Interestingly, all the obtained structures display E-configured C2 double bonds, contrasting with observations in 1 and 2, suggesting that V220 and L221 function as cis and trans stereochemistry gatekeepers as L285 in AsR6. In the new eunicellane structure of 3, the final deprotonation occurs at C10 rather than C18. This introduces a sp 2 hybridized carbon into the "bridge" of the eunicellane scaffolds, aligning three carbons of the macrocycle in the same plane, which would make the macrocycle nonflexible, as justified by the sharp NMR signals of 3 (Supplementary Figures and). In addition, the deprotonation in 5 also follows a different direction, with a proton of C13 being eliminated. Moreover, the relative stereochemistry of C14 and C1 in 3 are different from those in 1, indicating that a smaller residue substitution of V220 makes the bound GGPP exhibit a different pre-cyclized conformation. However, V220M and L221M did not present a cis-trans configuration switch observed in AsR6, indicating different enzymatic microenvironments among the two proteins (Supplementary Figure ). Further structural comparison of MicA and AsR6 allowed us to find that V220 and L221 exhibit relatively greater distances to C2 and C3 of GGPP than those in AsR6 (Extended Data Fig. ), while they are in close proximity to C4 and C5. Specifically, V220 exhibits distances of 5. (Extended Data Fig. ). Unexpectedly, 3 spontaneously converted into 4 during silica chromatography, a phenomenon not observed in the isolation of 1 but detected in chloroform-treated albireticulene . We repurified 3 with triethylamine-treated silica resulting in no 4 being detected after purification. Accordingly, the formation of 4 is likely driven by acid-based catalysis, where the C6 double bond attacks a proton, initiating cyclization, followed by C2-C7 cyclization and C20 deprotonation to generate 4 (Extended Data Fig. ).