Computer-assisted enzyme cocktails enhance fermentation by overcoming toxic inhibitors from pretreatment processes

Lignocellulosic biomass is the most abundant form of biomass available for fuel production, serving as the fourth leading energy source globally. However, inhibitors generated during pretreatment processes often hinder fermentation performance and conversion efficiency. In this study, we developed an enhanced computer-assisted enzyme cocktail strategy (ComEC 2.0) to mitigate the inhibitory effects. Through experimental studies and molecular dynamics simulations, eight optimization strategies were developed for enzyme cocktail formulation (comprising CBHI, EG, BG, XYN, LPMO). Notably, Strategy 4b, which accounts for both overall hydration and the synergistic effects between LPMO and CBHI/EG/BG/XYN, increased glucose and xylose yields by 20.7 % and 21 %, respectively, using corn stover, reducing Process Mass Intensity (PMI) by 70.78 % and water use by 80 % during ethanol fermentation. Applying Strategy 4b to industrial corn cob increased glucose and xylose yields by 22.1 % and 21.6 %, surpassing the commercial Ctec3 blend. This scalable approach significantly enhances biomass conversion and resource efficiency, offering broad industrial potential.