Targeted Capture and Heterologous Expression of the <i>Pseudoalteromonas</i> Alterochromide Gene Cluster in <i>Escherichia coli</i> Represents a Promising Natural Product Exploratory Platform

Abstract

Marine pseudoalteromonads represent a very promising source of biologically important natural product molecules. To access and exploit the full chemical capacity of these cosmopolitan Gram-(−) bacteria, we sought to apply universal synthetic biology tools to capture, refactor, and express biosynthetic gene clusters for the production of complex organic compounds in reliable host organisms. Here, we report a platform for the capture of proteobacterial gene clusters using a transformation-associated recombination (TAR) strategy coupled with direct pathway manipulation and expression in <i>Escherichia coli</i>. The ∼34 kb pathway for production of alterochromide lipopeptides by <i>Pseudoalteromonas piscicida</i> JCM 20779 was captured and heterologously expressed in <i>E. coli</i> utilizing native and <i>E. coli</i>-based T7 promoter sequences. Our approach enabled both facile production of the alterochromides and <i>in vivo</i> interrogation of gene function associated with alterochromide’s unusual brominated lipid side chain. This platform represents a simple but effective strategy for the discovery and biosynthetic characterization of natural products from marine proteobacteria