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Integrated recombinant mycobacterium smegmatis producing nicotinic acid and construction method thereof

A technology of Mycobacterium smegmatis and integration type is applied in the field of integration type recombinant Mycobacterium smegmatis and its construction to achieve the effects of low cost, simple production conditions and easy amplification

Active Publication Date: 2018-08-10
上海晶诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation of nicotinic acid by biocatalysis mainly relies on the production of nitrilase by microbial fermentation. Through this enzyme to catalyze the conversion of 3-cyanopyridine into nicotinic acid, the raw material 3-cyanopyridine still needs to be added.

Method used

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  • Integrated recombinant mycobacterium smegmatis producing nicotinic acid and construction method thereof
  • Integrated recombinant mycobacterium smegmatis producing nicotinic acid and construction method thereof
  • Integrated recombinant mycobacterium smegmatis producing nicotinic acid and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 C Gene amplification and construction of plasmid pMV361-NudC ms

[0045] 1, C gene amplification

[0046] Mycobacterium smegmatis mc 2 155 genomic DNA as a template, using the primer pair 5'-ATCGGAATTCATGAGCGAACACCGCACGT-3' / 5'-TGCAAAGCTTTCAGTCGAGTGCGGCCCAGG-3', PCR amplification C Gene ORF sequence. The PCR product was subjected to nucleic acid gel electrophoresis to separate the target DNA fragment, and the Omega company gel back kit was used to recover the target DNA fragment, which was obtained after sequencing C The ORF sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2. The recovered DNA was digested with EcoRI and HindIII restriction endonucleases, and the digested DNA was recovered by the Cycle-pure recovery kit from Omega Company for future use.

[0047] 2. Digestion

[0048] Escherichia coli DH5α strain containing the pMV361 plasmid was cultured in LB, and the plasmid was ...

Embodiment 2

[0053] Example 2 Construction of NudC Overexpression Mycobacterium smegmatis strain

[0054] 1. Preparation of Mycobacterium smegmatis mc 2 155 competent cells

[0055] Preparation of Mycobacterium smegmatis mc 2 155 competent cells, pick fresh Mycobacterium smegmatis mc 2 155 single colonies were inoculated in 5 ml 7H9 liquid medium, cultured at 37°C with shaking at 200 rpm until the logarithmic growth phase (OD0.5-1.0); the culture was inoculated in fresh 100 ml 7H9 liquid medium at a ratio of 1:100 , cultured overnight at 37°C to OD 600 Centrifuge at 5000 rpm for 10 min at 4°C to collect the bacteria, wash the bacteria at least twice with pre-cooled 10% sterile glycerol, and finally add 10 ml (appropriate amount) of pre-cooled 10% glycerol, blow After the bacteria were homogenized, they were stored in separate devices at -80°C for later use.

[0056] 2. Transform Mycobacterium smegmatis mc 2 155

[0057] Take the correctly constructed positive plasmid pMV361-NudC ms...

Embodiment 3

[0060] Example 3 Mycobacterium smegmatis mc 2 155-pMV361-NudC ms secrete niacin

[0061] 1. Mycobacterium smegmatis mc 2 155-pMV361-NudC ms Inoculate 7H9 liquid medium, culture to logarithmic growth phase at 37°C, use Millipore 0.22μm sterile filter to filter the culture medium and collect the filtered filtrate, then use Millipore3-kDa ultrafiltration concentrator tube to remove protein and other macromolecules in the filtrate and Collect the filtered filtrate. The obtained filtrate was separated and analyzed for niacin using high performance liquid chromatography (HPLC).

[0062] Specific conditions for HPLC:

[0063] Equipment model: Thermo Fisher Scientific UltiMate 3000 ultrahigh-performance liquid chromatography

[0064] Chromatographic column: Thermo Fisher, Hypersil Gold aQ, 150×4.6 mm, 3 μm, column temperature: 30°C;

[0065] Mobile phase: water phase: deionized water (containing 0.1% formic acid);

[0066] Organic phase: methanol;

[0067] Gradient elution: 9...

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Abstract

The invention discloses an integrated recombinant mycobacterium smegmatis producing nicotinic acid and a construction method thereof. The invention requires the protection of a recombinant mycobacterium smegmatis producing nicotinic acid, the recombinant mycobacterium smegmatis producing nicotinic acid is characterized in that the recombinant mycobacterium smegmatis contains recombinant integrative plasmid, which contains nudC gene, and the nucleotide sequence is shown as SEQ ID NO:1. Nicotinic acid can be directly obtained by one step through the engineering strain prepared by the invention without adding 3-cyanopyridine, therefore various shortcomings in the existing chemical synthesis process of nicotinic acid can be avoided, and also the method has the characteristics of simple production condition, no pollution, simplicity and feasibility, easy enlargement and low cost, is suitable for large-scale industrial production and application, and has great popularization and applicationvalue.

Description

technical field [0001] The invention relates to the technical field of nicotinic acid production, and more specifically relates to a nicotinic acid-producing integrated recombinant mycobacterium smegmatis and a construction method thereof. Background technique [0002] Niacin is 3-pyridinecarboxylic acid, also known as vitamin B3, belongs to vitamin B group, is a water-soluble vitamin, belongs to vitamin B group, and is one of the 13 kinds of vitamins necessary for 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, nicotinami...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P17/12C12R1/34
CPCC07K14/32C12N15/74C12P17/12
Inventor 王绪德周亚凤刘雪宾
Owner 上海晶诺生物科技有限公司
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