Two CYP716A subfamily cytochrome P450 monooxygenases of sweet basil play similar but nonredundant roles in ursane‐ and oleanane‐type pentacyclic triterpene biosynthesis

Abstract

SummaryThe medicinal plant sweet basil (Ocimum basilicum) accumulates bioactive ursane-and oleanane-type pentacyclic triterpenes (PCTs), ursolic acid and oleanolic acid, respectively, in a spatio-temporal manner; however, the biosynthetic enzymes and their contributions towards PCT biosynthesis remain to be elucidated.Two CYP716A subfamily cytochrome P450 monooxygenases (CYP716A252 and CYP716A253) are identified from a methyl jasmonate-responsive expression sequence tag collection and functionally characterized, employing yeast (Saccharomyces cerevisiae) expression platform and adapting virus-induced gene silencing (VIGS) in sweet basil.CYP716A252 and CYP716A253 catalyzed sequential three-step oxidation at the C-28 position of a-amyrin and b-amyrin to produce ursolic acid and oleanolic acid, respectively. Although CYP716A253 was more efficient than CYP716A252 for amyrin C-28 oxidation in yeast, VIGS revealed essential roles for both of these CYP716As in constitutive biosynthesis of ursolic acid and oleanolic acid in sweet basil leaves. However, CYP716A253 played a major role in elicitor-induced biosynthesis of ursolic acid and oleanolic acid.Overall, the results suggest similar as well as distinct roles of CYP716A252 and CYP716A253 for the spatio-temporal biosynthesis of PCTs. CYP716A252 and CYP716A253 might be useful for the alternative and sustainable production of PCTs in microbial host, besides increasing plant metabolite content through genetic modification.

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.…”

Triterpene Structural Diversification by Plant Cytochrome P450 Enzymes

“…Besides, in vitro and in vivo assays using heterologous system also revealed the ability of the CYP716A12 in converting α-amyrin to ursolic acid and lupeol to betulinic acid, following three-step oxidation at the C28 position ( Carelli et al, 2011 ; Fukushima et al, 2011 ). Subsequent to these initial reports, several CYP716s were identified and biochemically characterized from the plants, mostly by exploring transcriptomic and genomic resources ( Misra et al, 2017 ; Miettinen et al, 2017 ; Tamura et al, 2017a ). To date, about 20 CYP716As are biochemically characterized for the amyrin/lupeol C28-oxidase activities ( Figures 1 , 3 – 5 ).…”

“…To date, about 20 CYP716As are biochemically characterized for the amyrin/lupeol C28-oxidase activities ( Figures 1 , 3 – 5 ). A majority of these CYP716As catalyzed sequential three-step oxidation of the amyrin/lupeol scaffolds, leading to the consecutive formation of the hydroxyl, aldehyde, and carboxyl moieties at the C28 position ( Carelli et al, 2011 ; Fukushima et al, 2011 ; Misra et al, 2017 ; Tamura et al, 2017a ). A few plant CYP716As also target a carbon atom of the amyrin skeletons other than the C28.…”

“…In recent years, a notable progress has been made in understanding the biochemical functions of the P450s involved in plant triterpene metabolism. Together with the genetic screening of the mutant lines impaired with triterpene biosynthesis, the availability of the genomic and transcriptomic resources led to the identification of a number of P450s that are involved in plant triterpene structural modifications ( Qi et al, 2006 ; Carelli et al, 2011 ; Augustin et al, 2015 ; Biazzi et al, 2015 ; Miettinen et al, 2017 ; Misra et al, 2017 ). This article highlights the diverse roles of the plant P450s in triterpene scaffold modifications.…”

Multi‐omics data‐driven investigations of metabolic diversity of plant triterpenoids

“…For instance, the CYP716 family from the CYP85 clan is conserved across land plants but has evolved specifically toward pentacyclic triterpene metabolism in eudicots (Miettinen et al ., ). CYP716As are conserved in higher plants and catalyze a consecutive three‐step oxidation at C28 of α‐amyrin, lupel and β‐amyrin (Carelli et al ., ; Fukushima et al ., ; Misra et al ., ; Tamura et al ., ). Other CYP716 subfamily members CYP716C/‐E/‐S/‐Y also participate in oxidation of the pentacyclic triterpene scaffolds (Ghosh, ).…”