Cardenolides, such as digoxin, are still produced by extraction from dried leaves of Digitalis lanata. One structural feature of all cardenolides is the gamma-lactone ring ("butenolide ring") attached to C-17 of a steroid scaffold. It is assumed that butenolide ring formation is initiated by the formation of 21-O-malonyl-5ß-pregnane-3ß,14ß-diol-20-one. This step is catalyzed by a malonyl coenzyme A:21-hydroxypregnane-21-O-malonyltransferase (21MaT) which transfers a malonyl moiety from malonyl-CoA to the 21-hydroxy group of an appropriate pregnane precursor. Butenolide ring formation was demonstrated to occur spontaneously in vitro by decarboxylation and subsequent dehydration of 14β-hydroxylated 21-O-malonylpregnanes without participation of an enzyme. The attempts to purify 21MaTs from cardenolide-producing plants resulted in the (partial) purification of several enzymes. Sequencing of purified 21MaTs so far failed. In Arabidopsis thaliana AtPMaT1 we identified a first candidate gene that might encode an enzyme able to malonylate 21-hydroxpregnanes. The recombinant form of AtPMaT1 (termed rAtPMaT1) was indeed demonstrated to encode an enzyme that catalyzes the 21-O-malonylation of 5ß-pregnane-14ß,21-diol-20-one, yielding 21-O-malonyl-5ß-pregnane-14ß-ol-20-one, the direct precursor of digitoxigenin. A homologuous gene in D. lanata has also been identified, cloned and functionally expressed in E.coli. Further candidate genes have been identified. These genes will be expressed in E.coli , characterized and compared. In expressing 21MaT also in Saccharomyces cerevisiae an existing biomanufacturing platform for cardenolides will further be extended. We presume that pregnane-21-O-malonylation is a central step in cardenolide biosynthesis providing - like in the metabolism of certain xenobiotics - a tonoplast-transportable metabolite, that can enter the vacuole, a storage site for cardenolides. The isolation and characterization of 21MaT enzymes and genes will enable us to study the metabolism of 21-O-malonylated pregnanes and their relevance for cardenolide biosynthesis, in order to finally realize a cardenolide pathway in yeast.
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