Xin Li, Yuanning Liu, Jiaxing Chen, Chen Wang, Bao Chen, Lijun Liu, Xuemeng Liu, Xiaochen Chen, Yao Zhao, Ting He, Fangyan Chen, Zhengren Xu, Baofu Xu
The sphaeroane diterpenoids are natural products with a tricyclic scaffold, yet the biosynthetic mechanism remains unclear. We characterized a sphaeroane diterpene synthase, CrMTPSL3, from Claopodium rostratum . Chemical computation together with crystallization experiments determined the absolute stereochemistry of the products. Further deuterium-scanning experiments supported CrMTPSL3 forms the sphaeroane scaffold sequentially as GGPP isomerization, 1,11- and 10,14-cyclizations, two successive 1,2-hydride shifts, 1,2-methyl shift, 1,2-hydride transfer of the pro -S hydrogen, 1,6-cyclization, and deprotonation of the pro -R hydrogen cascade. The structural basis was also investigated by a series of mutagenesis studies. An engineered cell factory for overproducing sphaeroane compounds at the hundred-milligram-per-liter level was achieved.