Genetic Approaches to Regulation and Identification of Bacterial Specialized Metabolites

Abstract

In natural habitats, microorganisms synthesize specialized metabolites and enzymes that mediate chemical interactions between organisms. Those natural products have various biological activities, acting as signaling chemicals, siderophores, and antibiotics. Bioinformatic predictions suggest that many undiscovered metabolites are encoded in the genomes of soil bacteria, especially Streptomyces. Genetic strategies to disrupt metabolite biosynthesis provide a comparative means to uncover new molecules and their activities. However, genetic manipulation of Streptomyces is inefficient due to low recovery from recombination. To overcome these limitations, in this study, we adapted CRISPR-Cas9 based mutagenesis in Streptomyces sp. Mg1. To further improve the efficiency of genetic engineering, we exploited a dual-sgRNA CRISPRi system, which uses double guide RNAs and catalytically inactive dCas9 to diminish the production of targeted specialized metabolic pathways. With the improved genetic strategies, we discovered lavendomycin biosynthetic pathway from S. Mg1 and a potential new thiopeptide from Streptomyces albus. Meanwhile, we identified the biological functions associated with lavendomycin and the novel thiopeptide. Using a genetic approach, we uncovered regulatory functions in the specialized metabolic pathways.

Genetic and chemical approaches and identifications demonstrated in this dissertation improve the processes of phenotypic detection and metabolic identification. This research also expands our understanding of bacterial specialized metabolism.