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Biosynthesis of the oxygenated diterpene nezukol in the medicinal plant Isodon rubescens is catalyzed by a pair of diterpene synthases.

ABSTRACT: Plants produce an immense diversity of natural products (i.e. secondary or specialized metabolites) that offer a rich source of known and potentially new pharmaceuticals and other desirable bioproducts. The Traditional Chinese Medicinal plant Isodon rubescens (Lamiaceae) contains an array of bioactive labdane-related diterpenoid natural products. Of these, the ent-kauranoid oridonin is the most prominent specialized metabolite that has been extensively studied for its potent antimicrobial and anticancer efficacy. Mining of a previously established transcriptome of I. rubescens leaf tissue identified seven diterpene synthase (diTPSs) candidates. Here we report the functional characterization of four I. rubescens diTPSs. IrTPS5 and IrTPS3 were identified as an ent-copalyl diphosphate (CPP) synthase and a (+)-CPP synthase, respectively. Distinct transcript abundance of IrTPS5 and the predicted ent-CPP synthase IrTPS1 suggested a role of IrTPS5 in specialized ent-kaurene metabolism possibly en route to oridonin. Nicotiana benthamiana co-expression assays demonstrated that IrTPS4 functions sequentially with IrTPS3 to form miltiradiene. In addition, IrTPS2 converted the IrTPS3 product (+)-CPP into the hydroxylated tricyclic diterpene nezukol not previously identified in I. rubescens. Metabolite profiling verified the presence of nezukol in I. rubescens leaf tissue. The proposed IrTPS2-catalyzed reaction mechanism proceeds via the common ionization of the diphosphate group of (+)-CPP, followed by formation of an intermediary pimar-15-en-8-yl+ carbocation and neutralization of the carbocation by water capture at C-8 to yield nezukol, as confirmed by nuclear magnetic resonance (NMR) analysis. Oxygenation activity is rare for the family of class I diTPSs and offers new catalysts for developing metabolic engineering platforms to produce a broader spectrum of bioactive diterpenoid natural products.

SUBMITTER: Pelot KA

PROVIDER: S-EPMC5405970 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Biosynthesis of the oxygenated diterpene nezukol in the medicinal plant Isodon rubescens is catalyzed by a pair of diterpene synthases.

Pelot Kyle A KA Hagelthorn Lynne M LM Addison J Bennett JB Zerbe Philipp P

PloS one 20170426 4

Plants produce an immense diversity of natural products (i.e. secondary or specialized metabolites) that offer a rich source of known and potentially new pharmaceuticals and other desirable bioproducts. The Traditional Chinese Medicinal plant Isodon rubescens (Lamiaceae) contains an array of bioactive labdane-related diterpenoid natural products. Of these, the ent-kauranoid oridonin is the most prominent specialized metabolite that has been extensively studied for its potent antimicrobial and an ...[more]

PMID: 28445526

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Project description:Isodon rubescens (Hemsley) H. Hara (Lamiaceae) is a traditional Chinese medicine plant that has been used to treat various human diseases and conditions such as inflammation, respiratory and gastrointestinal bacterial infections, and malignant tumors. However, the contents of the main active components of I. rubescens from different origins differ significantly, which greatly affected its quality. Therefore, a molecular method to identify and classify I. rubescens is needed. Here, we report the DNA sequence of the chloroplast genome of I. rubescens collected from Lushan, Henan province. The genome is 152,642 bp in length and has a conserved structure that includes a pair of IR regions (25,726 bp), a LSC region (83,527 bp) and a SSC region (17,663 bp). The chloroplast genome contains 113 unique genes, four rRNA genes, 30 tRNA genes, and 79 protein-coding genes, 23 of which contain introns. The protein-coding genes account for a total of 24,412 codons, and most of them are A/T biased usage. We identified 32 simple sequence repeats (SSRs) and 48 long repeats. Furthermore, we developed valuable chloroplast molecular resources by comparing chloroplast genomes from three Isodon species, and both mVISTA and DnaSP analyses showed that rps16-trnQ, trnS-trnG, and ndhC-trnM are candidate regions that will allow the identification of intraspecific differences within I. rubescens. Also 14 candidate fragments can be used to identify interspecific differences between species in Isodon. A phylogenetic analysis of the complete chloroplast genomes of 24 species in subfamily Nepetoideae was performed using the maximum likelihood method, and shows that I. rubescens clustered closer to I. serra than I. lophanthoides. Interestingly, our analysis showed that I. rubescens (MW018469.1) from Xianyang, Shaanxi Province (IR-X), is closer to I. serra than to the other two I. rubescens accessions. These results strongly indicate that intraspecific diversity is present in I. rubescens. Therefore, our results provide further insight into the phylogenetic relationships and interspecific diversity of species in the genus Isodon.

Dataset Information

Biosynthesis of the oxygenated diterpene nezukol in the medicinal plant Isodon rubescens is catalyzed by a pair of diterpene synthases.

Publications

Biosynthesis of the oxygenated diterpene nezukol in the medicinal plant Isodon rubescens is catalyzed by a pair of diterpene synthases.

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