUCLEOTIDE - Google Patents

STRAIN AND USE THEREOF IN PRODUCTION OF β-NICOTINAMIDE MONONUCLEOTIDE Download PDF

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WO2024008046A1
WO2024008046A1 PCT/CN2023/105573 CN2023105573W WO2024008046A1 WO 2024008046 A1 WO2024008046 A1 WO 2024008046A1 CN 2023105573 W CN2023105573 W CN 2023105573W WO 2024008046 A1 WO2024008046 A1 WO 2024008046A1
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buffer
strain
nicotinamide
nmn
culture
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吴燕
王舒
郑孝富
田振华
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弈柯莱生物科技(上海)股份有限公司
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the field of biological fermentation, and specifically relates to a bacterial strain and its application in the production of ⁇ -nicotinamide mononucleotide.
  • ⁇ -Nicotinamide mononucleotide also known as nicotinamide mononucleotide (NMN)
  • NMN nicotinamide mononucleotide
  • NAD + coenzyme I - nicotinamide adenine dinucleotide
  • NMN neuropeptide derived neuropeptide
  • pancreatic islet function protects the heart from ischemia-reperfusion injury, repair brain mitochondrial respiratory defects, and have a beneficial effect on senile degenerative diseases, retinal degenerative diseases, etc. All have certain therapeutic effects.
  • NMN is relatively expensive due to limitations in the synthesis process.
  • the technology for preparing NMN through chemical synthesis is relatively mature, such as the bromoacetyl ribose method, TMSOTF catalytic condensation method, AMP acid hydrolysis catalytic method, ketal protection synthesis method, etc.
  • TMSOTF catalytic condensation method TMSOTF catalytic condensation method
  • AMP acid hydrolysis catalytic method ketal protection synthesis method
  • NMN The biosynthesis of NMN includes enzymatic and fermentative methods. Enzymatic method route one uses phosphoribosyl pyrophosphate (PRPP) and nicotinamide (NAM) as substrates in nicotinamide phosphoribosyltransferase (NAMPT) NMN is generated under the catalysis of nicotinamide riboside (NR); route two uses nicotinamide riboside (NR) as the substrate, ATP is the phosphate donor, and phosphorylation occurs under the catalysis of nicotinamide riboside kinase (NRK) to generate NMN; route three uses D-5-phosphate ribose and nicotinamide are used as raw materials. In the presence of ATP, NMN is synthesized using a combination of phosphoribosyl pyrophosphate synthase (Prs) and NAMPT enzyme for catalysis.
  • Prs phosphorib
  • Fermentation methods 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.)
  • Ss A, Ty A, Hm A Metalotinamide mononucleotide from glucose and nicotinamide-ScienceDirect[J]. Metabolic Engineering, 2020.
  • the enzyme was genetically modified to produce an NMN-producing E. coli strain. This strain uses glucose and nicotinamide as substrates to ferment NMN, and its yield can reach 6.79g/L. The yield of this method is relatively low and is not conducive to large-scale industrial production.
  • the present invention provides a strain and its application in producing ⁇ -nicotinamide mononucleotide.
  • the present invention provides a new strain for producing ⁇ -nicotinamide mononucleotide (also known as nicotinamide mononucleotide, NMN).
  • NMN nicotinamide mononucleotide
  • Using the strain to prepare NMN has the advantages of high yield and simple method.
  • the present invention The preparation of NMN by the NMIS208 strain can bring high yields up to 14.50g/L.
  • a first aspect of the present invention provides a strain of Escherichia coli, the deposit number of which is CCTCC M2022922.
  • the Escherichia coli strain of the present invention is the NMIS208 strain.
  • a second aspect of the present invention provides a microbial cell, which is obtained by culturing the strain described in the first aspect of the present invention.
  • the culture medium used for the culture may be a conventional medium in the art suitable for the growth of E. coli.
  • the culture medium used for culture is TB medium or M9 medium.
  • the TB culture medium or the M9 culture medium can be a conventional culture medium in this field.
  • the M9 culture medium formula (concentration g/L) is: disodium hydrogen phosphate dodecahydrate 17.9; potassium dihydrogen phosphate 6.8; glycerol 5mL; 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.
  • the TB medium formula (g/L): tryptone 10; yeast extract 18; glycerol 4 mL; dipotassium hydrogen phosphate 16.43; potassium dihydrogen phosphate 2.31.
  • the medium when culturing the NMIS208 strain, contains 40 ⁇ g/mL Km, 40 ⁇ g/mL Spec, and 20 ⁇ g/mL Cm.
  • Km is kanamycin
  • Spec is streptomycin
  • Cm chloramphenicol
  • the culturing includes the following steps:
  • the culture medium used is TB medium or M9 medium for culture; the culture conditions are 25-40°C, 100-300rpm.
  • kanamycin Preferably, 40 ⁇ g/mL kanamycin, 40 ⁇ g/mL streptomycin, and 20 ⁇ g/mL chloramphenicol are also added to the culture medium.
  • the microbial cells are obtained by the following method:
  • the glycerol bacteria of the strain were inoculated into 5 mL LB medium (containing 50 ⁇ g/mL Km, 50 ⁇ g/mL Spec, 25 ⁇ g/mL Cm) at an inoculum volume of 4%, and cultured at 37°C and 250 rpm for 4 hours. Transfer 1% of the inoculum into TB medium or M9 medium (containing 40 ⁇ g/mL Km, 40 ⁇ g/mL Spec, 20 ⁇ g/mL Cm), and culture at 37°C and 250 rpm. When the OD 600 reaches about 0.8 , add IPTG with a final concentration of 0.1mM, and induce culture for 16h at 25°C and 250rpm.
  • 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 and a substrate to react in a reaction system to produce the NMN, and the substrate is glucose and nicotinamide.
  • the reaction system contains phosphate buffer and/or culture medium.
  • the culture medium may be a conventional culture medium in the art suitable for the growth of E. coli.
  • the culture medium is TB culture medium or M9 culture medium.
  • the phosphate buffer solution is PBS buffer and/or MB buffer
  • the MB buffer solution includes Na 2 HPO 4 , K 2 HPO 4 , NH 4 Cl and NaCl.
  • the MB buffer includes Na 2 HPO 4 6.8g/L, K 2 HPO 4 3.0g/L, NH 4 Cl 1.0g/L, and NaCl 0.5g/L.
  • the PBS buffer is routine in the art.
  • the phosphate buffer is a mixed buffer of PBS buffer and MB buffer.
  • the mixed buffer contains 300mM PBS buffer and 2*MB buffer.
  • the microbial cells are prepared into a cell suspension and then mixed with the substrate to obtain the reaction system.
  • the preparation of the bacterial cell suspension includes any of the following:
  • the microbial cells are added to the phosphate buffer for suspension to obtain the bacterial cell suspension; preferably, the mass-to-volume ratio of the microbial cells to the phosphate buffer is 1:(5-20) For example 1:10;
  • the bacterial cell suspension is obtained after mixing the microbial cells and the culture medium supernatant for culturing them; preferably, the mass-to-volume ratio of the microbial cells and the culture medium supernatant is 1:(5 ⁇ 20) For example, 1:10.
  • the substrate when the method of i is used to prepare the bacterial cell suspension, the substrate is dissolved in the PBS phosphate buffer and mixed with the bacterial cell suspension; when the method of ii is used to prepare the bacterial cell suspension.
  • the substrate is dissolved in the mixing buffer and mixed with the bacterial cell suspension.
  • the concentration of nicotinamide in the reaction system is 5 to 10 g/L, such as 8 g/L.
  • the mass ratio of nicotinamide to glucose is 1: (1.5-5), for example, 1:2.625.
  • the mass-volume ratio of the microbial cells in the reaction system is 1: (10-30), for example, 1:20.
  • the mass to volume ratio refers to g/mL.
  • the mass ratio of the microbial cells to the nicotinamide is 1: (0.1-0.3), such as 1:0.16.
  • the microbial cells are wet bacterial cells obtained by centrifuging the bacterial liquid cultured by the bacterial strain described in the first aspect. Specifically, it can be obtained by the following method: obtain the bacterial liquid of microbial cells and centrifuge it at 4°C and 4000rpm for 15 minutes, collect the supernatant and wet bacterial cells respectively, and store them in a refrigerator at 4°C until use.
  • reaction is carried out by stirring or shaking.
  • the reaction conditions are 25-35°C, 200-300rpm and 6-24h, more preferably 30°C, 220rpm and 8-24h.
  • the substrate when the reaction time reaches 4-6 hours, the substrate is supplemented so that the concentration of glucose in the reaction system is 7-15g/L, such as 10.5g/L, And the concentration of nicotinamide in the reaction system is 3 to 5 g/L, such as 4 g/L.
  • the fourth aspect of the present invention provides a use of the Escherichia coli strain described in the first aspect of the present invention or the microbial cells described in the second aspect of the present invention in the preparation of NMN.
  • NMN in the present invention refers to ⁇ -nicotinamide mononucleotide (also known as nicotinamide mononucleotide, NMN).
  • the reagents and raw materials used in the present invention are all commercially available.
  • the positive and progressive effect of the present invention is that the present invention provides a new strain for producing NMN.
  • the use of the strain to prepare NMN has the advantages of high yield and simple method.
  • the NMIS208 strain of the present invention can produce NMN up to 14.50g/L. high yield.
  • the Escherichia coli NMIS208 strain of the present invention was deposited in the China Type Culture Collection Center (CCTCC) on June 20, 2022.
  • the deposit address is: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei province, China, Postal Code: 430072,
  • the deposit number is CCTCC M2022922, and the culture name is Escherichia coli NMIS208 strain.
  • the M9 medium formula (concentration g/L) used 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.
  • MB buffer (g/L): Na 2 HPO 4 6.8, K 2 HPO 4 3.0, NH 4 Cl 1.0, NaCl 0.5.
  • Mobile phase A: 10mM ammonium acetate + 0.1% formic acid, B: acetonitrile.
  • NMIS208 glycerol bacteria into 5 mL LB medium (containing 50 ⁇ g/mL Km, 50 ⁇ g/mL Spec, 25 ⁇ g/mL Cm) at an inoculum volume of 4%, and culture it at 37°C and 250 rpm for 4 hours. Transfer 1% of the inoculum into TB or M9 medium (containing 40 ⁇ g/mL Km, 40 ⁇ g/mL Spec, 20 ⁇ g/mL Cm) and culture at 37°C and 250 rpm. When OD 600 reaches about 0.8, Add IPTG with a final concentration of 0.1mM and induce culture at 25°C and 250rpm for 16h.
  • reaction substrate solution Add glucose and nicotinamide to 300mM PBS buffer to obtain a reaction substrate solution, in which the glucose concentration is 42g/L and the nicotinamide concentration is 16g/L.
  • Transformation reaction and result detection Mix the prepared reaction substrate solution and bacterial suspension at a volume ratio of 1:1 to obtain a 20mL reaction system, and react at 30°C and 220rpm. Samples were taken for detection after 4 hours, and the HPLC detection results are shown in Table 2.
  • the detection method is: take 1mL of the reaction solution, centrifuge at 12000rpm for 10min, take 100 ⁇ L of the supernatant, add 880 ⁇ L of deionized water and 20 ⁇ L of 20% hydrochloric acid solution, mix and centrifuge at 12000rpm for 10min. The obtained supernatant is filtered and subjected to HPLC measurement. .
  • reaction substrate solution Mix and dilute 1.5M PBS buffer and 10*MB buffer to obtain a mixed buffer of 300mM PBS+2*MB, add glucose and nicotinamide to prepare a reaction substrate solution, where , the glucose concentration is 42g/L, and the nicotinamide concentration is 16g/L.
  • Transformation reaction and result detection Mix the prepared reaction substrate solution and bacterial suspension at a volume ratio of 1:1 to obtain a 20mL reaction system, and react at 30°C and 220rpm. After 4 hours of reaction, samples were taken for detection.
  • the detection method was the same as that described in Example 2.
  • the HPLC detection results are shown in Table 3.

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Abstract

A strain and a use thereof in the production of a β-nicotinamide mononucleotide (NMN). Disclosed is a strain of Escherichia coli, the accession number of the strain being CCTCC M2022922. Disclosed is a new strain for producing a β-NMN, and the use of the strain to prepare NMNs has the advantages of high yield and simple method.

Description

一种菌株及其在生产β-烟酰胺单核苷酸中的应用A strain and its application in producing β-nicotinamide mononucleotide
本申请要求申请日为2022/7/6的中国专利申请202210799686X的优先权。本申请引用上述中国专利申请的全文。This application claims priority to Chinese patent application 202210799686X with a filing date of 2022/7/6. This application cites the full text of the above-mentioned Chinese patent application.
技术领域Technical field
本发明属于生物发酵领域，具体涉及一种菌株及其在生产β-烟酰胺单核苷酸中的应用。The invention belongs to the field of biological fermentation, and specifically relates to a bacterial strain and its application in the production of β-nicotinamide mononucleotide.
背景技术Background technique
β-烟酰胺单核苷酸又称烟酰胺单核苷酸(nicotinamide mononucleotide，NMN)，是人体内天然存在的物质，是细胞能量重要来源之一。NMN作为哺乳动物体内辅酶I——烟酰胺腺嘌呤核苷酸(nicotinamide adenine dinucleotide，NAD+)合成的关键前体之一，逐渐被研究者了解和研究。NMN通过提高NAD+水平发挥抗衰老功能。随着对NMN研究的逐渐深入，研究人员认为补充NMN可以修复脑损伤、改善胰岛功能、保护心脏免于缺血再灌注损伤、修复脑线粒体呼吸缺陷，对老年退行性疾病、视网膜退行性疾病等均具有一定治疗作用。目前由于合成工艺的限制，NMN的价格较为昂贵。
β-Nicotinamide mononucleotide, also known as nicotinamide mononucleotide (NMN), is a naturally occurring substance in the human body and is one of the important sources of cellular energy. As one of the key precursors for the synthesis of coenzyme I - nicotinamide adenine dinucleotide (NAD + ) in mammals, NMN has been gradually understood and studied by researchers. NMN exerts anti-aging functions by increasing NAD + levels. With the gradual deepening of research on NMN, researchers believe that supplementing NMN can repair brain damage, improve pancreatic islet function, protect the heart from ischemia-reperfusion injury, repair brain mitochondrial respiratory defects, and have a beneficial effect on senile degenerative diseases, retinal degenerative diseases, etc. All have certain therapeutic effects. Currently, NMN is relatively expensive due to limitations in the synthesis process.
目前化学合成法制备NMN的技术较为成熟，如溴代乙酰核糖法、TMSOTF催化缩合法、AMP酸水解催化法、缩酮化保护合成法等，但存在制备条件苛刻、安全性低、生产成本高等缺陷。At present, the technology for preparing NMN through chemical synthesis is relatively mature, such as the bromoacetyl ribose method, TMSOTF catalytic condensation method, AMP acid hydrolysis catalytic method, ketal protection synthesis method, etc. However, there are harsh preparation conditions, low safety, and high production costs. defect.
NMN的生物合成包括酶促法和发酵法。酶促法路线一以磷酸核糖焦磷酸(PRPP)和烟酰胺(NAM)为底物在烟酰胺磷酸核糖转移酶(NAMPT) 的催化作用下生成NMN；路线二以烟酰胺核苷(NR)为底物，ATP为磷酸供体，在烟酰胺核苷激酶(NRK)的催化作用下发生磷酸化反应生成NMN；路线三以D-5-磷酸核糖和烟酰胺为原料，在ATP存在的条件下，利用磷酸核糖焦磷酸合成酶(Prs)和NAMPT酶组合进行催化，合成NMN。
The biosynthesis of NMN includes enzymatic and fermentative methods. Enzymatic method route one uses phosphoribosyl pyrophosphate (PRPP) and nicotinamide (NAM) as substrates in nicotinamide phosphoribosyltransferase (NAMPT) NMN is generated under the catalysis of nicotinamide riboside (NR); route two uses nicotinamide riboside (NR) as the substrate, ATP is the phosphate donor, and phosphorylation occurs under the catalysis of nicotinamide riboside kinase (NRK) to generate NMN; route three uses D-5-phosphate ribose and nicotinamide are used as raw materials. In the presence of ATP, NMN is synthesized using a combination of phosphoribosyl pyrophosphate synthase (Prs) and NAMPT enzyme for catalysis.
路线一
Route one
路线二
Route 2
路线三Route three
发酵法如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.)通过对代谢路径中与NMN生成有关的酶基因改造得到生产NMN的大肠杆菌菌株。该菌株以葡萄糖和烟酰胺为底物，发酵制备NMN，其产量可达6.79g/L。该方法的产量相对较低，不利于大规模的工业化生产。Fermentation methods 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.) By analyzing the metabolic pathways related to NMN production The enzyme was genetically modified to produce an NMN-producing E. coli strain. This strain uses glucose and nicotinamide as substrates to ferment NMN, and its yield can reach 6.79g/L. The yield of this method is relatively low and is not conducive to large-scale industrial production.
发明内容Contents of the invention
为了解决上述技术问题，本发明提供一种菌株及其在生产β-烟酰胺单核苷酸中的应用。本发明提供一种新的生产β-烟酰胺单核苷酸(又称烟酰胺单核苷酸，nicotinamide mononucleotide，NMN)的菌株，使用菌株制备NMN具有产量高、方法简单的优点，比如本发明的NMIS208菌株制备NMN能够带来可达14.50g/L的高产率。In order to solve the above technical problems, the present invention provides a strain and its application in producing β-nicotinamide mononucleotide. The present invention provides a new strain for producing β-nicotinamide mononucleotide (also known as nicotinamide mononucleotide, NMN). Using the strain to prepare NMN has the advantages of high yield and simple method. For example, the present invention The preparation of NMN by the NMIS208 strain can bring high yields up to 14.50g/L.
本发明第一方面提供一种大肠杆菌(Escherichia coli)的菌株，所述菌株的保藏编号为CCTCC M2022922。A first aspect of the present invention provides a strain of Escherichia coli, the deposit number of which is CCTCC M2022922.
本发明所述的大肠杆菌(Escherichia coli)的菌株即为NMIS208菌株。The Escherichia coli strain of the present invention is the NMIS208 strain.
本发明第二方面提供一种微生物菌体，其通过培养本发明第一方面所述的菌株获得。A second aspect of the present invention provides a microbial cell, which is obtained by culturing the strain described in the first aspect of the present invention.
所述培养使用的培养基可为适于大肠杆菌生长的本领域常规的培养基。The culture medium used for the culture may be a conventional medium in the art suitable for the growth of E. coli.
较佳地，培养使用的培养基为TB培养基或M9培养基。Preferably, the culture medium used for culture is TB medium or M9 medium.
所述TB培养基或所述M9培养基可为本领域常规培养基，较佳地，所述M9培养基配方(浓度g/L)为：十二水磷酸氢二钠17.9；磷酸二氢钾6.8；甘油5mL；胰化蛋白胨5；酵母提取物5；氯化铵2.7；无水硫酸钠0.71；无水硫酸镁0.24；四水氯化锰0.02；氯化铁0.016；氯化钙0.01。The TB culture medium or the M9 culture medium can be a conventional culture medium in this field. Preferably, the M9 culture medium formula (concentration g/L) is: disodium hydrogen phosphate dodecahydrate 17.9; potassium dihydrogen phosphate 6.8; glycerol 5mL; 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.
较佳地，所述TB培养基配方(g/L)：胰化蛋白胨10；酵母提取物18；甘油4mL；磷酸氢二钾16.43；磷酸二氢钾2.31。Preferably, the TB medium formula (g/L): tryptone 10; yeast extract 18; glycerol 4 mL; dipotassium hydrogen phosphate 16.43; potassium dihydrogen phosphate 2.31.
更佳地，培养所述NMIS208菌株时所述培养基中含有40μg/mL Km、40μg/mL Spec、20μg/mL Cm。More preferably, when culturing the NMIS208 strain, the medium contains 40 μg/mL Km, 40 μg/mL Spec, and 20 μg/mL Cm.
Km为卡那霉素，Spec为链霉素，Cm为氯霉素。Km is kanamycin, Spec is streptomycin, and Cm is chloramphenicol.
在某一较佳实施方案中，所述培养包括以下步骤：In a certain preferred embodiment, the culturing includes the following steps:
a)使用培养基为TB培养基或M9培养基进行培养；培养条件为25-40℃，100-300rpm。a) The culture medium used is TB medium or M9 medium for culture; the culture conditions are 25-40°C, 100-300rpm.
b)当a)中培养OD600达到0.6-1时，加入0.1mM的诱导剂进行诱导培养，其中所述诱导剂为IPTG，诱导培养条件为25-40℃，100-300rpm，12-24h。 b) When the OD 600 of culture in a) reaches 0.6-1, add 0.1mM inducer for induction culture, wherein the inducer is IPTG, and the induction culture conditions are 25-40°C, 100-300rpm, 12-24h.
优选地，所述培养基中还加入40μg/mL卡那霉素、40μg/mL链霉素、20μg/mL氯霉素。Preferably, 40 μg/mL kanamycin, 40 μg/mL streptomycin, and 20 μg/mL chloramphenicol are also added to the culture medium.
在某一较佳实施方案中，所述微生物菌体通过以下方法获得：In a certain preferred embodiment, the microbial cells are obtained by the following method:
取菌株的甘油菌按4％的接种量接种至5mL LB培养基(含50μg/mL Km、50μg/mL Spec、25μg/mL Cm)中，于37℃、250rpm条件下培养4h。按1％的接种量转接至TB培养基或M9培养基(含40μg/mL Km、40μg/mL Spec、20μg/mL Cm)中，于37℃250rpm条件下培养，当OD600达到0.8左右时，加入终浓度为0.1mM的IPTG，25℃、250rpm下诱导培养16h。The glycerol bacteria of the strain were inoculated into 5 mL LB medium (containing 50 μg/mL Km, 50 μg/mL Spec, 25 μg/mL Cm) at an inoculum volume of 4%, and cultured at 37°C and 250 rpm for 4 hours. Transfer 1% of the inoculum into TB medium or M9 medium (containing 40 μg/mL Km, 40 μg/mL Spec, 20 μg/mL Cm), and culture at 37°C and 250 rpm. When the OD 600 reaches about 0.8 , add IPTG with a final concentration of 0.1mM, and induce culture for 16h at 25°C and 250rpm.
本发明第三方面提供一种生产NMN的方法，其使用本发明第二方面所述的微生物菌体与底物在反应体系中发生反应生产所述NMN，所述底物为葡萄糖和烟酰胺。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 and a substrate to react in a reaction system to produce the NMN, and the substrate is glucose and nicotinamide.
所述方法中，较佳地，所述的反应体系含有磷酸缓冲液和/或培养基。In the method, preferably, the reaction system contains phosphate buffer and/or culture medium.
所述培养基可为适于大肠杆菌生长的本领域常规的培养基。The culture medium may be a conventional culture medium in the art suitable for the growth of E. coli.
较佳地，所述培养基为TB培养基或M9培养基。Preferably, the culture medium is TB culture medium or M9 culture medium.
较佳地，所述磷酸缓冲溶液为PBS缓冲液和/或MB缓冲液，所述MB缓冲溶液包括Na2HPO4、K2HPO4、NH4Cl和NaCl。Preferably, the phosphate buffer solution is PBS buffer and/or MB buffer, and the MB buffer solution includes Na 2 HPO 4 , K 2 HPO 4 , NH 4 Cl and NaCl.
在某一较佳实施方案中，所述MB缓冲液包括Na2HPO4 6.8g/L、K2HPO4 3.0g/L、NH4Cl 1.0g/L、NaCl 0.5g/L。In a certain preferred embodiment, the MB buffer includes Na 2 HPO 4 6.8g/L, K 2 HPO 4 3.0g/L, NH 4 Cl 1.0g/L, and NaCl 0.5g/L.
所述PBS缓冲液为本领域常规。The PBS buffer is routine in the art.
在某一较佳实施方案中，所述磷酸缓冲液为PBS缓冲液和MB缓冲液的混合缓冲液。In a certain preferred embodiment, the phosphate buffer is a mixed buffer of PBS buffer and MB buffer.
较佳地，所述混合缓冲液包含300mM PBS缓冲液和2*MB缓冲液。Preferably, the mixed buffer contains 300mM PBS buffer and 2*MB buffer.
在某一较佳实施方案中，将所述微生物菌体制备为菌体悬浮液后与所述底物混合得到所述反应体系。In a certain preferred embodiment, the microbial cells are prepared into a cell suspension and then mixed with the substrate to obtain the reaction system.
所述菌体悬浮液的制备包括以下任一项：The preparation of the bacterial cell suspension includes any of the following:
i：将所述微生物菌体加入磷酸缓冲液中进行悬浮即得到所述菌体悬浮液；优选地，所述微生物菌体与所述磷酸缓冲液的质量体积比为1:(5～20)例如 1:10；i: The microbial cells are added to the phosphate buffer for suspension to obtain the bacterial cell suspension; preferably, the mass-to-volume ratio of the microbial cells to the phosphate buffer is 1:(5-20) For example 1:10;
ii：将所述微生物菌体与培养其的培养基上清混合后得到所述菌体悬浮液；优选地，所述微生物菌体与所述培养基上清的质量体积比为1:(5～20)例如1:10。ii: The bacterial cell suspension is obtained after mixing the microbial cells and the culture medium supernatant for culturing them; preferably, the mass-to-volume ratio of the microbial cells and the culture medium supernatant is 1:(5 ~20) For example, 1:10.
较佳地，当使i的方法制备所述菌体悬浮液时，所述底物溶于所述PBS磷酸缓冲液中并与所述菌体悬浮液混合；当使ii的方法制备所述菌体悬浮液时，所述底物溶于所述混合缓冲液中并与所述菌体悬浮液混合。Preferably, when the method of i is used to prepare the bacterial cell suspension, the substrate is dissolved in the PBS phosphate buffer and mixed with the bacterial cell suspension; when the method of ii is used to prepare the bacterial cell suspension. When the bacterial cell suspension is obtained, the substrate is dissolved in the mixing buffer and mixed with the bacterial cell suspension.
所述方法中，较佳地，所述反应体系中：In the method, preferably, in the reaction system:
所述烟酰胺占所述反应体系的浓度为5～10g/L，例如8g/L。The concentration of nicotinamide in the reaction system is 5 to 10 g/L, such as 8 g/L.
所述烟酰胺与所述葡萄糖的质量比为1：(1.5～5)，例如1:2.625。The mass ratio of nicotinamide to glucose is 1: (1.5-5), for example, 1:2.625.
所述微生物菌体占所述反应体系的质量体积比为1：(10～30)，例如1:20。The mass-volume ratio of the microbial cells in the reaction system is 1: (10-30), for example, 1:20.
所述质量体积比指的是g/mL。The mass to volume ratio refers to g/mL.
所述微生物菌体与所述烟酰胺的质量比为1:(0.1～0.3)，例如1:0.16。The mass ratio of the microbial cells to the nicotinamide is 1: (0.1-0.3), such as 1:0.16.
在某一较佳实施方案中，所述微生物菌体是经离心第一方面所述菌株培养后的菌液后获得的湿菌体。具体可通过以下方法获得：获取微生物菌体的菌液于4℃、4000rpm下离心15min，分别收集上清液和湿菌体，并置于4℃冷藏，待用。In a certain preferred embodiment, the microbial cells are wet bacterial cells obtained by centrifuging the bacterial liquid cultured by the bacterial strain described in the first aspect. Specifically, it can be obtained by the following method: obtain the bacterial liquid of microbial cells and centrifuge it at 4°C and 4000rpm for 15 minutes, collect the supernatant and wet bacterial cells respectively, and store them in a refrigerator at 4°C until use.
在某一较佳实施方案中，所述反应通过搅拌或震荡的方式进行。In a preferred embodiment, the reaction is carried out by stirring or shaking.
优选地，所述反应的条件为25-35℃、200-300rpm和6-24h，更优选30℃、220rpm和8～24h。Preferably, the reaction conditions are 25-35°C, 200-300rpm and 6-24h, more preferably 30°C, 220rpm and 8-24h.
在某一较佳实施方案中，当所述反应的时间达到4-6h时，补充所述底物，使所述葡萄糖占所述反应体系的浓度为7～15g/L例如10.5g/L，且所述烟酰胺占所述反应体系的浓度为3～5g/L例如4g/L。In a certain 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 7-15g/L, such as 10.5g/L, And the concentration of nicotinamide in the reaction system is 3 to 5 g/L, such as 4 g/L.
本发明第四方面提供一种本发明第一方面所述的大肠杆菌(Escherichia coli)菌株或本发明第二方面所述的微生物菌体在制备NMN中的应用。The fourth aspect of the present invention provides a use of the Escherichia coli strain described in the first aspect of the present invention or the microbial cells described in the second aspect of the present invention in the preparation of NMN.
本发明所述的NMN指的是β-烟酰胺单核苷酸(又称烟酰胺单核苷酸，nicotinamide mononucleotide，NMN)。 NMN in the present invention refers to β-nicotinamide mononucleotide (also known as nicotinamide mononucleotide, NMN).
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。On the basis of common sense in the field, the above preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.
本发明所用试剂和原料均市售可得。The reagents and raw materials used in the present invention are all commercially available.
本发明的积极进步效果在于：本发明提供一种新的生产NMN的菌株，使用菌株制备NMN具有产量高、方法简单的优点，比如本发明的NMIS208菌株制备NMN能够带来可达14.50g/L的高产率。The positive and progressive effect of the present invention is that the present invention provides a new strain for producing NMN. The use of the strain to prepare NMN has the advantages of high yield and simple method. For example, the NMIS208 strain of the present invention can produce NMN up to 14.50g/L. high yield.
生物材料保藏信息Biological material preservation information
本发明的大肠杆菌(Escherichia coli)NMIS208菌株，于2022年6月20日保藏在中国典型培养物保藏中心(CCTCC)，保藏地址：中国湖北省武汉市武昌珞珈山武汉大学，邮编：430072，保藏编号为CCTCC M2022922，培养物名称为大肠杆菌(Escherichia coli)NMIS208菌株。The Escherichia coli NMIS208 strain of the present invention was deposited in the China Type Culture Collection Center (CCTCC) on June 20, 2022. The deposit address is: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, China, Postal Code: 430072, The deposit number is CCTCC M2022922, and the culture name is Escherichia coli NMIS208 strain.
具体实施方式Detailed ways
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。The present invention is further described below by means of examples, but the present invention is not limited to the scope of the described examples. Experimental methods that do not indicate specific conditions in the following examples should be selected according to conventional methods and conditions, or according to product specifications.
实施例中，所用M9培养基配方(浓度g/L)为：十二水磷酸氢二钠17.9；磷酸二氢钾6.8；甘油5mL；胰化蛋白胨5；酵母提取物5；氯化铵2.7；无水硫酸钠0.71；无水硫酸镁0.24；四水氯化锰0.02；氯化铁0.016；氯化钙0.01。In the embodiment, the M9 medium formula (concentration g/L) used 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.
TB培养基配方(g/L)：胰化蛋白胨10；酵母提取物18；甘油4mL；磷酸氢二钾16.43；磷酸二氢钾2.31。TB medium formula (g/L): tryptone 10; yeast extract 18; glycerol 4mL; dipotassium hydrogen phosphate 16.43; potassium dihydrogen phosphate 2.31.
MB缓冲液(g/L)：Na2HPO4 6.8、K2HPO4 3.0、NH4Cl 1.0、NaCl 0.5。MB buffer (g/L): Na 2 HPO 4 6.8, K 2 HPO 4 3.0, NH 4 Cl 1.0, NaCl 0.5.
实施例中实验结果的检测采用高效液相色谱(HPLC)，色谱柱：Welch Ultimate AQ-C18(5μm，250×4.6mm)，流速：1mL/min；柱温：35℃，检测波长：260nm进样体积：5μL。 The experimental results in the examples were detected using high performance liquid chromatography (HPLC). Chromatographic column: Welch Ultimate AQ-C18 (5 μm, 250×4.6 mm), flow rate: 1 mL/min; column temperature: 35°C, detection wavelength: 260 nm. Sample volume: 5 μL.
流动相：A：10mM乙酸铵+0.1％甲酸，B：乙腈。Mobile phase: A: 10mM ammonium acetate + 0.1% formic acid, B: acetonitrile.
梯度洗脱程序见下表1：The gradient elution procedure is shown in Table 1 below:
表1
Table 1
实施例1 NMIS208菌株的培养Example 1 Cultivation of NMIS208 strain
取NMIS208甘油菌按4％的接种量接种至5mL LB培养基(含50μg/mL Km、50μg/mL Spec、25μg/mL Cm)中，于37℃、250rpm条件下培养4h。按1％的接种量转接至TB或M9培养基(含40μg/mL Km、40μg/mL Spec、20μg/mL Cm)中，于37℃、250rpm条件下培养，当OD600达到0.8左右时，加入终浓度为0.1mM的IPTG，25℃、250rpm下诱导培养16h。Inoculate NMIS208 glycerol bacteria into 5 mL LB medium (containing 50 μg/mL Km, 50 μg/mL Spec, 25 μg/mL Cm) at an inoculum volume of 4%, and culture it at 37°C and 250 rpm for 4 hours. Transfer 1% of the inoculum into TB or M9 medium (containing 40 μg/mL Km, 40 μg/mL Spec, 20 μg/mL Cm) and culture at 37°C and 250 rpm. When OD 600 reaches about 0.8, Add IPTG with a final concentration of 0.1mM and induce culture at 25°C and 250rpm for 16h.
将上述菌液于4℃、4000rpm下离心15min，分别收集上清液和湿菌体，并置于4℃冷藏，待用。Centrifuge the above bacterial liquid at 4°C and 4000 rpm for 15 min, collect the supernatant and wet bacterial cells respectively, and store them in a 4°C refrigerator until use.
实施例2 发酵TB-催化法制备NMNExample 2 Preparation of NMN by fermentation TB-catalytic method
(1)配制反应底物溶液：向300mM PBS缓冲液中加入葡萄糖和烟酰胺得到反应底物溶液，其中葡萄糖浓度为42g/L，烟酰胺浓度为16g/L。(1) Prepare reaction substrate solution: Add glucose and nicotinamide to 300mM PBS buffer to obtain a reaction substrate solution, in which the glucose concentration is 42g/L and the nicotinamide concentration is 16g/L.
(2)菌体悬浮液的制备：(2) Preparation of bacterial suspension:
将10*MB缓冲溶液稀释5倍得到2*MB缓冲液，取实施例1所述NMIS208湿菌体按1:10(g/mL)的比例加入到2*MB缓冲液中进行悬浮，得到菌体悬浮液。Dilute the 10*MB buffer solution 5 times to obtain a 2*MB buffer solution. Take the NMIS208 wet bacterial cells described in Example 1 and add them to the 2*MB buffer solution at a ratio of 1:10 (g/mL) for suspension to obtain the bacteria. body suspension.
(3)转化反应及结果检测：将配制好的反应底物溶液与菌体悬浮液按照1:1的体积比混合得到20mL反应体系，于30℃、220rpm条件下反应。 4h后取样检测，HPLC检测结果如表2所示。(3) Transformation reaction and result detection: Mix the prepared reaction substrate solution and bacterial suspension at a volume ratio of 1:1 to obtain a 20mL reaction system, and react at 30°C and 220rpm. Samples were taken for detection after 4 hours, and the HPLC detection results are shown in Table 2.
检测方法为：取1mL反应液，12000rpm离心10min后，取100μL上清液加入880μL去离子水和20μL 20％盐酸溶液，混合后于12000rpm离心10min，得到的上清液经过滤后，进行HPLC测定。The detection method is: take 1mL of the reaction solution, centrifuge at 12000rpm for 10min, take 100μL of the supernatant, add 880μL of deionized water and 20μL of 20% hydrochloric acid solution, mix and centrifuge at 12000rpm for 10min. The obtained supernatant is filtered and subjected to HPLC measurement. .
(4)补料：反应转化6h后，向反应液中补加葡萄糖和烟酰胺，至其浓度分别为10.5g/L和4g/L。24h后再次取样检测，检测方法同上，检测结果如表2所示。(4) Feeding: 6 hours after the reaction transformation, add glucose and nicotinamide to the reaction solution until their concentrations are 10.5g/L and 4g/L respectively. After 24 hours, samples were taken again for testing. The testing method was the same as above. The testing results are shown in Table 2.
表2发酵TB-催化法制备NMN实验结果
Table 2 Experimental results of preparing NMN by fermentation TB-catalytic method
由结果可知，在本实施例的条件下，当反应转化24h，NMN的产量可达12.96g/L。It can be seen from the results that under the conditions of this example, when the reaction is converted for 24 hours, the output of NMN can reach 12.96g/L.
实施例3双阶段发酵法制备NMNExample 3 Preparation of NMN by two-stage fermentation method
(1)配制反应底物溶液：将1.5M PBS缓冲液和10*MB缓冲液混合并稀释，得到300mM PBS+2*MB的混合缓冲液，加入葡萄糖和烟酰胺配置成反应底物溶液，其中，葡萄糖浓度为42g/L，烟酰胺浓度为16g/L。(1) Prepare reaction substrate solution: Mix and dilute 1.5M PBS buffer and 10*MB buffer to obtain a mixed buffer of 300mM PBS+2*MB, add glucose and nicotinamide to prepare a reaction substrate solution, where , the glucose concentration is 42g/L, and the nicotinamide concentration is 16g/L.
(2)菌体悬浮液的制备：将实施例1所述的NMIS208湿菌体和培养基M9上清液按质量体积比1:10(g/mL)进行混合，得到菌体悬浮液。(2) Preparation of bacterial cell suspension: Mix the NMIS208 wet bacterial cells described in Example 1 and the culture medium M9 supernatant at a mass-volume ratio of 1:10 (g/mL) to obtain a bacterial cell suspension.
(3)转化反应及结果检测：将配制好的反应底物溶液与菌体悬浮液按照1:1的体积比混合得到20mL反应体系，于30℃、220rpm条件下反应。反应4h后取样检测，检测方法同实施例2所述，HPLC检测结果如表3所示。(3) Transformation reaction and result detection: Mix the prepared reaction substrate solution and bacterial suspension at a volume ratio of 1:1 to obtain a 20mL reaction system, and react at 30°C and 220rpm. After 4 hours of reaction, samples were taken for detection. The detection method was the same as that described in Example 2. The HPLC detection results are shown in Table 3.
(4)补料：反应转化6h后，向反应液中补加葡萄糖和烟酰胺，至其浓度分别为10.5g/L和4g/L。24h后再次取样检测，检测方法同上，HPLC检测结果如表3所示。 (4) Feeding: 6 hours after the reaction transformation, add glucose and nicotinamide to the reaction solution until their concentrations are 10.5g/L and 4g/L respectively. After 24 hours, samples were taken again for testing. The detection method was the same as above. The HPLC detection results are shown in Table 3.
表3双阶段发酵法制备NMN实验结果
Table 3 Experimental results of preparing NMN by two-stage fermentation method
由结果可知，当使用原培养液重悬菌体，反应转化24h时，NMN的产量可达14.50g/L。 It can be seen from the results that when the original culture medium is used to resuspend the bacterial cells and the reaction is transformed for 24 hours, the output of NMN can reach 14.50g/L.

Claims (10)

  1. 一种大肠杆菌(Escherichia coli)的菌株，其特征在于，所述菌株的保藏编号为CCTCC M2022922。A strain of Escherichia coli, characterized in that the deposit number of the strain is CCTCC M2022922.
  2. 一种微生物菌体，其特征在于，其通过培养如权利要求1所述的菌株获得。A microbial cell characterized in that it is obtained by cultivating the strain according to claim 1.
  3. 如权利要求2所述的微生物菌体，其特征在于，所述培养包括以下步骤：The microbial cell according to claim 2, wherein said culturing includes the following steps:
    a)使用培养基为TB培养基或M9培养基进行培养；培养条件为25-40℃，100-300rpm；优选地，所述培养基中还加入40μg/mL卡那霉素、40μg/mL链霉素、20μg/mL氯霉素；a) The culture medium is TB medium or M9 medium for culture; the culture conditions are 25-40°C, 100-300 rpm; preferably, 40 μg/mL kanamycin, 40 μg/mL chain Mycin, 20μg/mL chloramphenicol;
    b)当a)中培养OD600达到0.6-1时，加入0.1mM的诱导剂进行诱导培养，其中所述诱导剂为IPTG，诱导培养条件为25-40℃，100-300rpm，12-24h。b) When the OD 600 of culture in a) reaches 0.6-1, add 0.1mM inducer for induction culture, wherein the inducer is IPTG, and the induction culture conditions are 25-40°C, 100-300rpm, 12-24h.
  4. 一种生产NMN的方法，其特征在于，使用如权利要求2或3所述的微生物菌体与底物在反应体系中发生反应生产所述NMN，所述底物为葡萄糖和烟酰胺。A method for producing NMN, characterized in that the NMN is produced by reacting the microbial cells according to claim 2 or 3 with a substrate in a reaction system, and the substrate is glucose and nicotinamide.
  5. 如权利要求4所述的方法，其特征在于，所述的反应体系含有磷酸缓冲液和/或培养基；较佳地，所述培养基为TB培养基或M9培养基，所述磷酸缓冲溶液为PBS缓冲液和/或MB缓冲液，所述MB缓冲溶液包括Na2HPO4、K2HPO4、NH4Cl和NaCl，例如Na2HPO4 6.8g/L、K2HPO4 3.0g/L、NH4Cl 1.0g/L、NaCl 0.5g/L。The method of claim 4, wherein the reaction system contains phosphate buffer and/or culture medium; preferably, the culture medium is TB culture medium or M9 culture medium, and the phosphate buffer solution It is PBS buffer and/or MB buffer, and the MB buffer solution includes Na 2 HPO 4 , K 2 HPO 4 , NH 4 Cl and NaCl, for example, Na 2 HPO 4 6.8g/L, K 2 HPO 4 3.0g/ L, NH 4 Cl 1.0g/L, NaCl 0.5g/L.
  6. 如权利要求5所述的方法，其特征在于，所述磷酸缓冲液为PBS缓冲液和MB缓冲液的混合缓冲液；较佳地，所述混合缓冲液包含300mM PBS缓冲液和2*MB缓冲液。The method of claim 5, wherein the phosphate buffer is a mixed buffer of PBS buffer and MB buffer; preferably, the mixed buffer includes 300mM PBS buffer and 2*MB buffer. liquid.
  7. 如权利要求4-6任一项所述的方法，其特征在于，所述反应体系中：The method according to any one of claims 4 to 6, characterized in that, in the reaction system:
    所述烟酰胺占所述反应体系的浓度为5～10g/L，例如8g/L；和/或， The concentration of nicotinamide in the reaction system is 5-10g/L, such as 8g/L; and/or,
    所述烟酰胺与所述葡萄糖的质量比为1：(1.5～5)，例如1:2.625；和/或，The mass ratio of nicotinamide to glucose is 1: (1.5-5), such as 1:2.625; and/or,
    所述微生物菌体占所述反应体系的质量体积比为1：(10～30)，例如1:20；和/或，The mass volume ratio of the microbial cells in the reaction system is 1: (10-30), such as 1:20; and/or,
    所述微生物菌体与所述烟酰胺的质量比为1:(0.1～0.3)，例如1:0.16。The mass ratio of the microbial cells to the nicotinamide is 1:(0.1-0.3), for example, 1:0.16.
  8. 如权利要求4-7任一项所述的方法，其特征在于，所述反应通过搅拌或震荡的方式进行；较佳地，所述反应的条件为25-35℃、200-300rpm和6-24h，优选30℃、220rpm和8～24h。The method according to any one of claims 4 to 7, characterized in that the reaction is carried out by stirring or shaking; preferably, the reaction conditions are 25-35°C, 200-300rpm and 6- 24h, preferably 30°C, 220rpm and 8 to 24h.
  9. 如权利要求8所述的方法，其特征在于，当所述反应的时间达到4-6h时，例如6h时，补充所述底物，使所述葡萄糖占所述反应体系的浓度为7～15g/L例如10.5g/L，且所述烟酰胺占所述反应体系的浓度为3～5g/L例如4g/L。The method of claim 8, wherein when the reaction time reaches 4-6 hours, for example, 6 hours, the substrate is supplemented so that the concentration of glucose in the reaction system is 7-15 g. /L, such as 10.5g/L, and the concentration of nicotinamide in the reaction system is 3 to 5g/L, such as 4g/L.
  10. 如权利要求1所述的大肠杆菌(Escherichia coli)的菌株或如权利要求2或3所述的微生物菌体在制备NMN中的应用。 Use of the Escherichia coli strain as claimed in claim 1 or the microbial cell as claimed in claim 2 or 3 in the preparation of NMN.
PCT/CN2023/105573 2022-07-06 2023-07-03 STRAIN AND USE THEREOF IN PRODUCTION OF β-NICOTINAMIDE MONONUCLEOTIDE WO2024008046A1 (en)

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