Development and Application of a High-Throughput Functional Metagenomic Screen for Glycoside Phosphorylases.

Affiliation

Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada; Genome Science and Technology Program, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; ECOSCOPE Training Program, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada. Electronic address: [Email]

Abstract

Glycoside phosphorylases (GPs) catalyze the reversible phosphorolysis of glycosidic bonds, releasing sugar 1-phosphates. To identify a greater range of these under-appreciated enzymes, we have developed a high-throughput functional screening method based on molybdenum blue formation. In a proof-of-principle screen focused on cellulose-degrading GPs we interrogated ∼23,000 large insert (fosmid) clones sourced from microbial communities inhabiting two separate environments and identified seven novel GPs from carbohydrate active enzyme family GH94 and one from GH149. Characterization identified cellobiose phosphorylases, cellodextrin phosphorylases, laminaribiose phosphorylases, and a β-1,3-glucan phosphorylase. To demonstrate the versatility of the screening method, varying substrate combinations were used to identify GP activity from families GH13, GH65, GH112, and GH130 in addition to GH94 and GH149. These pilot screen and substrate versatility results provide a screening paradigm platform for recovering diverse GPs from uncultivated microbial communities acting on different substrates with considerable potential to unravel previously unknown degradative pathways within microbiomes.

Keywords

CAZy,GH149,GH94,functional metagenomics,glycoside hydrolase,phosphorylase,reverse phosphorolysis,

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