Molecular characterization of daurichromenic acid synthase from Rhododendron dauricum

Abstract

Rhododendron dauricum L. (Ericaceae) produces daurichromenic acid (DCA), an anti-HIV component [1]. DCA is a terpenophenol, and would be biosynthesized from grifolic acid via oxidative cyclization of the farnesyl group, the reaction analogous to those reported for cannabinoid biosynthesis [2]. We attempted to amplify cDNA fragments encoding DCA synthase by homology-based RT-PCR with degenerate primers designed from conserved sequences in cannabinoid synthases and related plant oxidases. Then, the 3' and 5'-end regions of cDNA were obtained by rapid amplifications of cDNA ends. Consequently, three cDNA clones, that encode polypeptides named RdOx 1˜3, were cloned. RdOx 1˜3 consisted of 533, 533 and 534 amino acids containing a FAD binding motif. In addition, these polypeptides had >50% identities with cannabinoid synthases. The heterologous expression system for RdOxs was established using Pichia pastoris as a host. The recombinant RdOx1 and 2 could produce DCA from grifolic acid, whereas RdOx-3 showed no DCA-producing activity, suggesting that RdOx 1 and 2 are active DCA synthase in R. dauricum. DCA synthase would be applied for biotechnological production of DCA because the substrate grifolic acid has been isolated from a mushroom Albatrellus dispansus in a large amount [3].