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.

Diterpene Synthase‐Catalyzed Biosynthesis of Distinct Clerodane Stereoisomers

“…The enzyme activity of the generated protein variants was analyzed in co‐expression assays in an Escherichia coli co‐expression system engineered for enhanced diterpenoid production . The resulting reaction products were readily extracted from the microbial co‐expression cultures in their dephosphorylated form and identified by gas chromatography–mass spectrometry (GC‐MS) through comparison to known enzyme products and, where applicable, by using NMR analysis . Mutation of PvCPS1:F251 to histidine, tyrosine, or leucine—residues of similar size—did not alter product specificity, and the protein variants largely retained wild‐type overall catalytic activity (Figure S1).…”

“…GC‐MS analysis : Enzyme products formed by microbial co‐expression were extracted directly from the culture with hexane (50 mL), air‐dried and resuspended in hexane (1 mL) for GC‐MS analysis . Extracted products were analyzed on an Agilent 7890B GC interfaced with a 5977 Extractor XL MS detector (70 eV, He flow 1.2 mL min −1 ) by using an Agilent HP5‐ms column (30 m, 250 μm i.d., 0.25 μm film).…”

The foxtail millet (Setaria italica) terpene synthase gene family

“…Monocot diterpenoids are almost invariably functionally modified by P450 enzymes, with members of the CYP701, CYP71, CYP76 and CYP99 subfamilies having been shown (Harris et al, 2005) or the (+)-CPP synthase, IrTPS3, from Isodon rubescens (Pelot et al, 2017). (c) The GC-MS traces of products resulting from Escherichia coli co-expression assays of SiTPS28 and SiTPS8 with E,E-farnesyl pyrophosphate (FPP) produced by the maize FPP synthase ZmFPPS (Cervantes-Cervantes et al, 2006).…”

Comparative transcriptome analysis of roots, stems and leaves of Isodon amethystoides reveals candidate genes involved in Wangzaozins biosynthesis

“…For Isodon species, Li et al [ 18 ] reported the role of two IeCPS genes in ent- copalyl diphosphate synthesis in I. eriocalyx via homologous cloning. Very recently, the diterpene synthases (diTPSs) including CPS and KSL have been identified in I. rubescens [ 19 , 20 ].…”