Activity-based protein profiling reveals dynamic substrate-specific cellulase secretion by saprotrophic basidiomycetes

Nicholas G S McGregor; Casper de Boer; Mikhaaeel Santos; Mireille Haon; David Navarro; Sybrin Schroder; Jean-Guy Berrin; Herman S Overkleeft; Gideon J Davies

doi:10.1186/s13068-022-02107-z

Activity-based protein profiling reveals dynamic substrate-specific cellulase secretion by saprotrophic basidiomycetes

Biotechnol Biofuels Bioprod. 2022 Jan 17;15(1):6. doi: 10.1186/s13068-022-02107-z.

Authors

Affiliations

¹ York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, YO10 5DD, York, UK.
² Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA, Leiden, The Netherlands.
³ UMR1163 Biodiversité et Biotechnologie Fongiques, Faculté des Sciences de Luminy, INRAE, Aix Marseille Univ, 13288, Marseille, France.
⁴ Polytech Marseille, Aix Marseille Univ, 13288, Marseille, France.
⁵ York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, YO10 5DD, York, UK. Gideon.davies@york.ac.uk.

Abstract

Background: Fungal saccharification of lignocellulosic biomass occurs concurrently with the secretion of a diverse collection of proteins, together functioning as a catalytic system to liberate soluble sugars from insoluble composite biomaterials. How different fungi respond to different substrates is of fundamental interest to the developing biomass saccharification industry. Among the cornerstones of fungal enzyme systems are the highly expressed cellulases (endo-β-glucanases and cellobiohydrolases). Recently, a cyclophellitol-derived activity-based probe (ABP-Cel) was shown to be a highly sensitive tool for the detection and identification of cellulases.

Results: Here we show that ABP-Cel enables endo-β-glucanase profiling in diverse fungal secretomes. In combination with established ABPs for β-xylanases and β-D-glucosidases, we collected multiplexed in-gel fluorescence activity-based protein profiles of 240 secretomes collected over ten days from biological replicates of ten different basidiomycete fungi grown on maltose, wheat straw, or aspen pulp. Our results reveal the remarkable dynamics and unique enzyme fingerprints associated with each species substrate combination. Chemical proteomic analysis identifies significant arsenals of cellulases secreted by each fungal species during growth on lignocellulosic biomass. Recombinant production and characterization of a collection of probe-reactive enzymes from GH5, GH10, and GH12 confirm that ABP-Cel shows broad selectivity towards enzymes with endo-β-glucanase activity.

Conclusion: Using small-volume samples with minimal sample preparation, the results presented here demonstrate the ready accessibility of sensitive direct evidence for fungal enzyme secretion during early stages of growth on complex lignocellulosic substrates.

Keywords: Activity-based probe; Activity-based protein profiling; Basidiomycete; Biomass; Cellulase; Cyclophellitol; Enzyme identification; Enzyme secretion; Filamentous fungi; Fluorescence; Glycoside hydrolase; Kinetics; Pichia pastoris; Secretome.

Abstract

Grants and funding