Engineering enhanced cellobiohydrolase activity

Glycoside Hydrolase Family 7 cellobiohydrolases (GH7 CBHs) catalyze cellulose depolymerization in cellulolytic eukaryotes, making them key discovery and engineering targets. However, there remains a lack of robust structure–activity relationships for these industrially important cellulases. Here, we compare CBHs from Trichoderma reesei ( Tr Cel7A) and Penicillium funiculosum ( Pf Cel7A), which exhibit a multi-modular architecture consisting of catalytic domain (CD), carbohydrate-binding module, and linker. We show that Pf Cel7A exhibits 60% greater performance on biomass than Tr Cel7A. To understand the contribution of each domain to this improvement, we measure enzymatic activity for a library of CBH chimeras with swapped subdomains, demonstrating that the enhancement is mainly caused by Pf Cel7A CD. We solve the crystal structure of Pf Cel7A CD and use this information to create a second library of Tr Cel7A CD mutants, identifying a Tr Cel7A double mutant with near-equivalent activity to wild-type Pf Cel7A. Overall, these results reveal CBH regions that enable targeted activity improvements.