Progesterone metabolism in recombinant yeast simultaneously expressing bovine cytochromes P450c17 (CYP17A1) and P450c21 (CYP21B1) and yeast NADPH-P450 oxidoreductase

• Published in: Pharmacogenetics (London) Vol. 1; no. 2; p. 86
• Main Authors: Sakaki, T, Akiyoshi-Shibata, M, Yabusaki, Y, Manabe, K, Murakami, H, Ohkawa, H
• Format: Journal Article
• Language: English
• Published: England 01.11.1991
• Subjects: 17-alpha-Hydroxyprogesterone; Aldehyde-Lyases - genetics; Aldehyde-Lyases - metabolism; Androstenedione - metabolism; Animals; Cattle; Cortodoxone - metabolism; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Desoxycorticosterone - metabolism; Hydroxyprogesterones - metabolism; Models, Biological; NADPH-Ferrihemoprotein Reductase - genetics; NADPH-Ferrihemoprotein Reductase - metabolism; Progesterone - metabolism; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Steroid 17-alpha-Hydroxylase; Steroid 21-Hydroxylase - genetics; Steroid 21-Hydroxylase - metabolism; Testosterone - metabolism; Transformation, Genetic
• ISSN: 0960-314X
• DOI: 10.1097/00008571-199111000-00005

Simultaneous expression plasmids were constructed for bovine adrenal cytochromes P450c17 and P450c21 (pA gamma alpha) and for both P450s together with NADPH-cytochrome P450 reductase (pAR gamma alpha). On introduction of each of the plasmids into Saccharomyces cerevisiae AH22 cells, the transformed yeast strains AH22/pA gamma alpha and AH22/pAR gamma alpha produced about 10(5) molecules per cell of P450c17 and 2 x 10(3) molecules per cell of P450c21. The expression levels of NADPH-cytochrome P450 reductase was about 3 x 10(4) and 6 x 10(5) molecules per cell in the strains AH22/pA gamma alpha and AH22/pAR gamma alpha, respectively. When progesterone was added to growing cell cultures of the transformed yeast strains, the substrate was metabolized more rapidly in the AH22/pAR gamma alpha cells than AH22/pA gamma alpha cells, probably due to overproduction of the reductase. In the AH22/pAR gamma alpha cells, progesterone was first converted into 17 alpha-hydroxyprogesterone to the extent of 82% by the catalysis of P450c17. 17 alpha-hydroxyprogesterone was further converted into 11-deoxycortisol by P450c21 to the extent of 60% of the added substrate. The conversion of progesterone into androstenedione through 17 alpha-hydroxyprogesterone was estimated to be less than 3%, suggesting very low C17,20-lyase activity of P450c17, although other hydroxylation products were detected. Androstenedione was further converted into testosterone by an unknown pathway present in S. cerevisiae cells.