Discovery, Mechanism, and Bioproduction Studies of a Moss-Derived Microbial-like Terpene Synthase CrMTPSL3 Generating the Tricyclic Sphaeroane Scaffold

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.