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

Abstract

Lutein is a high-value carotenoid with many human health benefits. Lycopene β- and ε-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of lycopene into distinct downstream branches, one of which is the lutein biosynthesis pathway, via α-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 β-cyclize both ends of lycopene to produce β-carotene. On the other hand, CsLCYE possessed a strong ε-monocyclase activity for the production of δ-carotene and a weak ε-bicyclic activity for the production of ε-carotene. In addition, CsLCYE was able to catalyze lycopene into β-monocyclic γ-carotene and ultimately produced α-carotene with a β-ring and an ε-ring via γ-carotene or δ-carotene. Moreover, the co-expression of CsLCYB and CsLCYE in E. coli revealed that α-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.

The Clinical Promise of Microalgae in Rheumatoid Arthritis: From Natural Compounds to Recombinant Therapeutics

“…Although microalgae can potentially substitute plants in the production of carotenoids like lutein [201,202], their yields are still lagging behind heterotrophic microorganisms [203][204][205][206]. Genetic engineering can greatly enhance pigment productivity in microalgae [207] but requires a detailed knowledge of algal genomes and of the transcriptional networks regulating carotenoid biosynthesis to manipulate key metabolic genes [208][209][210][211] (Figure 5A). In this respect, a model organism for ketocarotenoid biosynthesis is C. zofingiensis [212,213], from which the β-carotene ketolase (BKT) enzyme was identified as a rate-limiting factor for ASTX production [214], while the chlorophytes D. salina and Desmodesmus spp.…”

Haematococcus lacustris Carotenogensis: A Historical Event of Primary to Secondary Adaptations to Earth’s Oxygenation

“…The cyclization of lycopene by lycopene cyclases marks an important regulatory point in carotenoid biosynthesis. The ε-cyclization controlled by LCYE is a key point in regulating primary carotenoid levels and the ratio of primary to secondary carotenoids [14].…”