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Abstract
The ability to utilize the carbohydrates of lignocellulosic biomass as a substrate for growth is a relatively rare trait in nature due to the resistance of the plant cell wall to degradation. Organisms able to deconstruct this complex web of polymers are of interest from both academic and biotechnological perspectives. Plant biomass depolymerization is integral to the cycling of organic compounds in the terrestrial biosphere. The plant cell wall also represents a renewable source of compounds that can be converted into useful fuels, chemicals, and other materials. Thus studying the divergent enzymes, mechanisms, and strategies of lignocellulose degradation is of economic and environmental value.
Extremely thermophilic bacteria of the genus Caldicellulosiruptor are among the most efficient known degraders of lignocellulosic biomass. They rely primarily on a novel set of secreted multifunctional glycoside hydrolases. These enzymes contain two or more catalytic domains tethered together by flexible linkers and carbohydrate binding modules. They convert plant polymers into fermentable sugars more efficiently than equivalent fungal enzymes and have the advantage of being active at higher temperatures.In this dissertation, elements necessary for the production of these multi-domain enzymes are explored in the genetically-tractable and highly cellulolytic species, C. bescii.
Deleting a glycosyltransferase family 39 gene showed it to be necessary for the glycosylation of CelA, the most abundant multifunctional enzyme in the C. bescii secretome. The absence of glycosylation drastically reduced the organism’s ability to grow on crystalline cellulose and made CelA more susceptible to degradation. Deletion of a nearby gene encoding a PrsA proline isomerase also inhibited utilization of cellulose, apparently by impacting the solubility of secreted multidomain glycoside hydrolases. Understanding the cellular machinery associated with post-translational modification and secretion of these multidomain enzymes is important to their use for industrial plant biomass conversion and provides insight into the less-studied physiology of Gram(+) bacteria in a thermophilic, anaerobic organism.
Extremely thermophilic bacteria of the genus Caldicellulosiruptor are among the most efficient known degraders of lignocellulosic biomass. They rely primarily on a novel set of secreted multifunctional glycoside hydrolases. These enzymes contain two or more catalytic domains tethered together by flexible linkers and carbohydrate binding modules. They convert plant polymers into fermentable sugars more efficiently than equivalent fungal enzymes and have the advantage of being active at higher temperatures.In this dissertation, elements necessary for the production of these multi-domain enzymes are explored in the genetically-tractable and highly cellulolytic species, C. bescii.
Deleting a glycosyltransferase family 39 gene showed it to be necessary for the glycosylation of CelA, the most abundant multifunctional enzyme in the C. bescii secretome. The absence of glycosylation drastically reduced the organism’s ability to grow on crystalline cellulose and made CelA more susceptible to degradation. Deletion of a nearby gene encoding a PrsA proline isomerase also inhibited utilization of cellulose, apparently by impacting the solubility of secreted multidomain glycoside hydrolases. Understanding the cellular machinery associated with post-translational modification and secretion of these multidomain enzymes is important to their use for industrial plant biomass conversion and provides insight into the less-studied physiology of Gram(+) bacteria in a thermophilic, anaerobic organism.