Overexpression of the Saccharomyces cerevisiae mannosylphosphodolichol synthase-encoding gene in Trichoderma reesei results in an increased level of protein secretion and abnormal cell ultrastructure

Production of extracellular proteins plays an important role in the physiol ogy of Trichoderma reesei and has potential industrial application. To impr ove the efficiency of protein secretion, we overexpressed in T. reesei the DPM1 gene of Saccharomyces cerevisiae, encoding mannosylphosphodolichol (MP D) synthase, under homologous, constitutively acting expression signals. Fo ur stable transformants, each with different copy numbers of tandemly integ rated DPM1, exhibited roughly double the activity of MPD synthase in the: r espective endoplasmic reticulum membrane fraction. On a dry-weight basis, t hey secreted up to sevenfold-higher concentrations of extracellular protein s during growth on lactose, a carbon source promoting formation of cellulas es. Northern blot analysis shelved that the relative level of the transcrip t of cbh1, which encodes the major cellulase (cellobiohydrolase I [CBH I]), did not increase in the transformants. On the other hand, the amount of se creted 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 the DPM1 amplified strains revealed amorphous structure of the cell wal l and over three times as many mitochondria as in the control. Our data ind icate that molecular manipulation of glycan biosynthesis in Trichoderma can result in improved protein secretion.