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

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.植物可合成种类极其丰富的天然产物（即次级代谢物或特化代谢物），这类产物是已知乃至潜在新型药物与其他高价值生物制品的宝贵来源。传统中药植物冬凌草（Isodon rubescens，唇形科Lamiaceae）含有多种具有生物活性的半日花烷类二萜天然产物。其中，对映-贝壳杉烷类化合物冬凌草甲素（oridonin）是最具代表性的特化代谢物，其强效抗菌与抗癌活性已被广泛研究。对已建立的冬凌草叶片组织转录组进行挖掘，共鉴定出7个二萜合酶（diterpene synthase, diTPS）候选基因。本研究对其中4个冬凌草二萜合酶进行了功能表征。研究分别鉴定出IrTPS5为对映-柯巴基焦磷酸（ent-copalyl diphosphate, CPP）合酶，IrTPS3为(+)-CPP合酶。IrTPS5与预测的对映-CPP合酶IrTPS1具有截然不同的转录本丰度，这提示IrTPS5可能参与特化的对映-贝壳杉烯代谢通路，进而参与冬凌草甲素的生物合成。本氏烟草（Nicotiana benthamiana）共表达实验证实，IrTPS4与IrTPS3可依次发挥催化功能，生成米替拉二烯（miltiradiene）。此外，IrTPS2可将IrTPS3的催化产物(+)-CPP转化为羟基化三环二萜类化合物涅祖醇（nezukol），该化合物此前尚未在冬凌草中被鉴定到。代谢组分析证实，冬凌草叶片组织中确实存在涅祖醇。经核磁共振（nuclear magnetic resonance, NMR）分析证实，IrTPS2催化的反应机制如下：首先(+)-CPP的焦磷酸基团发生常规电离，随后形成中间体海松-15-烯-8-正离子碳正离子，最终通过在C8位捕获水分子中和该碳正离子，从而生成涅祖醇。I类二萜合酶家族中具有氧化活性的成员较为罕见，该发现可为构建代谢工程平台以合成更多种类的生物活性二萜天然产物提供全新的催化工具。