Overexpression of the Saccharomyces cerevisiae Mannosylphosphodolichol Synthase-Encoding Gene in Trichoderma reesei Results in an Increased Level of Protein Secretion and Abnormal Cell Ultrastructure

Abstract

Production of extracellular proteins plays an important role in the physiology of Trichoderma reesei and has potential industrial application. To improve the efficiency of protein secretion, we overexpressed in T. reesei the DPM1 gene ofSaccharomyces cerevisiae, encoding mannosylphosphodolichol (MPD) synthase, under homologous, constitutively acting expression signals. Four stable transformants, each with different copy numbers of tandemly integrated DPM1, exhibited roughly double the activity of MPD synthase in the respective endoplasmic reticulum membrane fraction. On a dry-weight basis, they secreted up to sevenfold-higher concentrations of extracellular proteins during growth on lactose, a carbon source promoting formation of cellulases. Northern blot analysis showed that the relative level of the transcript ofcbh1, which encodes the major cellulase (cellobiohydrolase I [CBH I]), did not increase in the transformants. On the other hand, the amount of secreted CBH I and, in all but one of the transformants, intracellular CBH I was elevated. Our results suggest that posttranscriptional processes are responsible for the increase in CBH I production. The carbohydrate contents of the extracellular proteins were comparable in the wild type and in the transformants, and no hyperglycosylation was detected. Electron microscopy of theDPM1-amplified strains revealed amorphous structure of the cell wall and over three times as many mitochondria as in the control. Our data indicate that molecular manipulation of glycan biosynthesis in Trichoderma can result in improved protein secretion.

Protein production and secretion in an Aspergillus nidulans mutant impaired in glycosylation.

“…In this study we expected changes in the morphology of A. nidulans cells upon transformation, especially in view of the increased cell wall permeability observed previously for the T. reesei transformants (Kruszewska et al, 1999;Lenart et al, 2003). In A. nidulans significant changes in ultrastructure were found in the secretory apparatus and concerned the secretory vesicles.…”

“…Every factor recognised as a potential stimulant of protein secretion appears to be highly valu-able for biotechnological production of biologically active proteins. Stimulation of O-glycosylation by overexpression of the yeast DPM1 gene in T. reesei offered promise of activating protein production and/or secretion (Kruszewska et al, 1999). The O-glycosylation stimulation resulted in the seven-fold higher secretion of proteins being glycosylated to the wild type level.…”

“…Previous results (Kruszewska et al,1999) indicated that protein secretion in the filamentous fungus T. reesei could be upregulated by overexpression of the yeast DPM1 gene encoding DPMS. To unravel the link between DPMS involved in protein O-mannosylation and protein secretion, we overexpressed the yeast gene in an A. nidulans mutant impaired in DPMS activity.…”

“…Choline and Tween 80, when added to the cultivation medium, stimulated secretion and, at the same time, elevated DPMS activity (Kruszewska et al, 1990). Moreover, overexpression of the yeast DPM1 gene in T. reesei increased protein secretion of wild type glycosylated proteins up to seven-fold (Kruszewska et al, 1999). Significant changes were also observed in the ultrastructure of the mutant cell wall and some organelles.…”

“…Different glycoforms of invertase were found insite the cells, in the periplasmic space and in the cultivation medium. Our data point to the importance of the cell wall as a barrier in protein secretion.Elucidation of the regulatory mechanisms and limiting factors in O-glycosylation of secreted proteins is an important task, since the efficiency of protein secretion appears to be related to their O-glycosylation (Kubicek et al., 1987;Kruszewska et al, 1999;Agaphonov et al, 2001). It has been shown for Trichoderma reesei that inhibition of O-glycosylation, in Vol.…”