Progesterone metabolism in recombinant yeast simultaneously expressing bovine cytochromes P450c17 (CYP17A1) and P450c21 (CYP21B1) and yeast NADPH-P450 oxidoreductase.
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.
Sakaki
T
T
Takarazuka Research Center, Sumitomo Chemical Co. Ltd., Hyogo, Japan.
Akiyoshi-Shibata
M
M
Yabusaki
Y
Y
Manabe
K
K
Murakami
H
H
Ohkawa
H
H
D019326:17-alpha-Hydroxyprogesterone; D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism*; D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003350:Cortodoxone / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D003900:Desoxycorticosterone / Q000378:metabolism; D006908:Hydroxyprogesterones / Q000378:metabolism; D008954:Models, Biological; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013254:Steroid 17-alpha-Hydroxylase; D013255:Steroid 21-Hydroxylase / Q000235:genetics / Q000378:metabolism*; D013739:Testosterone / Q000378:metabolism; D014170:Transformation, Genetic
D006908:Hydroxyprogesterones; D011994:Recombinant Proteins; D013739:Testosterone; D000735:Androstenedione; D003900:Desoxycorticosterone; D011374:Progesterone; D019326:17-alpha-Hydroxyprogesterone; D003577:Cytochrome P-450 Enzyme System; D013255:Steroid 21-Hydroxylase; D013254:Steroid 17-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D000446:Aldehyde-Lyases; D003350:Cortodoxone