Single-molecule imaging of lignocellulose deconstruction by SCATTIRSTORM microscopy

Cellulases play a central role in the conversion of lignocellulosic material into bioethanol. However, biofuels production is hindered by the crystallinity of cellulose, which makes it difficult to hydrolyze, and by the presence of lignin and hemicellulose, which act as steric impediments that prevent access of cellulase enzymes to their cellulose substrate. Improved understanding of how the composition and architecture of lignocellulose affect the activities of degradative enzymes will improve efforts to engineer enzymes optimized for efficient and cost-effective bioenergy production. The goal of this project is to build a multimodal optical microscope to measure the binding, processive degradation, and pausing behaviors of cellulases as they interact with and degrade both synthetic and naturally occurring lignocellulosic walls. To achieve this, we will use high spatio-temporal single-molecule imaging to track cellulases, while visualizing specific molecular components of cellulose, lignin and hemicellulose, that make up their lignocellulose substrate. The microscope will combine Interferometric Scattering (iSCAT), which provides unprecedented spatiotemporal resolution; Total Internal Reflection Fluorescence (TIRF), which provides single-molecule resolution of multiple fluorophore-labeled molecules; and Stochastic Reconstruction (STORM), which allows for three-dimensional super-resolution imaging of intact plant cell walls during degradation. Initial studies will investigate cellulase dynamics on in vitro-assembled cell wall analogs, and later work will progress to using native plant cell walls. In addition to revealing the molecular mechanisms underlying biomass conversion, this work will help to bring cutting-edge microscopy approaches into the plant biology and bioenergy communities.