UBC Theses and Dissertations
Functional metagenomic screening for glycoside hydrolases Mewis, Keith
Limitations on the cultivation of a majority of naturally occurring microbes have spurred the rise of culture-independent methods for the investigation of environmental microbial communities, a field known as metagenomics. This thesis addresses both functional and informatic approaches to metagenomics with the aim of improving our knowledge of carbohydrate degradation. A high throughput functional metagenomic screen was developed and applied to over 350,000 fosmid clones to search for glycoside hydrolases (GHs) in metagenomic libraries. Screening yielded 798 fosmid clones capable of hydrolyzing a model sugar compound, and the genes responsible were subcloned and biochemically characterized for pH and temperature stability, and substrate specificity. The combination of functional and in silico methods developed were used in a longitudinal study of the beaver (Castor canadensis) digestive tract, in order to gain insight into the sequential degradation of biomass. A linear model was used to identify enrichment of endo-acting versus exo-acting GH families at five locations throughout the digestive tract. The discovery of high numbers of GH43 family genes on functionally identified fosmids resulted in their combination with all other known GH43 genes in order to create subfamily classifications that provide finer resolution of enzyme activities. This classification system resulted in an improved ability to assign functional characteristics to enzymes identified through informatic studies. Of the 37 subfamilies created, only 22 contained a characterized enzyme. Fosmids identified earlier in this work harboured genes from four uncharacterized GH43 subfamilies, and future characterization efforts will further our understanding of the GH43 family. Altogether, the developed methods provide a framework for future studies of biomass degradation and improve the power of both functional and in silico metagenomics.
Item Citations and Data
Attribution-NonCommercial-NoDerivatives 4.0 International