Rosemary (Rosmarinus officinalis) produces the phenolic
diterpenes carnosic acid and
carnosol, which, in addition to their general
antioxidant activities, have recently been suggested as potential ingredients for the prevention and treatment of
neurodegenerative diseases. Little is known about the biosynthesis of these
diterpenes. Here we show that the biosynthesis of phenolic
diterpenes in rosemary predominantly takes place in the glandular trichomes of young leaves, and used this feature to identify the first committed steps. Thus, a
copalyl diphosphate synthase (RoCPS1) and two
kaurene synthase-like (RoKSL1 and RoKSL2) encoding genes were identified and characterized. Expression in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana demonstrate that RoCPS1 converts
geranylgeranyl diphosphate (GGDP) to
copalyl diphosphate (
CDP) of normal stereochemistry and that both RoKSL1 and RoKSL2 use normal
CDP to produce an
abietane diterpene. Comparison to the already characterized
diterpene synthase from Salvia miltiorrhiza (SmKSL) demonstrates that the product of RoKSL1 and RoKSL2 is
miltiradiene. Expression analysis supports a major contributing role for RoKSL2. Like SmKSL and the
sclareol synthase from Salvia sclarea, RoKSL1/2 are
diterpene synthases of the TPS-e group which have lost the internal gamma-domain. Furthermore, phylogenetic analysis indicates that RoKSL1 and RoKSL2 belong to a distinct group of KSL
enzymes involved in specialized metabolism which most likely emerged before the dicot-monocot split.