Discovery and Characterization of a cis-Casbene Diterpene Synthase from Streptomyces paromomycinus

Casbene-type diterpenoids are important bioactive natural products, but all known examples possess exclusively trans-configured double bonds. Here, we report the discovery of SpTS1 from Streptomyces paromomycinus, a diterpene synthase that produces the first naturally occurring cis-casbene. Through heterologous expression and in vitro characterization, we isolated four novel cis-diterpenes from the substrate nerylneryl diphosphate (NNPP): (1S,2Z,6Z,10Z,14S)-casbene (1) featuring the characteristic bicyclo[12.1.0]pentadecane scaffold, 14-membered macrocyclic diterpene 2, and two monocyclic diterpenes 3 and 4. The absolute configurations of 1 and 2 were established through ECD spectroscopy and quantum calculations. Notably, SpTS1 also accepts trans-substrate geranylgeranyl diphosphate (GGPP) to produce cembrene-type diterpenes, demonstrating unusual substrate promiscuity. This discovery not only fills an important gap in casbene natural product diversity but also reveals new possibilities for engineering stereochemically diverse terpenoids.