Screening for cytochrome P450 expression in Pichia pastoris whole cells by P450-carbon monoxide complex determination.
Cytochrome P450 (CYP) enzymes are useful biocatalysts for the pharmaceutical and biotechnological industries. A high-throughput method for quantification of CYP expression in yeast is needed in order to fully exploit the yeast expression system. Carbon monoxide (CO) difference spectra of whole cells have been successfully used for the quantification of heterologous CYP expressed in Escherichia coli in the 96-well format; however, very few researchers have shown whole-cell CO difference spectra with yeast cells using 1-cm path length. Spectral interference from the native hemoproteins often obscures the P450 peak, challenging functional CYP quantification in whole yeast cells. For the first time, we describe the high-throughput determination of CO difference spectra using whole cells in the 96-well format for the quantification of CYP genes expressed in Pichia pastoris. Very little interference from the hemoproteins of P. pastoris enabled CYP quantification even at relatively low expression levels. P. pastoris strains carrying a single copy or three copies of both hCPR and CYP2D6 integrated into the chromosomal DNA were used to establish the method in 96-well format, allowing to detect quantities of CYP2D6 as low as 6 nmol gCDW(-1 ) and 12 pmol per well. Finally, the established method was successfully demonstrated and used to screen P. pastoris clones expressing Candida CYP52A13.
Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria.
Rama Krishna
RK
Gudiminchi
Martina
M
Geier
Anton
A
Glieder
Andrea
A
Camattari
D002248:Carbon Monoxide / Q000032:analysis* / Q000378:metabolism; D019389:Cytochrome P-450 CYP2D6 / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D009251:NADPH-Ferrihemoprotein Reductase / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010843:Pichia / Q000737:chemistry* / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000379:methods
D011994:Recombinant Proteins; D002248:Carbon Monoxide; D019389:Cytochrome P-450 CYP2D6; D009251:NADPH-Ferrihemoprotein Reductase