Cascading recombinase memory switch for programmable and stable gene expression in Pseudomonas putida

Genetic circuits for model bacteria often perform poorly in non-canonical hosts, limiting the deployment of regulatory programs in industrially relevant organisms. Here, we engineered an integrase-based cascading toggle switch that enables consistent and programmable gene expression in Pseudomonas putida. The system couples a rhamnose-inducible trigger module driving the Bxb1 serine integrase with a compact, insulated inversion module that provides binary OFF and ON transcriptional states. To expand both output range and modularity, the switching system controls expression of an orthogonal T7 RNA polymerase gene, thereby activating interchangeable PT7-driven target modules. Translation-level tuning of integrase and T7 RNA polymerase gene expression yielded tight OFF behavior and strong induction. Switching was efficient, and phenotypes were maintained stably after inducer removal, supporting its use as a genetic memory element. The platform was extended to control a bioprocess-relevant phenotype by modulating a hyperactive diguanylate cyclase that triggers biofilm formation. Engineering the system in a 2-fluoro-cis,cis-muconic acid production strain enabled switch-mediated catalytic biofilms operated in continuous mode, where the biofilm configuration supported stable chemical production. This work expands the synthetic biology toolbox for Pseudomonas by linking heritable genetic memory to stable microbial phenotypes useful for bioprocesses.