Constructing nucleoside deaminase-assisted orthogonal DNA replication system for continuous evolution in prokaryotes.

Orthogonal DNA replication system-based continuous evolution enables user-defined genes to undergo a hypermutagenic evolutionary state through a replication process independent of the host genome, while maintaining overall cellular stability. However, its application is constrained by the relatively low mutation rate of orthogonal error-prone DNA polymerases (ep-DNAP) and by strong mutation bias during misincorporation. To address this, we developed nucleoside deaminase-assisted orthogonal DNA replication (NDAORep) systems, in which nucleoside deaminases (NDA) are coupled to orthogonal DNA replication to enhance and tune mutagenesis. NDAORep was implemented in engineered Bacillus thuringiensis and synthetic Escherichia coli systems, achieving mutation rates up to 1.16 × 10-4 substitutions per base pair per generation (s.p.b.), approximately 880,000-fold to the host genome with adjustable mutation bias. Using NDAORep, we rapidly evolved a strong constitutive P43 promoter to a variant with 6.88-fold higher activity after only 12 h, compared with 8 days using ep-DNAP alone. We also evolved more efficient orthogonal aminoacyl-tRNA synthetases (aaRSs) in B. thuringiensis and E. coli, enhancing p-azido-L-phenylalanine incorporation by up to 50% and 83%, respectively. Furthermore, NDAORep enabled optimization of a synthetic formate assimilation pathway in E. coli, increasing formate assimilation 5-fold. Collectively, these results demonstrate that NDAORep accelerates the evolution of regulatory elements, protein functions, and metabolic pathways, establishing a powerful platform for metabolic engineering and synthetic biology. By coupling programmable mutational diversity with continuous selection, NDAORep enables rapid optimization of complex biological systems that are difficult to engineer through conventional approaches.