Unveiling the Biosynthesis of Sphaerols A–E: Catalyzed by Two Uncommon Diterpene Synthases AsSS and CvSS

Diterpene synthase plays an important role in the biosynthesis of novel diterpenoids with diverse biological activities. In this study, two uncommon diterpene synthases, AsSS and CvSS, were discovered in bacteria, showing only a 26% sequence identity. Unexpectedly, sphaerols A (1) and B (2) with an interesting rearrangement scaffold, a pair of diastereoisomers with different hydroxyl configurations at C-7, were synthesized. Their structures were elucidated using spectroscopic data and quantum chemical computational methods. The proposed cyclization mechanism of these products was validated through isotopic labeling experiments and density functional theory calculations. Through amino acid residue swapping, it was revealed that AsSS-M81 and CvSS-C82 play crucial roles in determining the stereoselective hydroxylation at C-7. Furthermore, functional interconversion between AsSS and CvSS has been successfully achieved.