Discovery and Functional Characterization of a Diverse Diterpene Synthase Family in the Medicinal Herb Isodon lophanthoides Var. gerardiana
Discovery and Functional Characterization of a Diverse Diterpene Synthase Family in the Medicinal Herb Isodon lophanthoides Var. gerardiana
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药用植物 Isodon lophanthoides var. 中多种二萜合酶家族的发现和功能表征
DOI：
10.1093/pcp/pcab089
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发表时间：
2021-06-16
影响因子：
4.9
通讯作者：
Huang, Lili
中科院分区：
文献类型：
--
作者：
Yang, Ruikang;Du, Zuying;Huang, Lili
细花香茶菜老鹳草（gerardiana），又名西黄草（xixhuangcao），为唇形科（Lamiaceae）植物，是一种具有广泛药理活性的传统中草药。冷杉烷型二萜类化合物是其特征成分。lophanthoides，但其生物合成尚未阐明。虽然地上部分是I.通过气相色谱-质谱法分析Lophanthoides的代谢物，显示地下部分也含有大量的唇形科二萜类化合物，包括松香三烯、米替拉烯和ferruginol，其不同于在地上部分中检测到的13-羟基-8（14）-松香烯。根和叶样品的比较转录组分析鉴定了一个多样的二萜合酶家族，包括6个柯巴基二磷酸合酶（IlCPS 1 -6）和5个贝壳杉烯脱氢酶样（IlKSL 1 -5）。在这里，我们报告的功能特性，这些酶使用酵母异源表达系统。IlCPS 1和IlCPS 3都合成了（+）-柯巴基二磷酸（CPP），与IlKSL 1结合产生了米替雷烯，松香烷型二萜类化合物的前体，而与IlKSL 5偶联导致形成羟基化的二萜支架奈珠醇。表达谱分析和系统发育分析进一步支持IlCPS 1和IlCPS 3的不同进化关系和空间分布。IlCPS 2将GGPP转化为labda-7，13 E-二烯-15-醇二磷酸酯。IlCPS 6被鉴定为ent-CPS，表明在赤霉素代谢中的作用。我们进一步鉴定了确定奈珠酚合酶IlKSL 5的水添加的单个残基。用异亮氨酸取代丙氨酸513完全改变了产物结果，从羟基化nezukol变为异海松酸7，15-二烯。总之，这些发现阐明了具有生物活性的冷杉烷型二萜类化合物生物合成的早期步骤在I。和nezukol合成酶的催化机制。
Isodon lophanthoides var. gerardiana (Lamiaceae), also named xihuangcao, is a traditional Chinese medicinal herb that exhibits a broad range of pharmacological activities. Abietane-type diterpenoids are the characteristic constituents of I. lophanthoides, yet their biosynthesis has not been elucidated. Although the aerial parts are the most commonly used organs of I. lophanthoides, metabolite profiling by gas chromatography-mass spectrometry showed the underground parts also contain large amounts of labdane diterpenoids including abietatriene, miltiradiene and ferruginol, which are distinct from the 13-hydroxy-8(14)-abietene detected in the aerial parts. Comparative transcriptome analysis of root and leaf samples identified a diverse diterpene synthase family including 6 copalyl diphosphate synthase (IlCPS1-6) and 5 kaurene synthase-like (IlKSL1-5). Here we report the functional characterization of six of these enzymes using yeast heterologous expression system. Both IlCPS1 and IlCPS3 synthesized (+)-copalyl diphosphate (CPP), in combination with IlKSL1 resulted in miltiradiene, precursor of abietane-type diterpenoids, while coupling with IlKSL5 led to the formation of hydroxylated diterpene scaffold nezukol. Expression profiling and phylogenetic analysis further support the distinct evolutionary relationship and spatial distribution of IlCPS1 and IlCPS3. IlCPS2 converted GGPP into labda-7,13E-dien-15-ol diphosphate. IlCPS6 was identified as ent-CPS, indicating a role in gibberellin metabolism. We further identified a single residue that determined the water addition of nezukol synthase IlKSL5. Substitution of alanine 513 with isoleucine completely altered the product outcome from hydroxylated nezukol to isopimara7,15-diene. Together, these findings elucidated the early steps of bioactive abietane-type diterpenoid biosynthesis in I. lophanthoides and the catalytic mechanism of nezukol synthase.