A method for activating the expression of microbial recessive secondary metabolite biosynthetic gene clusters

A secondary metabolite and biosynthesis technology, applied in the fields of microbiology and genetic engineering, can solve problems such as low efficiency, difficult to predict results, and low universality

Active Publication Date: 2020-07-07
GUANGDONG INST OF MICROORGANISM +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Methods such as polysin, interspecies crosstalk (co-culture) and ribosome engineering mainly rely on past experience and exquisite design, but it is difficult to predict the results and rational design in advance, which is inefficient; heterologous expression, For methods such as promoter modification, it is necessary to have a clear enough understanding of the genetic background and metabolic regulatory network of the producing bacteria, and the universality is low.

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  • A method for activating the expression of microbial recessive secondary metabolite biosynthetic gene clusters
  • A method for activating the expression of microbial recessive secondary metabolite biosynthetic gene clusters
  • A method for activating the expression of microbial recessive secondary metabolite biosynthetic gene clusters

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Embodiment 1

[0045] Inactivation of major secondary metabolite biosynthesis gene clusters by deletion of regulatory genes

[0046]Using Streptomyces vietnamensis GIMV4.0001 genomic DNA as a template, using primers Xgra4390F (5'-ACGCCAAGGAGGTGCTCACGAC-3', as shown in SEQ ID NO.1) / Xgra8287R (5'-CTTCCTGTCCGGGCACAA-3', as shown in shown in SEQ ID NO.2) to perform PCR to amplify a 3898bp fragment containing the two-component regulatory gene gra-orf11 and its upstream and downstream sequences. LA-Taq was used for PCR, and a 25 μL reaction solution containing 10 ng of genomic DNA fragments, 20 pmol of primers Xgra4390F and 20 pmol each of Xgra8287R was prepared according to the attached instructions, and carried out under the following conditions: pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 35 seconds, Anneal at 64°C for 1 minute, extend at 72°C for 3.5 minutes, do denaturation-annealing-extension cycle 30 times, store at 72°C for 10 minutes.

[0047] The PCR amplification p...

Embodiment 2

[0053] Activation of microbial mutants activates recessive secondary metabolite biosynthesis gene clusters by altered culture conditions

[0054]Group A experiment: Streptomyces vietnamensis Δgra-orf11 strain and Streptomyces vietnamensis GIMV4.0001 as a control were inoculated in Gaoshi Synthetic No. 1 Medium, Potato Glucose Medium, Sucrose Ca In the eight kinds of culture mediums including Shi's medium, calcium chloride medium, ISP3, YD, YMS, and YEME, shake culture at 30°C and 200 rpm for 7 days. Group B experiment: Another Streptomyces vietnamensis Δgra-orf11 strain and Streptomyces vietnamensis GIMV4.0001 as a control were inoculated in the above-mentioned eight kinds of media respectively, at 30°C, 200 rpm , after 3 days of shaking culture, 42°C, 200 rpm, shaking culture for 3 hours, then transfer to 30°C, 200 rpm, and continue shaking culture until the 7th day. Group C experiment: take the above eight kinds of culture medium, add 5% dimethyl sulfoxide (DMSO), ethanol a...

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Abstract

The invention discloses a method for activating the expression of microbial recessive secondary metabolite biosynthetic gene clusters. The present invention uses genetic engineering technology to partially or completely inactivate the biosynthetic gene clusters of the secondary metabolites that are the main secondary metabolites of microorganisms, so that the microorganisms do not produce or rarely produce the corresponding secondary metabolites, and obtain a relatively simple background of secondary metabolites. Then, through changes in culture conditions or genetic modification, the yield of secondary metabolites that were not expressed or expressed in a low amount was significantly increased, and became the main secondary metabolites, and were detected by chemical or biological activity analysis until some new secondary metabolites. The invention can significantly improve the success rate of recessive secondary metabolite biosynthesis gene cluster activation and product detection, and provides a new and effective method for mining new active substances from a large number of microbial recessive secondary metabolite biosynthesis gene clusters. way.

Description

Technical field: [0001] The invention belongs to the technical fields of microbiology and genetic engineering, and in particular relates to a method for activating the expression of microbial recessive secondary metabolite biosynthetic gene clusters. Background technique: [0002] The emergence and spread of multidrug-resistant bacteria, the continuous emergence of new pathogenic bacteria and the lack of high-efficiency and low-toxicity anticancer drugs make the creation and development of new anti-infection and anti-cancer drugs a very necessary and urgent task. As important natural products, microbial secondary metabolites play an extremely important role in the process of modern drug discovery and development, and are an important way and source of drug innovation [Butler MS, et al., Natural Product Reports, 2014,31:1612 -1661]. However, after decades of excavation, high-frequency repeated discoveries have seriously affected the screening efficiency of active natural pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P1/04C12P1/06C12N15/76C12R1/465
CPCC07K14/36C12N15/76C12P1/04C12P1/06C12N1/205C12R2001/465
Inventor 邓名荣朱红惠王燕林海州郭俊李燕旋陈美标王永红
Owner GUANGDONG INST OF MICROORGANISM
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