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Construction and application of engineering strain mainly producing gentamicin C1a

A technology of gentamicin and engineering bacteria, applied in the direction of genetic engineering, bacteria, microorganism-based methods, etc., can solve the problems of unreported, difficult to separate, and gentamicin C1a fermentation unit failing to meet the requirements of industrialization, etc. problems, to achieve the effect of convenient extraction and refining, single component, and meet the requirements of industrialization

Active Publication Date: 2019-10-22
福州市鼓楼区荣德生物科技有限公司
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AI Technical Summary

Problems solved by technology

However, there is still no report on the construction of new species that mainly produce single-component gentamicin C2, C2a, and C1
The work of constructing and blocking the metabolic pathway of gentamicin C1 and obtaining the metabolic pathway of gentamicin C1a has been realized, but it is facing the industrial technical problem that gentamicin C1a and C2b are difficult to separate, and the gentamicin C1a The plight of fermentation units failing to meet the requirements of industrialization

Method used

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  • Construction and application of engineering strain mainly producing gentamicin C1a
  • Construction and application of engineering strain mainly producing gentamicin C1a
  • Construction and application of engineering strain mainly producing gentamicin C1a

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Effect test

Embodiment 1

[0028] Breeding of Gentamicin C1a Strain for Obtaining Metabolic Flux

[0029] Using gentamicin-producing bacteria G1008 as the starting strain, screening by traditional methods, isolation of single colonies, fermentation in shake flasks, spotting of fermentation broth, development of volatile iodine gas, combined with detection of biological potency of fermentation units, from 2568 single colonies The strains of Micromonospora magenta whose metabolic flux was dominated by gentamicin C1a were screened, and one of them was named Micromonospora magenta Gb1068.

Embodiment 2

[0031] A gene library of Micromonospora crimsonosa varietal Gb1068 was constructed, and the DNA sequence SEQ ID NO.1 related to C6'-N-methylation of crimson samine was screened. Its characteristic DNA sequence is 4829bp, including the key gene CD3 ( Gen L ) 。 The DNA sequence of this genetic signature is far from the gentamicin biosynthesis gene cluster.

Embodiment 3

[0033] Referring to the reference [Wenrong Hong*. Lingbin Yan. Identification of gntK, a gene required for the methylation of purpurosamine C-6′ in gentamicin biosynthesis. J. Gen. Appl. Microbiol., 58, 349-356 (2012).], knock remove Gen K Gene, to obtain engineering bacteria that block the metabolic flow of gentamicin C1: Micromonospora crimson rubrum Gb1088 (Gb1088 for short).

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Abstract

The invention adopts the technique of combining traditional breeding and molecular breeding. Firstly, a key gene of genK is knocked out, the metabolic flow of gentamicin C1 is blocked, a micromonospora purpurea engineering strain Gb1088 is obtained, and components, C1, C2, C2a and G418, of the metabolic pathway of gentamicin C1 are no longer synthesized; then a genL-knocked micromonospora purpureaengineering strain Gb1098 is built. The strain does not synthesize gentamicin C2b, nor synthesizes the components, C1, C2, C2a and G418, of the metabolic pathway of gentamicin C1, and specificity synthesize gentamicin C1a. The new species micromonospora purpurea engineering strain Gb1098 has a fermentation unit of 1098 units / mL, the components are monotonous, extraction and refining are convenient, chromatography separation is not adopted, industrial production of gentamicin C1a is greatly facilitated, the key technical problems which have not been solved internationally in industrializationare solved, and the requirements of industrialization are met.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to the invention of key technologies for the industrialization of aminoglycoside antibiotics, in particular to the construction and application of engineering bacteria mainly producing gentamicin C1a. Background technique [0002] Gentamicin is a multi-component anti-infective drug, each component has different physiological activities and curative effects. Therefore, pharmacologists have been looking forward to and emphasizing the acquisition of single-component drugs for clinical application; however, due to the similar structure of glucosamine, Separation and purification are extremely difficult; therefore, it is necessary to solve the problem of obtaining a single-component industrialized new species; however, so far, it is still an international problem to obtain a single-component gentamicin industrialized strain. The present invention adopts the method of combining traditional...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/90C12P19/48C12R1/31
CPCC12N15/52C12N15/902C12P19/485
Inventor 洪文荣
Owner 福州市鼓楼区荣德生物科技有限公司
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