Eunicellin-based compounds are characterized by a distinctive 6,10-bicyclic carbon skeleton and exhibit a range of intriguing bioactivities, including cytotoxicity, antiinflammation, antibacterial, or antifouling properties . Take a notable example: eleutherobin , discovered from marine corals, demonstrated significant potency comparable to paclitaxel in inducing tubulin polymerization and has been subjected to clinical research (Supplementary Figure ). However, corals contain a low abundance of eleutherobin and cannot be over-harvested because of their critical effect on ecology and climate , a common drawback in downstream pharmaceutical studies among natural products. Chemical synthesis has also been explored as an alternative. Nonetheless, the efficiency and yield of these synthetic routes were unfavorable . Biosynthesis might be an excellent option for circumventing this obstacle. Nevertheless, little is known about the biosynthesis of eunicellane diterpenoids until several eunicellane terpene synthases are discovered . The identification of Bnd4-the first eunicellane synthase found in nature, cloned from a soil-derived bacteria , marked a significant advancement in the eunicellane biosynthesis research and refuted the previously hypothesized cembranederived biogenesis. Subsequent AlbS, EcTPS1, along with BaTC-2, synthesizing different types of eunicellane skeletons, were then soon reported , further enriching the mechanisms of eunicellane biosynthesis (Fig. ). But all these reported eunicellane synthases share a common yet-to-be-solved issue: the biosynthesized eunicellane scaffolds exhibit flexibility in different conformational ratios , dramatically impeding NMR-based structural elucidations and bringing great challenges for chemical synthesis of eunicellane-type compounds. To address this issue and interrogate the fascinating mechanisms involved in governing conformational changes, we sought to identify a terpene synthase responsible for synthesizing a non-flexible eunicellane scaffold. To our surprise, microeunicellols A and B , two bacteria-derived eunicellanes, exhibit non-flexibility even without the presence of ether bridge bonds in the ten-membered ring, a feature commonly observed in coral-derived eunicellanes that might reduce flexibility via bridging. This observation suggests that the intrinsic eunicellane scaffold of microeunicellols might be non-flexible. In this study, the discovery and characterization of the first nonflexible eunicellane synthesizing terpene synthase-MicA generating the eunicellane carbon scaffold of microeunicellols is presented. Through elaborate investigations of the cryptic eunicellane synthase via isotope labeling, density functional theory (DFT) calculation, enzyme structural analysis, and substrate analogs synthesis, we reasonably postulated that the structural flexibility of eunicellane scaffolds is primarily determined by the combined stereo characteristics of the bridging carbons and the adjacent double bond.