UBC Theses and Dissertations
Harnessing natural diversity for the discovery of glycoside hydrolases and design of new glycosynthases Armstrong, Zachary
Plant biomass offers a sustainable source for energy and materials and an alternative to fossil fuels. However, the industrial scale production or biorefining of fermentable sugars from plant biomass is currently limited by the lack of cost effective and efficient biocatalysts. Microbes, the earth's master chemists - employing biocatalytic solutions to harvest energy, and transform this energy into useful molecules - offer a potential solution to this problem. However, a majority of microbes remain uncultured, limiting our access to the genetic potential encoded within their genomes. This has spurred the development of culture independent methods, termed metagenomics. In this thesis I harnessed high-throughput functional metagenomic screening to discover biomass deconstructing biocatalysts from uncultured microbial communities. Towards this goal, twenty-two clone libraries containing DNA sourced from diverse microbial communities inhabiting terrestrial and aquatic ecosystems were screened with 4-methylumbelliferyl cellobioside to detect glycoside hydrolase activity. This revealed 178 active clones containing glycoside hydrolases, often in gene clusters. This set of active clones was consolidated and further characterized through sequencing and rapid, plate-based, biochemical assays. Additionally, libraries sourced from beaver fecal and gut microbiomes were screened with four fluorogenic probes (6-chloro-4-methylumbelliferyl derivatives of cellobiose, xylobiose, xylose and mannose) for glycoside hydrolase activity. This revealed a total of 247 active fosmid-harbouring clones, that encoded many polysaccharide-degrading genes and gene cassettes. Specific candidate genes from the fecal library were sub-cloned, and the resulting purified enzymes were shown to be involved in synergistic degradation of arabinoxylan oligomers. The clone libraries that were generated through functional metagenomic screening were then employed to reveal the promiscuity of glycoside hydrolases towards unnatural azido- and aminoglycosides. Promiscuous enzymes identified from metagenomic and synthetic clone libraries were then used as a starting point for the generation of new glycosynthases capable of incorporating modified glucosides and galactosides. The resulting set of eight new glycosynthases are capable of synthesizing di- and trisaccharides, glycolipids and inhibitors such as 2,4-dinitrophenyl 4'-amino-2,4'-dideoxy-2-fluoro-cellobioside. Taken together this work has exploited the power of functional metagenomics to reveal new modes of biocatalysis and develop new synthetic tools.
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