Biotransformation of cardenolide-precursors by Saccharomyces cerevisiae
Digitalis plants are the most important source for the extraction of cardenolides used in the treatment of cardiac insufficiency in humans. In order to study putative biosynthetic genes and enzymes we intend to establish a yeast system were glycosylated enzymes and P450 enzymes involved in cardenolide biosynthesis can be functionally expressed. For example, we were unable to express progesterone 5α-reductase, a glycoprotein, in E. coli. We also aim at the reconstruction of consecutive steps of the putative cardenolide pathway in yeast to produce commercially unavailable pregnanes from bulk precursors such as progesterone. We already cloned three genes encoding enzymes that are supposed to transform pregnanes during cardenolide formation: the Δ5–3β-hydroxysteroid dehydrogenase (3β-HSD, EC 188.8.131.52) from Digitalis lanata, which converts pregnenolone to isoprogesterone , the Arabidopsis thalianaΔ4,5-steroid 5β-reductase (At5β-StR, EC 184.108.40.206/220.127.116.11) which reduces progesterone to 5β-pregnane-3,20-dione  and an enzyme capable of hydroxylating pregnanes in position 21, the steroid-21-hydroxylase (Cyp21A1, EC 18.104.22.168) from Mus musculus. These genes were cloned into the Gateway® pYES-DEST52 vector system and then transformed into Saccharomyces cerevisiae strain BY4741a. Our data indicate, that the Cyp21A1 gene is functionally expressed in the recombinant yeast strain as it is capable of transforming progesterone into 21-hydroxyprogesterone. Product formation was demonstrated by GC-MS and TLC. Further studies are ongoing to show the functional expression of 3β-HSD and At5β-StR in yeast and to cotransform and coexpress these genes.
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