Functional Characterization of Lycopene - and -Cyclases from a Lutein-Enriched Green Microalga Chlorella sorokiniana FZU60

Lutein is a high-value carotenoid with many human health benefits. Lycopene beta- and epsilon-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of lycopene into distinct downstream branches, one of which is the lutein biosynthesis pathway, via ff-carotene. Hence, LCYB and LCYE are key enzymes in lutein biosynthesis. In this study, the coding genes of two lycopene cyclases (CsLCYB and CsLCYE) of a lutein-enriched marine green microalga, Chlorella sorokiniana FZU60, were isolated and identified. A sequence analysis and computational modeling of CsLCYB and CsLCYE were performed using bioinformatics to identify the key structural domains. Further, a phylogenetic analysis revealed that CsLCYB and CsLCYE were homogeneous to the proteins of other green microalgae. Subcellular localization tests in Nicotiana benthamiana showed that CsLCYB and CsLCYE localized in chloroplasts. A pigment complementation assay in Escherichia coli revealed that CsLCYB could efficiently beta-cyclize both ends of lycopene to produce beta-carotene. On the other hand, CsLCYE possessed a strong epsilon-monocyclase activity for the production of delta-carotene and a weak "-bicyclic activity for the production of "-carotene. In addition, CsLCYE was able to catalyze lycopene into beta-monocyclic gamma-carotene and ultimately produced alpha-carotene with a beta-ring and an epsilon-ring via gamma-carotene or delta-carotene. Moreover, the co-expression of CsLCYB and CsLCYE in E. coli revealed that alpha-carotene was a major product, which might lead to the production of a high level of lutein in C. sorokiniana FZU60. The findings provide a theoretical foundation for performing metabolic engineering to improve lutein biosynthesis and accumulation in C. sorokiniana FZU60.