Lu Yun, Liu Yuan, Zhang Yifeng, Gao Haiyun, Chen Xiaochao, Tu Lichan, Luo Yunfeng, Jiang Zhouqian, Yin Yan, Zhou Jiawei, Hu Tianyuan, Wu Xiaoyi, Wang Jiadian, Gao Wei, Huang Luqi
School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China.
Chengdu Second People's Hospital, Chengdu 610017, China.
J Nat Prod. 2024 Feb 23;87(2):176-185. doi: 10.1021/acs.jnatprod.3c00674. Epub 2024 Jan 26.
Celastrol is a bioactive pentacyclic triterpenoid with promising therapeutic effects that is mainly distributed in Celastraceae plants. Although some enzymes involved in the celastrol biosynthesis pathway have been reported, many biosynthetic steps remain unknown. Herein, transcriptomics and metabolic profiles of multiple species in Celastraceae were integrated to screen for cytochrome P450s (CYPs) that are closely related to celastrol biosynthesis. The CYP716 enzyme, TwCYP716C52, was found to be able to oxidize the C-2 position of polpunonic acid, a precursor of celastrol, to form the wilforic acid C. RNAi-mediated repression of in suspension cells further confirmed its involvement in celastrol biosynthesis. The C-2 catalytic mechanisms of TwCYP716C52 were further explored by using molecular docking and site-directed mutagenesis experiments. Moreover, a modular optimization strategy was used to construct an engineered yeast to produce wilforic acid C at a titer of 5.8 mg·L. This study elucidates the celastrol biosynthetic pathway and provides important functional genes and sufficient precursors for further enzyme discovery.
雷公藤红素是一种具有潜在治疗作用的生物活性五环三萜类化合物，主要分布在卫矛科植物中。尽管已经报道了一些参与雷公藤红素生物合成途径的酶，但许多生物合成步骤仍然未知。在此，整合了卫矛科多个物种的转录组学和代谢谱，以筛选与雷公藤红素生物合成密切相关的细胞色素P450（CYP）。发现CYP716酶TwCYP716C52能够氧化雷公藤红素前体polpunonic酸的C-2位，形成wilforic酸C。RNAi介导的悬浮细胞中TwCYP716C52的抑制进一步证实了其参与雷公藤红素的生物合成。通过分子对接和定点诱变实验进一步探索了TwCYP716C52的C-2催化机制。此外，采用模块化优化策略构建了一种工程酵母，以5.8 mg·L的产量生产wilforic酸C。本研究阐明了雷公藤红素的生物合成途径，并为进一步发现酶提供了重要的功能基因和充足的前体。
