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[Cloning of mitomycin C resistance gene from Streptoverticillium caespitosum ATCC27422 and studying its function]
1Laboratory of Molecular Regulation for Microbial Secondary Metabolism, Shanghai Institute of Plant Physiology, Chinese Academy of Science, Shanghai 200032.
Abstract:
Streptoverticillium caespitosum is the major producer of anti-cancer drug mitomycin. It demonstrated that there exists a strong restriction system for the transformation of foreign DNA in this strain. Using the blocked mutant S-6 which is sensitive to mitomycin C (MC) and does not produce mitomycin as a recepient and plasmid pIJ699 as vector, the gene library of the strain ATCC27422 was constructed. A clone of mitomycin C resistance gene(mcr) was obtained by screening from this library. The mitomycin C resistance gene cloned on the 6.2 kb fragment of plasmid pLX5 could be expressed in the Strptonmyces lividans TK54. It was the first time to introduce successfully the plasmid pLX5 into the wild type strain ATCC27422 by electroporation technique. And the transformant's resistance to mitomycin C has risen greatly: the MIC has gone up from 200 micrograms/ml to more than 1000 micrograms/ml. The result of fermentation showed that the yield of mitomycin of ATCC27422 harboring plasmid pLX5 was more than the wild strain ATCC27422's. It suggested that there is some relationship between the level of the resistance to mitomycin C and its biosynthesis.
Insights
Researchers identified a mitomycin C resistance gene (mcr) in Streptoverticillium caespitosum. Introducing this gene into the strain significantly increased mitomycin C resistance and enhanced drug production.
Area of Science:
- * Microbiology and Genetic Engineering: Focuses on microbial genetics and the manipulation of Streptoverticillium caespitosum for biotechnological applications.
- * Drug Discovery and Development: Investigates the genetic basis of antibiotic resistance and biosynthesis, specifically for the anti-cancer drug mitomycin C.
Context:
- * Streptoverticillium caespitosum is a key producer of the anti-cancer compound mitomycin C.
- * Genetic manipulation of this strain is challenging due to a robust DNA restriction system.
- * Previous efforts to engineer the strain for improved mitomycin C production were limited.
Purpose:
- * To overcome the DNA restriction system in Streptoverticillium caespitosum and enable genetic transformation.
- * To identify and clone the mitomycin C resistance gene (mcr) from Streptoverticillium caespitosum.
- * To enhance mitomycin C production in Streptoverticillium caespitosum through genetic modification.
Summary:
- * A gene library from Streptoverticillium caespitosum ATCC27422 was constructed using a blocked mutant (S-6) and plasmid pIJ699.
- * A mitomycin C resistance gene (mcr) was identified and cloned onto the 6.2 kb fragment of plasmid pLX5.
- * Plasmid pLX5 was successfully introduced into Streptoverticillium caespitosum ATCC27422 via electroporation, significantly increasing mitomycin C resistance (MIC >1000 µg/ml).
- * Fermentation studies revealed that transformants harboring pLX5 exhibited higher mitomycin C yields compared to the wild-type strain.
Impact:
- * Demonstrates the successful introduction of foreign DNA into Streptoverticillium caespitosum, overcoming its restriction system.
- * Provides the first successful expression of a mitomycin C resistance gene (mcr) in Streptomyces lividans TK54.
- * Suggests a correlation between mitomycin C resistance levels and the biosynthesis of mitomycin C, opening avenues for yield improvement.