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Genetically modified microorganism and method both for producing nicotinamide derivative, and vector for use in same

A technology for recombining microorganisms and nicotinamide, applied in biochemical equipment and methods, botany equipment and methods, using vectors to introduce foreign genetic material, etc., can solve problems such as long time, less NMN, and inability to obtain yield

Pending Publication Date: 2021-08-13
TEIJIN LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this method, it takes a long time to produce NMN, and the amount of NMN obtained is small, and a yield sufficient for practical use cannot be obtained.

Method used

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  • Genetically modified microorganism and method both for producing nicotinamide derivative, and vector for use in same
  • Genetically modified microorganism and method both for producing nicotinamide derivative, and vector for use in same
  • Genetically modified microorganism and method both for producing nicotinamide derivative, and vector for use in same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0499] ・Example 1 (production of NMN using BL21 / pRSF-NAMPT CP strain):

[0500] The BL21 / pRSF-NAMPT CP strain was inoculated into a test tube containing 5 mL of LB medium, and cultured at 37° C. and 200 rpm for 12 hours. The culture solution was added to a 500mL Erlenmeyer flask containing 200mL LB medium to make the OD 600 0.03, cultured at 37°C, 200rpm, at OD 600 Add isopropyl for the time point of 0.4- β -Thiogalactopyranoside (manufactured by Nakalai Tesque Co., Ltd.) was cultured at 25° C. and 200 rpm for 16 hours at a final concentration of 0.1 mM. Thereafter, the culture solution was transferred to a 50 mL conical tube, centrifuged at 3000 g for 5 minutes, and bacterial cells were recovered. Then, 1×PBS was added for washing, centrifuged at 3000 g for 5 minutes, and bacterial cells were recovered. This operation was performed 2 times. The recovered bacteria were suspended in LB medium to make the OD 600 is 10, then added to a 100mL Erlenmeyer flask to reach 10mL...

Embodiment 11

[0524] ・Example 11 (using BL21 / pRSF-NAMPT CP+pnuC BM / pCDF-pgi→prs+niaP BC / Production of NMN by pACYC-prs→pgi strain):

[0525] Bacterial cells were recovered in the same manner as in Example 1, except that the BL21 / pRSF-NAMPT CP strain was changed to the BL21 / pRSF-NAMPT CP+pnuC BM / pCDF-pgi→prs+niaP BC / pACYC-prs→pgi strain. The recovered bacteria were suspended in M9 medium to make the OD 600 40, add it to a 100mL Erlenmeyer flask to reach 10mL, charge it so that the nicotinamide is 7g / L, the D-glucose is 21g / L, and the phosphate buffer (pH6.2) is 0.05mol / L. , The reaction was carried out at 200 rpm. After 8 hours, the reaction solution was collected, frozen at -30° C., dissolved, centrifuged at 12,000 rpm for 3 minutes, and the supernatant was collected. When the recovered liquid was analyzed by HPLC, the amount of NMN was 6.52 g / L.

[0526] ・Comparative Example 3 (using BL21 / pRSF-NAMPT HS+pnuC BM / pCDF-pgi→prs+niaP BC / pACYC- NMN production of prs→pgi strain):

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Abstract

Provided is a technique for synthesizing a nicotinamide derivative (NAm derivative) such as a nicotinamide mononucleotide (NMN) with high efficiency. A genetically modified microorganism is used, which can express, as nicotinamide phosphoribosyltransferase (NAMPT), NAMPT having a conversion efficiency of 5-folds or more that of human NAMPT.

Description

technical field [0001] The present invention relates to a new type of recombinant microorganism used to manufacture nicotinamide derivatives (NAm derivatives, nicotinamide derivative) such as nicotinamide mononucleotide (NMN, nicotinamide mononucleotide), and a novel production method for manufacturing NAm derivatives, and relates to Novel vectors used in them. Background technique [0002] Nicotinamide adenine dinucleotide (NAD, nicotinamide adenine dinucleotide) is a nucleotide derived from ribose and nicotinamide. It is known that NAD functions as a coenzyme in various oxidation-reduction reactions in a living body and plays a central role in aerobic respiration (oxidative phosphorylation). NAD can take the oxidized form (NAD + ) and the reduced form (NADH) in two states. In this specification, unless otherwise specified, the term "NAD" collectively means the oxidized form (NAD + ) and reduced (NADH) both. [0003] There are many biosynthetic pathways of NAD, but in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30C12P19/32C12P19/36C12N1/19C12N1/21C12N15/53C12N15/54C12N15/56C12N15/61C12N15/70C12N15/81
CPCC12P19/36C12N15/70C12P19/30C12P19/32C12Y204/02012C12N9/1077C12N15/81C12Y204/02014C12Y204/02045C12N9/0006C12N9/18C12N9/92C12Y101/01043C12Y101/01049C12Y301/01031C12Y503/01006C12Y503/01009
Inventor 庄司信一郎石井纯近藤昭彦渡边秀和狩野理延中岛良太
Owner TEIJIN LTD
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