Fungal Gene Expression on Demand: an Inducible, Tunable, and Metabolism-Independent Expression System for Aspergillus niger

Applied and Environmental Microbiology - Tập 77 Số 9 - Trang 2975-2983 - 2011
Vera Meyer1,2, Franziska Wanka1, Janneke van Gent1, Mark Arentshorst1,2, Cees A. M. J. J. van den Hondel1, Arthur F. J. Ram1,2
1Leiden University, Institute of Biology Leiden, Department of Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, Netherlands
2Netherlands and Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA Delft, Netherlands

Tóm tắt

ABSTRACT Filamentous fungi are the cause of serious human and plant diseases but are also exploited in biotechnology as production platforms. Comparative genomics has documented their genetic diversity, and functional genomics and systems biology approaches are under way to understand the functions and interaction of fungal genes and proteins. In these approaches, gene functions are usually inferred from deletion or overexpression mutants. However, studies at these extreme points give only limited information. Moreover, many overexpression studies use metabolism-dependent promoters, often causing pleiotropic effects and thus limitations in their significance. We therefore established and systematically evaluated a tunable expression system for Aspergillus niger that is independent of carbon and nitrogen metabolism and silent under noninduced conditions. The system consists of two expression modules jointly targeted to a defined genomic locus. One module ensures constitutive expression of the tetracycline-dependent transactivator rtTA2 S -M2, and one module harbors the rtTA2 S -M2-dependent promoter that controls expression of the gene of interest (the Tet-on system). We show here that the system is tight, responds within minutes after inducer addition, and allows fine-tuning based on the inducer concentration or gene copy number up to expression levels higher than the expression levels of the gpdA promoter. We also validate the Tet-on system for the generation of conditional overexpression mutants and demonstrate its power when combined with a gene deletion approach. Finally, we show that the system is especially suitable when the functions of essential genes must be examined.

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Applied and Environmental Microbiology

Tập 77 Số 9

2975-2983

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