Coding gene of nicotinamide mononucleotide adenylyltransferase, recombinant expression vector and application thereof

A single nucleotide adenylyltransferase and single nucleotide adenylyl transferase technology, applied in the fields of genetic engineering and microbial fermentation engineering, can solve the problem of insufficient production requirements, high expression strains, and low yield of NMNATs and other issues, to achieve the effect of good industrial application prospects, low cost and high output

Inactive Publication Date: 2018-02-02
GUANGZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the method of synthesizing NAD+ is mainly biological or chemical synthesis. The biosynthesis method is to obtain wild strains with high NMNATs activity through screening or mutagenesis, but the NMNATs yield of these strains is relatively low, which is not enough to meet production needs.
In addition, some studies have tried to express NMNATs recombinantly in Escherichia coli, but high-expression strains cannot be obtained.

Method used

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Examples

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Comparison scheme
Effect test

Embodiment 1

[0029] The nucleotide sequence of the coding gene of the nicotinamide mononucleotide adenylyltransferase provided by the present invention is shown in SEQ ID NO: 1, the amino acid coded by it or the amino acid sequence of the nicotinamide mononucleotide adenylyltransferase Shown as SEQ ID NO:2.

[0030] The coding gene of the nicotinamide mononucleotide adenylyltransferase is artificially synthesized.

Embodiment 2

[0031] Example 2 Construction of recombinant expression vector pPIC9k-nmnT

[0032] (1) Utilize artificial synthesis method to synthesize the coding gene of described nicotinamide mononucleotide adenylyltransferase, add EcoR I at the two ends of the coding gene sequence of nicotinamide mononucleotide adenylyltransferase in the synthetic process and Not I restriction endonuclease recognition site sequences.

[0033] (2) Carry out double enzyme digestion to the coding gene of the nicotinamide mononucleotide adenylyltransferase in step (1) and the plasmid vector pPIC9k respectively (enzyme cutting site is EcoR I and Not I), to both Digested products were identified, purified and ligated.

[0034] (3) The ligation product was transformed into E. coli DH5α clone strain, and after identification by colony PCR and sequencing, the recombinant expression vector pPIC9k-nmnT was obtained.

Embodiment 3

[0035] The preparation of the host cell of embodiment 3 genetic engineering

[0036] (1) performing Sal I linearization treatment on the recombinant expression vector pPIC9k-nmnT constructed in Example 2;

[0037] (2) The recombinant expression vector in step (1) was introduced into the host cell Pichia pastoris GS115 by a calcium chloride heat shock method. Spread the transformants on the screening YPD plate medium containing G418 antibiotics, and obtain the recombinant expression host after colony PCR screening and sequencing identification.

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Abstract

The invention discloses a coding gene of nicotinamide mononucleotide adenylyltransferase. The nucleotide sequence of the coding gene is shown as SEQ ID NO:1, and the amino acid sequence of the codinggene encoded amino acid or nicotinamide mononucleotide adenylyltransferase is shown as SEQ ID NO:2. The invention also discloses a recombinant expression vector containing the coding gene of nicotinamide mononucleotide adenylyltransferase. The invention also discloses application of the coding gene of nicotinamide mononucleotide adenylyltransferase in yeast fermentation production of nicotinamidemononucleotide adenylyltransferase. The coding gene of nicotinamide mononucleotide adenylyltransferase is obtained by yeast DNA sequence optimization on the nicotinamide mononucleotide adenylyltransferase gene of methane thermophilic bacillus according to the yeast codon bias and the principle of elimination of yeast endogenous protease recognition sites, and the encoded nicotinamide mononucleotide adenylyltransferase has good thermal stability and high yield at the same time,.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and microbial fermentation engineering, and specifically relates to a coding gene of nicotinamide mononucleotide adenylyltransferase, a recombinant expression vector and its application. Background technique [0002] Nicotinamide adenine dinucleotide (nicotinamide adenine dinucleotide, NAD+) widely exists in various organisms and plays an extremely important role in life activities. Nicotinamide mononucleotide adenylyltransferase (nicotinamide mononucleotide adenylyltransferase, NMNATs) participates in the adenylation of nicotinamide mononucleotide (NMN) in cells, and then synthesizes NAD+, which satisfies the energy transfer, material metabolism and signaling of living organisms. The demand for NAD+ in transduction can reduce the aging and death of biological cells to a certain extent, and can reduce the occurrence and development of tumors. [0003] At present, the method of synthes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/81C12N1/19C12P19/32C12R1/84
CPCC12N9/1241C12P19/32C12Y207/07001
Inventor 江学斌
Owner GUANGZHOU UNIVERSITY
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