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Production of extracellular proteins plays an important role in the physiology ofTrichoderma reesei and has potential industrial application. To improve the efficiency of protein secretion, we overexpressed inT. reesei theDPM1 gene ofSaccharomyces cerevisiae , encoding mannosylphosphodolichol (MPD) synthase, under homologous, constitutively acting expression signals. Four stable transformants, each with different copy numbers of tandemly integratedDPM1 , 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 inTrichoderma can result in improved protein secretion.

applied and environmental microbiology

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

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