title: Gene discovery of modular diterpene metabolism in nonmodel systems.
abstract: Plants produce over 10,000 different diterpenes of specialized (secondary) metabolism, and fewer diterpenes of general (primary) metabolism. Specialized diterpenes may have functions in ecological interactions of plants with other organisms and also benefit humanity as pharmaceuticals, fragrances, resins, and other industrial bioproducts. Examples of high-value diterpenes are taxol and forskolin pharmaceuticals or ambroxide fragrances. Yields and purity of diterpenes obtained from natural sources or by chemical synthesis are often insufficient for large-volume or high-end applications. Improvement of agricultural or biotechnological diterpene production requires knowledge of biosynthetic genes and enzymes. However, specialized diterpene pathways are extremely diverse across the plant kingdom, and most specialized diterpenes are taxonomically restricted to a few plant species, genera, or families. Consequently, there is no single reference system to guide gene discovery and rapid annotation of specialized diterpene pathways. Functional diversification of genes and plasticity of enzyme functions of these pathways further complicate correct annotation. To address this challenge, we used a set of 10 different plant species to develop a general strategy for diterpene gene discovery in nonmodel systems. The approach combines metabolite-guided transcriptome resources, custom diterpene synthase (diTPS) and cytochrome P450 reference gene databases, phylogenies, and, as shown for select diTPSs, single and coupled enzyme assays using microbial and plant expression systems. In the 10 species, we identified 46 new diTPS candidates and over 400 putatively terpenoid-related P450s in a resource of nearly 1 million predicted transcripts of diterpene-accumulating tissues. Phylogenetic patterns of lineage-specific blooms of genes guided functional characterization.
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.
authors.forename: Philipp
authors.initials: P
authors.lastname: Zerbe
authors.forename: Björn
authors.initials: B
authors.lastname: Hamberger
authors.forename: Macaire M S
authors.initials: MM
authors.lastname: Yuen
authors.forename: Angela
authors.initials: A
authors.lastname: Chiang
authors.forename: Harpreet K
authors.initials: HK
authors.lastname: Sandhu
authors.forename: Lina L
authors.initials: LL
authors.lastname: Madilao
authors.forename: Anh
authors.initials: A
authors.lastname: Nguyen
authors.forename: Britta
authors.initials: B
authors.lastname: Hamberger
authors.forename: Søren Spanner
authors.initials: SS
authors.lastname: Bach
authors.forename: Jörg
authors.initials: J
authors.lastname: Bohlmann
mesh_terms: D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D057225:Data Mining; D030541:Databases, Genetic; D004224:Diterpenes / Q000378:metabolism*; D019143:Evolution, Molecular; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D010944:Plants / Q000235:genetics* / Q000378:metabolism*; D059467:Transcriptome
chemical_list: D004224:Diterpenes; D010940:Plant Proteins; D003577:Cytochrome P-450 Enzyme System