Evolving Robust and Interpretable Enzymes for the Bioethanol Industry

Abstract Obtaining a robust and applicable enzyme for bioethanol production is a dream for biorefinery engineers. Herein, we describe a general method to evolve an all‐round and interpretable enzyme that can be directly employed in the bioethanol industry. By integrating the transferable protein evolution strategy InSiReP 2.0 (In Silico guided Recombination Process), enzymatic characterization for actual production, and computational molecular understanding, the model cellulase PvCel5A (endoglucanase II Cel5A from Penicillium verruculosum ) was successfully evolved to overcome the remaining challenges of low ethanol and temperature tolerance, which primarily limited biomass transformation and bioethanol yield. Remarkably, application of the PvCel5A variants in both first‐ and second‐generation bioethanol production processes (i. Conventional corn ethanol fermentation combined with the in situ pretreatment process; ii. cellulosic ethanol fermentation process) resulted in a 5.7–10.1 % increase in the ethanol yield, which was unlikely to be achieved by other optimization techniques.