Gene Discovery of Modular Diterpene Metabolism in Nonmodel Systems    

Abstract

(Bj.H., Br.H., S.S.B.)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.

Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize

“…To test for the activity of ZmKSL4 in specialized diterpenoid metabolism, we utilized the combinatorial functionality of class II and class I diTPSs of different plant species, which can be exploited to probe diTPS functions (Brückner and Tissier 2013;Zerbe et al, 2013;Kitaoka et al, 2015). An established Escherichia coli coexpression system (Morrone et al, 2010) was used to analyze the coupled activities of ZmKSL4 with (1) the ent-CPP synthase ZmAN2, (2) the rice syn-CPP synthase (OsCPS4; Xu et al, 2004), and (3) the grand fir (Abies grandis) abietadiene synthase variant D621A that produces CPP of normal [i.e.…”

Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis

“…We have recently identified a diverse suite of 40 class II and class I diTPSs from ten plant species, each harboring structurally exceptional diterpenoids 5. In plants, specific pairs of class I and class II diTPSs form distinct diterpene skeletons, which underpins the structural diversity of native diterpenoids 6.…”

Manoyl Oxide (13R), the Biosynthetic Precursor of Forskolin, Is Synthesized in Specialized Root Cork Cells in Coleus forskohlii

“…As with the class II diTPSs, deprotonation or water capture terminate the class I diTPS reaction. Water capture as a mechanism of diTPSs that leads to oxygen functionalities in the diterpene products has been described for the class II active site of bifunctional class I/II gymnosperm diTPSs (Keeling et al, 2011;Zerbe et al, 2012Zerbe et al, , 2013, class II angiosperm diTPSs (Falara et al, 2010;Caniard et al, 2012;Zerbe et al, 2013), and class I angiosperm diTPS (Caniard et al, 2012). Further oxidative functionalization of diterpenoids is typically catalyzed by cytochrome P450-dependent monooxygenase enzymes (P450s; Ro et al, 2005;Swaminathan et al, 2009;Hamberger et al, 2011;Wang et al, 2011;Guo et al, 2013;Hamberger and Bak, 2013), which then provide molecular handles for addition of auxiliary functional groups, leading ultimately to highly complex and decorated products such as forskolin and its derivatives.…”

“…Recently, Zerbe et al (2013) reported the sequencing and assembly of a root transcriptome of C. forskohlii using 454 and Illumina sequencing technologies. Mining of the transcriptome databases resulted in the identification of a panel of candidate CfTPS genes.…”

“…Given its importance as a pharmaceutical, we set out to discover genes involved in the biosynthesis of forskolin. Our general approach utilized and built on a recently established diterpene pathway gene discovery strategy in nonmodel systems (Zerbe et al, 2013). As guiding principles, we considered possible spatial separation of different diterpenoid pathways in C. forskohlii, the large diversity of diterpenoids found in C. forskohlii, and the particular heterocyclic structure of forskolin.…”