Cytochrome P450 Monooxygenase CYP716A141 is a Unique β-Amyrin C-16β Oxidase Involved in Triterpenoid Saponin Biosynthesis in Platycodon grandiflorus

Abstract

The roots of Platycodon grandiflorus are widely used as a crude drug. The active components include a variety of triterpenoid saponins. Recent studies have revealed that Cyt P450 monooxygenases (P450s) function as triterpene oxidases in triterpenoid saponin biosynthesis in many plant species. However, there have been no reports regarding triterpene oxidases in P. grandiflorus. In this study, we performed transcriptome analysis of three different P. grandiflorus tissues (roots, leaves and petals) using RNA sequencing (RNA-Seq) technology. We cloned six P450 genes that were highly expressed in roots, and classified them as belonging to the CYP716A, CYP716D and CYP72A subfamilies. We heterologously expressed these P450s in an engineered yeast strain that produces β-amyrin, one of the most common triterpenes in plants. Two of the CYP716A subfamily P450s catalyzed oxidation reactions of the β-amyrin skeleton. One of these P450s, CYP716A140v2, catalyzed a three-step oxidation reaction at C-28 on β-amyrin to produce oleanolic acid, a reaction performed by CYP716A subfamily P450s in a variety of plant species. The other P450, CYP716A141, catalyzed the hydroxylation of β-amyrin at C-16β. This reaction is unique among triterpene oxidases isolated to date. These results enhance our knowledge of functional variation among CYP716A subfamily enzymes involved in triterpenoid biosynthesis, and provide novel molecular tools for use in synthetic biology to produce triterpenoid saponins with pre-defined structures.

Comparative analysis of CYP716A subfamily enzymes for the heterologous production of C-28 oxidized triterpenoids in transgenic yeast

“…This study contributes to an increased understanding of the role of CYP716A enzymes in the diversification of the triterpenoid backbone (Figure 4; Miettinen et al 2017;Misra et al 2017;Tamura et al 2017aTamura et al , 2017bYasumoto et al 2016Yasumoto et al , 2017. We also identified previously reported putative compounds, oleanolic, ursolic, and betulinic aldehydes (3, 7, 11), by synthesizing authentic standards and comparing their Rts and mass spectra with those of chromatographic peaks obtained from heterologous yeast extracts.…”

“…Three step oxidations catalyzed by CYP716A enzymes at the C-28 position of three triterpene skeletons are shown. bAS, β-amyrin synthase; aAS, α-amyrin synthase; LUS, lupeol synthase Copyright © 2018. The Japanese Society for Plant Cell and Molecular Biology…”

Bayogenin 3-O-Cellobioside is a novel non-cultivar specific anti-blast metabolite produced in rice in response toPyricularia oryzaeinfection

“…This clustering, and the observation that a higher number of UDP-GT genes are expressed upon inoculation of resistant cultivars compared to susceptible cultivars suggesting that multiple UDP-GTs enhance rice immunity against blast fungus by producing diverse glycosides, likely including Bayogenin 3-O-cellobioside. Disruption of a β-amyrin synthase and UDP-GTs blocks avenacin biosynthesis in oat (30, 31). However, the extent to which β-amyrin synthases and UDP-GTs influence Bayogenin 3-O-cellobioside biosynthesis, as well as the resistance or susceptibility of different rice cultivars to P oryzae , remains to be determined.…”

Transcriptome sequencing and identification of cytochrome P450 monooxygenases involved in the biosynthesis of maslinic acid and corosolic acid in <i>Avicennia marina</i>

“…CYP716 family enzymes, divided into five subfamilies (A, C, E, S, and Y), are widely distributed among eudicots and considered to be a major contributor to the diversification of eudicot triterpenoid biosynthesis (Miettinen et al 2017). Most CYP716A subfamily enzymes have been identified to catalyze oxidation at the C-28 position of pentacyclic triterpene skeletons ( Figure 5), such as β-amyrin, α-amyrin, and lupeol, except for several CYP716As, including CYP716A14v2 from Artemisia annua catalyzing oxidation at the C-3 position (Moses et al 2015) and CYP716A141 from Platycodon grandiflorus catalyzing oxidation at the C-16β position (Miettinen et al 2017;Tamura et al 2017). In accordance with these observations, CYP716A259 catalyzed oxidation at C-28 in β-amyrin, α-amyrin, and lupeol ( Figure 3).…”