CAREER: Proteomic Tools for the Study of Nonribosomal Peptide Biosynthesis

University Of California-San Diego, La Jolla CA

Investigators

Abstract

Non-ribosomal peptides are a large class of secondary metabolites produced in both eukaryotes and prokaryotes as molecular vectors for the regulation of natural environments. These metabolites have been studied for over 100 years and are commonly used in a variety of practical applications. The future discovery of non-ribosomal peptides lies within two avenues: 1) continued sampling of native producer organisms and 2) metabolic engineering. This project opens access to a novel collection of domain activity assays for non-ribosomal peptide (NRP) synthases. The project forwards a bold new means to selectively functionalize NRP domains and, thereby, provides a collection of tools that further the techniques used to decode biosynthetic machinery. These tools are developed through the synthesis of a series small molecule probes that are tuned to recognize and interact with specific functional aspects of NRP synthases. In particular, novel approaches are forwarded to identify peptidyl carrier protein, adenylation, and condensation domains. The tyrocidine A synthase from Bacillus brevis, the organism producing the cyclic peptide antibiotic tyrocidine A, will be used as the primary model for this study. It is anticipated that the methods described herein represent the beginnings of a functional system to identify, isolate, and manipulate modular synthase enzymes from both native and engineered NRP pathways. The lessons learned serve to further the understanding of enzyme mechanisms, natural product isolation, and metabolic engineering. These approaches also make understanding chemistry and biology of the natural world more accessible and exciting to students. Broader Impacts: In addition to the research component, this award supports educational and training activities. The project offers new groundwork to teach secondary metabolism as the integration of chemistry and biology. This discipline has changed enormously in the last fifteen years, and its instruction must include new advances in genomics, proteomics, and computational methods thereon. Proteomic methods developed in this project can be adapted to identify and optimize natural product biosynthesis from a functional perspective. For teaching purposes, these tools provide an ideal opportunity for students to come in contact with complex enzymatic systems. These tools necessitate an interdisciplinary approach for studying familiar natural products from the interface of chemistry and biology.