|Title||Targeted capture and heterologous expression of the pseudoalteromonas alterochromide gene cluster in Escherichia coil represents a promising natural product exploratory platform|
|Publication Type||Journal Article|
|Year of Publication||2015|
|Authors||Ross AC, Gulland L.ES, Dorrestein PC, Moore BS|
|Journal||ACS Synthetic Biology|
|Type of Article||Article|
|Keywords||antagonistic interactions; biosynthesis; biosynthetic pathways; cloning; coli; discovery; drug; heterologous expression; lipopeptides; marine natural products; marine-bacteria; metabolites; peptides; transformation-associated recombination; tunicata|
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 dusters using a transformation associated recombination (TAR) strategy coupled with direct pathway manipulation and expression in Escherichia coli. The similar to 34 kb pathway for production of alterochromide lipopeptides by Pseudoalteromonas piscicida JCM 20779 was captured and heterologously expressed in Escherichia coli utilizing native and Escherichia 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.