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

(1) Background: Oxygen has exerted a great effect in shaping the environment and driving biological diversity in Earth's history. Green lineage has evolved primary and secondary carotenoid biosynthetic systems to adapt to Earth's oxygenation, e.g., Haematococcus lacustris, which accumulates the highest amount of secondary astaxanthin under stresses. The two systems are controlled by lycopene ε-cyclase (LCYE) and ß-cyclase (LCYB), which leave an important trace in Earth's oxygenation. (2) Objectives: This work intends to disclose the underlying molecular evolutionary mechanism of Earth's oxygenation in shaping green algal carotenogensis with a special focus on lycopene cyclases. (3) Methods: The two kinds of cyclases were analyzed by site-directed mutagenesis, phylogeny, divergence time and functional divergence. (4) Results: Green lineage LCYEs appeared at ~1.5 Ga after the first significant appearance and accumulation of atmospheric oxygen, the so-called Great Oxygenation Event (GOE), from which LCYBs diverged by gene duplication. Bacterial ß-bicyclases evolved from ß-monocyclase. Enhanced catalytic activity accompanied evolutionary transformation from ε-/ß-monocyclase to ß-bicyclase. Strong positive selection occurred in green lineage LCYEs after the GOE and in algal LCYBs during the second oxidation, the Neoproterozoic Oxygenation Event (NOE). Positively selected sites in the catalytic cavities of the enzymes controlled the mono-/bicyclase activity, respectively. Carotenoid profiling revealed that oxidative adaptation has been wildly preserved in evolution. (5) Conclusions: the functionalization of the two enzymes is a result of primary to secondary adaptations to Earth's oxygenation.

GhLCYε-3 characterized as a lycopene cyclase gene responding to drought stress in cotton.

Drought stress significantly affects crop productivity. Carotenoids are essential photosynthetic pigment for plants, bacteria, and algae, with signaling and antioxidant functions. Lutein is a crucial branch product in the carotenoid synthesis pathway, which effectively improves the stress tolerance of higher plants. lycopene cyclase, a central enzyme for lutein synthesis, holds great significance in regulating lutein production. This research establishes a correlation between lutein content and stress resistance by measuring the drought resistance and lutein content of various cotton materials. To identify which crucial genes are associated with lutein, the lycopene cyclase family (LCYs) was analyzed. The research found that LCYs form a highly conserved family divided into two subfamilies, LCY-ε (lycopene ε-cyclase) and LCY-ß (lycopene ß-cyclase). Most members of the LCY family contain photoresponsive elements and abscisic acid elements. qRT-PCR demonstrates showed that most genes responded positively to drought stress, and GhLCYε-3 was expressed significantly differently under drought stress. Virus-induced gene silencing (VIGS) assay showed that the content of GhLCYε-3 was significantly increased with MDA and PRO, and the contents of chlorophyll and lutein were significantly decreased in pYL156 plants. The decrease in GhLCYε-3 expression is speculated to lead to reduced lutein content in vivo, resulting in the accumulation of reactive oxygen species (ROS) and decreased drought tolerance. This research enriched the understanding of LCY gene family and lutein function, and provided a new reference for cotton planting in arid areas. Synopsis: Lycopene cyclase plays an important role in enhancing the ability of scavenging ROS and drought resistance of plants.

Gene structure and potential regulation of the lycopene cyclase genes in Bixa orellana L.

Lycopene cyclases (LCYs) are a key branching point in regulating the carotenoid biosynthesis pathway in plants. Bixa orellana L. is characterized by the presence in its seed of bixin, an apocarotenoid of significant importance in the food, pharmaceutical, and cosmetic industries. Gene analysis provides the opportunity to investigate the LCY gene structure in plant species and its relationship with the synthesis of carotenoids. Coding sequences of the LCY genes were retrieved from a B. orellana genome DNA. Boß-LCY1 and Boß-LCY2 genes exhibit 100% of identity to their respective cDNA accessions, and exhibit a single coding region of 1512 bp (504 aa) and 1495 bp (498 aa), respectively. In contrast, Boε-LCY gene shows a coding region of 1581 bp (527 aa) with 10 introns of diverse lengths. Putative Transcription Factors (TFs) binding sites were upstream (3000 bp) identified for each LCY gene. TFs cover two groups, one with the categories of photosynthesis, reproduction, and oxidative processes that are frequent. The second one with the categories of defense, cell cycle, signaling, and carbohydrate metabolism, which are poorly represented. Besides, repetitive DNA elements showed motifs and proteins related to LTR from the Ty3/Gypsy family, were associated with the TFs regions. In general, TFs vary in the different BoLCY genes, being more abundant in the Boε-LCY gene. LCY expression analyzed from a transcriptome database, and validated by RT-qPCR, shows an upregulation of the three LCYs, mainly oriented to the synthesis of essential carotenoids in photosynthetic tissues (leaves), as well as an upregulation of the Boß-LCY2 gene in the non-photosynthetic tissues (firsts seed development stages) related to the bixin accumulation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01384-8.

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

Lutein is a high-value carotenoid with many human health benefits. Lycopene ß- 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 ß-cyclize both ends of lycopene to produce ß-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 ß-monocyclic γ-carotene and ultimately produced α-carotene with a ß-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.

Lycopene ε-cyclase mediated transition of α-carotene and ß-carotene metabolic flow in carrot fleshy root.

The accumulation of carotenoids, such as xanthophylls, lycopene, and carotenes, is responsible for the color of carrot (Daucus carota subsp. sativus) fleshy roots. The potential role of DcLCYE, encoding a lycopene ε-cyclase associated with carrot root color, was investigated using cultivars with orange and red roots. The expression of DcLCYE in red carrot varieties was significantly lower than that in orange carrots at the mature stage. Furthermore, red carrots accumulated larger amounts of lycopene and lower levels of α-carotene. Sequence comparison and prokaryotic expression analysis revealed that amino acid differences in red carrots did not affect the cyclization function of DcLCYE. Analysis of the catalytic activity of DcLCYE revealed that it mainly formed ε-carotene, while a side activity on α-carotene and γ-carotene was also observed. Comparative analysis of the promoter region sequences indicated that differences in the promoter region may affect the transcription of DcLCYE. DcLCYE was overexpressed in the red carrot 'Benhongjinshi' under the control of the CaMV35S promoter. Lycopene in transgenic carrot roots was cyclized, resulting in the accumulation of higher levels of α-carotene and xanthophylls, while the ß-carotene content was significantly decreased. The expression levels of other genes in the carotenoid pathway were simultaneously upregulated. Knockout of DcLCYE in the orange carrot 'Kurodagosun' by CRISPR/Cas9 technology resulted in a decrease in the α-carotene and xanthophyll contents. The relative expression levels of DcPSY1, DcPSY2, and DcCHXE were sharply increased in DcLCYE knockout mutants. The results of this study provide insights into the function of DcLCYE in carrots, which could serve as a basis for creating colorful carrot germplasms.

Horizontal Gene Transfer and Fusion Spread Carotenogenesis Among Diverse Heterotrophic Protists.

Thraustochytrids (phylum: Labyrinthulomycota) are nonphotosynthetic marine protists. Some thraustochytrids have crtIBY, a trifunctional fusion gene encoding a protein capable of ß-carotene biosynthesis from geranylgeranyl pyrophosphate. Here we show that crtIBY is essential in, and encodes the sole pathway for, carotenoid biosynthesis in the thraustochytrid Aurantiochytrium limacinum ATCC MYA-1381. We explore the evolutionary origins of CrtIBY and discover that the closest related protein domains are present in a small but diverse group of other heterotrophic protists, including the apusomonad Thecamonas trahens and the dinoflagellates Oxyrrhis marina and Noctiluca scintillans. Each organism within this cluster also contains one or more ß-carotene 15-15' oxygenase genes (blh and rpe65), suggesting that the acquisition of ß-carotene biosynthesis genes may have been related to the production of retinal. Our findings support a novel origin of eukaryotic (apo)carotenoid biosynthesis by horizontal gene transfer from Actinobacteria, Bacteroidetes, and/or Archaea. This reveals a remarkable case of parallel evolution of eukaryotic (apo)carotenogenesis in divergent protistan lineages by repeated gene transfers.

Carotenoid biosynthesis is associated with low-temperature adaptation in Rhodosporidium kratochvilovae.

BACKGROUND: Low temperatures greatly limit the growth of microorganisms. Low-temperature adaptation in microorganisms involves multiple mechanisms. Carotenoids are naturally occurring lipid-soluble pigments that act as antioxidants and protect cells and tissues from the harmful effects of free radicals and singlet oxygen. However, studies on the regulation of carotenoid biosynthesis at low temperatures in microorganisms are limited. In this study, we investigated the correlation between carotenoids and low-temperature adaptation in the cold-adapted strain of Rhodosporidium kratochvilovae YM25235. RESULTS: Carotenoid biosynthesis in YM25235 was inhibited by knocking out the bifunctional lycopene cyclase/phytoene synthase gene (RKCrtYB) using the established CRISPR/Cas9 gene-editing system based on endogenous U6 promoters. The carotenoids were extracted with acetone, and the content and composition of the carotenoids were analyzed by spectrophotometry and HPLC. Then, the levels of reactive oxygen species (ROS) and the growth rate in YM25235 were determined at a low temperature. The results indicated that the carotenoid biosynthesis and ROS levels were increased in the YM25235 strain at a low temperature and inhibition of carotenoid biosynthesis was associated with higher ROS levels and a significant decrease in the growth rate of YM25235 at a low temperature. CONCLUSIONS: The regulation of carotenoid biosynthesis was associated with low-temperature adaptation in YM25235. Our findings provided a strong foundation for conducting further studies on the mechanism by which YM25235 can adapt to low-temperature stress.

Concurrent Production of α- and ß-Carotenes with Different Stoichiometries Displaying Diverse Antioxidative Activities via Lycopene Cyclases-Based Rational System.

α- and ß-carotenes belong to the most essential carotenoids in the human body and display remarkable pharmacological value for health due to their beneficial antioxidant activities. Distinct high α-/ß-carotene stoichiometries have gained increasing attention for their effective preventions of Alzheimer's disease, cardiovascular disease, and cancer. However, it is extremely difficult to obtain α-carotene in nature, impeding the accumulations of high α-/ß-carotene stoichiometries and excavation of their antioxidant activities. Herein, we developed a dynamically operable strategy based on lycopene cyclases (LCYB and LCYE) for concurrently enriching α- and ß-carotenes along with high stoichiometries in E. coli. Membrane-targeted and promoter-centered approaches were firstly implemented to spatially enhance catalytic efficiency and temporally boost expression of TeLCYE to address its low competitivity at the starting stage. Dynamically temperature-dependent regulation of TeLCYE and TeLCYB was then performed to finally achieve α-/ß-carotene stoichiometries of 4.71 at 37 °C, 1.65 at 30 °C, and 1.06 at 25 °C, respectively. In the meantime, these α-/ß-carotene ratios were confirmed to result in diverse antioxidative activities. According to our knowledge, this is the first time that both the widest range and antioxidant activities of high α/ß-carotene stoichiometries were reported in any organism. Our work provides attractive potentials for obtaining natural products with competitivity and a new insight on the protective potentials of α-/ß-carotenes with high ratios for health supply.

Suppression of the Lycopene Cyclase Gene Causes Downregulation of Ascorbate Peroxidase Activity and Decreased Glutathione Pool Size, Leading to H2O2 Accumulation in Euglena gracilis.

Carotenoids are photosynthetic pigments and hydrophobic antioxidants that are necessary for the survival of photosynthetic organisms, including the microalga Euglena gracilis. In the present study, we identified an uncharacterized gene encoding the E. gracilis ß-carotene synthetic enzyme lycopene cyclase (EgLCY) and discovered a relationship between EgLCY-mediated carotenoid synthesis and the reactive oxygen species (ROS) scavenging system ascorbate-glutathione cycle. The EgLCY cDNA sequence was obtained via homology searching E. gracilis transcriptome data. An enzyme assay using Escherichia coli demonstrated that EgLCY converts lycopene to ß-carotene. E. gracilis treated with EgLCY double-stranded RNA (dsRNA) produced colorless cells with hypertrophic appearance, inhibited growth, and marked decrease in carotenoid and chlorophyll content, suggesting that EgLCY is essential for the synthesis of ß-carotene and downstream carotenoids, which are abundant and physiologically functional. In EgLCY dsRNA-treated cells, the ascorbate-glutathione cycle, composed of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR), was unusually modulated; APX and GR activities significantly decreased, whereas DHAR and MDAR activities increased. Ascorbate content was significantly increased and glutathione content significantly decreased in EgLCY dsRNA-treated cells and was correlated with their recycling enzyme activities. Fluorescent imaging demonstrated that EgLCY dsRNA-treated cells accumulated higher levels of H2O2 compared to wild-type cells. Taken together, this study revealed that EgLCY-mediated synthesis of ß-carotene and downstream carotenoid species upregulates APX activity and increases glutathione pool size for H2O2 scavenging. Our study suggests a possible relationship between carotenoid synthesis and the ascorbate-glutathione cycle for ROS scavenging in E. gracilis.

Carotenoid biosynthesis and the evolution of carotenogenesis genes in rust fungi.

Diseases caused by rust fungi pose a significant threat to global plant production. Although carotenoid pigments are produced in spores of nearly all rust species, the corresponding biosynthesis pathway(s) have not been investigated. Here, candidate genes for carotenoid biosynthesis in Puccinia graminis f. sp. tritici (Pgt) were identified, cloned and functionally complemented using specifically engineered strains of Escherichia coli. A part of the carotenoid biosynthesis pathway in rust fungi was elucidated, with only two genes, CrtYB and CrtI, catalysing the reactions from geranyl-geranyl diphosphate (GGPP) to γ-carotene. The CrtYB gene encodes a bi-functional lycopene cyclase/phytoene synthase, which catalyses the condensation of two GGPP into phytoene, as well as the cyclisation of the ψ-end of lycopene to form γ-carotene. The CrtI gene encodes a phytoene desaturase that carries out four successive desaturations of phytoene, through the intermediates phytofluene and neurosporene to lycopene. The evolution of carotenoid pigmentation in rust fungi, including Pgt, P. graminis avenae, P. graminis secalis (Pgs), P. graminis lolli, P. striiformis f. sp. tritici, P. striiformis f. sp. pseudohordei, P. striiformis f. sp. hordei, the "scabrum" rust (putative hybrids between Pgt and Pgs), P. triticina, and P. hordei, was investigated by phylogenetic analysis. Both CrtYB and CrtI were found to be closely related among rust fungi, other pathogenic fungi, and some aphids. Our results provide a springboard to increase the understanding of the physiological role(s) of carotenoid pigments in rust fungi, to better understand evolution within the Pucciniales, and to develop robust molecular diagnostics for rust fungi.

Carotenogenesis in cyanobacteria: CruA/CruP-type and CrtL-type lycopene cyclases.

Cyanobacteria are oxygenic photoautotrophic prokaryotes containing chlorophylls and carotenoids, and the latter play important roles in light-harvesting, protection of excess light, assembly of pigment-protein complexes, and stabilization of lipid membranes. Cyanobacteria produce many kinds of carotenoids, such as ß-carotene, zeaxanthin, echinenone, and myxol glycosides, which have a cyclic structure at one or both end(s). Cyclization of lycopene is a branch point in carotenoid biosynthesis to ß-carotene and γ-carotene. Two types of lycopene cyclases, CruA/CruP-type and CrtL-type, are functionally confirmed in only five species, while homologous genes are found in the genomes of most cyanobacteria. This review summarizes the carotenogenesis pathways and the functional enzymes along with genes, focusing particularly on the cyclization of lycopene by distinct types of lycopene cyclases in cyanobacteria.

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 ß- 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 ß,ß-branch (e.g., ß-carotene) are found in all photosynthetic organisms, but those of the ß,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 ß-carotene and lutein biosynthesis. Our phylogenetic analysis suggests that LCYEs of the green lineage and the red algae originated separately during evolution.

The bifunctional identification of both lycopene ß- and ε-cyclases from the lutein-rich Dunaliella bardawil.

The halophilic green alga Dunaliella bardawil FACHB-847 is rich in lutein and α-carotene, which has great potential for carotenoid production in open ponds. In this study, genes encoding lycopene ß- and ε-cyclases (DbLcyB and DbLcyE) from D. bardawil FACHB-847 were functionally identified by genetic complementation in E. coli. The bifunctional DbLcyB not only catalyzed the formation of both mono- and bi-cyclic ß-rings with a major ß-cyclase activity, but also possessed a weak ε-cyclase activity. In contrast, DbLcyE preferred to convert lycopene into monocyclic δ-carotene, and possessed a weak ß-monocyclase activity. Lutein and α-carotene were the prominent carotenoids in D. bardawil FACHB-847, which was in agreement with the result of genetic complementation of co-expression of DbLcyB and DbLcyE in E. coli with α-carotene as the prominent product. The bifunctional DbLcyB and DbLcyE may contribute to the high accumulation of α-carotene in D. bardawil FACHB-847. Interestingly, the accumulation of lutein in D. bardawil FACHB-847 was more sensitive to salt stress, while the accumulation of ß-carotene in D. salina CCAP 19/18 was induced by salt stress. In brief, the production of different carotenoid compositions from these two Dunaliella species can be induced by different growth conditions.

Manipulation of Carotenoid Metabolic Flux by Lycopene Cyclization in Ripening Red Pepper ( Capsicum annuum var. conoides) 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 ß-rings on both ends or ß- and ε-rings separately on two ends of their molecules and are synthesized from the acyclic lycopene as the precursor. Lycopene ß- and ε-cyclases (LCYB and LCYE, respectively) catalyze the ß- and ε-cyclization of lycopene, respectively, and regulate the metabolic flux from lycopene to its downstream ß,ß-branches (by LCYB alone) and ß,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 ß-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 ß,ß-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 ß,ß-branch. Our results provide important information for future metabolic engineering studies to manipulate carotenoid biosynthesis and accumulation in fruits.

Lycopene cyclases determine high α-/ß-carotene ratio and increased carotenoids in bananas ripening at high temperatures.

Bananas are a recommended food source to alleviate vitamin A deficiency because they contain a high ratio of provitamin A precursors. The objective of this study was to investigate carotenoid accumulation pattern in banana fruits during postharvest ripening and the mechanisms regulating this process. Ripe banana pulp had an unusually high α-/ß-carotene ratio (1.05), and the carotenoid contents increased (p ≤ 0.05) under light and high temperature. We analyzed the sequences, transcript levels, and functions of genes involved in carotenoid synthesis. The high ratio of α-/ß-carotene in ripe banana fruit was explained by the high flux to the α-carotene biosynthetic pathway, as reflected by high transcript levels of LCYE, and the weak flux to the ß-carotene branch of the biosynthetic pathway due to inactive MaLCYB1.2. High temperature during ripening up-regulated the transcript levels of genes involved in the α- and ß-carotene biosynthesis pathways and the activities of their encoded enzymes.

[Construction of high-yield strain by optimizing lycopene cyclase for ß-carotene production].

To optimize key enzymes, such as to explore the gene resources and to modify the expression level, can maximize metabolic pathways of target products. ß-carotene is a terpenoid compound with important application value. Lycopene cyclase (CrtY) is the key enzyme in ß-carotene biosynthesis pathway, catalyzing flavin adenine dinucleotide (FAD)-dependent cyclization reaction and ß-carotene synthesis from lycopene precursor. We optimized lycopene cyclase (CrtY) to improve the synthesis of ß-carotene and determined the effect of CrtY expression on metabolic pathways. Frist, we developed a ß-carotene synthesis module by coexpressing the lycopene ß-cyclase gene crtY with crtEBI module in Escherichia coli. Then we simultaneously optimized the ribosome-binding site (RBS) intensity and the species of crtY using oligo-linker mediated DNA assembly method (OLMA). Five strains with high ß-carotene production capacity were screened out from the OLMA library. The ß-carotene yields of these strains were up to 15.79-18.90 mg/g DCW (Dry cell weight), 65% higher than that of the original strain at shake flask level. The optimal strain CP12 was further identified and evaluated for ß-carotene production at 5 L fermentation level. After process optimization, the final ß-carotene yield could reach to 1.9 g/L. The results of RBS strength and metabolic intermediate analysis indicated that an appropriate expression level of CrtY could be beneficial for the function of the ß-carotene synthesis module. The results of this study provide important insight into the optimization of ß-carotene synthesis pathway in metabolic engineering.

Functional Characterization of a Missing Branch Component in Haematococcus pluvialis for Control of Algal Carotenoid Biosynthesis.

Cyclization of acyclic lycopene by cyclases marks an important regulatory point in carotenoid biosynthesis. Though some algal lycopene epsilon cyclases (LCYEs) have been predicted computationally, very few have been functionally identified. Little is known about the regulation mechanisms of algal LCYEs. Recent comparative genomic analysis suggested that Haematococcus pluvialis contained only the ß type cyclase (HpLCYB). However, in this study, carotenoid profiling found trace α-carotene in the salt-treated cells, indicating the in vivo activity of HpLCYE, a missing component for α-branch carotenoids. Thus, genes coding for HpLCYB and HpLCYE were isolated and functionally complemented in Escherichia coli. Substrate specificity assays revealed an exclusive cyclization order of HpLCYE to HpLCYB for the biosynthesis of heterocyclic carotenoids. Expression pattern studies and bioinformatic analysis of promoter regions showed that both cyclases were differentially regulated by the regulatory cis-acting elements in promoters to correlate with primary and secondary carotenoid biosynthesis under environmental stresses. Characterization of the branch components in algal carotenoid biosynthesis revealed a mechanism for control of metabolic flux into α- and ß-branch by the competition and cooperation between HpLCYE and HpLCYB; and supplied a promising route for molecular breeding of cyclic carotenoid biosynthesis.

Functional Lycopene Cyclase (CruA) in Cyanobacterium, Arthrospira platensis NIES-39, and its Role in Carotenoid Synthesis.

The genus Arthrospira is filamentous, non-nitrogen-fixing cyanobacteria that is commercially important. We identified the molecular structures of carotenoids in Arthrospira platensis NIES-39. The major carotenoid identified was ß-carotene. In addition, the hydroxyl derivatives of ß-cryptoxanthin and (3R,3'R)-zeaxanthin were also found to be present. The carotenoid glycosides were identified as (3R,2'S)-myxol 2'-methylpentoside and oscillol 2,2'-dimethylpentoside. The methylpentoside moiety was a mixture of fucoside and chinovoside in an approximate ratio of 1 : 4. Trace amounts of the ketocarotenoid 3'-hydroxyechinenone were also found. Three types of lycopene cyclases have been functionally confirmed in carotenogenesis organisms. In cyanobacteria, the functional lycopene cyclases (CrtL, CruA and CruP) have only been found in four species. In this study, we found that CruA exhibited lycopene cyclase activity in transformed Escherichia coli, which contains lycopene, but CruP exhibited no lycopene cyclase activity and crtL was absent. This is the third cyanobacterial species in which CruA activity has been confirmed. Neurosporene was not a substrate of CruA in E. coli, whereas lycopene cyclases of CrtY (bacteria), CrtL (plants) and CrtYB (fungi) have been reported to convert neurosporene to 7,8-dihydro-ß-carotene. ß-Carotene hydroxylase (CrtR) was found to convert ß-carotene to zeaxanthin in transformed E. coli, which contains ß-carotene. Among the ß-carotene hydroxylases, bacterial CrtZ and eukaryotic CrtR and BCH have similarities, whereas cyanobacterial CrtR appears to belong to another clade. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the A. platensis NIES-39 genome, we propose a biosynthetic pathway for the carotenoids as well as the corresponding genes and enzymes.

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

Lycopene cyclases cyclize the open ends of acyclic lycopene (ψ,ψ-carotene) into ß- or ε-ionone rings in the crucial bifurcation step of carotenoid biosynthesis. Among all carotenoid constituents, ß-carotene (ß,ß-carotene) is found in all photosynthetic organisms, except for purple bacteria and heliobacteria, suggesting a ubiquitous distribution of lycopene ß-cyclase activity in these organisms. In this work, we isolated a gene (BfLCYB) encoding a lycopene ß-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 ß- and ε-rings, including ß-carotene, zeaxanthin, α-carotene (ß,ε-carotene) and lutein. Functional complementation in Escherichia coli demonstrated that BfLCYB is able to catalyze cyclization of lycopene into monocyclic γ-carotene (ß,ψ-carotene) and bicyclic ß-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.

Synechocystis sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll a for activity.

The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces ß-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA 6803) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or ß-carotene, respectively. However, expression of this orthologous cruA 6803 gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA6803 was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and ß-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA6803 was assayed in vitro with either lycopene or γ-carotene as substrate, ß-carotene was synthesized. These data establish that CruA6803 is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.