Discovery and Characterization of a <i>cis</i> -Casbene Diterpene Synthase from <i>Streptomyces paromomycinus</i>

Casbene-type diterpenoids are important bioactive natural products, but all known examples possess exclusively <i>trans</i>-configured double bonds. Here, we report the discovery of <i>Sp</i>TS1 from <i>Streptomyces paromomycinus</i>, a diterpene synthase that produces the first naturally occurring <i>cis</i>-casbene. Through heterologous expression and <i>in vitro</i> characterization, we isolated four novel <i>cis</i>-diterpenes from the substrate nerylneryl diphosphate (NNPP): (1<i>S</i>,2<i>Z</i>,6<i>Z</i>,10<i>Z</i>,14<i>S</i>)-casbene (<b>1</b>) featuring the characteristic bicyclo[12.1.0]pentadecane scaffold, 14-membered macrocyclic diterpene <b>2</b>, and two monocyclic diterpenes <b>3</b> and <b>4</b>. The absolute configurations of <b>1</b> and <b>2</b> were established through ECD spectroscopy and quantum calculations. Notably, <i>Sp</i>TS1 also accepts <i>trans</i>-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.