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Polyketide and nonribosomal peptide retro-biosynthesis …
There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1-alb6. Bioinformatic analysis of the proteins encoded by alb1-6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2-Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism.
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.