CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts
CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts
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CYP76AH1 催化丹参酮生物合成中 Miltiradiene 的周转，并能够在酵母中异源生产铁甘油
DOI：
10.1073/pnas.1218061110
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发表时间：
2013-07-16
影响因子：
11.1
通讯作者：
Huang, Luqi
中科院分区：
文献类型：
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作者：
Guo, Juan;Zhou, Yongjin J.;Huang, Luqi
细胞色素 P450 酶 (CYP) 在生成高功能化萜类化合物方面发挥着重要作用，但确定植物 CYP 在萜类化合物生物合成中催化的确切生物转化步骤极具挑战性。丹参酮是松香烷型去甲二萜萘醌，是中药丹参（丹参）的主要亲脂性生物活性成分。尽管最近已经描述了负责将(E,E,E)-香叶基香叶基二磷酸转化为松香烷miltiradiene（丹参酮的潜在前体）的二萜合酶，但miltiradiene进一步转化的分子表征仍然不可用。在这里，我们报告了稳定同位素标记结果，证明了 Miltiradiene 在丹参酮生物合成中的中介作用。我们进一步使用下一代测序方法来鉴定与根茎和丹参毛状根中的二萜合酶基因共同调控的六个候选CYP基因，并证明其中之一CYP76AH1在miltiradiene上催化独特的四电子氧化级联，在体外和体内产生铁鲁醇。然后，我们以之前在酿酒酵母中建立的 Miltiradiene 生产为基础，掺入 CYP76AH1 和植物-CYP 还原酶基因，从而异源生产 10.5 mg/L 的铁甘油。由于铁甘油醇已在包括丹参在内的许多植物中被发现，因此本文描述的结果和方法为进一步阐明丹参酮和相关二萜类化合物的生物合成奠定了坚实的基础。此外，这些结果应该有助于建造用于生产植物萜类化合物的微生物细胞工厂。
Cytochrome P450 enzymes (CYPs) play major roles in generating highly functionalized terpenoids, but identifying the exact biotransformation step(s) catalyzed by plant CYP in terpenoid biosynthesis is extremely challenging. Tanshinones are abietane-type norditerpenoid naphthoquinones that are the main lipophilic bioactive components of the Chinese medicinal herb danshen (Salvia miltiorrhiza). Whereas the diterpene synthases responsible for the conversion of (E,E,E)-geranylgeranyl diphosphate into the abietane miltiradiene, a potential precursor to tanshinones, have been recently described, molecular characterization of further transformation of miltiradiene remains unavailable. Here we report stable-isotope labeling results that demonstrate the intermediacy of miltiradiene in tanshinone biosynthesis. We further use a next-generation sequencing approach to identify six candidate CYP genes being coregulated with the diterpene synthase genes in both the rhizome and danshen hairy roots, and demonstrate that one of these, CYP76AH1, catalyzes a unique four-electron oxidation cascade on miltiradiene to produce ferruginol both in vitro and in vivo. We then build upon the previous establishment of miltiradiene production in Saccharomyces cerevisiae, with incorporation of CYP76AH1 and phyto-CYP reductase genes leading to heterologous production of ferruginol at 10.5 mg/L. As ferruginol has been found in many plants including danshen, the results and the approaches that were described here provide a solid foundation to further elucidate the biosynthesis of tanshinones and related diterpenoids. Moreover, these results should facilitate the construction of microbial cell factories for the production of phytoterpenoids.