A mutant Mycobacterium smegmatis secreting niacin and its construction method

A technology of Mycobacterium smegmatis and construction method, which is applied in the field of bioengineering to achieve the effects of low cost, simple production conditions and easy amplification

Active Publication Date: 2021-08-03
FOSHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report to directly use microbial strains to prepare niacin without relying on the addition of 3-cyanopyridine and enzyme catalysis

Method used

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  • A mutant Mycobacterium smegmatis secreting niacin and its construction method
  • A mutant Mycobacterium smegmatis secreting niacin and its construction method
  • A mutant Mycobacterium smegmatis secreting niacin and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Construction of plasmid pMHS-NrtR ms

[0033] 1) Take Mycobacterium smegmatis mc 2 155 genomic DNAs were used as templates, and the primer pairs shown in SEQ ID No. 1-4 were used for PCR amplification. Such as figure 1 As shown, the PCR product nucleic acid gel electrophoresis was used to separate the target DNA fragments, and the Omega company gel back kit was used to recover the target DNA fragments shown in SEQ ID No. 5 and 6. The recovered DNA was digested with Van91I restriction endonuclease, and the digested DNA was recovered by Omega's Cycle-pure recovery kit for use;

[0034] 2) Escherichia coli DH5α strain containing the pMHS plasmid was inoculated with LB liquid medium (adding 150 μg / mL hygromycin B) and cultured overnight at 37°C, and then the bacteria were collected to extract the plasmid using the Omega plasmid extraction kit to obtain the pMHS plasmid Carry out nucleic acid gel electrophoresis after Van91I restriction endonuclease digestion ...

Embodiment 2

[0037] Example 2: Construction of plasmid pHAGE-NrtR ms

[0038] 1) LB were cultured with pHAGE (amphicillin resistance) and pMHS-NrtR ms (Hygromycin B resistance) Escherichia coli DH5α strain of the plasmid and use the plasmid extraction kit of Omega company to extract the plasmid. React overnight. The ligation product was packaged and transformed into Escherichia coli HB101 competent cells using a packaging kit (EPICENTREBiotechnologies: MaxPlax Lambda Packaging Extracts), and then coated on an LB solid plate (containing 150 μg / mL hygromycin B), and the plate was placed in a 37°C constant temperature incubator After culturing overnight, single clones grown on the plate were picked, inoculated with LB liquid medium (containing 150 μg / mL hygromycin B) and cultured overnight on a shaker at 200 rpm at 37°C, and the plasmid was extracted using Omega’s plasmid extraction kit. Validated by PacI restriction endonuclease digestion, positive plasmid pHAGE-NrtR ms Store in -20°C re...

Embodiment 3

[0039] Embodiment 3: Preparation of recombinant TM4 phage

[0040] 1) Preparation of Mycobacterium smegmatis mc 2 155 competent cells: Mycobacterium smegmatis mc 2 155 was inoculated in 5ml 7H9 liquid medium, cultured with shaking at 200rpm at 37°C until the logarithmic growth phase (OD0.5-1.0); the culture was inoculated in fresh 100mL 7H9 liquid medium at a ratio of 1:100, overnight at 37°C Cultivate until the OD600 reaches about 0.6, collect the bacteria by centrifugation at 5000rpm at 4°C for 10 minutes, wash the bacteria with pre-cooled 10% sterile glycerol at least twice, and finally add 10 mL (appropriate amount) of pre-cooled 10% glycerol, weigh After suspending the bacteria, freeze them in separate devices at -80°C for later use;

[0041] 2) will construct the correct positive plasmid pHAGE-NrtR ms Add DNA to 200 μL of M. smegmatis mc 2 155 electroporation-competent cells were incubated on ice for 10 minutes, then transferred to a 2mm BTX electroporation cup, wip...

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Abstract

The present invention provides a method for constructing a mutant Mycobacterium smegmatis secreting niacin, comprising steps: 1). Constructing plasmid pMHS-NrtR ms ; 2). Construct plasmid pHAGE-NrtR ms ; 3). Preparation of recombinant TM4 phage; 4). Construction of nrtR ms Mycobacterium smegmatis mc 2 100. The beneficial effects of the present invention are: by constructing nrtR ms The gene deletion strain Mycobacterium smegmatis enables the strain to directly prepare nicotinic acid without relying on the addition of 3-cyanopyridine and enzyme catalysis, not only avoiding various shortcomings in the existing chemical synthesis process of nicotinic acid, but also the production conditions Simple, pollution-free, easy to operate, easy to enlarge, low cost, suitable for large-scale industrial production and application, and has great value for popularization and application.

Description

technical field [0001] The invention relates to the technical field of bioengineering. Background technique [0002] Niacin is 3-picolinic acid, also known as vitamin B3, a water-soluble vitamin, belonging to the vitamin B family, one of the 13 vitamins necessary for the human body. Niacin is an important hydrogen transporter in body tissues and an anti-pellagra factor. It can maintain skin and nerve health and promote digestion. If it is lacking, pellagra can occur, manifested as symptoms such as dermatitis, glossitis, oropharynx, diarrhea, irritability, and insomnia. Together with nicotinamide, it is called vitamin PP, which is used for anti-pellagra and can also be used as a blood dilator. As a pharmaceutical intermediate, it is used in the production of isoniazid, nicotinamide, nicosyl and inositol nicotinate, etc. Niacin is also used in food products, meat additives and feed additives to prevent pellagra. In particular, the addition of nicotinic acid in the feed can...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/66C12R1/34
CPCC07K14/35C12N15/66C12N15/74
Inventor 王绪德周亚凤毕利军周盈张晓丽
Owner FOSHAN UNIVERSITY
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