Studies on the Biosynthesis and Isolation of the Biosynthetic Gene Cluster for the Salinosporamides of the Marine Bacterium Salinospora tropica

Abstract

Salinosporamide A is a potent inhibitor of the 20S proteasome and has recently entered phase I human clinical trials for the treatment of multiple myeloma. This novel marine natural product is produced by the recently described marine bacterium Salinispora tropica. Salinosporamide A contains an unusual fused gamma-lactam-beta-lactone ring structure that is uniquely functionalized. Its biosynthesis has been examined through feeding experiments with 13C-labeled precursors and a biosynthetic gene cluster has been isolated. Feeding experiments indicate that salinosporamide A is assembled from three distinct precursor molecules, namely acetate, a non-proteinogenic amino acid arising from a shunt in the shikimic acid pathway and a chlorinated tetrose molecule. Analysis of the biosynthetic gene cluster revealed that a mixed polyketide synthase (PKS) / nonribosomal peptide synthetase (NRPS) pathway is present. Three ORFs encode a large multifunctional type I PKS with a C-terminal NRPS C-domain (salA) an NRPS (salB) and a stand-alone type I KS domain (salC). Genes are also present for biosynthesis of the unusual amino acid including genes encoding a deoxy-arabino-heptulosonate-7-phosphate (DAHP) synthase (salU), a cytochrome P450 oxygenase (salD) and a potential prephenate dehydratase (salX). An unprecedented pathway for biosynthesis of chlorobutyrate is present that utilizes 5'chloro-5'-deoxyadenosine synthase (salL) in a pathway analogous to the fluorinase from Streptomyces cattleya. The cyclization and offloading may be facilitated by salO and salF that encode a cyclase and a thioesterase respectivy. A gene encoding a proteasome 20S -subunit (salJ) may be involved in resistance and genes encoding transcriptional regulatory proteins in addition to genes with no known function are also present in the cluster. Based on results from feeding experiments and the putative gene cluster, a biosynthetic scheme for salinosporamide A and some of the lesser produced salinosporamide analogs is proposed.