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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 Escherichia coli. The ~34 kb pathway for production of alterochromide lipopeptides by Pseudoalteromonas piscicida JCM 20779 was captured and heterologously expressed in E. coli utilizing native and E. coli-based T7 promoter sequences. Our approach enabled both facile production of the alterochromides and in vivo 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.

acs synthetic biology

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

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