Tracking localization and secretion of cellulase spatiotemporally and directly in living Trichoderma reesei

Chengcheng Li,Ai-Ping Pang,Zhihua Zhou,Fengming Lin,Fu-Gen Wu,Hang Yang,Roujing Lv

doi:10.1186/s13068-019-1538-0

Abstract

BackgroundFilamentous fungi secret hydrolytic enzymes like cellulase and hemicellulase outside the cells, serving as important scavengers of plant biomass in nature and workhorses in the enzyme industry. Unlike the extensive study on the mechanism of cellulase production in fungi, research on spatiotemporal distribution and secretion of cellulase in fungi is lacking, retarding the deeper understanding of the molecular mechanism behind the fungal cellulase production.ResultRecombinant Trichoderma reesei strains RBGL, RCBH, and RCMC were successfully constructed from T. reesei RUT-C30, expressing red fluorescent protein DsRed-tagged versions of β-glucosidase (BGL), cellobiohydrolase (CBH), and endoglucanase (CMC), respectively. With the assistance of these strains, we found that all three cellulase components BGL, CBH, and CMC diffused throughout the whole fungal mycelium with major accumulation at the hyphal apexes. These enzymes located in ER, Golgi, vacuoles and cell membrane/wall, but not septum, and secreted abundantly into the culture medium. Moreover, the major secretion of CBH and CMC started more early than that of BGL. Brefeldin A (BFA) completely blocked cellulase expression and secretion in T. reesei.ConclusionBased on recombinant T. reesei RBGL, RCBH, and RCMC expressing DsRed-fused versions of BGL, CBH, and CMC, respectively, the distribution and secretion of cellulase production in T. reesei were first visualized directly in a dynamic way, preliminarily mapping the location and secretion of T. reesei cellulase and providing evidence for revealing the secretion pathways of cellulase in T. reesei. The obtained results suggest that cellulase excretion majorly occurs via the conventional ER–Golgi secretory pathway, and might be assisted through unconventional protein secretion pathways.

Highlights

Filamentous fungi secret hydrolytic enzymes like cellulase and hemicellulase outside the cells, serv‐ ing as important scavengers of plant biomass in nature and workhorses in the enzyme industry
The obtained results suggest that cellulase excretion majorly occurs via the conventional endoplasmic reticulum (ER)–Golgi secretory pathway, and might be assisted through unconventional protein secretion pathways
Cellulase is located throughout the whole hyphal cell with tip‐high gradient, but not septum Expression of cellulases BGL, CBH, and CMC as a fusion protein with red fluorescence protein DsRed under a modified CBH1 promoter (Fig. 1a) in T. reesei RUT-C30 was carried out, leading to recombinant strains RBGL, RCBH, and RCMC, respectively

Summary

Introduction

Filamentous fungi secret hydrolytic enzymes like cellulase and hemicellulase outside the cells, serv‐ ing as important scavengers of plant biomass in nature and workhorses in the enzyme industry. Filamentous fungi like Trichoderma, Penicillium, and Aspergillus are extraordinary in their capability of cellulase/hemicellulase secretion for the efficient deconstruction of recalcitrant polymers in plant biomass including cellulose, hemicellulose, pectin, Protein secretion in filamentous fungi can occur either by the conventional pathway traversing the endomembrane system or via other alternative routes termed as unconventional secretions [5]. Once in Golgi, the proteins undergo further glycosylation by the addition and/or removal of specific sugar resides, and are again packed in secretory vesicles and directed to the endosomal/vacuolar system, the Spitzenkörper, or the plasma membrane for exocytosis. The mechanism for this unconventional pathway was not very clear

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