Engineering enhanced cellobiohydrolase activity

Abstract

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 (TrCel7A) and Penicillium funiculosum (PfCel7A), which exhibit a multi-modular architecture consisting of catalytic domain (CD), carbohydrate-binding module, and linker. We show that PfCel7A exhibits 60% greater performance on biomass than TrCel7A. 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 PfCel7A CD. We solve the crystal structure of PfCel7A CD and use this information to create a second library of TrCel7A CD mutants, identifying a TrCel7A double mutant with near-equivalent activity to wild-type PfCel7A. Overall, these results reveal CBH regions that enable targeted activity improvements.

Fig. 1

Multi-modular structure of Family 7 cellobiohydrolases. The GH Family 7 CBH from T. reesei is shown in the catalytically active complex on a cellulose microfibril. Shown in gray are the enzyme domains: at right is the catalytic domain (CD), at left is the carbohydrate-binding module (CBM), and connecting the two is the linker domain bound to the cellulose surface. Structure adapted from Zhong et al. The cellulose microfibril is shown in green ‘surface’ representation; ‘sticks’ are also shown for the strand upon which Cel7A is complexed. O-glycans are shown on the linker and CBM in yellow; N-glycans are shown in dark blue on the catalytic domain

Fig. 2

Activity data on dilute acid pre-treated corn stover. Glucan conversion is shown as a function of time on PCS for a wild-type PfCel7A and wild-type TrCel7A and b the domain swap chimera library. The P and T stand for P. funiculosum and T. reesei, respectively in the domain architecture in the order of CD, linker, and CBM. The lines represent double-exponential fits to the data. These assays were performed at T = 40 °C and pH = 5.0. The inset graph shows the time to 80% conversion (in hours) of the double exponential fit to each data trend. Graphs with these fits are available in Supplementary Fig. 2. Experiments were performed in triplicate; error bars represent the standard error of the mean (SEM) and are smaller than the data markers

Fig. 3

The structure of PfCel7A. The structure of PfCel7A (PDB code 4XEB), shown in orange, along with that of the canonical GH7 CBH TrCel7A (PDB code 4C4C) in light gray. The nine substrate binding sites (−7 to + 2) and the eight loops that form the substrate binding tunnel (A1, B1, etc.) are labeled according to the standard convention. The cellononaose ligand from the TrCel7A Michaelis complex (PDB code 4C4C) is shown in green ‘sticks’. For key areas of differences in these structures that inspired the construction of a library of CD mutants, see Supplementary Fig. 5

Fig. 4

Selected catalytic domain (CD) mutant activity data. T1 and T3 (TrCel7A parent enzyme) show slight improvement in activity on pre-treated corn stover (PCS) over WT TrCel7A, whereas combining these into the double mutant T1/T3 demonstrates activity comparable to WT PfCel7A. Experiments were performed in triplicate; error bars represent the SEM and are smaller than the data markers

Fig. 5

Mechanistic insight from molecular simulations. a The RMSF profile for the glucosyl residue at each binding site in solution. This contrast in flexibility can be seen b in the WT TrCel7A, and c in the mutant enzyme, particularly at the −5, −6, and −7 subsites. The ligand is shown every 8 ns over 1 µs. d Whereas Gln7 in WT TrCel7A maintains a hydrogen bond with the glucosyl moiety in the −7 binding site, the mutant is more flexible, essentially abolishing this enzyme-substrate interaction at the tunnel entrance. e In the WT enzyme, a persistent salt bridge forms between Lys102 (A1 loop) and Glu408 (A2 loop). f The deletion of Lys102 results in the loss of this salt bridge in the mutant enzyme, resulting in significant rearrangements for loops A1, A2, and A3