School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, PR China.
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, PR China.
Int J Biol Macromol. 2018 Dec;120(Pt A):203-212. doi: 10.1016/j.ijbiomac.2018.08.073. Epub 2018 Aug 17.
Squalene epoxidase, thought to be one of the rate-limiting enzymes in the biosynthetic pathways of both membrane sterols and triterpenes (e.g., celastrol), catalyses the formation of oxidosqualene as the common precursor of sterols and triterpenoids. In this work, we first found five squalene epoxidase genes (TwSEs) from Tripterygium wilfordii. Tissue expression pattern, consistent with methyl jasmonate induction study, showed that TwSEs1-4 were involved in the production of special metabolites. In contrast, TwSE5 showed a different tissue expression pattern and was not induced by methyl jasmonate. To probe the functions of the TwSEs, we first tried using a prokaryotic system by constructing an engineered bacterium, but we failed to detect their products. Next, we used the CRISPR/Cas9 tool to construct an erg1 mutant yeast by knocking out the ERG1 gene of yeast strain BY4741 and then applied this mutant to identify the function of TwSEs. We found that only TwSEs1-4 can functionally complement the erg1 mutant yeast. This study laid the foundation for the heterologous biosynthesis of special metabolites in Tripterygium wilfordii.
鲨烯环氧化酶被认为是膜甾醇和三萜（如雷公藤红素）生物合成途径中的限速酶之一，催化氧化鲨烯的形成，作为甾醇和三萜类化合物的共同前体。在这项工作中，我们首先从雷公藤中发现了五个鲨烯环氧化酶基因（TwSEs）。组织表达模式与茉莉酸甲酯诱导研究一致，表明 TwSEs1-4 参与了特殊代谢产物的产生。相比之下，TwSE5 表现出不同的组织表达模式，且不受茉莉酸甲酯诱导。为了探究 TwSEs 的功能，我们首先尝试使用原核系统通过构建工程菌来实现，但未能检测到它们的产物。接下来，我们使用 CRISPR/Cas9 工具敲除酵母菌株 BY4741 的 ERG1 基因，构建 erg1 突变酵母，并应用该突变酵母来鉴定 TwSEs 的功能。我们发现只有 TwSEs1-4 可以在功能上互补 erg1 突变酵母。这项研究为雷公藤特殊代谢物的异源生物合成奠定了基础。
