A lycopene β-cyclase/lycopene ε-cyclase/light-harvesting complex-fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α-carotene and β-carotene at different ratios.

Biosynthesis of asymmetric carotenoids such as α-carotene and lutein in plants and green algae involves the two enzymes lycopene β-cyclase (LCYB) and lycopene ε-cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C-terminal light-harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α-carotene and β-carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C-terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α-carotene-to-β-carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α-carotene or lutein in bacteria or fungi.

Blatt A ，Bauch ME ，Pörschke Y ，Lohr M ... -  《-》

Carotenoid analysis of a liverwort Marchantia polymorpha and functional identification of its lycopene β- and ε-cyclase genes.

Carotenoid biosynthesis in bryophytes has yet to be clarified. The liverwort Marchantia polymorpha L. is known to be an early land plant and is an emerging bryophyte model. In order to gain insight into the evolution of carotenoid biosynthesis in plants, we studied carotenoid biosynthesis in this liverwort. As is the case in higher plants, liverwort thalli contain lutein and β-carotene, as major carotenoids, as well as zeaxanthin, antheraxanthin, violaxanthin and 9'-cis-neoxanthin. Based on liverwort expressed sequence tag (EST)/cDNA and genome sequences, we isolated two cyclase genes encoding lycopene β-cyclase (LCYb) and lycopene ε-cyclase (LCYe), which were involved in the synthesis of β-carotene and α-carotene. These enzymes were phylogenetically positioned between corresponding proteins of a green alga (Chlorophyta) and higher plants. Functional analysis of the two genes was performed using a heterologous Escherichia coli expression system, in which the Pantoea ananatis lycopene biosynthesis genes were co-expressed. The results indicated liverwort LCYb activity for the synthesis of β-carotene from lycopene, which was the same as that of higher plants. On the other hand, liverwort LCYe was able to form two ε-rings from lycopene to ε-carotene via δ-carotene, which was different from the Arabidopsis LCYe enzyme which generates only one ε-ring from lycopene.

Takemura M ，Maoka T ，Misawa N 《-》

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

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, 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 showed that CsLCYB and CsLCYE localized in chloroplasts. A pigment complementation assay in 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 revealed that α-carotene was a major product, which might lead to the production of a high level of lutein in FZU60. The findings provide a theoretical foundation for performing metabolic engineering to improve lutein biosynthesis and accumulation in FZU60.

Fang H ，Liu J ，Ma R ，Zou Y ，Ho SH ，Chen J ，Xie Y ... -  《Marine Drugs》

Enhanced Lutein Production in Chlamydomonas reinhardtii by Overexpression of the Lycopene Epsilon Cyclase Gene.

Tokunaga S ，Morimoto D ，Koyama T ，Kubo Y ，Shiroi M ，Ohara K ，Higashine T ，Mori Y ，Nakagawa S ，Sawayama S ... -  《-》

Cloning and Functional Characterization of a Lycopene β-Cyclase from Macrophytic Red Alga Bangia fuscopurpurea.

Lycopene cyclases cyclize the open ends of acyclic lycopene (ψ,ψ-carotene) into β- or ε-ionone rings in the crucial bifurcation step of carotenoid biosynthesis. Among all carotenoid constituents, β-carotene (β,β-carotene) is found in all photosynthetic organisms, except for purple bacteria and heliobacteria, suggesting a ubiquitous distribution of lycopene β-cyclase activity in these organisms. In this work, we isolated a gene () encoding a lycopene β-cyclase from , a red alga that is considered to be one of the primitive multicellular eukaryotic photosynthetic organisms and accumulates carotenoid constituents with both β- and ε-rings, including β-carotene, zeaxanthin, α-carotene (β,ε-carotene) and lutein. Functional complementation in demonstrated that BfLCYB is able to catalyze cyclization of lycopene into monocyclic γ-carotene (β,ψ-carotene) and bicyclic β-carotene, and cyclization of the open end of monocyclic δ-carotene (ε,ψ-carotene) to produce α-carotene. No ε-cyclization activity was identified for BfLCYB. Sequence comparison showed that BfLCYB shares conserved domains with other functionally characterized lycopene cyclases from different organisms and belongs to a group of ancient lycopene cyclases. Although also synthesizes α-carotene and lutein, its enzyme-catalyzing ε-cyclization is still unknown.

Cao TJ ，Huang XQ ，Qu YY ，Zhuang Z ，Deng YY ，Lu S ... -  《Marine Drugs》