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

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.

Study on the electro-transformation conditions of improving transformation efficiency for Bacillus subtilis.

AIMS: To optimize the transformation conditions and improve the transformation efficiency of Bacillus subtilis WB800 and DB104. METHODS AND RESULTS: Trehalose, which could decrease the damage of electric shock to the cells, was added to the electroporation medium containing sorbitol and mannitol. The factors affecting the transformation efficiency, such as the growth phase of bacteria, cell concentration, electric field strength and plasmid variety, were examined and improved. The new method increased the transformation efficiency of B. subtilis by nearly 100-fold compared with the conventional one. CONCLUSIONS: With the optimized method, the transformation efficiency came up to 3.64 × 10(5) transformants µg(-1) DNA for WB800, and 2.10 × 10(5) transformants µg(-1) DNA for DB104. SIGNIFICANCE AND IMPACT OF THE STUDY: This improvement in transformation efficiency will be largely attributed to the research of expression of exogenous genes in B. subtilis, gene library construction for directed evolution and transformation of wild-type B. subtilis strains.