Vector host system for expressing antibiotic gene cluster and its application

An antibiotic, gene cluster technology, applied in the field of genetic engineering or microbiology, can solve the problem of slow growth of Streptomyces at room temperature

Active Publication Date: 2011-12-14
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, compared with E. coli and Bacillus, Streptomyces mesophilus grows slowly (doubling time in M145 exponential phase is 2.2 hours at 28°C in SMM medium)

Method used

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  • Vector host system for expressing antibiotic gene cluster and its application
  • Vector host system for expressing antibiotic gene cluster and its application
  • Vector host system for expressing antibiotic gene cluster and its application

Examples

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Embodiment approach

[0052] As an embodiment of the present invention, a method for producing antibiotics is provided, comprising: transforming antibiotic synthesis gene clusters derived from room temperature and high temperature into the Streptomyces of the present invention, and then obtaining the transformed Streptomyces. The transformed Streptomyces is cultured in the culture medium, and then the target antibiotic is obtained by fermenting the transformed Streptomyces after cultivation. This provides a host bacteria platform that can grow at high temperature for heterologous expression.

[0053] Methods for introducing foreign genes or gene clusters into Streptomyces are also known to those skilled in the art, and the present invention is not particularly limited. Preferably, a method for transforming an exogenous gene or gene cluster in Streptomyces is provided, comprising: preparing protoplasts of Streptomyces, and transferring the vector containing the exogenous gene or gene cluster by Esch...

Embodiment 1

[0086] Embodiment 1, isolate and identify Streptomyces hyperthermia from soil samples of various sources

[0087] The inventor collected soil from high temperature environment sources from various places in China in summer. Streptomyces was selectively enriched and cultured with SC medium at 50°C and isolated and purified. The inventors isolated 20, 11 and 8 strains of bacteria in vegetable garden soil, weed compost and pig manure respectively. The 16S rRNA of the isolated and purified strains were amplified, cloned and sequenced. The sequence homology analysis showed that they had high homology with various Streptomyces hyperthermophilus. For example, the 16S rRNA of 2C (CCTCC M 2010091) (as shown in SEQ ID NO: 2) is 99% similar to the 16S rRNA of S. glaucescens DSM40716 (Genbank X79322.1 GI: 488843), and is 99% similar to that of S. thermocarboxydus NBRC 16323 The 16S rRNA (Genbank AB249926.1GI: 109945079) similarity was 98%. The 16S rRNA of 4F (CCTCC M 2010092) (shown as...

Embodiment 2

[0090] Embodiment 2, identification of 4F and 2C growth characteristics of fast-growing moderate Streptomyces thermophilus

[0091] The inventors diluted the spore suspension of the isolated Streptomyces hyperthermicus strain 10 times serially, spread it on MS medium, cultured it at 30, 37, and 45°C respectively, and took pictures and recorded observations at different time points . Such as image 3 showed that 4F grew in the temperature range of 30-50°C, respectively, while the 3 strains of mesophilic Streptomyces grew at 30°C and 37°C. 4F grows very slowly at 55°C, M145, ISP5230 and GLA 4-26 cannot grow at 45°C and 50°C. Thus 4F belongs to Streptomyces moderate thermophilus.

[0092] 4F can start sporulation in 20 hours on MS medium at 37°C and 45°C, while M145, ISP5230 and GLA 4-26 have almost no visible signs of growth at this time. The growth of 4F at 37°C or 45°C was significantly better than that at 30°C or 50°C, and ISP5230 began to produce spores at about 30 hours...

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Abstract

The invention relates to a vector-host system for expressing antibiotic gene clusters and application thereof. According to the invention, high temperature streptomycete which has rapid growth and is moderately mesophile is found, and the streptomycete is able to carry out genetic transformation and can successfully express exogenous genes or gene clusters. The growth speed of the high temperature streptomycete is obviously greater than that of sky blue streptomycete, and the high temperature streptomycete is moderately mesophile, having a good production application value.

Description

technical field [0001] The invention relates to the field of genetic engineering or microbiology, in particular to a vector host system expressing an antibiotic gene cluster and its application. Background technique [0002] Streptomyces are a group of high GC-content Gram-positive bacteria, and half of the known microbial antibiotics are derived from this group. The slow-growing mesophilic Streptomyces is the source strain of many important antibiotics, but the fast-growing Streptomyces hyperthermicus resources are rarely discovered. Since the Streptomyces type strain Streptomyces coelicolor has strong restriction modifications for the transformation of double-stranded DNA derived from E. coli, for decades, the taxonomic closeness of Streptomyces coelicolor has lost the lack of strong restriction modification of the endogenous plasmid. A systematic strain of Streptomyces lividans was used as a heterologous host for gene cloning and expression. However, compared with Esche...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/76C12R1/465
Inventor 覃重军陈威华
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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