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Construction and Application of Engineering Bacteria Mainly Producing Gentamicin c1a

A technology of gentamicin and engineering bacteria, which is applied in the direction of genetic engineering, bacteria, and microorganism-based methods, etc. It can solve the problems that no reports have been seen, the fermentation unit of gentamicin C1a cannot meet the requirements of industrialization, and it is difficult to separate, etc. question

Active Publication Date: 2021-04-20
福州市鼓楼区荣德生物科技有限公司
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  • Description
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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 Bacteria Mainly Producing Gentamicin c1a
  • Construction and Application of Engineering Bacteria Mainly Producing Gentamicin c1a
  • Construction and Application of Engineering Bacteria 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, agene 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 present invention adopts the technique of combining traditional breeding and molecular breeding, and first knocks out Gen K The key gene is to block the metabolic flow in the direction of gentamicin C1, and obtain the micromonospora magenta engineering bacterium Gb1088, which no longer synthesizes the components C1, C2, C2a and G418 of the metabolic pathway of gentamicin C1; and then constructs Gen L Gene knockout of Micromonospora magenta engineered strain Gb1098. The strain does not synthesize gentamicin C2b, nor does it synthesize C1, C2, C2a and G418 components of the metabolic stream of gentamicin C1, and specifically synthesizes gentamicin C1a. The new species of micromonospora crimson engineering bacteria Gb1098 has a fermentation unit of 1098 units / mL, a single component, convenient extraction and purification, and does not involve chromatographic separation, which greatly facilitates the industrial production of gentamicin C1a and solves the The key technical problems that have not been solved internationally in the industrialization meet the requirements of industrialization.

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 Patents(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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