Molecular cloning and functional characterization of CvLCYE, a key enzyme in lutein synthesis pathway in Chlorella vulgaris

Abstract

Human completely rely on food and nutrition supply to absorb enough lutein, which is an important antioxidant beneficial to health of eyes and heart. In plants, lycopene epsilon-cyclase (LCYE) is the key enzyme in lutein synthesis pathway, where lycopene is converted into δ-carotene, α-carotene and lutein. In this study, for the first time, we successfully cloned CvLCYE, the evolutionarily conserved lycopene cyclase in the genome of Chlorella vulgaris that is a green alga rich in lutein. Homology analysis showed that the amino acid sequence of CvLCYE protein is factually high identical with that of other aquatic green algae including Chlorella variabilis, Auxenochlorella protothecoides strain CS-41, Chromochloris zofingiensis and Chlamydomonas reinhardtii. Meanwhile, the UPGMA tree analysis of CvLCYE protein sequence showed the consistent results. Bioinformatic analysis of CvLCYE protein revealed a typical lycopene cyclase domain (Pfam05834) between the 64th–475th amino acid. The 3D structure prediction of CvLCYE protein displayed a structure of beta strand-alpha helix-beta strand, which is conserved in all known lycopene epsilon-cyclases. Subcellular localization tests demonstrated that mature CvLCYE-GFP fused protein localized to cytoplasm in Arabidopsis protoplasts. Importantly, targeted HPLC analysis on lycopene content are performed with Escherichia coli strain expressing CvLCYE, which displayed faded pink of consumption of lycopene, in comparison to strain expressing empty vector, which maintained pink because of accumulation of lycopene. HPLC data strongly suggested the capability of CvLCYE to catalyze lycopene. Furthermore, enzymatic activity of CvLCYE was also clearly verified in Chlamydomonas reinhardtii that is the model organism for algae research. Interestingly, we found that the lutein content in transgenic C. reinhardtii expressing CvLCYE increased by about 2.3 times than that of wild type strain. All these results provide insight into the positive role played by CvLCYE in lutein synthesis and lay a promising foundation for the molecular breeding of lutein metabolic pathway in Chlorella vulgaris and other algae.