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bioRxiv · 10.1101/2021.03.30.437764

Furanoditerpenoid biosynthesis in the bioenergy crop switchgrass is catalyzed by an alternate metabolic pathway

Muchlinski, A. J.·Jia, M.·Tiedge, K. J.·Fell, J. S.·Pelot, K. A.·Chew, L.·Davisson, D. L.·Chen, Y.·Siegel, J.·Lovell, J. T.·Zerbe, P.
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Abstract

Specialized diterpenoid metabolites are important mediators of stress resilience in monocot crops. A deeper understanding of how species-specific diterpenoid-metabolic pathways and functions contribute to plant chemical defenses can enable crop improvement strategies. Here, we report the genomics-enabled discovery of five cytochrome P450 monooxygenases (CYP71Z25-29) that form previously unknown furanoditerpenoids in the monocot bioenergy crop switchgrass (Panicum virgatum). Combinatorial pathway reconstruction showed that CYP71Z25-29 catalyze furan ring addition to diterpene alcohol intermediates derived from distinct class II diterpene synthases, thus bypassing the canonical role of class I diterpene synthases in plant diterpenoid metabolism. Transcriptional co-expression patterns and presence of select diterpenoids in droughted switchgrass roots support possible roles of CYP71Z25-29 in abiotic stress responses. Integrating molecular dynamics, structural analysis, and targeted mutagenesis, identified active site determinants controlling distinct CYP71Z25-29 catalytic specificities and, combined with broad substrate promiscuity for native and non-native diterpenoids, highlights the potential of these P450s for natural product engineering. Significance StatementDiterpenoids play important roles in stress resilience and chemically mediated interactions in many plant species, including major food and bioenergy crops. Enzymes of the cytochrome P450 monooxygenase family catalyze the various functional decorations of core diterpene scaffolds that determine the large diversity of biologically active diterpenoids. This study describes the identification and mechanistic analysis of an unusual group of cytochrome P450 monooxygenases, CYP71Z25-29, from the bioenergy crop switchgrass (Panicum virgatum). These enzymes catalyze the furan ring addition directly to class II diterpene synthase products, thus bypassing the conserved pairwise reaction of class II and class I diterpene synthases in labdane diterpenoid metabolism. Insight into the distinct substrate-specificity of CYP71Z25-29 offers opportunity for engineering of furanoditerpenoid bioproducts.

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Muchlinski, A. J., Jia, M., Tiedge, K. J., Fell, J. S., Pelot, K. A., Chew, L., Davisson, D. L., Chen, Y., Siegel, J., Lovell, J. T., Zerbe, P.. 2021-03-31. Furanoditerpenoid biosynthesis in the bioenergy crop switchgrass is catalyzed by an alternate metabolic pathway. https://doi.org/10.1101/2021.03.30.437764

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