Continuous Evolution System Based on Toxin–Antitoxin System Combined with Base Deaminase Accelerates the In Vivo Modification of Target Proteins

ABSTRACT To overcome the labor‐intensive nature of traditional directed evolution, the promoted Escherichia coli ‐assisted continuous evolution (PEACE) system has been developed. PEACE 1.0 employs a cytidine deaminase‑T7 RNA polymerase (T7 RNAP) fusion for targeted mutagenesis and integrates a toxin–antitoxin selection module for spontaneous, growth‑coupled enrichment of evolved variants. Through dual optimization of mutagenesis and selection, PEACE 2.0 achieves an average mutation frequency of 3.49 × 10 −3 per base pair within a 60‑h cycle (compared to 1.54 × 10 −3 in 120 h for PEACE 1.0), while shortening the cycle reduces host background mutation accumulation. A dual‑factor (growth‑fluorescence) selection/screening mechanism eliminates a 12%–20% absolute fraction of undesired non‑specific survivors, and coupling with fluorescence‑activated cell sorting enables ultrahigh‑throughput screening exceeding 10 6 variants per day. PEACE rapidly evolves T7 RNAP to recognize non‑canonical promoters and efficiently reprograms the transcription factor PsiR from a d ‑psicose inducer to a d ‑fructose/ d ‑mannose repressor, establishing a novel regulatory mode. These results demonstrate that PEACE is an efficient, versatile, and robust platform for in vivo continuous evolution with broad potential in protein engineering.