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Recombinant microorganism for producing beta-nicotinamide mononucleotide and method for producing NMN by using recombinant microorganism

A technology for recombinant microorganisms and single nucleotides, applied in the biological field, can solve problems such as long time-consuming, low yield, and low enzyme activity

Active Publication Date: 2021-12-07
SUZHOU BIOSYNTHETICA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of Nampt to catalyze Nam to generate NMN not only requires enzymatic synthesis of phosphoribosyl pyrophosphate, but also requires a large amount of expensive ATP in the entire synthesis, and due to the low enzymatic activity of the existing nicotinamide phosphoribosyltransferase (Nampt), the enzymatic reaction There are problems such as long time-consuming, high cost, and low yield, and it is difficult to achieve large-scale factory production, thus limiting the large-scale application of NMN

Method used

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  • Recombinant microorganism for producing beta-nicotinamide mononucleotide and method for producing NMN by using recombinant microorganism
  • Recombinant microorganism for producing beta-nicotinamide mononucleotide and method for producing NMN by using recombinant microorganism
  • Recombinant microorganism for producing beta-nicotinamide mononucleotide and method for producing NMN by using recombinant microorganism

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Effect test

Embodiment 1

[0030] Example 1 The method for gene knockout in Escherichia coli

[0031] The present invention adopts Datsenko's method to carry out gene knockout in Escherichia coli (Datsenko 2000.ProcNatl Acad Sci USA, 97 (12): 6640-6645), corresponding gene knockout primer sees Baba 2006.Mol SystBiol, 2 (1) 0008.

Embodiment 2

[0032] Embodiment 2 shaking flask fermentation verifies the method for recombinant bacterial strain

[0033] To verify the ability of recombinant strains to produce NMN in shake flask fermentation, the specific components are 30g of glucose per liter of medium, 200ml of 5N-5 times salt solution, 1ml of TM2 solution, 10mg of ferric citrate, 246mg of magnesium sulfate heptahydrate, chloride Calcium 111mg, thiamine 1ug, dilute to 1L with sterile deionized water. Among them, the 5N-5 times salt solution is 75.6g per liter of disodium hydrogen phosphate dodecahydrate, 15g per liter of potassium dihydrogen phosphate, 2.5g per liter of sodium chloride, 25g of ammonium chloride, and dilute to 1L with ionized water; TM3 solution is zinc chloride tetrahydrate 2.0g, calcium chloride hexahydrate 2.0g, sodium molybdate dihydrate 2.0g, copper sulfate pentahydrate 1.9g, boric acid 0.5g, hydrochloric acid 100ml, deionized water to 1L. The above solution was sterilized by high pressure steam ...

Embodiment 3

[0035] Embodiment 3 5L fermentor utilizes the method for fermenting and producing NMN of recombinant bacterial strain

[0036] The fermentation medium is a semi-synthetic medium, containing 5g of ammonium sulfate, 2g of sodium chloride, 4g of potassium dihydrogen phosphate, 2g of magnesium sulfate heptahydrate, 15g of glucose, 0.105g of calcium chloride, and 0.01g of zinc chloride per liter of medium , TM3 1mL, ferric citrate 94mg, corn steep liquor 5g, VB 1 2.5mg, NA 40mg, bubble enemy 1g, constant volume with deionized water, feed medium containing 500g of glucose per liter, adjust pH to 6.9 with ammonia water; TM3 solution is 2.0g of zinc chloride tetrahydrate, chlorination of hexahydrate Calcium 2.0g, sodium molybdate dihydrate 2.0g, copper sulfate pentahydrate 1.9g, boric acid 0.5g, hydrochloric acid 100ml, deionized water to 1L.

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Abstract

The invention provides a recombinant microorganism for producing beta-nicotinamide mononucleotide (NMN) and a method for producing NMN by using the recombinant microorganism, the strain of the recombinant microorganism contains one or more or all of the following characteristics: (1) adding nicotinamide into a fermentation culture medium, and transforming by the recombinant microorganism to generate NMN; and (2) the nicotinamide phosphoribosyltransferase is overexpressed. And (3) deletion or inactivation or enzyme activity reduction of genes for encoding alkaline phosphatase and nucleotidase on the recombinant microorganism genome.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the construction of one or more recombinant microorganisms producing β-nicotinamide mononucleotide, and the production of β-nicotinamide mononucleotide by using the recombinant microorganism. Background technique [0002] Nicotinamide mononucleotide (nicotinamide mononucleotide, NMN CAS: 1094-61-7) is a naturally occurring biologically active nucleotide, which has two irregular forms of α and β; and the β-isomer is the NMN Active form, molecular formula C11H15N2O8P, molecular weight 334.22. It contains a molecule of nicotinamide, because nicotinamide belongs to vitamin B3, so NMN is also classified as a vitamin B derivative, which is catalyzed by nicotinamide phosphoribosyltransferase (Nampt, EC 2.4.2.12). The product of the reaction of amide (Nam) with phosphoribosyl pyrophosphate (PRPP), it is also a key precursor of the nicotinamide adenine dinucleotide (NAD+) salvage ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P19/30C12R1/19
CPCC12N15/52C12N9/16C12N9/80C12N9/1205C12N9/1077C12N9/14C12Y301/03005C12Y305/01042C12Y207/01022C12Y305/01019C12Y204/02019C12Y204/02012C12Y306/01016C12Y301/03001C12Y301/03002C12N15/70C12P19/30
Inventor 万丽花江君君田锋陆红霞胡志浩
Owner SUZHOU BIOSYNTHETICA CO LTD
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