the bifunctional diterpene synthase isopimara-7,15-diene synthase (diTPS-ISO) catalyzes the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate (cf. EC 5.5.1.12) and the formation of isopimara-7,15-diene from (+)-copalyl diphosphate. Compound identification and quantification by GC-MS analysis
IrKSL3 and IrKSL6 have the gammabetaalpha tridomain structure, as these proteins tend to possess the bidomain structure of IrKSL1, highlighting the evolutionary history of KSL gene domain loss and further elucidating chemical diversity evolution from a macroevolutionary stance in Lamiaceae. Identified IrCPS4 is predicted to be involved in oridonin biosynthesis, three KSL genes are involved in ent-CPP interactions, and a normal-CPP mediated miltiradiene and isopimaradiene biosynthesis pathway. The three KSL genes bearing different domain structures are involved in the normal-CPP mediated biosynthesis. The tridomain enzymes (IrKSL3 and IrKSL6) belong to an ancestral KSL clade that has the loss of g domain, a widespread occurrence in the Lamiaceae. Evolutionary origin of the gamma-domain loss
the chloroplastidic bifunctional diterpene synthase, isopimara-7,15-diene synthase (diTPS-ISO) from Picea abies and a cytosolic multifunctional cytochrome P450 (CYP720B4, UniProt ID E5FA70) from Picea sitchensis, which is associated to the endoplasmic reticulum. The diterpene synthase diTPS-ISO also converts geranylgeranyl diphosphate (GGPP) to the tricyclic diterpene isopimara-7,15-diene (isopimaradiene)
ent-kaurene diterpenoids are the largest group of known Isodon diterpenoids, five copalyl diphosphate synthase (CPS) and six kaurene synthase-like (KSL) genes are identified by transcriptome profiling of Isodon rubescens leaves. IrKSL6 reacts with normal-CP, synthesized by (+)-copalyl diphosphate synthases IrCPS1 or IrCPS2 (EC 5.5.1.12) to produce isopimaradiene. Enzyme IrKSL2 generates a product with identical EI mass spectrum to isopimaradiene (isopimara-7,15-diene), from ent-copalyl diphosphate
the bifunctional diterpene synthase, isopimara-7,15-diene synthase (diTPS-ISO) and a multifunctional cytochrome P450, CYP720B4 perform the biosynthesis of isopimaric acid. The diterpene synthase converts geranylgeranyl diphosphate (GGPP) to the tricyclic diterpene isopimara-7,15-diene (isopimaradiene) via formation of (+)-copalyl diphosphate. In three subsequent oxygenation reactions, the endoplasmic reticulum-associated enzyme CYP720B4 catalyzes the conversion of isopimaradiene into isopimaric acid, pathway overview
transient heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana. Expressing DXS and GGPPs from Plectranthus barbatus in Nicotiana benthamiana are efficient in boosting diterpenoid levels compared to coexpression of the Nicotiana endogenous DXS and GGPPs genes, which have no effect on boosting diterpenoid level. Pigment analysis of infiltrated leaves, overview
functional expression of Picea abies isopimara-7,15-diene synthase (diTPS-ISO) in Nicotiana benthamiana leaves via Agrobacterium tumefaciens transfection method, coexpression with a multifunctional cytochrome P450 (CYP720B4) from Picea sitchensis, subcloning in Escherichia coli strain NEB10beta
gene KSL6, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis, recombinant expression of His-tagged codon-optimized KSL6 in Escherichia coli strain Tuner(DE3)
Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme