Abstract
To address the challenge of microbial tolerance in industrial biomanufacturing, we developed an adaptive evolution strategy for Escherichia coli W3110 to enhance its salicylic acid (SA) tolerance. Utilizing a CmeR-P(cmeO) biosensor-enabled high-throughput screening system, we isolated an SA-tolerant variant (W3110K-4) that exhibited a 2.3-fold increase in tolerance (from 0.9 to 2.1 g/L) and a 2.1-fold improvement in SA production (from 283 to 588.1 mg/L). Subsequently, the designed sensors were combined with multi-pathway sgRNA arrays to dynamically modulate the other three branched-chain acid derivatives, achieving a balance between biomass growth and rapid SA production in the adaptively evolved strain, resulting in a maximum SA yield of 1477.8 mg/L, which represents a 30% improvement over the non-evolved control strain W3110K-W2 (1138.2 mg/L) using the same metabolic strategy. Whole-genome sequencing revealed that adaptive mutations in genes such as ducA* and anti-drug resistance C2 mutation genes (ymdA*, ymdB*, clsC*, csgB*, csgA*, and csgC*) play a key role in enhancing SA tolerance and productivity. Notably, the evolved strain W3110K-4 exhibits significant resistance to bacteriophages, making it a promising candidate for large-scale SA fermentation. This work develops and expands the CmeR-P(cmeO) system, proposes new insights into improved strains through biosensor screening, guided multi-pathway metabolism, and adaptive evolution, and provides a paradigm for engineers to obtain engineered strains.