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Method for producing nicotinamide mononucleotide by utilizing recombinant bacillus subtilis

A technology of Bacillus subtilis and nicotinamide is applied in the field of recombinant Bacillus subtilis to produce nicotinamide mononucleotide, and can solve the problems of low product yield, failure to meet commercial production requirements, and high substrate cost

Pending Publication Date: 2020-11-27
南宁邦尔克生物技术有限责任公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In vitro enzymatic conversion requires the collection of enzyme preparations required for purification, and the use of various substrates for the enzyme conversion reaction, and the cost of the substrate is relatively high; the microbial fermentation method only needs to use nicotinamide as the reaction substrate, which is low in cost, but currently The reported yield of Escherichia coli fermentation and yeast fermentation is low, only less than 16mg / L fermentation broth, which cannot meet the requirements of commercial production

Method used

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  • Method for producing nicotinamide mononucleotide by utilizing recombinant bacillus subtilis
  • Method for producing nicotinamide mononucleotide by utilizing recombinant bacillus subtilis
  • Method for producing nicotinamide mononucleotide by utilizing recombinant bacillus subtilis

Examples

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Embodiment 1

[0024] In this example, the nicotinamide phosphoribosyltransferase expression element composed of the overlapping promoter P43 promoter derived from Bacillus subtilis (Bacillus subtilis) and the nicotinamide phosphoribosyltransferase gene derived from Haemophilus ducreyi was cloned into the integrated Plasmid pMLK83.

[0025] 1. Construction of recombinant plasmid pMLK83-P43

[0026] According to the promoter P43 sequence annotated in Genbank, the upstream primer was designed as 5'attgctggacgcttatggac3' and the downstream primer as 5'cgggatccattcctctcttacctataat 3'. PCR reaction system 100ul: DNA template (Bacillus subtilis 1A751 total DNA) 1ul (about 20ng), 5×PrimeSTAR Buffer 20ul, 10pmol / ul dNTP 2ul, 10pmol / ul forward and reverse primers 2ul each, 2.5U / ul PrimeSTAR HS DNA polymerase 1ul, add ddH 2 0 to 100ul. PCR reaction program: 94°C for 5min; 30 cycles of 94°C for 30s, 60°C for 30s, 72°C for 1min; 72°C for 10min; store at 4°C. The PCR fragment and plasmid pMLK83 were ...

Embodiment 2

[0033] This example will be derived from the promoter of the maltose amylase gene amyM derived from Bacillus licheniformis (Baclicus lincheniformis) and the trehalose synthase expression element derived from the gene of nicotinamide phosphoribosyltransferase from Thermus ruber (Meiothermus ruber) into the integration plasmid pMLK83.

[0034] 1. Construction of recombinant plasmid pMLK83-amyM

[0035] According to the promoter amyM sequence annotated in Genbank, the upstream primer was designed as 5'cccaagcttctgtacacttgcgtcctcca 3' and the downstream primer as 5'cgggatcctctcctcccctttcaatgtg3'. PCR reaction system 100ul: DNA template (Bacillus licheniformis ATCC 14580 total DNA) 1ul (about 20ng), 5×PrimeSTAR Buffer 20ul, 10pmol / ul dNTP 2ul, 10pmol / ul forward and reverse primers 2ul each, 2.5U / ul PrimeSTAR HS DNA polymerase 1ul, add ddH 2 0 to 100ul. PCR reaction program: 94°C for 5min; 30 cycles of 94°C for 30s, 60°C for 30s, and 72°C for 30s; 72°C for 10min; store at 4°C. T...

Embodiment 3

[0042] Construction of Derivative Strains of Bacillus subtilis 168 Deleting Transketolase Gene

[0043] The following DNA fragments are artificially synthesized:

[0044]

[0045]

[0046] The synthesized fragment was cloned on the Puc57 plasmid and named Puc57-TST.

[0047] Digest the Puc57-TST plasmid with EcoR I / Pst I, gel-purify the 1.7kb fragment, transform Bacillus subtilis 1A751 with conventional transformation methods, and screen spectinomycin-resistant (60ug / ml) colonies. The homologous double crossover transformants were screened by PCR method to obtain the Bacillus subtilis 168 derivative strain with deletion of transketolase gene. The PCR method for screening homologous double crossover transformants is as follows:

[0048] The following primers were synthesized: DT1: 5'gacccagaaggcaatgatgt 3'; DT2: 5'cttcaagccccgtgtatttg3'; DTC: 5'caccatcaccgcaaatactg 3'. After the transformants were cultured overnight in LB medium, the total DNA was extracted, and then th...

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Abstract

The invention discloses a method for producing nicotinamide mononucleotide by utilizing recombinant bacillus subtilis. Particularly, a nicotinamide phosphoribosyl transferase expression element is integrated into optimized modified bacillus subtilis chromosomes; and nicotinamide mononucleotide is produced through fermentation by taking the bacteria as a strain, using an optimized culture medium and adding nicotinamide as a substrate. The method in the invention has the advantages that: the nicotinamide mononucleotide is produced by utilization an expression system of food safety; the foreign gene contained in the integrated expression recombinant strain could be stably passaged and expressed; and, by utilization of the optimized recombinant strain and culture medium, the yield of the nicotinamide mononucleotide is higher than that reported in other literatures.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for producing nicotinamide mononucleotide by recombinant bacillus subtilis. Background technique [0002] Cardiovascular disease is one of the most threatening diseases to human health and life in modern society. At the same time, common diseases and symptoms of aging, such as Alzheimer's disease, osteoporosis, sarcopenia, and decreased liver function, are all associated with reduced capillaries. Studies have found that nicotinamide adenine dinucleotide (nad+, also known as coenzyme I) is a key substance, and in normal age growth in animals and humans, the bioavailability and related metabolism of nad+ in cells are Gradual decline, and then lead to physiological aging. [0003] A number of studies have begun to explore how to delay aging by increasing the activity of nad+ in the body. A nad+ precursor, nicotinamide mononucleotide (NMN), shows great promise. In animal mod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/90C12N15/67C12N15/54C12N1/21C12P19/30C12R1/125
CPCC12N15/75C12N15/902C12N15/67C12N9/1077C12P19/30C12Y204/02012
Inventor 李晓明韦航梁树华卢运琨李丛周志强韦旭钦陆迪
Owner 南宁邦尔克生物技术有限责任公司
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