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Ievdokymenko, Kateryna

University of British Columbia

We live in a world dominated by microorganisms that encode a vast yet still largely unexplored diversity of metabolic functions. Using function-driven approaches it is possible to mine this diversity for both basic science insights and industrial applications. In this thesis, a functional metagenomic screening paradigm using large-insert environmental DNA (fosmid) library pool selection was developed in Escherichia coli EPI300 background enabling users to design and build pool selection experiments, sequence pooled clones, assemble and annotate resulting sequences and begin to characterize activities of interest encoded within pools. Tests were conducted on a collection of nine fosmid libraries to select for conversion or tolerance traits enabling growth in the presence of inhibitory concentrations of vanillin to identify biological parts useful in design of industrial chassis for lignin valorization. Relevant fitness traits related to monoaromatic conversion as well as tolerance to monoaromatic compounds, including stress responses, transporters and cell surface modification functions were recovered. Based on these results, a larger selection experiment was conducted using seven lignin-derived monoaromatic compounds. Comparative analysis of enrichment pools revealed that the same clones were recovered on multiple aromatic compounds consistent with the presence of genes encoding non-specific tolerance traits. Efforts to grow fosmid pools on lignin-derived monoaromatics as sole carbon sources were unsuccessful pointing to workflow modifications including adoption of new screening hosts, overexpression of transporters and regulatory elements, or the insertion of genes in E. coli EPI300 encoding upstream or downstream conversion steps within multi-component catabolic pathways. Finally, the screening paradigm was extended to search for hydrocarbon tolerance phenotypes using toluene in three fosmid pools sourced from anaerobic hydrocarbon degrading enrichment cultures resulting in recovery of fitness traits including transport and cell surface modification. Pool growth was unstable prompting a time course experiment to better constrain pool diversity and dynamics. Fosmid pool sequencing recovered numerous genes encoding tolerance traits that varied between initial and later exposure phases consistent with clone succession. Looking ahead, integration of high-throughput functional metagenomics with strain engineering promises to promote functional characterization and these biological parts and will ultimately inform the development of microbial cell factories for sustainable biotechnology innovation.

Text

2022-08-11

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/82321

Microbiology and Immunology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/