A generalizable framework for genetic code expansion in bacterial systems beyond E. coli.

Genetic code expansion (GCE) enables the site-specific incorporation of noncanonical amino acids into proteins, providing a powerful platform to investigate and engineer biological functions with molecular precision. While GCE has facilitated transformative studies in Escherichia coli, its utility in bacteria other than E. coli has been hindered by the lack of universal, high-efficiency methods for its implementation and optimization. To address this limitation, we introduce a modular, extensible platform for the generalizable implementation and optimization of GCE in phylogenetically diverse bacteria, based on the host-agnostic serine recombinase-assisted genome engineering toolkit. We apply this approach to five bacterial species and demonstrate robust incorporation of structurally diverse noncanonical amino acids, including a key bacterial post-translational modification. This work provides a generalizable and scalable strategy to deploy expanded genetic systems in bacteria, advancing programmable microbial engineering across diverse applications.IMPORTANCEExpanding the genetic code allows scientists to introduce new chemical functionalities into proteins, creating powerful opportunities to study and engineer biological systems. However, these technologies have remained largely confined to the laboratory bacterium Escherichia coli because adapting them to other bacterial species has been slow and organism-specific. We developed a general, chromosomally integrated framework that makes it easier to establish and optimize genetic code expansion across diverse bacteria. Using this approach, we identify practical design principles for improving performance, demonstrate transferability between distantly related species, and enable site-specific incorporation of modified amino acids into native bacterial proteins. This work expands access to advanced protein engineering technologies for studying bacterial post-translational regulation and developing engineered microbes for biotechnology, environmental applications, and synthetic biology.