Conjugation-based genome engineering enables rapid prototyping and bioproduction in non-model bacteria
Guzman, M. S.; Kiattisewee, C. I.; Comes, J.; Robert, A. M.; Cardiff, R. A. L.; Cook, M.; Scott, A. V.; Alba Burbano, D.; Anastasakis, S.; Grube, S.; Heiberg, K.; Darst, B. H.; Howell, D.; Dexter, G. N.; Egbert, R. G.; Zalatan, J. G.; Guss, A.; Elmore, J. R.; Beliaev, A. S.; Carothers, J. M.
Show abstract
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 broad-host-range platform that enables predictable, iterative genomic integration in transformation-resistant bacteria. cSAGE combines conjugative DNA delivery, standardized low-copy vectors, orthogonal recombinases, and modular genetic parts to support rapid pathway assembly and cross-host benchmarking. Using purple nonsulfur bacteria as a testbed, we integrate promoter engineering, multi-payload genome modification, and genome-scale metabolic modeling to empirically evaluate host-dependent pathway performance. Applying this workflow, we identify strain-specific differences in photosynthetic conversion of lignin-derived p-coumarate to the thermoplastic precursor p-vinylphenol. By enabling genome engineering and functional comparison across diverse bacteria using a single plasmid system, cSAGE provides a general framework for non-model strain prototyping and biotransformation discovery.
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