Genetic Manipulation and Heterologous Expression of Silent Biosynthetic Gene Clusters in Actinomycetes

Abstract

The escalating global crisis of antimicrobial resistance (AMR) poses a severe threat to human health, creating an urgent need to discover novel antimicrobial compounds. Natural products derived from actinobacteria represent a rich and diverse repertoire of bioactive molecules. However, biosynthetic gene clusters (BGCs) encoding these compounds ae often poorly expressed under standard laboratory conditions. As the conventional antibiotic discovery pipeline has dried up, synthetic biology techniques have emerged as a promising alternative to unlock this untapped biosynthetic potential. This thesis aimed to activate putative BGCs to discover novel antimicrobial natural products through two activation strategies, namely heterologous expression and transcriptional activation in the native host via promoter engineering and regulatory gene overexpression. These strategies involved genome mining to detect putative BGCs, genetic engineering and cloning of BGCs into expression vectors, and heterologous expression to isolate and characterise encoded natural products. Seven BGCs were provided at the outset of this project. Subsequent genome mining of 40 actinomycete strains from the Owen laboratory collection identified nine additional putative BGCs. Of these eleven BGCs were successfully assembled into plasmid vectors. Ten SARP regulatory genes were identified across five of these clusters, and one was successfully conjugated. This research provides a foundation for future isolation and characterisation studies to discover novel bioactive natural products