Glycoprotein Hypersecretion Alters the Cell Wall in Trichoderma reesei Strains Expressing the Saccharomyces cerevisiae Dolichylphosphate Mannose Synthase Gene

Abstract

Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increased amount of N-acetylglucosamine, suggesting an increase in chitin content. Calcofluor white staining followed by fluorescence microscopy indicated changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble ␤-(1,6) glucan. A comparison of protein secretion from protoplasts with that from mycelia showed that the cell wall created a barrier for secretion in the DPM1 transformants. We also discuss the relationships between the observed changes in the cell wall, increased protein glycosylation, and the greater secretory capacity of T. reesei strains expressing the yeast DPM1 gene.The saprobic fungus Trichoderma reesei secretes a wide range of hydrolytic enzymes, such as cellulases and hemicellulases, which are widely used in the food, animal feed, and paper industries (10). Hence, stimulation of its secretory capacity is of considerable interest for biotechnology. Many, if not all, of these extracellular proteins are glycosylated. Our previous study showed a close correlation between protein secretion and the activity of dolichylphosphate mannose (DPM)-synthase (EC 2.4.1.83), a key enzyme in O glycosylation in T. reesei (15,17). We have shown that in T. reesei, DPM, which is synthesized by DPM-synthase, donates the mannosyl residue that is transferred to the hydroxyl group of serine or threonine in protein O mannosylation (16). Moreover, T. reesei DPM-synthase, like its counterpart from rat liver (3), is activated in vitro by cyclic AMP-dependent protein kinase (18). An obligatory requirement for DPM-synthase in O mannosylation was demonstrated for Saccharomyces cerevisiae by the finding that a temperature-sensitive DPM-synthase mutant (dpm1) was completely blocked in O mannosylation of the model protein chitinase (24). Loss of DPM1 expression in yeast is lethal (24). DPM-synthase also participates in N glycosylation of protein, supplying the last four mannosyl residues during the assembly of the lipid-linked precursor oligosaccharide dolichol diphosphate-GlcNAc 2 Man 9 Glc 3 , and is required for the biosynthesis of glycosylphosphatidylinositol membrane anchors (11).Our earlier data indicated that overexpression of the S. cerevisiae DPM1 gene encoding DPM-synthase in T. reesei elevated the enzyme activity twofold and resulted in an increased level of protein secretion. The secreted proteins were glycosylated to the same extent as in the control, although at a level up to seven times higher (15).We have also isolated the dpm1 gene encoding DPM-synthase from T. reesei and tr...

Influence of sorbitol on protein production and glycosylation and cell wall formation in Trichoderma reesei

“…Cultivation of T. reesei with 1 M sorbitol caused a decrease in the activity of DPM synthase and at the same time a significant decrease in protein secretion, in agreement with earlier data showing that activity of DPM synthase was tightly connected with protein secretion (Kruszewska et al 1999;Perli nska-Lenart et al 2006). This low concentration of secretory proteins caused by osmotic stress made their intensive processing non-necessary.…”

Saprotrophic competitiveness and biocontrol fitness of a genetically modified strain of the plant-growth-promoting fungus Trichoderma hamatum GD12

“…Similar reductions in sporulation efficiency were found in Aspergillus mutants altered in chitin deposition (Borgia et al, 1996;Horiuchi et al, 1999;Müller et al, 2002). Alterations in chitin deposition could also account for the increased secretion of the MF2 antigen in DThnag : : hph, since secretion in fungi is a process that occurs at the flexible growing tip (Wösten et al, 1991;Wessels, 1994;Gordon et al, 2000;Thornton, 2004) and is governed by the structure of the cell wall (Kruszewska et al, 1999;Perliń ska-Lenart et al, 2006). This is consistent with the increased secretion of glycoproteins observed in the related fungus Trichoderma reesei as a consequence of mutations in chitin distribution in the hyphal cell wall (Perliń ska- Lenart et al, 2006).…”

Integration of additional copies of Trichoderma reesei gene encoding protein O-mannosyltransferase I results in a decrease of the enzyme activity and alteration of cell wall composition

“…Moreover, all those proteins were glycosylated at the wild type level (Kruszewska et al 1999). Similarly, expression of the DPM1 gene in Aspergillus nidulans stimulated protein production, but the proteins were arrested between the cell wall and cell membrane (Perli nska-Lenart et al 2005). This result pointed at a fundamental role of cell wall permeability for protein secretion in Aspergillus.…”

“…At the same time, secretion from protoplasts and mycelia of the control strain was nearly identical, showing that not only the cell wall was a barrier for secretion but also efficient processing of newly synthesized glycoproteins (Perli nska- Lenart et al 2006b). …”

“…Some species of Trichoderma secrete up to 40 g of protein per litre of culture (Durand et al 1988). Our previous study indicated that protein production and secretion could be up-regulated simultaneously with the activity of the O-glycosylation pathway (Kruszewska et al 1999;Perli nska-Lenart et al 2005, 2006b. Overexpression of the Saccharomyces cerevisiae DPM1 gene coding for dolichyl phosphate mannose synthase (DPM synthase) in Trichoderma reesei resulted in up to sevenfold higher secretion of proteins.…”