Accelerated Adaptive Evolution of Gram‐Negative Bacteria With a Broad‐Host‐Range Dual Base Editor

Adaptive laboratory evolution (ALE) is a powerful strategy for exploring functional solution spaces, yet its pace is fundamentally constrained by low spontaneous mutation rates. Here, we present a broadly applicable genetic device that accelerates genome diversification in Gram-negative bacteria through the transient action of a dual base editor (ACBE3). This construct combines cytidine and adenosine deaminase activities with a single-stranded DNA-binding module, enabling efficient genome-wide mutagenesis without reliance on host-specific replication or repair systems. The expression of ACBE3 increased mutation rates by up to four orders of magnitude in Escherichia coli and Pseudomonas putida, outperforming classical random mutagenesis approaches such as UV irradiation and chemical treatment. Whole-genome sequencing revealed extensive and uniformly distributed mutations, predominantly consistent with deamination signatures, as well as a broader spectrum of nucleotide changes and small indels. The system displayed strong orthogonality, functioning across diverse bacterial taxa. As a proof of concept, coupling ACBE3-mediated diversification with selective growth conditions enabled the rapid evolution of Acinetobacter calcoaceticus P320, yielding mutant strains with enhanced growth on n-dodecane within only a few induction cycles. This approach decouples diversification from selection, enabling the controlled exploration of adaptive landscapes at unprecedented speeds. While high mutational loads impose cellular burdens and require tight regulatory control, the platform offers a versatile route to accelerate microbial evolution at both single-strain and community levels. Our results thus document dual base editing as a powerful framework for fostering evolution in microbial biotechnology.