AUTHOR=Park Heung-Soon , Park Ji-Hee , Kim Hye-Jin , Kang Seung-Hoon , Choi Si-Sun , Kim Eung-Soo 

TITLE=BAC cloning and heterologous expression of a giant biosynthetic gene cluster encoding antifungal neotetrafibricin in streptomyces rubrisoli

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=10

YEAR=2022

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2022.964765

DOI=10.3389/fbioe.2022.964765

ISSN=2296-4185

ABSTRACT=<p>Polyene natural products including nystatin A1, amphotericin B, ECO-02301, and mediomycin belong to a large family of valuable antifungal polyketide compounds typically produced by soil actinomycetes. A previous study (Park et al., Front. Bioeng. Biotechnol., 2021, 9, 692340) isolated <italic>Streptomyces rubrisoli</italic> Inha501 with strong antifungal activity and analyzed a large-sized biosynthetic gene cluster (BGC) of a linear polyene compound named Inha-neotetrafibricin (I-NTF) using whole genome sequencing and bioinformatics. In the present study, an entire I-NTF BGC (∼167 kb) was isolated through construction and screening of <italic>Streptomyces</italic> BAC library. Overexpression of the cloned I-NTF BGC in the wild-type <italic>S. rubrisoli</italic> Inha501 and its heterologous expression in <italic>S. lividans</italic> led to 2.6-fold and 2.8-fold increase in I-NTF yields, respectively. The qRT-PCR confirmed that the transcription levels of I-NTF BGC were significantly increased in both homologous and heterologous hosts containing the BAC integration of I-NTF BGC. In addition, the I-NTF aglycone-producing strains were constructed by a target-specific deletion of glycosyltransferase gene present in I-NTF BGC. A comparison of the <italic>in vitro</italic> biological activities of I-NTF and I-NTF aglycone confirmed that the rhamnose sugar motif of I-NTF plays a critical role in both antifungal and antibacterial activities. These results suggest that the <italic>Streptomyces</italic> 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.</p>