Discovery, expression, and characterization of biosynthetic gene clusters encoding antimicrobial secondary metabolites from a soil metagenome and cultured Actinobacteria
Type of DegreePhD Dissertation
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Dating back to the discovery of penicillin in 1928, nature and its diverse microbial life has been the most prolific source of antimicrobial compounds. These bioactive compounds are often derived from microbial secondary metabolites whose biosynthesis is encoded by a group of genes that cluster together in the genome known as biosynthetic gene clusters (BGCs). Advancements in sequencing and bioinformatics has revealed a wealth of uncharacterized BGCs from environmental microorganisms. Accessing these untapped gene clusters is important for the discovery of novel therapeutic compounds for human health, and to advance our understanding on their function, diversity, and evolution. The purpose of this dissertation was to identify, express, and characterize novel gene clusters encoding antimicrobial secondary metabolites using culture-dependent and –independent techniques. Although different in their methodologies, each chapter is centralized on identifying BGCs from and/or expressing clusters in taxa from the Gram-positive bacterial phylum Actinobacteria. Members of the phylum Actinobacteria, particularly from the genus Streptomyces, are widely distributed in soil and marine environments and are the source of most clinically used antibiotics. The established biosynthetic potential of Actinobacteria taxa makes them an ideal target to find new BGCs and to serve as hosts for the expression of gene clusters encoding bioactive secondary metabolites. In Chapter 1, I discuss the role of Actinobacteria, with particular emphasis on the genus Streptomyces, in past and modern strategies to find natural products and offer insights into their advantages and limitations. For Chapter 2, a culture-based approach was taken to isolate a new species of the genus Streptomyces from marine sponges collected in the Trondheim fjord of Norway. This new species, Streptomyces poriferorum P01-B04T, was found to harbor many BGCs encoding secondary metabolites in its genome and expressed antibacterial activity against a drug-resistant Staphylococcus aureus pathogen. For Chapter 3, a culture-independent (i.e. metagenomics-based) strategy was used to identify and express BGCs predicted to encode novel chemistry from a soil metagenome in an engineered Streptomyces coelicolor heterologous host. A high hit rate of S. coelicolor clones were found to inhibit the growth of a multidrug-resistant Acinetobacter baumannii pathogen. The expression of a BGC by a particular clone, P17B06, could be enhanced using a dual-inducible expression vector and attempts were made to characterize the antimicrobial secondary metabolites encoded by this gene cluster through a bioactivity-guided fractionation approach. Finally, in Chapter 4, I explored the function and taxonomic origin of another BGC (P12B21 BGC) encoding antimicrobial metabolites derived from the soil metagenomic library. I found that the P12B21 BGC encodes a hybrid biosynthetic pathway with homology to a gene cluster from Microbacterium hatanonis, a member of the phylum Actinobacteria. Collectively, this dissertation work demonstrates the value of culture-dependent and metagenomics-based approaches to access and study biologically-relevant gene clusters from microorganisms.