There is an "I" in team: individual improvements in supercharged cellulase cocktail facilitates cooperative cellulose degradation.

Lignocellulosic biomass is an abundant renewable carbon source for biofuel production, but its conversion to fermentable sugars is hindered by poor cellulase activity on highly crystalline and insoluble cellulose. While pretreatment makes biomass more amenable to enzymatic degradation, several issues linger related to productive enzyme binding and efficient catalytic turnover. To address this bottleneck, we employed protein supercharging to rationally design a glycosyl hydrolase (GH) family-6 exocellulase (Cel6B) and its native family-2a carbohydrate binding module (CBM2a) from the thermophilic cellulolytic microbe Thermobifida fusca. A chimeric library of 32 supercharged constructs rationally designed across both GH/CBM domains was synthesized and expressed in E. coli. Screening of the entire library of supercharged enzymes on several cellulosic substrates identified one key construct, D5 CBM2a-WT Cel6B, containing a positively supercharged CBM2a that showed 2-threefold higher activity on all substrates tested at pH 5.5. Purified enzyme assays confirmed that exocellulases behave quite differently from their endocellulase counterparts when supercharged using similar protocols. Still, the purified D5 CBM2a-WT Cel6B mutant showed a 2.3-fold improvement in specific activity compared to native enzyme on crystalline cellulose. Analysis of melt curves depicts that, while all other constructs tested have one distinct melt peak near the expected CBM melting point, domain melting is decoupled for the D5 CBM2a mutant. This effect reveals an intrinsic melting temperature of the Cel6B CD nearly 18 °C higher than the coupled melting temperature of the full-length enzyme. This unexpected stabilization effect of supercharged CBM2a domain is likely the driving force for activity improvements seen for this exocellulase that is otherwise prone to stalling and denaturation on the cellulose surface during processive catalytic turnover cycles. When combining this supercharged exocellulase construct with its endocellulase counterpart, our results showed that supercharged enzymes, exhibiting the highest activity alone, produced the best synergistic partners. This study highlights another successful implementation of protein supercharging for cellulases and provides another key piece toward building an effective synergistic cellulase cocktail for lignocellulosic biomass deconstruction.