New methods for the discovery and characterization of bacterial natural products

Abstract

Actinomycetes are renowned for their ability to produce a wide array of natural products (NPs), with a broad range of applications. NPs are typically encoded by biosynthetic gene clusters (BGCs). Bioinformatic analysis of sequenced Actinomycetes genomes have shown that they possess many BGCs whose potential products are not detected under standard laboratory culture conditions. Advances in genome mining and molecular biology have led to the development of diverse strategies to activate these “cryptic” BGCs, but further improvements in these methods are warranted. This thesis explores multiple approaches for discovering novel bioactive compounds from Actinomycetes and investigating their BGCs. Chapters 3 and 4 focus on the development of a new bacterial artificial chromosome (BAC) vector, designated pJE2, and optimization of a protocol for genomic library construction enabling the isolation of large BGCs. This approach was applied to Actinomadura madurae T576, a producer of unusual sulphated metabolites, resulting in a BAC library from which a clone harbouring the complete 80 kbp BGC responsible for these metabolites was identified. Heterologous expression of this clone in Streptomyces albus successfully led to production of the sulphated metabolites. Screening of additional clones also revealed the successful capture of several previously uncharacterized BGCs. Chapter 5 focuses on a chemical elicitation method, using plant extracts to stimulate NP production in soil-derived Actinomycetes. Notably, hibiscus flower extract induced the production of the antibiotic thiolutin by Streptomyces strain MBN 2-2. Further analysis identified hibiscus acid and hydroxycitric acid as the elicitor compounds. The final chapter centres on associating putative BGCs with the synthesis of three novel antibiotics demurilactone A, persiathiacin, and quinovosamycin. In all three cases, insertional mutagenesis led to the abolition of antibiotic synthesis, thereby validating assignment of the BGCs to their respective antibiotics.