Single-molecule studies of cellulose, cellulose-binding domains, and cellulose biosynthesis machinery

Abstract

Cellulose is the most abundant biopolymer on Earth and provides several kingdoms of life with structure, energy and protection. Life biomanufactures and degrades cellulose on the order of a hundred billion tons annually making cellulose synthesis and degradation two of the most prolific processes performed by life. In sole relation to humans, cellulose production directly or indirectly provides food, shelter, clothing, and energy, or poses issues such as disease or pipe fouling. Because of this, cellulose, its synthesis and its degradation are of enormous interest in biotechnology with the possibilities to vastly improve agricultural yield, biofuel production, anti-microbial treatments and process engineering. A molecular understanding of enzymatic cellulose hydrolysis and polymerization is essential for advancement. In this dissertation, optical tweezers probe cellulose molecular machinery to investigate both cellulose degradation and synthesis at the single-molecule level. The carbohydrate binding module associated with cellobiohydrolase, the main contributor in enzymatic hydrolysis for biofuel production, displays multimodal, complex binding mechanics to cellulose that alters with changes to substrate crystal structure and exchanging protein binding residues. The bacterial cellulose synthase complex, responsible for manufacturing a main component of bacterial biofilms, maintains heavy biochemical and temperature dependence while also displaying a stall force and a tight grip on the product. Single-strand cellulose is a relatively elastic polymer with a high propensity to fold on itself. Cellulose-cellulose association is likely a driving force behind cellulose biosynthesis. Additionally, this dissertation details the construction of a high-resolution optical tweezers microscope with drift correction and temperature control.