Discovery of new natural products from Actinobacteria using Cas12a-directed cloning

Abstract

Microbial natural products are the source of over 70% of all known antibiotics, yet the pace of their discovery has slowed significantly since its peak in the mid-20th century. This stagnation is largely due to the repeated isolation of known compounds from readily culturable microorganisms, while the vast majority of microbial biosynthetic gene clusters (BGCs) remain silent and unexpressed under typical laboratory conditions. The convergence of genomics, synthetic biology, and high resolution analytical chemistry now provides a powerful toolkit to unlock this cryptic biosynthetic potential. This thesis presents a strategy that integrates these disciplines to awaken silent BGCs and discover novel bioactive molecules. A genome mining approach was utilised to identify 22 promising BGCs from diverse actinobacteria, prioritised for their predicted novelty. To activate their expression, a suite of synthetic biology and molecular cloning strategies was implemented in both native and engineered heterologous hosts. This systematic activation campaign yielded several significant outcomes: (i) the linking of three known compounds to their previously unknown BGCs; and (ii) the discovery and structural elucidation of two novel natural products. Notably, one of the compounds represents a new class of calcium-dependent antibiotics with potent antimicrobial activity. In conclusion, this research demonstrates the efficacy of a genome-led approach to drug discovery. It has successfully translated genomic data into tangible chemical matter, functionally characterised previously cryptic BGCs, and contributed a novel class of antibiotics to the global pipeline for combating infectious diseases.