Luo Fei, Zhou Qian, Chen Feilong, Liu Xinyu, Chiu Tsan-Yu, Zhu Guo-Yuan, Huang Ancheng C
Shenzhen Key Laboratory of Plant Genetic Engineering and Molecular Design, SUSTech-PKU Institute of Plant and Food Science, Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, Guangdong, China.
BGI Research, Shenzhen, China.
Nat Commun. 2025 Jul 1;16(1):5857. doi: 10.1038/s41467-025-61188-0.
Diterpenoids exhibit remarkable structural diversity and bioactivities, shaped primarily by the tandem actions of skeleton-forming terpene synthases (TPSs) and cytochrome P450 monooxygenases. The ent-kaurene and ent-atiserene are labdane-derived diterpene scaffolds for the biosynthesis of diverse bioactive diterpenoids and diterpene alkaloids, including the clinically used analgesic 3-acetylaconitine and anti-arrhythmic guan-fu base A in Aconitum spp., yet what and how P450s drive their structural and functional diversification remain largely unexplored. Here, via mining the transcriptomes of Aconitum carmichaelii and Aconitum coreanum followed by functional validation, we discover nine TPSs capable of furnishing the ent-kaurene, ent-atiserene or 16α-hydroxy-ent-kaurene scaffold and 14 divergent P450s, of which eight are multifunctional, catalyzing oxidation at seven different sites of the scaffolds. Protein analysis and mutagenesis experiments reveal key residues tuning the P450 activity and product profiles, shedding light on how they diverge functionally. The discovered TPSs and P450s enable combinatorial biosynthesis of tripterifordin, guan-fu diterpenoid A, and 14 novel atiserenoids, with some exhibiting allelopathic activity. Intriguingly, Tripterygium wilfordii-derived tripterifordin was detected in A. coreanum, revealing convergent biosynthesis of tripterifordin. Our findings highlight P450 plasticity in selective diterpene oxidation and allow for accessing tripterifordin and derivatives, paving ways for elucidating downstream diterpenoid pathways via synthetic biology.
二萜类化合物具有显著的结构多样性和生物活性，其形成主要受骨架形成萜烯合酶（TPSs）和细胞色素P450单加氧酶的协同作用影响。对映贝壳杉烯和对映阿替生烯是半日花烷型二萜骨架，用于多种生物活性二萜类化合物和二萜生物碱的生物合成，包括临床使用的镇痛药3-乙酰乌头碱和乌头属植物中的抗心律失常药关附甲素，然而P450s如何驱动它们的结构和功能多样化在很大程度上仍未得到探索。在这里，通过挖掘乌头和朝鲜乌头的转录组，随后进行功能验证，我们发现了9种能够提供对映贝壳杉烯、对映阿替生烯或16α-羟基对映贝壳杉烯骨架的TPSs和14种不同的P450s，其中8种是多功能的，可催化骨架7个不同位点的氧化。蛋白质分析和诱变实验揭示了调节P450活性和产物谱的关键残基，阐明了它们在功能上的差异。所发现的TPSs和P450s能够实现雷公藤红素、关附二萜A和14种新型阿替生烯类化合物的组合生物合成，其中一些具有化感活性。有趣的是，在朝鲜乌头中检测到了来源于雷公藤的雷公藤红素，揭示了雷公藤红素的趋同生物合成。我们的研究结果突出了P450在选择性二萜氧化中的可塑性，并为获得雷公藤红素及其衍生物提供了可能，为通过合成生物学阐明下游二萜类途径铺平了道路。
