Systematically Engineering for Efficient Production of 3‐Methyl‐1‐Butanol in Escherichia coli

ABSTRACT 3‐Methyl‐1‐butanol (3‐MB), a promising next‐generation biofuel, has garnered significant interest owing to its superior combustion characteristics and fuel compatibility. However, current 3‐MB biosynthesis faces major challenges, including low production efficiency and severe toxicity‐induced growth inhibition, which significantly limit its industrial feasibility. In this study, we systematically developed an integrated metabolic engineering approach for high‐level 3‐MB production in Escherichia coli . Through semi‐rational engineering of the rate‐limiting enzyme dihydroxyacid dehydratase (DHAD), combined with molecular dynamics simulations, we identified and addressed previously unrecognized catalytic bottlenecks. The engineered strain exhibited a 32.3‐fold increase in 3‐MB production, reaching 2.20 g/L in shake‐flask cultures. Subsequent adaptive laboratory evolution further improved strain robustness, while genomic analysis revealed novel regulatory targets for metabolic optimization. In a scaled‐up bioreactor fermentation system, the final strain achieved a record titer of 6.24 g/L, representing the highest reported titer for engineered microbial systems. This work not only establishes a scalable platform for 3‐MB biosynthesis but also provides a modular engineering framework applicable to other advanced biofuels.