Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution

Abstract

Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 °C increase in T_m and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 °C. The X-ray crystal structure of the catalytic domain was determined at 2.1 Å resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites.

Keywords: Aspergillus; Trichoderma reesei; bioenergy; biotechnology; cellobiohydrolase; cellulase; crystal structure; directed evolution; molecular dynamics; protein engineering.

Figure 1.

Hydrolysis of PASC by the most stable Cel7A variant FCA398 (filled symbols) compared with wild type (FCA301; open symbols) at 53 °C (diamonds), 65 °C (triangles), and 75 °C (squares), using 1% PASC and 0.5 mg enzyme/g cellulose at pH 5.5. The amount of soluble sugar was determined by HPLC.

Figure 2.

Overview of the 18 mutation sites (magenta) in the H. jecorina Cel7A FCA398 variant and comparison of the crystal structure of the catalytic domain of FCA398 (yellow) with that of Cel7A (light gray) in complex with cellononaose (green; PDB code 4C4C) (83). A, the linker–CBM region (light blue) from a previous model of the full-length Cel7A enzyme from MD simulation (102) shows the location of the T462I mutation at the linker–CBM junction. B, view showing the cellulose-binding tunnel entrance side of the catalytic domain. C, view showing the product binding side of the catalytic domain.

Figure 3.

RMSF of the wild-type Cel7A and FCA398 variant backbone from molecular dynamics simulations at 300, 475, and 525 K. In all cases, the RMSF shown was determined by averaging values from three independent simulation trajectories. At 300 K, both wild type and the variant exhibit nearly identical dynamic flexibility, but at high temperatures, wild-type Cel7A is prone to higher backbone fluctuation relative to the FCA398 variant. In the top right panel, the wild-type Cel7A structure is shown colored by its RMSF at 300 K. The red regions represent backbone segments with the greatest fluctuation, whereas blue indicates the lowest fluctuation. The RMSF values shown on the structure have been scaled to a maximum of 2.5. Loops are labeled in black type on the structure.

Figure 4.

Total number of native contacts retained by wild-type Cel7A and FCA398 mutant over the course of MD simulations at 300, 475, and 525 K. The lines represent the average of three independent MD simulations for each enzyme at a given temperature.

Figure 5.

Comparison of the Cel7A FCA398 (yellow) and 4C4C (white) structures around the P227L mutation site. Selected side chains are shown in stick representations, with mutated residues in FCA398 in magenta.

Figure 6.

The S113N mutation in the Cel7A FCA398 variant introduces a new N-glycosylation site at a surface hairpin turn. An N-acetyl glucosamine residue attached to Asn¹¹³ is visible in the electron density map of the FCA398 crystal structure. The 2F_oF_c − F_c map is contoured at a σ level of 1.8. The Cel7A 4C4C structure is in light gray, and FCA398 is shown in yellow with mutated sites in magenta.