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. 2007 Mar;6(3):533-45.
doi: 10.1128/EC.00265-06. Epub 2006 Nov 3.

Carotenoid biosynthesis in the primitive red alga Cyanidioschyzon merolae

Francis X Cunningham Jr  1 , Hansel Lee, Elisabeth Gantt
Affiliations

Carotenoid biosynthesis in the primitive red alga Cyanidioschyzon merolae

Francis X Cunningham Jr et al. Eukaryot Cell. 2007 Mar.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
The pathway of carotenoid biosynthesis in Cyanidioschyzon merolae. The pathway shown here was inferred from the presence of candidate genes for the requisite pathway enzymes in the nuclear and plastid genomes of C. merolae (Table 3) and from an analysis of the carotenoid pigments present in cells of this red alga (Table 4). C. merolae candidate gene designations (Table 3) are in parentheses to the right of the designated pathway enzymes. Note that although certain cis-geometrical isomers of phytoene (15-cis) and ζ-carotene (9,9′-cis and/or 9,9′,15-cis) are likely intermediates in the C. merolae carotenoid pathway (see references and 26), the all-trans forms of these carotenoids are shown in this figure for simplicity. GGPP, geranylgeranyl diphosphate; CrtR, cyanobacterial-type β-carotene hydroxylase.
FIG. 2.
FIG. 2.
HPLC separation of carotenoids and chlorophylls of C. merolae. (A) Elution profile with detection at A437 to monitor both chlorophylls and carotenoids. (B) Detection at A465 to illustrate carotenoids only. The major pigments were identified (Table 4) as zeaxanthin (peak 4), chlorophyll a (peak 6), β-cryptoxanthin (peak 8), and β-carotene (peak 10). Pigments were extracted from mid-log-phase cultures grown under a relatively low light intensity (30 μE·m−2·s−1). HPLC was with a mobile phase gradient of 10 to 60% B (ethyl acetate) in A (acetonitrile:water:triethylamine, 90:10:0.1) over the course of 35 min.
FIG. 3.
FIG. 3.
Neighbor-joining tree for polypeptides encoded by plant, algal, and cyanobacterial members of the lycopene cyclase (LCY) gene family. Structures and carbon numbering for β- and ɛ-rings are illustrated at the upper left. The putative C. merolae LCYb is highlighted by placement within a rectangular box at the top of the figure. The encircled cluster designated “LCYb/e,” within the larger “Cyanobacterial LCYb” cluster, contains enzymes of mixed function that convert lycopene into products with both β-and ɛ-rings (59). The cluster labeled “CCS/NSY,” within the “Plant LCYb” cluster, includes plant enzymes that function as capsanthin-capsorubin synthases (CCS) and/or neoxanthin synthases (NSY) but that also, in some cases at least, retain the ability to function as a lycopene β-ring cyclase. Bootstrap values for the major branches are indicated.
FIG. 4.
FIG. 4.
Assay of the candidate C. merolae lycopene β-cyclase enzyme in lycopene-accumulating E. coli. (A) HPLC elution profile for an extract of E. coli cells that contained plasmid pAC-LYCipi, leading to the synthesis and accumulation of the lycopene. (B) HPLC elution profile for an extract of E. coli cells that contained plasmid pAC-LYCipi together with plasmid pCmLCY, expressing the putative C. merolae lycopene β-cyclase gene (CMK050C). (C) HPLC elution profile for an extract of E. coli cells that contained the plasmids pAC-BETA-At and pAtLCYe. The combination of genes within these two plasmids leads to the production of α-carotene (β,ɛ-carotene) and β-carotene (14), which here serve as chromatographic standards. Panels D and E display absorption spectra in the HPLC mobile phase for the indicated peaks in panels A to C, except that the absorption spectrum for β-carotene is for a peak obtained by HPLC separation of an extract of E. coli cells that contained the plasmid pAC-BETAipi alone (; Table 2). HPLC was with an isocratic mobile phase of 35% B (ethyl acetate) in A (acetonitrile:water:triethylamine, 90:10:0.1).
FIG. 5.
FIG. 5.
Neighbor-joining tree for products of the cyanobacterial and red algal crtR gene family. The three red algal sequences are encompassed by a box at the top of the figure. Bootstrap values of greater than 50% are indicated.
FIG. 6.
FIG. 6.
Alignment of the deduced amino acid sequence of the CrtR-type β-carotene hydroxylase of the cyanobacterium Synechocystis sp. strain PCC6803 (Sy) and related polypeptides specified by genes in the red algae G. sulphuraria (Gs), C. caldarium (Cc), and C. merolae (Cm). Residues are in white text on a black background where identical for all four sequences and in black text on a gray background where identical for three of the four sequences.
FIG. 7.
FIG. 7.
Assay of the candidate C. merolae β-carotene hydroxylase enzyme in β-carotene-accumulating E. coli. Panels A through D show HPLC elution profiles for extracts of E. coli cells that contained the plasmid pAC-BETAipi, leading to the synthesis and accumulation of β-carotene. (B) Cells also contained pCmCrtR, with the C. merolae crtR (CMV041C) cloned in frame in the expression vector pTrcHisB. (C) Cells also contained pSynCrtR with a Synechocystis PCC 6803 gene (sll1468), encoding a CrtR-type β-carotene hydroxylase, cloned in frame in the expression vector pTrcHisB. (D) Cells also contained pSynCrtRdel26N, producing a Synechocystis CrtR truncated by 26 amino acids at the N terminus (see Fig. 6; the polypeptide begins with MNVAMFGIAIL). Identities of known carotenoids are indicated. HPLC separation was with a gradient of 10 to 60% mobile phase B (ethyl acetate) in A (acetonitrile:water:triethylamine, 90:10:0.1) over 35 min.
FIG. 8.
FIG. 8.
Analysis of products formed in E. coli from lycopene through the action of an Arabidopsis thaliana lycopene ɛ-ring monocyclase. Results obtained using a lycopene β-ring monocyclase from the marine bacterium P99-3 are shown for comparison. (A) HPLC elution profile for an extract of E. coli cells that contained pAC-DELTA, a plasmid constructed by insertion of an A. thaliana LCYe cDNA into plasmid pAC-LYC, the latter of which otherwise leads to the synthesis and accumulation of lycopene. (B) HPLC elution profile for an extract of E. coli cells that contained pAC-DELTA together with pHpKetoSK, a plasmid that contains and expresses a cDNA from Haematococcus pluvialis that encodes an enzyme that adds a carbonyl to the number 4 carbon of β-rings (34). (C) HPLC elution profile for an extract of E. coli cells that contained pAC-GAMMA, a plasmid constructed by insertion of a gene encoding the lycopene β-ring monocyclase of the marine bacterium P99-3 into plasmid pAC-LYC. (D) HPLC elution profile for an extract of E. coli cells that contained pAC-GAMMA together with pHpKetoSK. Panels E and F display absorption spectra for the indicated peaks in panels A to D. Mid-log-phase cultures were harvested for pigment extraction in order to minimize the relative amounts of cis-geometrical isomers, which increase in proportion in older cultures. HPLC separation was with an isocratic mobile phase of 35% B (ethyl acetate) in A (acetonitrile:water:triethylamine, 90:10:0.1) over 35 min. The detector was set to a wavelength of 510 nm in order to enhance the prominence of those peaks containing γ-carotene and 4-keto-γ-carotene.
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References

    1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. - PubMed
    1. Antoine, R., and C. Locht. 1992. Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. Mol. Microbiol. 6:1785-1799. - PubMed
    1. Barbier, G., C. Oesterhelt, M. D. Larson, R. G. Halgren, C. Wilkerson, R. M. Garavito, C. Benning, and A. P. M. Weber. 2005. Comparative genomics of two closely related unicellular thermo-acidophilic red algae, Galdieria sulphuraria and Cyanidioschyzon merolae, reveals the molecular basis of the metabolic flexibility of Galdieria sulphuraria and significant differences in carbohydrate metabolism of both algae. Plant Physiol. 137:460-474. - PMC - PubMed
    1. Bede, J. C., P. E. Teal, W. G. Goodman, and S. S. Tobe. 2001. Biosynthetic pathway of insect juvenile hormone III in cell suspension cultures of the sedge Cyperus iria. Plant Physiol. 127:584-593. - PMC - PubMed
    1. Bjørnland, T., and M. Aguilar-Martinez. 1976. Carotenoids in red algae. Phytochemistry 15:291-296.

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