EXPLORING THE REGULATION OF SECONDARY METABOLITES IN STREPTOMYCES

Abstract

The genus Streptomyces harbors a wealth of untapped pharmaceutical potential within uncharacterized biosynthetic clusters whose transcription may be silent or their product undetected. Regulation of these clusters is, in part, managed by global transcription factors, nucleoid-associated proteins, and pathway-specific regulatory proteins. Consequently, a complicated interplay of regulatory elements governs secondary metabolite production and a lack of understanding of this has undermined efforts to screen for novel drug candidates. In an effort to better understand this regulation, we have focussed on one highly expressed NAP, and one silent and cryptic biosynthetic cluster. In parallel, we have leveraged our existing knowledge to improve our screening of Streptomyces for novel bioactive compounds. Here, we investigate the nucleoid-associated protein Streptomyces integration host factor (sIHF), which non-specifically binds DNA and impacts development and antibiotic production. Through targeted mutations, we probe the interfaces of sIHF thought responsible for its interactions with DNA and assess the relevance of these interfaces to in vivo activity. Further, we examine the transcriptionally silent SCO6429-SCO6438 biosynthetic cluster and attempt to activate transcription from its promoter by manipulating its potential regulatory proteins. Binding sites for the putative global regulator cAMP-receptor protein (Crp) in close proximity to this cluster and a potential pathway specific regulator are examined. Finally, we overexpress Crp in a library of Streptomyces and assay for novel bioactivity against known clinical pathogens. Collectively, this work establishes a foundation for further exploring the regulatory networks within Streptomyces and how they may be influenced to discover novel pharmaceuticals.