The biological world is the source of numerous naturally produced chemicals of significance to human health, including the life saving antibiotics penicillin and vancomycin. Recent technological and intellectual advances in the genetic and biochemical understanding of how the multitude of plants, fungi and bacteria produce such natural products has revolutionized their discovery. Critical to the growth and impact of natural product drug development is the continued discovery of new natural products and the elucidation of the mechanisms of their biosynthesis. The pyrroloquinoline alkaloids are a diverse class of natural products which exhibit broad biological activity. Despite their production in a number of organisms, little is known about the biosynthesis of the pyrroloquinoline alkaloids. Recently studies of lymphostin, a pyrroloquinoline alkaloid with potent immunosuppressant properties, have begun to shed light on a unique biosynthesis. I hypothesize, based upon an analysis of the lymphostin gene cluster and my recent work characterizing the gene cluster for the biosynthesis of two structurally related anticancer compounds, ammosamide A and B, that these natural products arise from the chemical modification of a peptide produced by ribosomal synthesis. This unusual mechanism may prove to be general for the pyrroloquinoline alkaloids, and a genetic and biochemical understanding may open entirely new directions for the discovery of related natural product drugs. The goal of this proposal is to further elucidate the biosynthesis of pyrroloquinoline alkaloids by the comprehensive genetic and biochemical characterization of the biosynthesis of ammosamides A and B. Because lymphostin and the ammosamides are produced by two different bacteria, a comparative genetic analysis will be highly informative of the generality of the biosynthetic mechanism, especially with regard to the putative role that ribosomal peptide synthesis plays in the process. For the proposed work, the ammosamide biosynthetic gene cluster will be cloned, expressed in a heterologous host and validated using directed gene deletions, which will knockout the production of the ammosamides. The functional role that each gene plays in the biosynthesis will be interrogated by introducing chemical complements to the knocked out genes to ultimately restore ammosamide production. Further mechanistic detail will be achieved through in vitro biochemical studies of the early stages of ammosamide biosynthesis. The knowledge of the biosynthesis will then be used to bioengineer the production of new analogues of the ammosamides, which will be assayed for their biological potency.
In this proposal I described a plan to elucidate the biosynthetic mechanism by which a class of potent anticancer compounds, the ammosamides, is produced in bacteria. An understanding of this biosynthetic process may greatly accelerate the discovery of new compounds relevant to cancer treatment. This proposal outlines a strategy to exploit the biological mechanism for the production of ammosamides to ultimately bioengineer new and potentially more potent versions of these molecules for development as future anticancer drugs.