Evidence is presented that temperature-sensitive Saccharomyces cerevisiae mutants, impaired in dolichol kinase (Sec59p) or dolichyl phosphate mannose synthase (Dpm1p) activity have an aberrant cell wall composition and ultrastructure. The mutants were oversensitive to Calcofluor white, an agent interacting with the cell wall chitin. In accordance with this, chemical analysis of the cell wall alkali-insoluble fraction indicated an increased amount of chitin and changes in the quantity of β1,6-and β1,3-glucan in sec59-1 and dpm1-6 mutants. In order to unravel the link between the formation of dolichyl phosphate and dolichyl phosphate mannose and the cell wall assembly, we screened a yeast genomic library for a multicopy suppressors of the thermosensitive phenotype. The RER2 and SRT1 genes, encoding cis-prenyltransferases, were isolated. In addition, the ROT1 gene, encoding protein involved in β1,6-glucan synthesis (Machi et al., 2004) and protein folding (Takeuchi et al., 2006) acted as a multicopy suppressor of the temperature-sensitive phenotype of the sec59-1 mutant. The cell wall of the mutants and of mutants bearing the multicopy suppressors was analysed for carbohydrate and mannoprotein content. We also examined the glycosylation status of the plasma membrane protein Gas1p, a β1,3-glucan elongase, and the degree of phosphorylation of the Mpk1/Slt2 protein, involved in the cell wall integrity pathway.
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...
Due to its natural properties, Trichoderma reesei is commonly used in industry-scale production of secretory proteins. Since almost all secreted proteins are O-glycosylated, modulation of the activity of enzymes of the O-glycosylation pathway are likely to affect protein production and secretion or change the glycosylation pattern of the secreted proteins, altering their stability and biological activity. Understanding how the activation of different components of the O-glycosylation pathway influences the glycosylation pattern of proteins and their production and secretion could help in elucidating the mechanism of the regulation of these processes and should facilitate creation of engineered microorganisms producing high amounts of useful proteins. In this review we focus on data concerning Trichoderma, but also present some background information allowing comparison with other fungal species.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.