Fucoxanthin is a major light-harvesting pigment in ecologically important algae such as diatoms, haptophytes, and brown algae (Phaeophyceae). Therefore, it is a major driver of global primary productivity. Species of these algal groups are brown colored because the high amounts of fucoxanthin bound to the proteins of their photosynthetic machineries enable efficient absorption of green light. While the structure of these fucoxanthin-chlorophyll proteins has recently been resolved, the biosynthetic pathway of fucoxanthin is still unknown. Here, we identified two enzymes central to this pathway by generating corresponding knockout mutants of the diatom Phaeodactylum tricornutum that are green due to the lack of fucoxanthin. Complementation of the mutants with the native genes or orthologs from haptophytes restored fucoxanthin biosynthesis. We propose a complete biosynthetic path to fucoxanthin in diatoms and haptophytes based on the carotenoid intermediates identified in the mutants and in vitro biochemical assays. It is substantially more complex than anticipated and reveals diadinoxanthin metabolism as the central regulatory hub connecting the photoprotective xanthophyll cycle and the formation of fucoxanthin. Moreover, our data show that the pathway evolved by repeated duplication and neofunctionalization of genes for the xanthophyll cycle enzymes violaxanthin de-epoxidase and zeaxanthin epoxidase. Brown algae lack diadinoxanthin and the genes described here and instead use an alternative pathway predicted to involve fewer enzymes. Our work represents a major step forward in elucidating the biosynthesis of fucoxanthin and understanding the evolution, biogenesis, and regulation of the photosynthetic machinery in algae.

Pepper (Capsicum annuum) fruits are a rich source of carotenoids. Geranylgeranyl diphosphate (GGPP) is the precursor for carotenoid biosynthesis and is produced by GGPP synthase (GGPPS), which belongs to the prenyl transferase (PTS) family. In this study, we identified from the pepper genome a total of eight PTS homologues. Our subcellular localization, enzymatic activity, and expression level analyses proved that among these homologues Capana04g000412 is the only functional GGPPS (CaGGPPS1) for carotenoid biosynthesis in pepper fruits. We demonstrated that CaGGPPS1 interacts with a catalytically inactive small subunit homologue protein CaSSUII, and such an interaction promotes CaGGPPS1 enzymatic activity. We also revealed a protein−protein interaction between CaSSUII and a putative phytoene synthase and the repression of carotenoid accumulation by silencing CaSSUII in pepper fruits. Taken together, our results suggest an essential contribution of the CaGGPPS1/CaSSUII interaction to carotenoid biosynthesis in ripening pepper fruits.

Carotenoids are essential phytonutrients for the human body. Higher plants usually synthesize and accumulate carotenoids in their leaves, flowers, and fruits. Most carotenoids have either two β-rings on both ends or β- and ε-rings separately on two ends of their molecules and are synthesized from the acyclic lycopene as the precursor. Lycopene β- and ε-cyclases (LCYB and LCYE, respectively) catalyze the β- and ε-cyclization of lycopene, respectively, and regulate the metabolic flux from lycopene to its downstream β,β-branches (by LCYB alone) and β,ε-branches (by LCYE and LCYB). In this study, we identified and characterized genes for two LCYBs (CaLCYB1 and CaLCYB2), one LCYE (CaLCYE1), and a capsanthin/capsorubin synthase (CaCCS1) which is also able to β-cyclize lycopene from the red pepper (Capsicum annuum var. conoides) genome. By quantifying transcript abundances of these genes and contents of different carotenoid components in ripening fruits, we observed a correlation between the induction of both CaLCYBs and the accumulation of carotenoids of the β,β-branch during ripening. Although capsanthin was accumulated in ripened fruits, our quantification demonstrated a strong induction of CaCCS1 at the breaker stage, together with the simultaneous repression of CaLCYE1 and the decrease of lutein content, suggesting the involvement of CaCCS1 in competing against CaLCYE1 for synthesizing carotenoids of the β,β-branch. Our results provide important information for future metabolic engineering studies to manipulate carotenoid biosynthesis and accumulation in fruits.

Carotenoids are essential phytonutrients synthesized by all photosynthetic organisms. Acyclic lycopene is the first branching point for carotenoid biosynthesis. Lycopene βand 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 LCY genes from Porphyra umbilicalis and Pyropia yezoensis (susabi-nori) of Bangiales, the most primitive red algal order that synthesizes lutein. Our functional characterization in both Escherichia coli and Arabidopsis thaliana 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.

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 (BfLCYB) encoding a lycopene β-cyclase from Bangia fuscopurpurea, 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 Escherichia coli 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 B. fuscopurpurea also synthesizes α-carotene and lutein, its enzyme-catalyzing ε-cyclization is still unknown.