초록
<P>The labdanoid diterpene alcohol <I>cis</I>-abienol is a major component of the aromatic oleoresin of balsam fir (<I>Abies balsamea</I>) and serves as a valuable bioproduct material for the fragrance industry. Using high-throughput 454 transcriptome sequencing and metabolite profiling of balsam fir bark tissue, we identified candidate diterpene synthase sequences for full-length cDNA cloning and functional characterization. We discovered a bifunctional class I/II <I>cis</I>-abienol synthase (<I>Ab</I>CAS), along with the paralogous levopimaradiene/abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily. The <I>Ab</I>CAS-catalyzed formation of <I>cis</I>-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site. This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. Comparative homology modeling suggested the active site residues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially important for the specificity of <I>Ab</I>CAS. As a class I/II bifunctional enzyme, <I>Ab</I>CAS is a promising target for metabolic engineering of <I>cis</I>-abienol production.</P>