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》

Carotenoid biosynthesis in the primitive red alga Cyanidioschyzon merolae.

Cyanidioschyzon merolae is considered to be one of the most primitive of eukaryotic photosynthetic organisms. To obtain insights into the origin and evolution of the pathway of carotenoid biosynthesis in eukaryotic plants, the carotenoid content of C. merolae was ascertained, genes encoding enzymes of carotenoid biosynthesis in this unicellular red alga were identified, and the activities of two candidate pathway enzymes of particular interest, lycopene cyclase and beta-carotene hydroxylase, were examined. C. merolae contains perhaps the simplest assortment of chlorophylls and carotenoids found in any eukaryotic photosynthetic organism: chlorophyll a, beta-carotene, and zeaxanthin. Carotenoids with epsilon-rings (e.g., lutein), found in many other red algae and in green algae and land plants, were not detected, and the lycopene cyclase of C. merolae quite specifically produced only beta-ringed carotenoids when provided with lycopene as the substrate in Escherichia coli. Lycopene beta-ring cyclases from several bacteria, cyanobacteria, and land plants also proved to be high-fidelity enzymes, whereas the structurally related epsilon-ring cyclases from several plant species were found to be less specific, yielding products with beta-rings as well as epsilon-rings. C. merolae lacks orthologs of genes that encode the two types of beta-carotene hydroxylase found in land plants, one a nonheme diiron oxygenase and the other a cytochrome P450. A C. merolae chloroplast gene specifies a polypeptide similar to members of a third class of beta-carotene hydroxylases, common in cyanobacteria, but this gene did not produce an active enzyme when expressed in E. coli. The identity of the C. merolae beta-carotene hydroxylase therefore remains uncertain.

Cunningham FX Jr ，Lee H ，Gantt E 《-》

Functional Characterization of Lycopene Cyclases Illustrates the Metabolic Pathway toward Lutein in Red Algal Seaweeds.

Carotenoids are essential phytonutrients synthesized by all photosynthetic organisms. Acyclic lycopene is the first branching point for carotenoid biosynthesis. Lycopene β- and ε-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of its open ends and direct the metabolic flux into different downstream branches. Carotenoids of the β,β-branch (e.g., β-carotene) are found in all photosynthetic organisms, but those of the β,ε-branch (e.g., lutein) are generally absent in cyanobacteria, heterokonts, and some red algae. Although both LCYBs and LCYEs have been characterized from land plants, there are only a few reports on LCYs from cyanobacteria and algae. Here, we cloned four genes from and (susabi-nori) of Bangiales, the most primitive red algal order that synthesizes lutein. Our functional characterization in both and demonstrated that each species has a pair of LCYB and LCYE. Similar to LCYs from higher plants, red algal LCYBs cyclize both ends of lycopene, and their LCYEs only cyclize a single end. The characterization of LCYEs from red algae resolved the first bifurcation step toward β-carotene and lutein biosynthesis. Our phylogenetic analysis suggests that LCYEs of the green lineage and the red algae originated separately during evolution.

Deng YY ，Cheng L ，Wang Q ，Ge ZH ，Zheng H ，Cao TJ ，Lu QQ ，Yang LE ，Lu S ... -  《-》

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》

Functional analysis of the beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation.

Carotenoids with cyclic end groups are essential components of the photosynthetic membranes in all plants, algae, and cyanobacteria. These lipid-soluble compounds protect against photooxidation, harvest light for photosynthesis, and dissipate excess light energy absorbed by the antenna pigments. The cyclization of lycopene (psi, psi-carotene) is a key branch point in the pathway of carotenoid biosynthesis. Two types of cyclic end groups are found in higher plant carotenoids: the beta and epsilon rings. Carotenoids with two beta rings are ubiquitous, and those with one beta and one epsilon ring are common; however, carotenoids with two epsilon rings are rare. We have identified and sequenced cDNAs that encode the enzymes catalyzing the formation of these two rings in Arabidopsis. These beta and epsilon cyclases are encoded by related, single-copy genes, and both enzymes use the linear, symmetrical lycopene as a substrate. However, the epsilon cyclase adds only one ring, forming the monocyclic delta-carotene (epsilon, psi-carotene), whereas the beta cyclase introduces a ring at both ends of lycopene to form the bicyclic beta-carotene (beta, beta-carotene). When combined, the beta and epsilon cyclases convert lycopene to alpha-carotene (beta, epsilon-carotene), a carotenoid with one beta and one epsilon ring. The inability of the epsilon cyclase to catalyze the introduction of a second epsilon ring reveals the mechanism by which production and proportions of beta,beta- and beta, epsilon-carotenoids may be controlled and adjusted in plants and algae, while avoiding the formation of the inappropriate epsilon,epsilon-carotenoids.

Cunningham FX Jr ，Pogson B ，Sun Z ，McDonald KA ，DellaPenna D ，Gantt E ... -  《-》