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

Spencer S Macdonald; Zachary Armstrong; Connor Morgan-Lang; Magdalena Osowiecka; Kyle Robinson; Steven J Hallam; Stephen G Withers

doi:10.1016/j.chembiol.2019.03.017

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

Cell Chem Biol. 2019 Jul 18;26(7):1001-1012.e5. doi: 10.1016/j.chembiol.2019.03.017. Epub 2019 May 9.

Authors

Spencer S Macdonald¹, Zachary Armstrong², Connor Morgan-Lang³, Magdalena Osowiecka⁴, Kyle Robinson⁵, Steven J Hallam⁶, Stephen G Withers⁷

Affiliations

¹ 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.
² Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada; Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Genome Science and Technology Program, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.
³ Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Graduate Program in Bioinformatics, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.
⁴ Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada.
⁵ Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada; ECOSCOPE Training Program, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
⁶ Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Graduate Program in Bioinformatics, University of British Columbia, Vancouver, BC V6T 1Z4, 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; Peter Wall Institute for Advanced Studies, University of British Columbia, Vancouver, BC V6T 1Z1, Canada.
⁷ 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: withers@chem.ubc.ca.

PMID: 31080075
DOI: 10.1016/j.chembiol.2019.03.017

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.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Carbohydrate Metabolism
Glucosyltransferases / metabolism
Glycosides / chemistry
Glycosides / metabolism*
High-Throughput Screening Assays / methods
Metagenome / physiology
Microbiota
Molybdenum / chemistry
Phosphorylases / chemistry
Phosphorylases / metabolism*
Proof of Concept Study
Substrate Specificity

Substances

Glycosides
molybdenum blue
Molybdenum
Glucosyltransferases
Phosphorylases
cellobiose phosphorylase
laminaribiose phosphorylase
cellodextrin phosphorylase