Natural Product Biosynthesis: Friend or Foe? From Anti-tumor Agent to Disease Causation

Biosynthetic natural products are invaluable resources that have been gleaned from the environment for generations, and they play an essential role in drug development. Natural product biosynthesis also possesses the latent ability to affect biological systems adversely. This work implements recent advances in genomic, proteomic and microbiological technologies to understand further biosynthetic molecules that may influence progression of human disease. Azinomycin A and B are antitumor metabolites isolated from the terrestrial bacterium Streptomyces sahachiroi. The azinomycins possess an unusual aziridine [1,2-a] pyrrolidine ring that reacts in concert with an epoxide moiety to produce DNA interstrand cross-links. Genomic sequencing of S. sahachiroi revealed a putative azinomycin resistance protein (AziR). Overexpression of AziR in heterologous hosts demonstrated the protein increases cell viability and decreases DNA damage response in the presence of azinomycin. Fluorescence titration indicated AziR functions as an azinomycin binding protein. An understanding of azinomycin resistance is important for future engineering and drug delivery strategies. Additionally, the S. sahachiroi draft genome obtained via 454 pyrosequencing and Illumina sequencing revealed several silent secondary metabolic pathways that may provide new natural products with biomedical application. β-lactoglobulin (BLG), the most abundant whey protein in bovine milk, has been observed to promote the self-condensation of retinal and similar α,β-unsaturated aldehydes. BLG is a possible non-genetic instigator of cycloretinal and A2E accumulation in the macula, a condition associated with age-related macular degeneration. BLG-mediated terpenal condensation has been optimized for in vitro study with the retinal mimic citral. In rabbits fed retinal and BLG or skim milk, cycloretinal formation was detected in the blood by 1H-NMR, and SDS-PAGE analysis indicated BLG was present in blood serum, suggesting the protein survives ingestion and retains catalytic activity. Mass spectrometry and site-directed mutagenesis provided mechanistic insight toward this unusual moonlighting behavior. The experiments described in this dissertation serve to further natural product biosynthesis discovery and elucidation as they relate to consequences for human health. Efforts to solve azinomycin biosynthesis via enzymatic reconstitution, characterize compounds produced by orphan gene clusters within S. sahachiroi, and obtain a clear mechanism for BLG-promoted cycloterpenal formation are immediate goals within the respective projects