Bacterial natural products are a rich source of pharmaceutical compounds with significant therapeutic potential. Recent advancements in genomics and bioinformatics have revolutionized the prediction of biosynthetic gene clusters (BGCs) that encode the natural products, enabling the correlation of chemical structures with the molecular machinery responsible for the biosynthesis of natural products in bacteria. The exponential growth of genomic sequencing data has generated an immense but untapped reservoir of BGCs, offering vast opportunities for novel compound discoveries. With rapid increase in the number of identified microbial natural products and their associated BGCs, the identification of novel bioactive compounds became more challenging. To overcome these obstacles, the development of innovative strategies for natural product discovery, coupled with advanced genetic tools for manipulating biosynthetic pathways, is imperative.
In this thesis study, we addressed these challenges by introducing two novel genome mining strategies for uncovering prenyltransferase-associated cyclodipeptides and heterocyclic nonribosomal peptides modified by in-trans oxidases, coupled with a metabolic profiling approach to prioritize novel compounds. Utilizing these methodologies, we identified and characterized two families of bioactive compounds: the neuroprotective griseocazines and the cytotoxic bathiapeptides. Through biotransformation experiments, we elucidated the specific roles of individual prenyltransferases in the biosynthesis of griseocazines. Through HiFi sequencing, gene knockout, mutagenesis, and phylogenetic analysis, we deciphered the biosynthetic machinery of bathiapeptides, revealing the essential functions of BatE in thiazoline oxidation and BatF in substrate transfer between the peptidyl carrier protein and the thioesterase domain.
Furthermore, we developed a novel Bacillus TAR cloning vector with enhanced cloning efficiency and stability. We optimized its parameters for efficient direct cloning and demonstrated its application by successfully cloning four nonribosomal peptide synthetase (NRPS) BGCs containing in-trans NRPS oxidases. Collectively, our findings in this thesis work provide valuable strategies and a robust direct cloning tool for natural product discovery, demonstrating their efficacy through the identification of novel bioactive compounds and the comprehensive characterization of their biosynthetic pathways. These findings advance our understanding of bacterial natural product biosynthesis and provide a foundation for future drug discovery efforts.
 

All Are Welcome!