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, 19 (1), 114

Functional Characterization of the Cytochrome P450 Monooxygenase CYP71AU87 Indicates a Role in Marrubiin Biosynthesis in the Medicinal Plant Marrubium Vulgare

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Functional Characterization of the Cytochrome P450 Monooxygenase CYP71AU87 Indicates a Role in Marrubiin Biosynthesis in the Medicinal Plant Marrubium Vulgare

Prema S Karunanithi et al. BMC Plant Biol.

Abstract

Background: Horehound (Marrubium vulgare) is a medicinal plant whose signature bioactive compounds, marrubiin and related furanoid diterpenoid lactones, have potential applications for the treatment of cardiovascular diseases and type II diabetes. Lack of scalable plant cultivation and the complex metabolite profile of M. vulgare limit access to marrubiin via extraction from plant biomass. Knowledge of the marrubiin-biosynthetic enzymes can enable the development of metabolic engineering platforms for marrubiin production. We previously identified two diterpene synthases, MvCPS1 and MvELS, that act sequentially to form 9,13-epoxy-labd-14-ene. Conversion of 9,13-epoxy-labd-14-ene by cytochrome P450 monooxygenase (P450) enzymes can be hypothesized to facilitate key functional modification reactions in the formation of marrubiin and related compounds.

Results: Mining a M. vulgare leaf transcriptome database identified 95 full-length P450 candidates. Cloning and functional analysis of select P450 candidates showing high transcript abundance revealed a member of the CYP71 family, CYP71AU87, that catalyzed the hydroxylation of 9,13-epoxy-labd-14-ene to yield two isomeric products, 9,13-epoxy labd-14-ene-18-ol and 9,13-epoxy labd-14-ene-19-ol, as verified by GC-MS and NMR analysis. Additional transient Nicotiana benthamiana co-expression assays of CYP71AU87 with different diterpene synthase pairs suggested that CYP71AU87 is specific to the sequential MvCPS1 and MvELS product 9,13-epoxy-labd-14-ene. Although the P450 products were not detectable in planta, high levels of CYP71AU87 gene expression in marrubiin-accumulating tissues supported a role in the formation of marrubiin and related diterpenoids in M. vulgare.

Conclusions: In a sequential reaction with the diterpene synthase pair MvCPS1 and MvELS, CYP71AU87 forms the isomeric products 9,13-epoxy labd-14-ene-18/19-ol as probable intermediates in marrubiin biosynthesis. Although its metabolic relevance in planta will necessitate further genetic studies, identification of the CYP71AU87 catalytic activity expands our knowledge of the functional landscape of plant P450 enzymes involved in specialized diterpenoid metabolism and can provide a resource for the formulation of marrubiin and related bioactive natural products.

Keywords: Cytochrome P450 monooxygenase; Diterpene synthase; Diterpenoid biosynthesis; Marrubiin; Marrubium vulgare; Plant natural products.

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Figures

Fig. 1
Fig. 1
Proposed marrubiin-biosynthetic pathway. MvCPS1 and MvELS form a diterpene synthase pair that converts the central precursor geranylgeranyl diphosphate (GGPP) into 9,13-epoxy labd-14-ene 1 (and possibly additional closely related products including labda-13(16),14-dien-9-ol 2) via the prenyl diphosphate intermediate peregrinol diphosphate. Cytochrome P450 monooxygenases are hypothesized to catalyze position-specific functional modifications to yield premarrubiin possibly via the CYP71AU87 product 9,13-epoxy labd-14-en-19-ol 5 and other pathway intermediates such as premarrubenol previously identified in M. vulgare tissues. Subsequent lactone formation would form premarrubiin, from which marrubiin will derive through enzymatic or spontaneous ring opening to yield the free hydroxyl group at C-9. Dashed lines represent enzyme products identified in co-expression assays of MvCPS1 and MvELS in this study
Fig. 2
Fig. 2
Identification of P450 candidates in the Marrubium vulgare leaf transcriptome. a Maximum-likelihood phylogenetic tree of P450 candidates (Additional file 2: Table S1) identified in the M. vulgare leaf transcriptome [23]. Bars illustrate transcript abundance of full-length P450 candidates as based on calculated FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values, and color-coded to represent different CYP families. b Maximum-likelihood phylogenetic tree of select M. vulgare CYP71 family candidates as compared to functionally characterized CYP71 enzymes. A CYP71 family member of the ancestral dicot Amborella trichopoda was used to root the tree. Black dots highlight bootstrap support of ≥75% (1000 repetitions). Mv, Marrubium vulgare; Tp, Tanacetum parthenium; Gm, Glycine max; Pa, Persea americana; At, Arabidopsis thaliana; Cr, Catharanthus roseus; Am, Ammi majus; Mxp, Mentha x piperita, Atr, Amborella trichopoda
Fig. 3
Fig. 3
Analysis of reaction products of the pairwise activity of MvCPS1 and MvELS. a Total ion chromatogram of reactions products derived from co-expression assays of MvCPS1 and MvELS in N. benthamiana. Compound 1, 9,13-epoxy labd-14-ene; compound 2 [16], labda-13(16),14-dien-9-ol (as verified by NMR analysis); compound 3, unidentified diterpenoid closely related to 9,13-epoxy labd-14-ene. b Mass spectra of compounds 1, 2, and 3. c Structure of labda-13(16),14-dien-9-ol as verified by NMR analysis (see Additional file 3: Figure S1 for details)
Fig. 4
Fig. 4
Functional characterization of CYP71AU87. a Extracted ion chromatograms (m/z 151) of enzyme products resulting from Agrobacterium-mediated transient co-expression of MvCPS1, MvELS, and CYP71AU87 in N. benthamiana. Compound 1, 9,13-epoxy labd-14-ene; compound 2 [16], labda-13(16),14-dien-9-ol; compound 3, unidentified diterpenoid closely related to 9,13-epoxy labd-14-ene; compound 4, 9,13-epoxy labd-14-ene-18-ol (as verified by NMR analysis); compound 5, 9,13-epoxy labd-14-ene-19-ol (as verified by NMR analysis). b GC-MS total ion chromatogram (TIC) of the purified CYP71AU87 products 4 and 5 obtained via metabolite extraction from ~ 50 N. benthamiana leaves transfected with MvCPS1, MvELS and CYP71AU87. c GC-MS-based quantification of 9,13-epoxy labd-14-ene 1 and 9,13-epoxy labd-14-ene-18/19-ol 4/5 (combined amounts) extracted from single-leaves of N. benthamiana co-transformed with MvCPS1, MvELS and CYP71AU87. Co-expression assays performed in triplicate with error bars denoting one standard deviation and * denoting a P-value < 0.05 using Tukey’s test. d GC-MS mass spectra and structures of 9,13-epoxy labd-14-ene-18-ol and 9,13-epoxy labd-14-ene-19-ol as based on NMR analyses (see Additional file 5: Figure S3 for details on NMR analyses)
Fig. 5
Fig. 5
Substrate-specificity of CYP71AU87. Shown are total ion chromatograms (TIC) and extracted ion chromatograms (EIC, individual mass ions) of reaction products resulting from transient N. benthamiana co-expression assays of CYP71AU87 with different class II and class diTPSs that produce distinct diterpenoid scaffolds. Tested products included the gibberellin precursor ent-kaurene 9 formed by the maize (Zea mays) ent-CPP synthase ZmAN2 and the ent-kaurene synthase GrEKS from Grindelia robusta [45], manoyl oxide 10 produced by the LPP synthase GrLPPS from G. robusta [23] and MvELS, and miltiradiene 11 formed by the (+)-CPP synthase IrTPS3 from Isodon rubescens [44] and MvELS. Co-expression of MvCPS1, MvELS and CYP71AU87 was used as a positive control. Mass spectra and structures of the formed diterpenoids are given in Additional file 7: Figure S5
Fig. 6
Fig. 6
Analysis of MvELS and CYP71AU87 products in planta. Extracted ion chromatograms (m/z 151) resulting from GC-MS analysis of metabolite extracts from M. vulgare leaf and flower tissues as compared to purified standards of 9,13-epoxy labd-14-ene 1, labda-13(16),14-dien-9-ol 2, 9,13-epoxy labd-14-ene-18-ol 4, and 9,13-epoxy labd-14-ene-19-ol 5, and marrubiin. Diterpenoids were extracted from 12-week-old plants using hexane and detected using electron ionization (EI) GC-MS analysis
Fig. 7
Fig. 7
Relative Transcript abundance of CYP71AU87 in M. vulgare tissues. Transcript abundance was measured by qPCR using gene-specific oligonucleotides (Additional file 9: Table S2) and normalization to the Elongation Factor 1α (EF1α) from M. vulgare as a reference. Error bars represent standard errors based on triplicate measurements of three biological replicates. Gene expression was calculated on the basis of efficiency-corrected oligonucleotides, and the reaction specificity was verified by dissociation curves and sequence verification of representative products

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