Dehydroepiandrosterone (DHEA) metabolism in Saccharomyces cerevisiae expressing mammalian steroid hydroxylase CYP7B: Ayr1p and Fox2p display 17beta-hydroxysteroid dehydrogenase activity.
We have engineered recombinant yeast to perform stereospecific hydroxylation of dehydroepiandrosterone (DHEA). This mammalian pro-hormone promotes brain and immune function; hydroxylation at the 7alpha position by P450 CYP7B is the major pathway of metabolic activation. We have sought to activate DHEA via yeast expression of rat CYP7B enzyme. Saccharomyces cerevisiae was found to metabolize DHEA by 3beta-acetylation; this was abolished by mutation at atf2. DHEA was also toxic, blocking tryptophan (trp) uptake: prototrophic strains were DHEA-resistant. In TRP(+) atf2 strains DHEA was then converted to androstene-3beta,17beta-diol (A/enediol) by an endogenous 17beta-hydroxysteroid dehydrogenase (17betaHSD). Seven yeast polypeptides similar to human 17betaHSDs were identified: when expressed in yeast, only AYR1 (1-acyl dihydroxyacetone phosphate reductase) increased A/enediol accumulation, while the hydroxyacyl-CoA dehydrogenase Fox2p, highly homologous to human 17betaHSD4, oxidized A/enediol to DHEA. The presence of endogenous yeast enzymes metabolizing steroids may relate to fungal pathogenesis. Disruption of AYR1 eliminated reductive 17betaHSD activity, and expression of CYP7B on the combination background (atf2, ayr1, TRP(+)) permitted efficient (>98%) bioconversion of DHEA to 7alpha-hydroxyDHEA, a product of potential medical utility.
Transgene SA, 11 Rue de Molsheim, 67000 Strasbourg, France. rlathe@ed.ac.uk
Pedro
P
Vico
Gilles
G
Cauet
Ken
K
Rose
Richard
R
Lathe
Eric
E
Degryse
D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism; D000107:Acetylation; D000123:Acetyltransferases / Q000235:genetics; D015114:Androstenediol / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D000072474:Cytochrome P450 Family 7; D003687:Dehydroepiandrosterone / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D009154:Mutation; D051381:Rats; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000235:genetics*; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan
C400127:FOX2 protein, Glomus mosseae; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D003687:Dehydroepiandrosterone; D014364:Tryptophan; D003577:Cytochrome P-450 Enzyme System; D015114:Androstenediol; D015067:17-Hydroxysteroid Dehydrogenases; D013401:Sugar Alcohol Dehydrogenases; C022312:acylglycerone-phosphate reductase; D013250:Steroid Hydroxylases; D000072474:Cytochrome P450 Family 7; C490784:Cyp7b1 protein, rat; D000123:Acetyltransferases; C116217:ATF2 protein, S cerevisiae