Biosynthesis of sterols and ecdysteroids in Ajuga hairy roots

Hairy roots of Ajuga reptans var. atropurpurea produce clerosterol, 22‐dehydroclerosterol, and cholesterol as sterol constituents, and 20‐hydroxyecdysone, cyasterone, isocyasterone, and 29‐norcyasterone as ecdysteroid constituents. To better understand the biosynthesis of these steroidal compounds, we carried out feeding studies of variously 2H‐ and 13C‐labeled sterol substrates with Ajuga hairy roots. In this article, we review our studies in this field. Feeding of labeled desmosterols, 24‐methylenecholesterol, and 13C2‐acetate established the mechanism of the biosynthesis of the two C29‐sterols and a newly accumulated codisterol, including the metabolic correlation of C‐26 and C‐27 methyl groups. In Ajuga hairy roots, 3α‐, 4α‐, and 4β‐hydrogens of cholesterol were all retained at their original positions after conversion into 20‐hydroxyecdysone, in contrast to the observations in a fern and an insect. Furthermore, the origin of 5β‐H of 20‐hydroxyecdysone was found to be C‐6 hydrogen of cholesterol exclusively, which is inconsistent with the results in the fern and the insect. These data strongly support the intermediacy of 7‐dehydrocholesterol 5α,6α‐epoxide. Moreover, 7‐dehydrocholesterol, 3β‐hydroxy‐5β‐cholest‐7‐en‐6‐one (5β‐ketol), and 3β,14α‐dihydroxy‐5β‐cholest‐7‐en‐6‐one (5β‐ketodiol) were converted into 20‐hydroxyecdysone. Thus, the pathway cholesterol→7‐dehydrocholesterol→7‐dehydrocholesterol 5α,6α‐epoxide→5β‐ketol→5β‐ketodiol is proposed for the early stages of 20‐hydroxyecdysone biosynthesis. 3β‐Hydroxy‐5β‐cholestan‐6‐one was also incorporated into 20‐hydroxyecdysone, suggesting that the introduction of a 7‐ene function is not necessarily next to cholesterol. C‐25 Hydroxylation during 20‐hydroxyecdysone biosynthesis was found to proceed with ca. 70% retention and 30% inversion. Finally, clerosterol was shown to be a precursor of cyasterone and isocyasterone.