Covalent and Orthogonal Cohesin–Dockerin Interactions Enabled by Intermolecular Disulfide Bonds for Hyperthermostable Cellulase Assembly

Cellulosic biofuel represents a sustainable alternative to fossil fuels, yet high cellulase costs hinder its development. Thermostable cellulosomes, which function at elevated temperatures, increase reaction rates and reduce cooling costs by using cohesin–dockerin interactions to colocalize hyperthermostable cellulases. Due to the noncovalent nature of the cohesin–dockerin interaction, cellulosome stability has been limited to 75 °C. Our study leverages computational design and rapid screening to introduce two different intermolecular disulfide bridges between the same cohesin and dockerin, creating two disulfide cohesin–dockerin pairs. Both disulfide pairs withstood 100 °C and denaturing conditions. Furthermore, the two disulfide bridges retained their orthogonality, expanding the number of orthogonal cohesin–dockerin interactions. Finally, at the cellulase optimal temperature of 80 °C, disulfide assembly improved the activity of a bivalent cellulosome by 26% compared to that of its noncovalent counterpart. These disulfide cohesin–dockerin interactions can be used as building blocks to construct covalent protein complexes that can endure extreme temperatures.