Continuous Evolution System Based on Toxin-Antitoxin System Combined with Base Deaminase Accelerates the In Vivo...

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 10⁶ 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.