ISOLATION AND HETEROLOGOUS EXPRESSION OF A LARGE-SIZED POLYENE BGC IN STREPTOMYCES RUBRISOLI, A SOIL ISOLATE ACTIVE AGAINST PHYTOPATHOGENIC FUNGI

초록

Microbial-based eco-friendly biological substances are needed to protect crops from phytopathogenic fungi and replace toxic chemical fungicides that cause serious environmental issues. To develop the substances, bioassay-based antifungal screening of approximately 2,400 Streptomyces strains led to the isolation of 149 strains as tentative antifungal producers. Among them, one Streptomyces strain (called S. rubrisoli Inha501) was selected as a potential novel fungicide producer to protect various crops in the soil environment through an in vitro antifungal assay against various phytopathogenic fungi. Whole-genome sequencing of the Streptomyces strain and an anti-SMASH genome mining approach revealed a large-sized biosynthetic gene cluster (BGC) of a linear polyene compound named Inha-neotrafibricin (I-NTF) in the chromosome. An entire I-NTF BGC (~ 167 kb) was isolated through construction and screening of Streptomyces BAC library. Overexpression of the cloned I-NTF BGC in the wild-type S. rubrisoli Inha501 and its heterologous expression in S. lividans led to 2.6-fold and 2.8-fold increase in I-NTF yields, respectively. In addition, the I-NTF aglycone-producing strains were constructed by deletion of glycosyltransferase gene in I-NTF BGC. A comparison of the in vitro biological activities of I-NTF and I-NTF aglycone confirmed that the rhamnose sugar motif plays a critical role in both antifungal and antibacterial activities. As a result, the newly-screened actinomycetes species could be the promising candidates for development of novel eco-friendly microbial antifungals and the Streptomyces BAC cloning of a large-sized natural product BGC is a valuable approach for natural product titer improvement and biological activity screening of natural product in actinomycetes.