Characterisation of a silent biosynthetic biosynthetic gene cluster in Streptomyces

Natural products produced by organisms are molecules that are structurally and functionally diverse. They play an important role in modern medicine, with many being used as treatments against diseases such as cancer and cardiovascular disease. Many also possess antimicrobial activity and have been prescribed as antibiotics to treat and prevent bacterial and fungal infections. Unfortunately, the last century has seen an increase of bacteria gaining resistance against antibiotics. Paired with the slow discovery rate of novel antibiotics, antimicrobial resistance has become a significant problem and threatens modern medicine as routine operations and procedures become high risks for new infections. Advancements in DNA sequencing technologies and computational tools have uncovered a trove of silent biosynthetic gene clusters (BGCs) in Streptomyces species. Compounds encoded by silent BGCs are not produced under standard laboratory conditions and require manipulation through chemical and genetic methods to activate them. This has lead to the discovery of novel products and potential therapeutics to combat antimicrobial resistance. The work described in this project focusses on the characterisation of a silent BGC identified in Streptomyces. A multi-disciplinary approach was adopted to probe the BGC. A multi-disciplinary approach was adopted to identify the function of enzymes and proteins, ranging from computational analysis to biochemical characterisation in vitro and in vivo. Confirmation of enzyme function led to insights about compound structure and growth conditions were identified where the BGC was transcriptionally active and enabled attempts for production, purification, and functional characterisation of the compound. This approach informed about various aspects of natural product biosynthesis, revealing an enzyme class present that is responsible for a rare amino acid derivative and identifying substrates of multiple biosynthetic enzymes, which in turn yielded structural insights of the mature compound, which will aid in identification and purification