Investigations of the effects of stereochemistry on flexibility among eunicellanes At last, we arrived at the central question: what makes the MicA-synthesized eunicellane scaffold non-flexible? We began by thoroughly examining all previously reported eunicellane scaffolds. We discovered that benditerpe-2,6,15-triene (6), albireticulene (7), klysimplexin R (8), and 1 displayed a range of flexibility, from flexible to less flexible, and finally to non-flexible. This pattern suggests that the combined stereochemistry likely governs the conformational changes. As such, a combined analysis of the four eunicellane synthases, Bnd4, AlbS, EcTPS1, and MicA, could help clarify the underlying mechanisms. To this end, we synthesized three additional genes, expressed them in E. coli strains using the same MKI4 system, and successfully isolated their respective cyclization products. The NMR spectra of these eunicellane products revealed varying degrees of broadening (Fig. and Supplementary Figure ). We then meticulously analyzed the resulting structures, including the epoxidized analogs (1a-1d). The distinction between 7 and 1 lies in the configuration and positioning of the double bond. Based on these observations, we hypothesized that the stereochemistry of the bridging carbons, C1 and C10, and the adjacent double bond, Δ 2,3 , might primarily determine the flexibility of these compounds.