Lanosterol 14 alpha-demethylase-encoding gene: systematic analysis of homologous overexpression in Saccharomyces cerevisiae using strong yeast promoters.
The Saccharomyces cerevisiae 14DM gene, encoding cytochrome P450 lanosterol 14 alpha-demethylase (14DM), was overexpressed in various S. cerevisiae strains under the control of three strong heterologous yeast transcription promoters (pADC1, pGPD, pPHO5) and under the control of its own promoter. Striking, strain-specific differences in 14DM transcription and in 14DM contents have been observed. The relative abundances of 14DM-specific mRNA and protein derived from a series of different expression plasmids were compared. It was found that the inducible PHO5 promoter in combination with the JL745 host led to the highest expression levels. 14DM-specific RNA reached up to 2% of the total cellular mRNA in this strain and approx. 3% of the total soluble yeast-cell protein was determined to be 14DM by quantitative Western blotting. By comparing the abundances of the different fusion transcripts with the transcript originating from the corresponding endogenous gene from which the promoter was derived, it could be concluded that the expression levels of the different 14DM fusion genes were far below the theoretically attainable values.
Institut für Biotechnologie, ETH-Hönggerberg, Zürich, Switzerland.
J M
JM
Weber
J
J
Reiser
O
O
Käppeli
D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D005656:Fungal Proteins / Q000032:analysis; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D006418:Heme / Q000032:analysis; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D058886:Sterol 14-Demethylase; D014158:Transcription, Genetic
D005656:Fungal Proteins; D012333:RNA, Messenger; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase