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. 2019 Dec;127(6):1698-1705.
doi: 10.1111/jam.14417. Epub 2019 Sep 2.

Characterization of three regulatory genes involved in enduracidin biosynthesis and improvement of enduracidin production in Streptomyces fungicidicus

Y-W Chen  1   2 , X-C Liu  1   3 , F-X Lv  3 , P Li  1   4
Affiliations

Characterization of three regulatory genes involved in enduracidin biosynthesis and improvement of enduracidin production in Streptomyces fungicidicus

Y-W Chen et al. J Appl Microbiol. 2019 Dec.

Abstract

Aims: To increase enduracidin production in Streptomyces fungicidicus ATCC 31731 by overexpressing positive regulators in enduracidin biosynthesis.

Methods and results: Genes orf22 and orf42 were knocked out by in-frame deletion based on CRISPR/Cas9 strategy, while the orf41 gene was inactivated by replacing it with the apramycin resistance gene cassette aac(3)IV using a fast screening blue/white system. The integrative plasmid pSET152ermE was used for the overexpression of orf22, orf41 and orf42 individually. The constructed plasmids were transformed into wild-type strain Streptomyces fungicidicus ATCC 31731. Three gene inactivation mutants Δorf22, Δorf41 and Δorf42 and three recombinant strains overexpressing orf22, orf41 and orf42 were all fermented and the enduracidin production of each strain was detected and compared by HPLC analysis. Two resulting engineered strains were generated through overexpression of gene orf22 and orf42 in Streptomyces fungicidicus, respectively, and in these strains the enduracidins titres were increased by approximately 4·0-fold and 2·3-fold higher than that of the wild-type strain.

Conclusions: The functions of three regulatory genes orf22, orf41 and orf42 in the enduracidin gene cluster in Streptomyces fungicidicus ATCC 31731 were examined. The orf22 gene, encoding a SARP family protein, was proposed to act in a positive manner. The proteins encoded by genes orf41 and orf42 were proposed to compose a two-component regulation system, in which the response protein Orf41 was characterized as a repressor, and the kinase Orf42 was shown to be an activator. The production of enduracidins was improved considerably by overexpression of the two positive regulatory genes orf22 and orf42 respectively.

Significance and impact of the study: The production of enduracidins was successfully improved by manipulating the regulatory genes involving in enduracidin biosynthesis, providing an efficient approach to improve enduracidin production further for fermentation industry and synthetic biological research.

Keywords: Streptomyces fungicidicus; biosynthesis; enduracidins; regulator; titre improvement.

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