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

Biotechnology for Biofuels and Bioproducts, vol. 15, pp. 6

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.

Authors 9

1. Nicholas G. S. McGregor

   University of York

   Affiliation as printed

   York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, YO10 5DD, York, UK

2. Casper de Boer Aachen

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA, Leiden, The Netherlands

3. Mikhaaeel Santos

   University of York

   Affiliation as printed

   York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, YO10 5DD, York, UK

4. Mireille Haon

   Aix-Marseille Université · Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement · Biodiversité et Biotechnologie Fongiques

   Affiliation as printed

   Polytech Marseille, Aix Marseille Univ, 13288, Marseille, France

   UMR1163 Biodiversité et Biotechnologie Fongiques, Faculté des Sciences de Luminy, INRAE, Aix Marseille Univ, 13288, Marseille, France

5. David Navarro

   Aix-Marseille Université · Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement · Biodiversité et Biotechnologie Fongiques

   Affiliation as printed

   Polytech Marseille, Aix Marseille Univ, 13288, Marseille, France

   UMR1163 Biodiversité et Biotechnologie Fongiques, Faculté des Sciences de Luminy, INRAE, Aix Marseille Univ, 13288, Marseille, France

6. Sybrin P. Schröder Aachen

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA, Leiden, The Netherlands

7. Jean‐Guy Berrin

   Aix-Marseille Université · Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement · Biodiversité et Biotechnologie Fongiques

   Affiliation as printed

   Polytech Marseille, Aix Marseille Univ, 13288, Marseille, France

   UMR1163 Biodiversité et Biotechnologie Fongiques, Faculté des Sciences de Luminy, INRAE, Aix Marseille Univ, 13288, Marseille, France

8. Herman S. Overkleeft Aachen

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA, Leiden, The Netherlands

9. G.J. Davies corresponding

   University of York

   Affiliation as printed

   York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, YO10 5DD, York, UK. Gideon.davies@york.ac.uk

   York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, YO10 5DD, York, UK