A Strictly Inducible and Orthogonal Dre-rox System for Precise and Markerless Genome Editing in Bacillus subtilis

Site-specific recombination enables precise and modular genome engineering in microbial systems. In Bacillus subtilis, Cre is the most commonly used site-specific recombinase (SSR) and has been widely applied in genome engineering. Developing SSRs with comparable performance to Cre that can also function orthogonally would significantly expand the genome engineering toolkit. We established a resistance gene-based reporter in B. subtilis to assess the genome editing potential of the Dre-rox system. A theophylline-inducible riboswitch tightly controlled Dre expression to minimize leaky recombination, improving the specificity of rox-mediated recombination. Notably, Dre and Cre function without crosstalk at their respective recognition sites. This orthogonal combination enabled a modular workflow: Cre-mediated integration followed by Dre-mediated markerless deletion. Dual and triple-site models confirmed that Dre-rox supports synchronized multi-locus excision with a single induction. Optimized Dre-rox architecture highlighted its reliability for genome engineering in B. subtilis. The system features high-fidelity recombination, low toxicity, and strong host adaptability. This work extends Dre-rox utility to prokaryotic systems. The standardized Dre-rox platform provides a foundation for hierarchical pathway engineering, mutant library generation, and modular chassis development in synthetic biology.