OSC2 and CYP716A14v2 Catalyze the Biosynthesis of Triterpenoids for the Cuticle of Aerial Organs of <i>Artemisia annua</i>

Moses, Tessa; Pollier, Jacob; Shen, Qian; Soetaert, Sandra; Reed, James; Erffelinck, Marie‐Laure; Nieuwerburgh, Filip Van; Bossche, Robin Vanden; Osbourn, Anne; Thevelein, Johan M.; Deforce, Dieter; Tang, Kexuan; Goossens, Alain

doi:10.1105/tpc.114.134486

Cited by 97 publications

(78 citation statements)

References 52 publications

(82 reference statements)

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“…This resulted in the identification of CYP450s that showed sequence homology with the CYP82, CYP97 and CYP716 family members (Misra et al ., ). CYP716s are the potential candidates for amyrin C‐28 oxidase activity (Carelli et al ., ; Moses et al ., ,b; Andre et al ., ). Thus, candidate CYP716s, representing three ESTs (ObMU203, ObMU260 and ObMU385), were further considered for the detailed functional analysis.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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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.

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Section: Resultsmentioning

confidence: 99%

“…The biosynthesis and accumulation of PCTs in plants are subject to tissue‐, organ‐ and developmental stage‐specificity, and also to environmental cues and phytohormone elicitations (Phillips et al ., ; Misra et al ., ; Moses et al ., ,b). PCTs are suggested to play specialized (i.e.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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“…Other CYP716 family enzymes such as CYP716Y1 from Bupleurum falcatum and CYP716A14v2 from Artemisia annua oxidize the C-16a and C-3 positions of the oleanane backbone, respectively [12,13]. As members of the CYP716 family, we expected that CYP716A1 and CYP716A2 would oxidize triterpene skeletons, such as a-amyrin, b-amyrin, and lupeol.…”

Section: Triterpene Oxidizing Activities Of Cyp716a1 and Cyp716a2mentioning

confidence: 99%

“…Nuclear magnetic resonance (NMR) spectra ( 1 H, 13 C, HMBC, HSQC, DEPT135, DQF-COSY, HSQC-TOCSY, and NOESY) of purified compound 4 were reported on a JMN-ECS400 system (400 MHz; JEOL, Tokyo, Japan) in deuterated chloroform. Tetramethylsilane was added as an internal standard.…”

Section: Chemical Structure Determinationmentioning

confidence: 99%

Novel triterpene oxidizing activity of Arabidopsis thaliana CYP716A subfamily enzymes

et al. 2016

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Edited by Ulf-Ingo Fl€ uggeTriterpenoids have diverse chemical structures and bioactivities. Cytochrome P450 monooxygenases play a key role in their structural diversification. In higher plants, CYP716A subfamily enzymes are triterpene oxidases. In this study, Arabidopsis thaliana CYP716A1 and CYP716A2 were characterized by heterologously expressing them in simple triterpene-producing yeast strains. In contrast to the C-28 oxidative activity of CYP716A1 shown in several CYP716A subfamily enzymes, remarkably, CYP716A2 displayed 22a-hydroxylation activity against a-amyrin that has not been previously reported, which produces the cytotoxic triterpenoid, 22a-hydroxy-a-amyrin. Our results contribute to the enrichment of the molecular toolbox that allows for the combinatorial biosynthesis of diverse triterpenoids.Keywords: 22a-hydroxylation; Arabidopsis thaliana; combinatorial biosynthesis; CYP716A; cytochrome P450 monooxygenase; triterpenoids HighlightsThe full-length CDS of CYP716A2, miss-annotated on TAIR, is successfully cloned. Novel 22a-hydroxylation activity of CYP716A subfamily enzymes is reported. The chemical structure of 22a-hydroxy-a-amyrin is confirmed by NMR analysis.Triterpenoids represent one of the largest groups of plant specialized metabolites and are composed of six C5 (isopentenyl diphosphate) units [7]. Their carbon skeletons are synthesized from the common precursor 2,3-oxidosqualene by oxidosqualene cyclases (OSCs), and these skeletons are further oxidized by cytochrome P450 monooxygenases (P450s) and glycosylated by UDP-dependent glycosyltransferases (UGTs), which generate diverse triterpenoid/triterpenoid saponin structures [17,19]. Around 80 OSCs, 30 P450s, and 10UGTs are reportedly involved in triterpenoid biosynthesis in plants [22,25]. Among them, P450s have been described as the key players in diversifying triterpenoid scaffolds, and were recently identified in many triterpenoid-producing plant species [22]. For example, CYP88D6 and CYP72A154 from Glycyrrhiza uralensis were identified as components in the biosynthesis of glycyrrhetinic acid, the aglycone of glycyrrhizin [20,21]. In addition, CYP716A12 and other CYP716A subfamily enzymes have been identified as multifunc-

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“…dendroidea [38] using the nucleotide sequence of the characterized A . annua cycloartenol synthase ( CAS ) (GenBank accession KM670093 [39]) as query. The domain composition of the sequence coding for the OSC from L .…”

Section: Methodsmentioning

confidence: 99%

Cloning and Functional Characterization of Cycloartenol Synthase from the Red Seaweed Laurencia dendroidea

et al. 2016

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The red seaweed Laurencia dendroidea belongs to the Rhodophyta, a phylum of eukaryotic algae that is widely distributed across the oceans and that constitute an important source of bioactive specialized metabolites. Laurencia species have been studied since 1950 and were found to contain a plethora of specialized metabolites, mainly halogenated sesquiterpenes, diterpenes and triterpenes that possess a broad spectrum of pharmacological and ecological activities. The first committed step in the biosynthesis of triterpenes is the cyclization of 2,3-oxidosqualene, an enzymatic reaction carried out by oxidosqualene cyclases (OSCs), giving rise to a broad range of different compounds, such as the sterol precursors cycloartenol and lanosterol, or triterpene precursors such as cucurbitadienol and β-amyrin. Here, we cloned and characterized the first OSC from a red seaweed. The OSC gene was identified through mining of a L. dendroidea transcriptome dataset and subsequently cloned and heterologously expressed in yeast for functional characterization, which indicated that the corresponding enzyme cyclizes 2,3-oxidosqualene to the sterol precursor cycloartenol. Accordingly, the gene was named L. dendroidea cycloartenol synthase (LdCAS). A phylogenetic analysis using OSCs genes from plants, fungi and algae revealed that LdCAS grouped together with OSCs from other red algae, suggesting that cycloartenol could be the common product of the OSC in red seaweeds. Furthermore, profiling of L. dendroidea revealed cholesterol as the major sterol accumulating in this species, implicating red seaweeds contain a ‘hybrid’ sterol synthesis pathway in which the phytosterol precursor cycloartenol is converted into the major animal sterol cholesterol.

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OSC2 and CYP716A14v2 Catalyze the Biosynthesis of Triterpenoids for the Cuticle of Aerial Organs of Artemisia annua

Cited by 97 publications

References 52 publications

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

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

Novel triterpene oxidizing activity of Arabidopsis thaliana CYP716A subfamily enzymes

Cloning and Functional Characterization of Cycloartenol Synthase from the Red Seaweed Laurencia dendroidea

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