Adaptive laboratory evolution of Saccharomyces cerevisiae enhances butyric acid tolerance and enables co-cultivation with Clostridium tyrobutyricum

Adaptive laboratory evolution (ALE) was successfully applied to improve the tolerance of Saccharomyces cerevisiae toward butyric acid, enabling its use in co-culture with Clostridium tyrobutyricum for the simultaneous production of ethanol and butyric acid as ester precursors. S. cerevisiae was adapted through serial transfer at progressively increasing butyric acid concentrations up to 20 g L⁻ 1 . The evolved yeast population exhibited significantly enhanced butyric acid tolerance and maintained ethanol production under acid-stress conditions. Interestingly, the evolved population also displayed increased maximum glucose consumption rate and ethanol productivity under non-stress conditions. Whole-genome variant analysis was performed by comparing the wild-type strain, an intermediate evolved population and the final evolved population obtained during ALE. The results suggest that adaptation may be associated with changes in regulatory processes, cellular homeostasis, and membrane and cell wall remodeling. The co-culture with the acid producer C. tyrobutyricum demonstrated efficient and balanced substrate utilization, indicating a stable division of labor between the two organisms. Fed-batch cultivation yielded 20.31 ± 2.43 g L⁻ 1 butyric acid and 28.18 ± 3.37 g L⁻ 1 ethanol. With the evolved yeast, ethanol concentrations increased fourfold compared to those achieved with the wild-type strain in previous studies, underscoring the potential of ALE to overcome limitations in co-culture systems.