UBC Theses and Dissertations
Discovery of new glycoside phosphorylases through rational, genomic and metagenomic exploration Macdonald, Spencer
Carbohydrates are abundant throughout nature, play key roles in diverse biological processes and form fundamental components of a wide range of functional materials. Their inherent complexity is a double edge sword in that it allows carbohydrates to adopt a diverse range of structures and functions, but in an industrial setting, that complexity can also make them especially difficult and costly to synthesize in a strictly defined manner. To help ensure uniform production, carbohydrate manufacturing processes are known to utilize Carbohydrate Active Enzymes (CAZymes), which have innate substrate specificities and conformational control. A class of CAZymes, known as glycoside phosphorylases (GPs) catalyze the reversible phosphorolysis of glycosidic bonds, releasing sugar 1-phosphates and have considerable potential as catalysts for the assembly of useful carbohydrates for products ranging from functional foods and prebiotics to novel materials. However, the substrate diversity of currently identified GPs is relatively small, limiting their practical applications. To address this limitation, I used a combined approach drawing upon rational, metagenomic and genomic exploration methods to discover new GPs. First, using a rational approach, I identified a new GP activity, the first reported with a β-retaining mechanism, while investigating a mechanistic oddity observed in the GH3 CAZy family. Next, I developed and deployed two metagenomic screening methodologies that targeted GP activity in the vast reservoir of uncultivated genetic diversity encoded in microbial communities inhabiting natural and engineered ecosystems. This approach yielded eight new GPs and established a screening paradigm that can be applied to an even greater range of GP activities. Lastly, a phylogenomically diverse, synthetic gene GP library was characterized, which led to the discovery of another previously undiscovered GP activity, a new biopolymer (that I named acholetin), and established a high throughput (HT) substrate specificity assay for GPs. In total, 11 new GPs were discovered, two of those representing previously unknown activities. The research presented in this dissertation will result in a better understanding of GP diversity, provide insights into how we can engineer GPs, establish a HT functional-characterization method, and provide a platform to explore genomic and metagenomic sequence space for more novel GP activities.
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