To decode the biosynthesis, molecular evolution and biological functions of plant terpenoid metabolism for agricultural and bioproduct applications, our group developes and applies approaches of:
- Functional Genomics-enabled Gene, Enzyme, & Pathway Discovery
- Metabolomics- & NMR-based Terpenoid Identification
- Enzymology & Protein Structure-Function Studies
- Analysis of Molecular Plant-Pathogen & Plant-Microbiome Interactions
- Functional Genomics-enabled Gene, Enzyme, & Pathway Discovery
- Metabolomics- & NMR-based Terpenoid Identification
- Enzymology & Protein Structure-Function Studies
- Analysis of Molecular Plant-Pathogen & Plant-Microbiome Interactions
Modularity of terpenoid metabolism fuels plant's chemical diversity
The tens of thousands of plant terpenoids derive from only two five-carbon (C5) isoprenoids, isopentenyl diphosphate (IPP), and dimethylallyl diphosphate (DMAPP), produced via the cytosolic mevalonate (MVA) and the plastidial 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. Sequential condensation of these units by prenyl transferases yields a handful of central prenyl diphosphate intermediates in terpenoid biosynthesis, geranyl diphosphate (GPP, C10), farnesyl diphosphate (FPP, C15), and geranylgeranyl diphosphate (GGPP, C20) as the general precursors for all terpenoids. Plant terpenoid metabolism is often organized in form of modular networks, where combining a few enzyme modules along a common blueprint, facilitates the formation of thousands of different metabolites. Terpene synthases (TPS) and cytochrome P450-dependent monooxygenases (P450) are major enzyme modules driving terpenoid chemical diversity and bioactivity. TPS facilitate the committed carbocation-driven cyclization and/or rearrangement of prenyl dihphophate precursors GGPP into various diterpene scaffolds, which are then modified through P450-catalyzed oxygenations and further functional decorations. On account of their critical role in terpenoid production, our group is particularly interested in the molecular evolution, distribution, and catalytic specificity of the large TPS and P450 families. [Zerbe & Bohlmann (2015) Trends Biotechnol 33, 419-28]
Functional genomics enable efficient disocvery of terpenoid metabolic pathways
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In order to investigate and ultimately harness the vast chemical repertoire of plant terpenoid metabolism, a core strength of our lab is the efficient identification of terpenoid-metabolic genes, enzymes and pathways by combining genomics-enabled gene discovery using in-house protein databases, rapid enzyme biochemical characterization through microbial and plant co-expression assays, and de novo identification of novel metabolites using mass spectrometry and NMR approaches. Using these tools, we have identified more than 50 functionally distinct TPS and P450 enzymes in over a dozen plant species with relevance for food, bioenergy and medicine. We integrate these biochemical insights with in planta terpenoid profiling via GC- and LC-MS analyses, genetic gene function studies using CRISPR/Cas9-enabled pathway alteration, as well as plant-environment interaction studies using in vitro and in vivo plant-pathogen and plant-microbiome analyses to investigate the bioactivity of teprenoid metabolites and evaluate their potential for agricultural and other biotechnology applications. [e.g. Zerbe et al. (2013) Plant Physiol. 162, 1073-91; Zerbe & Bohlmann (2015) Trends Biotechnol. 33, 419-28]
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Stress resistance in food crops - the corn example
Terpenoids are major components of maize (Zea mays) chemical defenses that mediate responses to herbivores, pathogens and other environmental challenges. We recently used our genomics-enabled pathway disocvery strategy to identify yet unrecognized defense compounds in maize. We identified a new class of maize diterpenoids, named dolabralexins. Dolabralexin biosynthesis involves the sequential activity of two diterpene synthases, the ent-copalyl diphosphate synthase (ZmAN2) and kaurene synthase-like 4 (ZmKSL4). In addition, we biochemically characterized a cytochrome P450 monooxygenase, namely ZmCYP71Z16, which catalyzes the oxygenation of dolabradiene to afford the epoxides 15,16-epoxy-dolabradiene and 3β-epoxy-dolabranol. Absence of dolabradiene and 3β-epoxy-dolabranol in Zman2 mutants under elicited conditions confirms the in vivo biosynthetic requirement of ZmAN2. Combined mass spectrometry and nuclear magnetic resonance experiments demonstrate that much of the 3β-epoxy-dolabranol is further converted into 3β,15,16-trihydroxydolabrene (THD). Metabolite profiling of common maize cultivars under field conditions supports the widespread biosynthesis of dolabralexins in roots with THD as the predominant diterpenoid accumulating. Oxidative stress induces dolabralexin accumulation and transcript expression of ZmAN2 and ZmKSL4 in root tissues. Likewise, metabolite and transcript accumulation are up-regulated in response to elicitation with the fungal pathogens Fusarium verticillioides and F. graminearum. Consistently, 3β-epoxy-dolabranol and THD significantly inhibit growth of both pathogens in vitro at 10 µg/mL, although THD-mediated inhibition is specific to F. verticillioides. These findings support defense-related roles for dolabralexins in maize stress interactions and expand the known chemical space of diterpenoid defenses as genetic targets for understanding and ultimately improving maize resilience. [Mafu et al. (2018) Plant Physiol 176, 2677-90]
Decoding the metabolic diversity in medicinal plants - the marrubiin example
Horehound (Marrubium vulgare) is a medicinal plant of the mint family (Lamiaceae) whose major bioactive compounds, marrubiin and related diterpenoids, have potential uses as anti-diabetics or analgesics. Metabolome and transcriptome profiling of Marrubium vulgare leaves identified five different candidate diterpene synthases (diTPSs). We describe the in vitro and in vivo functional characterization of the Marrubium vulgare diTPS family. We identified three diTPSs of specialized metabolism: MvCPS3 (+)-copalyl diphosphate synthase, and the functional diTPS pair MvCPS1 and MvELS. In a sequential reaction, MvCPS1 and MvELS produce a unique oxygenated diterpene scaffold 9,13-epoxy-labd-14-ene en route to marrubiin and an array of related compounds. In contrast with previously known diTPSs that introduce a hydroxyl group at carbon C-8 of the labdane backbone, the MvCPS1-catalyzed reaction proceeds via oxygenation of an intermediate carbocation at C-9, yielding the bicyclic peregrinol diphosphate. MvELS is active with three different prenyl diphosphate substrates forming the marrubiin precursor 9,13-epoxy-labd-14-ene, manoyl oxide and miltiradiene. MvELS fills a central position in the biosynthetic system that forms the foundation for the diverse repertoire of Marrubium diterpenoids. Co-expression of MvCPS1 and MvELS in engineered E. coli and Nicotiana benthamiana offers future opportunities for producing precursors for an array of biologically active diterpenoids. [Zerbe et al. (2014) Plant J 79, 914-27]
Improved biofuel production through discovery and engineering of terpene metabolism in the bioenergy crop switchgrass
Of the myriad specialized metabolites that plants deploy to adapt to environmental challenges, terpenes form the largest group. In many major crops, unique terpene blends serve as key stress defenses that directly impact plant fitness and yield. In addition, terpenes, such as bisabolene and pinene, are used for biofuel manufacture. Thus, engineering of terpene metabolism provides a versatile resource for advancing biofuel feedstock production, but requires a system-wide knowledge of the diverse biosynthetic machinery and defensive potential of often species-specific terpene blends. This DOE Early Career Research Program funded project would merge genome-wide enzyme discovery with comparative –omics, and protein structural studies to define the biosynthesis and stress-defensive functions of switchgrass (Panicum virgatum) terpene metabolism. These insights would be combined with the development of non-transgenic genome editing tools to design plants with desirable terpene blends for improved biofuel production on marginal lands. As a dedicated lignocellulosic biofuel feedstock with high net energy yield, stress tolerance, and available genome resources, switchgrass is well-suited for devising new avenues for U.S. biofuel production. [Pelot et al. (2018) Plant Physiol]
