Cytochrome P450‐catalyzed biosynthesis of furanoditerpenoids in the bioenergy crop switchgrass (Panicum virgatum L.)

Abstract

Specialized diterpenoid metabolites are important mediators of plant-environment interactions in monocot crops. To understand metabolite functions in plant environmental adaptation that ultimately can enable crop improvement strategies, a deeper knowledge of the underlying species-specific biosynthetic pathways is required. Here, we report the genomics-enabled discovery of five cytochrome P450 monooxygenases (CYP71Z25-CYP71Z29) that form previously unknown furanoditerpenoids in the monocot bioenergy crop Panicum virgatum (switchgrass). Combinatorial pathway reconstruction showed that CYP71Z25-CYP71Z29 catalyze furan ring addition directly to primary diterpene alcohol intermediates derived from distinct class II diterpene synthase products. Transcriptional co-expression patterns and the presence of select diterpenoids in switchgrass roots support the occurrence of P450-derived furanoditerpenoids in planta. Integrating molecular dynamics, structural analysis and targeted mutagenesis identified active site determinants that contribute to the distinct catalytic specificities underlying the broad substrate promiscuity of CYP71Z25-CYP71Z29 for native and non-native diterpenoids.

Complex scaffold remodeling in plant triterpene biosynthesis

“…Although likely the result of convergent evolution, furan rings are found in a variety of natural products ( 36, 37 ). Although furan-forming enzymes have been reported from other plants ( 38, 39 ), ( 40 ), the AKR, CYP716AD and 2-ODD module described here represents a new mechanism of furan formation via the oxidative cleavage of a C-4 moiety. Along with the sterol isomerases (MOIs), the AKR and 2-ODDs add to the growing pool of enzyme families ( 41, 42 ) associated with primary sterol metabolism that appear to have been recruited to plant secondary triterpene metabolism, possibly due to the structural similarities between sterols and tetracyclic triterpenes.…”

Comparative transcriptomics and metabolomics reveal specialized metabolite drought stress responses in switchgrass (Panicum virgatumL.)

“…In addition to predicted phospholipids, a few features were enriched in Alamo leaves under drought stress, whereas many unidentified compounds were enriched in drought-treated Cave-in-Rock leaves (Supporting Information Table S2). In contrast, several compounds identified as diterpenoids and triterpenoids by comparison of RT and fragmentation patterns to previously identified compounds (Li et al, 2020;Muchlinski et al, 2021) accumulated in roots of both Alamo and Cave-in-Rock plants under drought stress, with a stronger increase in Alamo (Fig. 7).…”

“…Our prior research identified expansive, species-specific diterpene synthase (diTPS) and P450 families in switchgrass that form complex metabolic networks toward a range of labdane-related diterpenoids, including syn -pimarane and furanoditerpenoid compounds that occur, perhaps uniquely, in switchgrass (Fig. S5) (Pelot et al 2018; Muchlinski et al 2021). This pathway knowledge enabled a detailed analysis of transcriptomic alterations related to diterpenoid metabolism.…”

“…5). In Alamo roots, the cis-trans-clerodienyl pyrophosphate (CLPP) synthase PvCPS1 and the P450 genes, CYP71Z25 , CYP71Z26 , and CYP71Z28 shown to form furanoditerpenoids (Muchlinski et al 2021), showed patterns of co-expression at two and four weeks of drought. Similarly, the predicted syn-CPP synthases , PvCPS9 and PvCPS10 , as well as two class I diTPS, PvKSL4 and PvKSL5 , shown to form syn -pimaradiene compounds (Pelot et al 2018), co-expressed in roots, albeit without significant drought-inducible transcript changes.…”

“…In addition, accumulation in both Alamo and Cave-in-Rock support a role of these metabolites in drought responses rather than general stress responses associated with the more pronounced wilting phenotype observed in Cave-in-Rock. Structural analysis identified three compounds as oxygenated clerodane furanoditerpenoids, named here panicoloid A-C, which likely represent derivatives of furanoditerpenoid scaffolds recently identified in switchgrass (Pelot et al 2018; Muchlinski et al 2021). Notably, the enantiomeric stereochemistry of panicoloid B and C is likely derived from the activity of yet unidentified diTPS functionally related to the CLPP synthase PvCPS1.…”