WEBINAR
October 2025

Uncovering microbial N-glycan degradation pathways through ultrahigh-throughput single emulsion droplet screening.

Rajneesh K. Bains(1), Charlotte Olagnon(1), Jacob F. Wardman(1), Stephen G. Withers(1),
(1)Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z3, Canada

N-glycans play a critical role within the human glycome. To advance our understanding of these glycans, it is essential to expand the current enzymatic toolkit. Functional metagenomics offers a promising approach, significantly accelerating the discovery of novel enzymes, particularly glycoside hydrolases with desirable activities. However, a common challenge in such screenings is that the substrates used to identify target enzymes often fail to accurately represent those encountered in vivo. To address this limitation, our work focuses on designing substrates that better represent the structural complexity of glycans, using complex-type N-glycans as a proof of concept.

We have developed a novel fluorescence-quenched N-glycan probe that, when cleaved by enzymes capable of breaking any linkage within complex N-glycans, activates fluorescence. This enables the facile identification of genes encoding N-glycan-degrading enzymes. Our lab has also recently introduced a robust ultrahigh-throughput droplet-based platform that significantly accelerates the screening of metagenomic libraries. This platform offers several advantages, including a significant reduction in the amount of substrate needed—particularly valuable when working with synthetically complex substrates like the fluorescence-quenched probe described here. We have successfully integrated the fluorescence-quenched N-glycan probe into this platform, enabling the screening of several metagenomic libraries to identify N-glycan-degrading pathways from diverse environments. Overall, our findings highlighting the power of ultrahigh-throughput screening platforms in discovering enzymes with the desired activities and open new possibilities for developing probes that can identify such enzymes.