Construction of a pathway to C(50)-ε-carotene

Substrate tolerance of bacterial cyclases has been demonstrated in various contexts, but little is known about that of plant cyclases. Here, we tested two plant ε-cyclases to convert C(50)-lycopene, which we previously established by rounds of directed evolution. Unlike bacterial β-cyclases, two-end cyclase from lettuce exhibited complete specificity against this molecule, indicating that this enzyme has some mechanism that exerts size-specificity. Arabidopsis one-end cyclase At-y2 showed detectable activity to C(50)-lycopene. Interestingly, we found that it functions as a two-end cyclase in a C(50) context. Based on this observation, a possible model for substrate discrimination of this enzyme is proposed.