(723b) “Dock and Lock” Covalent Assembly of Designer Cellulosomes

Bugada, L. F., University of Michigan
Pushpam, P. L., University of Michigan
Bailey, R., University of Michigan
Wen, F., University of Michigan
Lignocellulosic bioethanol offers an alternative energy source to fossil fuels while reducing lignocellulosic waste. Regrettably, this process is not yet economical due to the recalcitrant nature of lignocellulosic biomass. Microbes have evolved mechanisms to efficiently degrade lignocellulosic biomass through large enzyme assemblies termed cellulosomes. In cellulosomes, enzyme assembly is mediated through the interaction between cohesin domains that make up the noncatalytic scaffoldin proteins and dockerin domains that are fused to enzymes. The use of orthogonal cohesin-dockerin interactions has further led to the development of designer cellulosomes in which the number, ratio, and position of protein components can be controlled. However, the natural cohesin-dockerin assembly is mediated through noncovalent, reversible interactions that inevitably result in the partial dissociation of designer cellulosome components, an issue that will be compounded as the size of designer cellulosomes increases.

In this study, we developed a “dock and lock” method to enable irreversible assembly of cellulosomes by introducing a covalent bond to the cohesin-dockerin interaction. Specifically, homology modeling, docking, and structural analysis were performed to design cohesins and dockerins with single cysteine mutations so that cohesin-dockerin guided assembly causes the formation of a disulfide bond between the two proteins. Our data showed that introducing the cysteine mutations did not affect the cohesin-dockerin interaction, but created a covalent complex that has a greatly reduced dissociation rate and much improved stability without dissociation even under boiling conditions. As a result, these engineered cohesin-dockerin pairs improved the assembly efficiency of designer cellulosomes, which is further translated to enhanced catalytic properties. The method reported here provides a simple way to form covalent designer cellulosomes and can be seamlessly incorporated into any large protein assemblies with minimal design implications.