Conjugation-based genome engineering enables rapid prototyping and bioproduction in non-model bacteria
Non-model bacteria offer unique metabolic capabilities for sustainable bioproduction, yet their limited genetic accessibility hinders systematic strain development. Here we present conjugation-based serine recombinase-assisted genome engineering (cSAGE), a portable platform for sequential, site-specific genomic integration in transformation-resistant bacteria. Using Rhodobacter sphaeroides as a testbed, we show that genome integration fails by electroporation and is not rescued by disruption of three candidate restriction endonucleases. Conjugative delivery of the same integration vectors recovers genomic integrants at frequencies of approximately 10-4 per recipient cell, an improvement of at least five orders of magnitude over electroporation. We demonstrate site-specific genomic integration across eight bacterial hosts spanning four classes and two phyla, as well as three sequential payload integrations using a standardized, non-replicating plasmid toolkit. We then use cSAGE to install the same bioproduction pathway in two Rhodopseudomonas palustris strains: the reference strain CGA009 and the environmental isolate P4, for which genetic engineering has not previously been reported. Under anoxygenic photosynthetic growth conditions, engineered P4 produces p -vinylphenol from p -coumarate, whereas CGA009 engineered with the same construct, does not. By enabling standardized genome engineering and cross-host evaluation of bioproduction pathways, cSAGE provides a general framework for non-model strain prototyping and biotransformation discovery.