Comparative Genomics in Actinomycetes

Actinomycetes are prolific producers of specialized metabolites many of which have found use as pharmaceuticals and agrochemicals. Yet complete assembly of this class of bacteria has been challenging, due to their large guanine-cytosine-rich genomes, encumbering accurate biosynthetic gene cluster discovery. This thesis demonstrates strategies for improvement, which integrate optimized gDNA isolation and sequencing workflows, high-quality genome assemblies, comparative genomics and experimental validation to accelerate natural-product discovery. In this work, a robust pipeline was developed for extracting high-molecular-weight genomic DNA from hard-to-lyse filamentous Actinomycetes, coupled with long-read sequencing and polishing to routinely generate analysis-grade assemblies. Second, leveraging both newly generated and public data, a large quality-controlled repository of Actinomycete genomes spanning diverse genera was built, curated and analyzed. Comparative analyses accounting for assembly integrity, topology and chromosomal organization, revealed non-random biosynthetic gene cluster localization and synteny and highlighted biases and pitfalls in public datasets that impact mining accuracy. Guided by these insights, genome-mining approaches that unify domain architecture, gene cluster context and conserved neighborhoods to prioritize candidate biosynthetic gene clusters with elevated novelty
and translational potential, was implemented. Finally in silico predictions were validated through case studies including identification and heterologous expression of the globomycin biosynthetic gene cluster and elucidation of the Azodyrecin biosynthesis. These validations confirm gene cluster–molecule linkages, uncover mechanistic features relevant to structure–activity relationships, with the intent of providing further insight into the in silico pipeline to refine natural products pathway predictions. Collectively, this work establishes a reproducible framework for producing complete Actinomycete genomes, exposes principles of BGC organization that improve mining and demonstrates how targeted experimentation shortens the path from sequence to bioactive molecule. The resulting resources and methods enable more reliable discovery of new bioactive molecules for e.g. therapeutic and agricultural applications.