Dissecting Cellular Function and Distribution of β-Glucosidases in Trichoderma reesei

Abstract

Trichoderma reesei has 11 putative β-glucosidases in its genome, playing key parts in the induction and production of cellulase. Nevertheless, the reason why the T. reesei genome encodes so many β-glucosidases and the distinct role each β-glucosidase plays in cellulase production remain unknown. In the present study, the cellular function and distribution of 10 known β-glucosidases (CEL3B, CEL3E, CEL3F, CEL3H, CEL3J, CEL1A, CEL3C, CEL1B, CEL3G, and CEL3D) were explored in T. reesei, leaving out BGL1 (CEL3A), which has been well investigated. We found that the overexpression of cel3b or cel3g significantly enhanced extracellular β-glucosidase production, whereas the overexpression of cel1b severely inhibited cellulase production by cellulose, resulting in nearly no growth of T. reesei. Four types of cellular distribution patterns were observed for β-glucosidases in T. reesei: (i) CEL3B, CEL3E, CEL3F, and CEL3G forming clearly separated protein secretion vesicles in the cytoplasm; (ii) CEL3H and CEL3J diffusing the whole endomembrane as well as the cell membrane with protein aggregation, like a reticular network; (iii) CEL1A and CEL3D in vacuoles; (iv) and CEL3C in the nucleus. β-glucosidases CEL1A, CEL3B, CEL3E, CEL3F, CEL3G, CEL3H, and CEL3J were identified as extracellular, CEL3C and CEL3D as intracellular, and CEL1B as unknown. The extracellular β-glucosidases CEL3B, CEL3E, CEL3F, CEL3H, and CEL3G were secreted through a tip-directed conventional secretion pathway, and CEL1A, via a vacuole-mediated pathway that was achieved without any signal peptide, while CEL3J was secreted via an unconventional protein pathway bypassing the endoplasmic reticulum (ER) and Golgi. IMPORTANCE Although β-glucosidases play an important role in fungal cellulase induction and production, our current understanding does not provide a global perspective on β-glucosidase function. This work comprehensively studies all the β-glucosidases regarding their effect on cellulase production and their cellular distribution and secretion. Overexpression of cel3b or cel3g significantly enhanced β-glucosidase production, whereas overexpression of cel1b severely inhibited cellulase production on cellulose. In addition, overexpression of cel3b, cel3e, cel3f, cel3h, cel3j, cel3c, or cel3g delayed endoglucanase (EG) production. We first identified four cellular distribution patterns of β-glucosidases in Trichoderma reesei. Specially, CEL3C was located in the nucleus. CEL3J was secreted through the nonclassical protein secretion pathway bypassing endoplasmic reticulum (ER) and Golgi. CEL1A was secreted via a vacuole-mediated conventional secretion route without a signal peptide. These findings advance our understanding of β-glucosidase properties and secretory pathways in filamentous fungi, holding key clues for future study.

Transmembrane transport process and endoplasmic reticulum function facilitate the role of gene cel1b in cellulase production of Trichoderma reesei

“…This data was consistent with the reported finding that cel1b was highly induced in T. reesei cultivated on cellulose or sophorose together with cel1a [ 31 ], whose transcriptional levels were only second to the major extracellular β-glucosidase CEL3A [ 22 ], demonstrating that cel1b might be actively involved in the cellulase biosynthesis in T. reesei . Our previous study has shown that cel1b overexpression using an inducible cbh1 promoter led to a dramatic decrease in cellulase production on cellulose as observed in T. reesei Rcel1B, while the overexpression of other β-glucosidases did not [ 22 ]. We wondered if the decrease on cellulase production was due to that the promoter cbh1 was too strong, which was considered as the strongest promoter in T. reesei on cellulose [ 40 , 41 ].…”

“…To investigate its cellular distribution, the C terminus of CEL1B were fused with red fluorescent protein (DsRed) as reported previously (Additional file 1 : Fig. S2) [ 22 ]. Among the three cel1b -overexpressing strains cultivated in TMM + 2% cellulose, the cellular distribution of CEL1B-DsRed could only be observed in strain OEcel1B during 7 days’ fermentation process by confocal laser scanning microscopy (CLSM) (Fig.…”

“…Gene cel1b is predicted to possess the binding sites for several transcriptional factors relevant to cellulase biosynthesis [ 34 , 36 – 39 ] and the mRNA level of cel1b was reported to be related with the cellulase production [ 33 – 35 ], demonstrating that cel1b is of great importance for cellulase production in filamentous fungi. Our previous study showed that among 11 β-glucosidases predicted in T. reesei , only putative β-glucosidase cel1b overexpression almost completely abolished cellulase production in T. reesei [ 22 ], which triggered our great interest. Therefore, in this study, we further evidenced that the cellulase inhibition was indeed caused by the overexpression of cel1b and explored extensively the mechanisms behind this phenomenon to reveal the role of cel1b in cellulase production in T. reesei (Fig.…”

“…They are distributed at different genomic sites with genes cel3e , cel3f and cel3g in chromosome I, cel1b in chromosome II, cel3a , cel1a and cel3d in chromosome III, cel3h , cel3j and cel3c in chromosome IV, and cel3b in chromosome VI [ 21 ]. Their effects on cellulase production, and their cellular distribution and secretion have been comprehensively studied, identifying that β-glucosidases CEL1A, CEL3B, CEL3E, CEL3F, CEL3G, CEL3H, and CEL3J are extracellular, CEL3C and CEL3D intracellular, CEL1B unknown [ 22 ]. The well characterized β-glucosidase was CEL3A, responsible for most of the extracellular β-glucosidase activity [ 23 ].…”

Glutamine involvement in nitrogen regulation of cellulase production in fungi

“…(Hemi)cellulase activity assay, confocal imaging, spore counting, RNA-seq analysis, and RT-PCR were performed as described in our previous research [ 41 , 65 ]. T. reesei RUT-C30 cultured for 24 h with or without 2.92 g/L glutamine were utilized for RNA-seq.…”