A Refined Adaptive Laboratory Evolution Strategy With Biosensor-Assisted Selection Resolves the Tolerance-Efficiency Trade-Off in Toxic Chemical Biosynthesis.

Enhancing microbial tolerance to target chemicals through conventional adaptive laboratory evolution (ALE) is time-consuming, labor-intensive, and further constrained by the challenge of balancing improved tolerance with maintaining optimal biosynthetic efficiency. Here, this work proposes a refined ALE strategy that combines initial mutagenesis with an automated microdroplet cultivation (MMC) system, thereby expediting the acquisition of tolerance phenotypes. Integrating a biosensor-assisted high-throughput screening platform enables identification of strains exhibiting advantageous "win-win" phenotypes, characterized by simultaneous improvements in both tolerance and biosynthetic capacity. Using E. coli for the biosynthesis of 3-hydroxypropionic acid (3-HP) as a model system, this work rapidly evolves strains capable of tolerating 720 mM 3-HP within 12 days. Leveraging a newly developed and validated 3-HP-responsive biosensor, this work efficiently screens and isolates superior strains. The top-performing strain produced 86.3 g L 3-HP with a yield of 0.82 mol mol glycerol. Transcriptomic analysis provide insights into mechanisms underlying this "win-win" phenotype. Collectively, this study establishes an effective ALE framework for accelerating the development of microbial chassis tailored for high-efficiency biochemical production.