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Applied and Environmental Microbiology, June 1999, p. 2382-2387, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Joanna S. Kruszewska,1 Arno H. Butterweck,2 Wieslaw Kurzatkowski,3 Andrzej Migdalski,1 Christian P. Kubicek,2 and Grazyna Palamarczyk1,*

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw,1 and State Institute of Hygiene, 00-791 Warsaw,3 Poland, and Institute of Biochemical Technology and Microbiology, TU Wien, A-1060 Vienna, Austria2

Received 21 September 1998/Accepted 2 April 1999

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 of Saccharomyces 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 of cbh1, 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 the DPM1-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.

* Corresponding author. Mailing address: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland. Phone: 48 22 658 47 02. Fax: 48 39 121 623. E-mail: gp{at}ibbrain.ibb.waw.pl.

Applied and Environmental Microbiology, June 1999, p. 2382-2387, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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