Replication-coupled dual-deaminase mutagenesis (RCDM) enables continuous genomic evolution in Bacillus subtilis

Bacillus subtilis is an important Generally Recognized as Safe (GRAS) chassis widely used for industrial enzyme production, yet systematic improvement of complex host phenotypes—such as protein secretion capacity—remains difficult using rational engineering alone. Continuous evolution strategies offer a powerful alternative but remain underdeveloped in B. subtilis , largely due to the organism’s efficient DNA repair systems that suppress mutagenic intermediates. Here, we report a replication-coupled dual-deaminase mutagenesis platform (RCDM) that enables efficient and continuous genome-wide diversification in B. subtilis . In this system, complementary cytidine and adenine deaminases are tethered to the helicase-loading factor DnaB to access replication-associated single-stranded DNA. Systematic architectural optimization revealed a striking topology dependence of mutagenic activity, where cytidine deamination requires C-terminal fusion whereas adenine deamination is most efficient when fused to the N-terminus of DnaB. Importantly, we show that host DNA repair strongly shapes the mutational output: uracil excision by Ung effectively masks cytidine-derived mutations in dual-deaminase constructs. Co-expression of the uracil-DNA glycosylase inhibitor Ugi alleviates this repair constraint and restores balanced C: G→T: A and A: T→G: C transitions, producing mutation frequencies of (2.80 ± 0.10) × 10⁻⁴ across the genome. To demonstrate the utility of this platform, we coupled α-amylase secretion to growth on soluble starch and performed growth-coupled continuous evolution. Within 20 iterative enrichment cycles, the evolved population exhibited a ~ 30-fold increase in extracellular α-amylase activity relative to the parental strain. Together, these results establish a replication-targeted and repair-aware continuous evolution platform for B. subtilis , providing a versatile strategy for rapid optimization of complex host phenotypes and expanding the continuous evolution toolbox for industrial Gram-positive microbes.