Engineering bacteria for producing gentamicin C1a and application thereof

A gentamicin, engineering bacteria technology, applied in the direction of enzymes, bacteria, microorganism-based methods, etc., can solve the problems of serious pollution, slow progress in the application of Micromonospora, and long chromatographic separation cycles.

Active Publication Date: 2012-10-17
FUZHOU UNIV +1
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Streptomyces genetic engineering, which emerged in the 1980s, has made some progress in the cloning of antibiotic biosynthesis genes, yield improvement, component improvement, and hybrid antibiotic production. However, the application in Micromonospora Slow progress
These complexes have similar chemical structures and similar physical and chemical properties. It is very difficult to separate single-component gentamicin C1a from them, resulting in a shortage of gentamicin C1a, high production costs, long chromatographic separation periods, and low yields. The consumption is high, the pollution is serious, the extraction and refining process is complex, and the product quality is unstable. The by-products with a structure similar to gentamicin C1a may interfere at any time, which has caused great uncertainty to the quality control of etimicin products. Serious It affects the stability, safety and effectiveness of drugs, and is an urgent scientific problem to be solved

Method used

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  • Engineering bacteria for producing gentamicin C1a and application thereof
  • Engineering bacteria for producing gentamicin C1a and application thereof
  • Engineering bacteria for producing gentamicin C1a and application thereof

Examples

Experimental program
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Embodiment 1

[0029] The present invention comprises following main steps:

[0030] 1. Construct wxya gene replacement plasmid

[0031] According to the gentamicin biosynthetic gene cluster (refer to GenBank Accession Number AY524043), respectively in wxya Two pairs of primers LB1 and LB2 and LB3 and LB4 were designed upstream and downstream of the gene (SEQ.NO.1) to start the strain S-1212 (Zhou Xiaolan, Huang Jianzhong, , Shi Bihong, Shi Qiaoqin. Affecting Micromonospora magenta ( Micromonospora purpurea) S-1212 spore germination of several main factors. Fujian Normal University Journal (Natural Science Edition), Vol.18 No.1, p72-75) chromosomal DNA (CTAB method) as a template, PCR, amplified The DNA sequence at both ends of gntK was added as a homologous exchange arm; the upstream exchange arm was called KB1 (LB1 / LB2) with a length of 2076 bp; the downstream exchange arm was called KB2 (LB3 / LB4) with a length of 2044 bp. Plasmid pAGe was used as template (Zhu Biyin, Hong Wenr...

Embodiment 2

[0040] Example 2: Preparation of Metabolite C1a of Micromonospora magenta GK1101

[0041] provided by the invention wxya The inactivated engineering bacteria Micromonospora crimson GK1101 can be directly used to manufacture gentamicin C1a. The preparation of gentamicin C1a is as follows (Hong Wenrong. Optimization of gentamicin fermentation process. Chinese Journal of Pharmaceutical Industry. 1994, 25(1).p1-3,):

[0042] 1. Fermentation of Micromonospora magenta strain GK1101

[0043] Seed medium: glucose 0.1%, corn starch 1.0%, corn flour 1.5%, peptone 0.2%, soybean cake powder 1.0%, KNO3 0.05%, CaCO3 0.5%, pH7.0.

[0044] Fermentation medium: corn starch 6.0%, corn flour 1.0%, peptone 0.4%, soybean cake powder 2.0%, KNO 3 0.01%, (NH 4 ) 2 SO 4 0.1%, CaCO 3 0.5%, amylase 0.025%, pH7.5.

[0045] Shake flask fermentation: Micromonospora magenta GK1101 obtained in step 3 of Example 1 was fermented. Before fermentation, isolate the single colony with rich sporulatio...

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Abstract

The invention belongs to the technical field of medicines and relates to engineering bacteria which are used for producing gentamicin C1a and have a function of deactivating gntK and application of the engineering bacteria. The engineering bacteria are micromonospora purpurea GK1101 which were registered and collected in China General Microbiological Culture Collection Center (CGMCC) in September13, 2011 and have the collection number of CGMCC No.5245. The engineering bacteria are applied to preparation of antibacterial medicines. The engineering bacteria for producing the gentamicin C1a are obtained; the production process is simplified; production cost is reduced; and quality control over the products is facilitated.

Description

technical field [0001] The invention belongs to the field of antibiotic pharmacy, and relates to the construction and application of an engineering bacterium. Specifically, the invention relates to an engineering bacterium producing gentamicin C1a, which is used in the manufacture of antibiotics. Background technique [0002] Micromonospora can produce abundant secondary metabolites, especially aminoglycoside antibiotics, such as gentamicin, sisomicin and fortimicin. The distribution of these micromonospora is peculiar, the growth is slow and the cell wall structure is special, so the research progress is slow. In addition, due to the lack of a general gene operating system, the genetic manipulation of Micromonospora is difficult, making the research on its molecular biology lag behind that of Streptomyces. [0003] Micromonospora is a gentamicin-producing bacterium, and gentamicin is an aminoglycoside antibiotic that has been clinically used for nearly half a century and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P19/50C12R1/01
CPCC12N9/1007C12P19/485C12N1/205C12R2001/31
Inventor 洪文荣严凌斌林玉双封成军
Owner FUZHOU UNIV
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