Investigation of Transcriptional Regulation of the Fermentative ADH in Pichia stipitis Using an EGFP Reporter Gene
To show the application of the yEGFP gene as a reporter of promoter activities of hypoxia-induced genes in Pichia stipitis.
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Investigation of Transcriptional Regulation of the Fermentative ADH in Pichia stipitis Using an EGFP Reporter Gene Volkmar Passoth, Ulrich Klinner, and Bärbel Hahn-Hägerdal
Aim To show the application of the yEGFP gene as a reporter of promoter activities of hypoxia-induced genes in Pichia stipitis.
Introduction A reporter gene usually encodes a protein, whose activity is easily to detect. If this gene is expressed under the control of a specific promoter, expression from this promoter is simple to investigate. Thus, reporter genes are sophisticated tools for the investigation of transcriptional gene regulation (Mount et al. 1996; Koloteva et al. 1998). For P. stipitis, only few reports about the introduction of reporter genes exist. Cho and Jeffries (1999) and Weierstall et al. (1999) both described attempts to use the widely applied lacZ gene of E. coli as a reporter in P. stipitis; however, both studies yielded contradictory results regarding the Volkmar Passoth (✉), Klinik für innere Medizin B, Ernst-Moritz-Arndt-Universität Present address: Swedish University of Agricultural Sciences (SLU), Dept. of Microbiology, Uppsala Genetic Center, Box 7025, S 750 07 Uppsala, Sweden Phone: +46-18-673207, Fax: +46-18-673392, email: [email protected] Ulrich Klinner, Institut für Biologie IV (Mikrobiologie), Lehr- und Forschungsgebiet Angewandte Mikrobiologie, RWTH Aachen, Worringer Weg, 56056 Aachen, Germany, Tel.: +49-241-806616; Fax: +49-241-8888180, e-mail: [email protected] Bärbel Hahn-Hägerdal, Applied Microbiology, Lund University, P.O. Box 124, 22100 Lund, Sweden, Tel.: +46-46-2228325; Fax: +46-46-2224203, e-mail: [email protected]
Springer Lab Manual K. Wolf, K. Breunig, G. Barth (Eds.) Non-Conventional Yeasts in Genetics, Biochemistry and Biotechnology © Springer-Verlag Berlin Heidelberg 2003
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Volkmar Passoth, Ulrich Klinner, and Bärbel Hahn-Hägerdal
formation of an active gene product. Possibly, there are problems involved in trying to express bacterial genes in P. stipitis. Therefore, the yEGFP gene, whose codon bias is specifically adapted to expression in yeast (Cormack et al. 1997), has been established as a reporter system in this yeast (Passoth et al. 2003). Besides the robust expression of the reporter, this system is easy to analyze without breaking the cells. In P. stipitis, alcoholic fermentation is specifically induced by oxygen limitation (Passoth et al.1996,1998),which is similar to the induction of fermentation in higher organisms (Hochachka et al. 1996). Therefore, the identification of components of the oxygen-sensing system in this yeast may contribute to general progress in understanding oxygen sensing in eukaryotes. In this chapter, we demonstrate methods for parallel screening of strains under oxygen limitation and aerobic condition and for oxygencontrolled aerobic cultivation. The effect of promoter mutations or drugs such as Co2+ or cyanide (Passoth et al. 2003) can be studied using the described reporter system.
Materials Strain
P.
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