title: Bifunctional cis-abienol synthase from Abies balsamea discovered by transcriptome sequencing and its implications for diterpenoid fragrance production.
abstract: The labdanoid diterpene alcohol cis-abienol is a major component of the aromatic oleoresin of balsam fir (Abies balsamea) 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 cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily. The AbCAS-catalyzed formation of cis-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 AbCAS. As a class I/II bifunctional enzyme, AbCAS is a promising target for metabolic engineering of cis-abienol production.
authors.affiliation: Michael Smith Laboratories, University of British Columbia, 301-2185 East Mall, Vancouver, British Columbia V6T 1Z4, Canada.
authors.forename: Philipp
authors.initials: P
authors.lastname: Zerbe
authors.forename: Angela
authors.initials: A
authors.lastname: Chiang
authors.forename: Macaire
authors.initials: M
authors.lastname: Yuen
authors.forename: Björn
authors.initials: B
authors.lastname: Hamberger
authors.forename: Britta
authors.initials: B
authors.lastname: Hamberger
authors.forename: Jason A
authors.initials: JA
authors.lastname: Draper
authors.forename: Robert
authors.initials: R
authors.lastname: Britton
authors.forename: Jörg
authors.initials: J
authors.lastname: Bohlmann
mesh_terms: D028202:Abies / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D055162:Biocatalysis; D020134:Catalytic Domain; D004224:Diterpenes / Q000737:chemistry* / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009284:Naphthols / Q000737:chemistry* / Q000378:metabolism; D010476:Perfume; D010802:Phylogeny; D024301:Plant Bark / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D059467:Transcriptome
chemical_list: D004224:Diterpenes; D009284:Naphthols; D010476:Perfume; D010940:Plant Proteins; C085761:abienol