Exploring Microbial Small-Molecule Chemistry Using 2D Nmr- And Lc/Esi-Ms-Based Comparative Metabolomics
Microbial biology is integrated with a seemingly endless number of pathways involving small organic molecules. The coordinated regulation of biosynthesis, export, and detection of metabolites confer fitness for the producing microbes in nearly every environment. Continued, rapid evolution has yielded small-molecule biosynthetic enzymes capable of catalyzing an array of reactions that produce chemical species of high structural complexity, diversity, and target specificity. The procurement rate of new chemical entities (NCEs) over the recent decades has decreased, largely due to the redundant discovery of previously characterized chemical species and an incomplete understanding of the regulation and biosynth etic steps involved in microbial metabolic pathways. Methodology to establish the full diversity of metabolites produced by a particular microbial species, its metabolome, and associated biosynthetic genes will greatly accelerate our ability to procure NCEs and explore their biological roles. Metabolomics requires dedicated tools to characterize the small molecule products and biosynthetic potential of an organism. NMR spectroscopy and mass spectrometry are powerful analytical tools for unambiguously characterizing the structures of organic molecules. Therefore, the development of these methods for exploring microbial metabolism should foster access to the yet to be described metabolites of bacterial and fungal small molecule chemistry. Described herein is the development of comparative metabolomics approaches, combining differential analysis by 2D NMR spectroscopy (DANS), LC/ESI-MS profiling, with specific gene overexpression, knock-out, or knock-down strategies to associate and characterize secondary metabolites with their corresponding biosynthetic genes or gene clusters. Applied to the fungus Aspergillus fumigatus's gli cluster, DANS has revealed nine novel gliZ-dependent diketopiperazines, and their identification provides insight into gliotoxin biosynthesis. In a project involving the bacterium Streptomyces clavuligerus's hlm gene cluster, comparative NMR and LC/ESI+-MS demonstrated that the organism largely relies on alkylation though dimerization and/or methylation to detoxify deleteriously reactive thiol hlm pathway intermediates. Finally, as an extension to the A. fumigatus study, DANS is demonstrated to be a powerful tool for systematically examining the products of orphan secondary metabolic gene clusters , supporting the discovery of seven novel alkaloids in Aspergillus flavus.
comparative metabolomics; non-ribosomal peptide synthetases; secondary metabolism
Lin, Hening; Crane, Brian
Chemistry and Chemical Biology
Ph.D. of Chemistry and Chemical Biology
Doctor of Philosophy
dissertation or thesis