Nicotinamide nucleoside kinase whole yeast cells and process for biocatalytically synthesizing NMN from nicotinamide nucleoside kinase whole yeast cells

Abstract

The invention provides nicotinamide nucleoside kinase whole yeast cells and a process for biocatalytically synthesizing NMN by the nicotinamide nucleoside kinase whole yeast cells, and relates to the technical field of enzyme engineering. The process for biocatalytically synthesizing the NMN comprise the following steps: S1, constructing a nicotinamide nucleoside kinase recombinant display expression vector of saccharomyces cerevisiae: designing an encoding DNA sequence of NrK1 according to an amino acid sequence (SEQ ID NO: 1) of an NrK1 mutant, adopting preferred codons of the saccharomyces cerevisiae as codons, and synthesizing encoding DNA (SEQ ID NO: 2) of NrK1 in a whole-gene synthesis manner; and according to the nicotinamide nucleoside kinase whole yeast cell and the NMN biocatalytic synthesis process thereof, the nicotinamide nucleoside kinase whole yeast cell and the NMN biocatalytic synthesis process thereof can anchor and express high-activity enzymes capable of catalyzing NMN synthesis from different biological sources on the cell wall surface of saccharomyces cerevisiae through a genetic engineering technology; and the whole-yeast engineering cell enzyme capable of efficiently synthesizing the NMN is formed.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to whole yeast cells with nicotinamide riboside kinase and its biocatalytic synthesis of NMN. Background technique [0002] NMN (nicotinamide mononucleotide) plays an important role in the energy metabolism of human cells. It participates in the synthesis of the most important electron transfer carrier NAD+ (oxidized nicotinamide adenine dinucleotide) in the cell, and is also a cellular NAD+ remedy. Synthetic intermediates, in mammals, NMN is generated from NMN under the catalysis of Nampt by Nicotinamide (Nam), and then NMN is catalyzed by Nicotinamide mononucleotide adenosinetransferase (Nicotinamide mononucleotideadenosinetransferase, Nmnat) To generate NAD+, extracellular NMN needs to be dephosphorylated to NR (nicotinamide riboside) to enter the liver cells, phosphorylated under the action of nicotinamide riboside kinase to regenerate NMN, and then NMN reacts with AT...

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Problems solved by technology

[0003] However, in the prior art, the traditional NMN production is a chemical synthesis method, that is, nicotinamide ribose is used as a raw material and phosphorylated with phosphorus oxychloride to obtain it. The productivity is low, the product purity is low, and it contains about 50% of hand α-NMN, which is toxic and difficult to separate, so the yield is very low. In addition, a large amount of organic solvents need to be used in the synthesis process, which seriously damages the environment. In recent years, biocatalytic technology has been used in the production of NMN. Through Nicotinamide riboside kinase (NRK) produced by recombinant DNA technology can efficiently convert nicotinamide glycoside (NR) to produce NMN in vitro, which is a green biotransformation with almost zero environmental pollution and no chiral molecule α-NMN , high yield, low cost, is a major revolution in NMN production technology, but NRK is almost all from the free enzyme expressed in E. It has always been a safety threat in the production of medicines and health food, which makes the actual production process more cumbersome, the resource reuse rate is low, and the actual production effect is not good.

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