Overexpression and RNAi-mediated Knockdown of Two 3β-hydroxy-Δ5-steroid dehydrogenase Genes in Digitalis lanata Shoot Cultures Reveal Their Role in Cardenolide Biosynthesis.

3β-hydroxy-Δ5-steroid dehydrogenases (3βHSDs) are supposed to be involved in -cardenolide biosynthesis. Here, a novel () was isolated from shoot cultures and expressed in . Recombinant 3βHSD1 and 3βHSD2 shared 70% amino acid identity, reduced various 3-oxopregnanes and oxidised 3-hydroxypregnanes, but only 3βHSD2 converted small ketones and secondary alcohols efficiently. To explain these differences in substrate specificity, we established homology models using borneol dehydrogenase of (6zyz) as the template. Hydrophobicity and amino acid residues in the binding pocket may explain the difference in enzyme activities and substrate preferences. Compared to is weakly expressed in shoots. High constitutive expression of was realised by -mediated transfer of genes fused to the CaMV-35S promotor into the genome of wild type shoot cultures. Transformed shoots (35S: and 35S:) accumulated less cardenolides than controls. The levels of reduced glutathione (GSH), which is known to inhibit cardenolide formation, were higher in the 35S: lines than in the controls. In the 35S: lines cardenolide levels were restored after adding of the substrate pregnane-3,20-dione in combination with buthionine-sulfoximine (BSO), an inhibitor of GSH formation. RNAi-mediated knockdown of the yielded several shoot culture lines with strongly reduced cardenolide levels. In these lines, cardenolide biosynthesis was fully restored after addition of the downstream precursor pregnan-3β-ol-20-one, whereas upstream precursors such as progesterone had no effect, indicating that no shunt pathway could overcome the knockdown. These results can be taken as the first direct proof that 3βHSD1 is indeed involved in -cardenolide biosynthesis.