Sustainable Biosynthesis of Butyl Acetate From Glucose: Metabolic Engineering and Process Optimization

Butyl acetate, a valuable industrial chemical, is traditionally produced via energy-intensive processes. In this study, we engineered Clostridium tyrobutyricum for the efficient de novo biosynthesis of butyl acetate from glucose. By introducing a heterologous pathway combining adhE1 and ATF1 and systematically rewiring the central carbon metabolism, we achieved high-level butyl acetate production. A key metabolic engineering insight was that eliminating cryptic chloramphenicol acetyltransferase activity prevented unintended substrate competition, while the overexpression of the ctfAB-adc cluster to reassimilate acetate expanded the acetyl-CoA pool. Through the integration of targeted metabolic engineering and optimized fermentation strategies, a final butyl acetate titer of 43.27 g/L was achieved in a 5 L bioreactor, representing the highest level reported to date, with a yield of 0.20 mol/mol glucose and >98% selectivity. This work not only demonstrates the potential of C. tyrobutyricum as an efficient cell factory for ester synthesis but also establishes a versatile metabolic engineering framework applicable to the sustainable production of a broad range of ester compounds.