Identification of clerodane diterpene modifying cytochrome P450 (CYP728D26) in Salvia divinorum ‐ en route to psychotropic salvinorin A biosynthesis

Salvia divinorum is a hallucinogenic plant native to the Oaxaca in Mexico. The active ingredient for psychotropic effects in this plant is salvinorin A, a potent and highly selective κ‐opioid receptor agonist. Salvinorin A is distinct from other well‐known opioids, such as morphine and codeine, in that it is a non‐nitrogenous diterpenoid with no affinity for μ‐opioid receptor, the prime receptor of alkaloidal opioids. A terpene opioid that selectively targets a new opioid receptor (κ‐opioid receptor) can be instrumental in developing alternative analgesics. Elucidation of the salvinorin A biosynthetic pathway can help bio‐manufacture diverse semi‐synthetic derivatives of salvinorin A but, to date, only two enzymes in the Salvinorin A pathway have been identified. Here, we identify CYP728D26 that catalyzes a C18 oxygenation on crotonolide G, which bears a clerodane backbone. Biochemical identity of CYP728D26 was validated by in vivo reconstitution in yeast, 1H‐ and 13C‐NMR analyses of the purified product, and kinetic analysis of CYP728D26 with a Km value of 13.9 μM. Beyond the single oxygenation on C18, collision‐induced dissociation analysis suggested two additional oxygenations are catalyzed by CYP728D26 to form crotonoldie G acid, although this carboxylic acid form is a minor product. Its close homologue CYP728D25 exhibited a C1‐hydroxylation on the clerodane backbone in a reconstituted yeast system. However, CYP728D25 showed no activity in in vitro assays. This result implies that catalytic activities observed from overexpression systems should be interpreted cautiously. This work identified a new CYP catalyst and advanced our knowledge of salvinorin A biosynthesis.