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

Introduction

The genus Arthrospira, also known as ‘Spirulina’, includes filamentous, non-nitrogen-fixing cyanobacteria. This microalga has been commercially produced as human food and animal feed owing to its considerable nutritional content. This includes a high content of proteins (60–70% by dry weight), the presence of all essential amino acids and its richness in essential fatty acids (γ-linoleic acid), vitamins, minerals and nutritional pigments such as carotenoids, Chl and phycocyanin (Spolaore et al. 2006). Arthrospira platensis has suitable properties for industrial production; it prefers to live in highly alkaline and high pH water; this prevents contamination with other organisms, and it is relatively easy to harvest by filtration because of its long spiral-shaped filament (Ciferri 1983). The whole-genome DNA sequences of six species of Arthrospira, i.e. A. platensis NIES-39 (Fujisawa et al. 2010), Arthrospira sp. PCC 8005 (Janssen et al. 2010), A. platensis C1 (PCC 9438) (Cheevadhanarak et al. 2012), A. platensis strain Paraca (Lefort et al. 2014), A. platensis strain YZ (Xu et al. 2016) and A. maxima CS-328 (cited in in Cheevadhanark 2012, http://genome.ornl.gov/microbial/amax), have been completed and the identification and manipulation of genes involved in producing important gene products in Arthrospira is expected.

Carotenoids are essential isoprenoid pigments synthesized by all photosynthetic and many non-photosynthetic organisms, including bacteria, algae, fungi and plants. They are known to play roles in light harvesting, photoprotection, assembly of pigment–protein complexes in phototrophs and stabilization of lipid membranes (Takaichi 2013). Cyanobacteria contain ketocarotenoids (e.g. echinenone and 4-ketomyxol) and carotenoid glycosides (e.g. myxol glycosides and oscillol diglycosides) that are unique among the photosynthetic organisms (Takaichi and Mochimaru 2007). Carotenoids are also beneficial to human health. Some carotenoids with a β-end group, including β-carotene, are the precursors of vitamin A, which is a fundamental nutrient for humans. Zeaxanthin is the predominant xanthophyll in human eyes and may reduce the risk of cataracts and age-related macular degeneration (Abdel-Aal et al. 2013).

The biosynthetic pathway of carotenoids begins with the synthesis of phytoene through the condensation of two molecules of geranylgeranyl diphosphate (GGPP) by phytoene synthase (CrtB in cyanobacteria, PSY in plants). Phytoene desaturase (CrtP, PDS) catalyzes the first two desaturation steps from phytoene to ζ-carotene via phytofluene, while ζ-carotene desaturase (CrtQ, ZDS) catalyzes the next two desaturation steps from ζ-carotene to lycopene via neurosporene. Cis-carotene isomerase (CrtH, CrtISO) then isomerizes poly-cis lycopene to all-trans lycopene in cyanobacteria and plants (Takaichi and Mochimaru 2007). In contrast, other bacteria (apart from cyanobacteria and green sulfur bacteria) and fungi use only one enzyme, phytoene desaturase (CrtI), to convert phytoene to all-trans lycopene (Takaichi 2013).

The cyclization of lycopene is a branch point in carotenoid biosynthesis pathways. Three distinct types of lycopene cyclase have been functionally identified in carotenogenic organisms (Maresca et al. 2007, Takaichi 2011). The first type of lycopene cyclase is CrtY in bacteria and CrtL (CrtL-b, Lcy-b) in algae and plants. Lycopene ɛ-cyclases (CrtL-e, Lcy-e) from plants and lycopene β-monocyclases (CrtYm, CrtLm) from bacteria are also included in this type. The second type is a heterodimer (CrtYc and CrtYd) from bacteria or a monomer (CrtYc-Yd from archaea and CrtYB from fungi), but not from phototrophs (Takaichi 2011). The third type of CruA/CruP has been found in green sulfur bacteria and cyanobacteria (Takaichi 2013). In cyanobacteria, four enzymes have been functionally confirmed (Takaichi and Mochimaru 2007); CrtL from Synechococcus elongatus PCC 7942 (Cunningham et al. 1994) and Prochlorococcus marinus MED-4 (Stickforth et al. 2003), CruA and CruP from Synechococcus sp. PCC 7002 (Maresca et al. 2007) and CruA from Synechocystis sp. PCC 6803 (Xiong et al. 2016). Their homologous genes are widely distributed in the genome of some cyanobacteria, such as Anabaena sp. PCC 7120; however, the lycopene cyclase activity in these gene products has not been detected despite the strong efforts of some laboratories. Recently, Xiong et al. (2016) have reported that CruA from Synechocystis sp. PCC 6803 requires bound Chl a for activity.

β-Carotene hydroxylase (CrtR) from cyanobacteria catalyzes the hydroxylation of β-carotene to zeaxanthin, echinenone to 3′-hydroxyechinenone and deoxymyxol 2′-dimethyl-fucoside to myxol 2′-dimethyl-fucoside (Masamoto et al. 1998). Two distinct β-carotene ketolases, CrtO and CrtW, catalyze the conversion of β-carotene to echinenone and myxol 2′-fucoside to 4-ketomyxol 2′-fucoside, respectively, in Anabaena sp. PCC 7120 (Takaichi and Mochimaru 2007).

Arthrospira platensis produces some carotenoids, such as β-carotene, zeaxanthin and myxol-glycoside (Aakermann et al. 1992). However, its carotenoid biosynthetic pathway and the corresponding enzymes have not been elucidated. In this study, lycopene β-cyclase (CruA) and β-carotene hydroxylase (CrtR) genes were isolated from A. platensis NIES-39 and characterized by functional complementation analysis in transformed Escherichia coli, which contains lycopene and β-carotene, respectively. Our results indicate that this bacterium was the third cyanobacterium and the fourth phototroph to possess functional CruA. These findings should provide new gene sources for carotene engineering and improve the understanding of carotene biosynthesis.

Results and Discussion

Identification of carotenoids

Fig. 1 shows an elution profile of C₁₈-HPLC for the organic solvent-soluble pigments extracted from A. platensis NIES-39. The peak-5 pigment was identified as Chl a by its absorption spectrum and specific retention time on HPLC. The minor peak-6 pigment also had a Chl a-like spectrum.

Fig. 1

HPLC elution profiles of pigments extracted from Arthrospira platensis NIES-39. The elution (1.8 ml min^–1) was started with a linear gradient from methanol/water (9 : 1, v/v) to 100% methanol at 20 min, and then continued with 100% methanol. Absorbance at 470 nm (red line) and 618 nm (green line) is shown. Peaks are referred to in the text, figures and table.

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The absorption maxima of each pigment in the HPLC eluent are summarized in Table 1. Only the peak-4 pigment had a broad carbonyl-type spectrum. The retention times of each pigment on HPLC were compared with those of Thermosynechococcus elongatus strain BP-1 (Iwai et al. 2008). Their relative molecular masses after purification were also analyzed (Table 1). To determine the stereochemistry, cirular dichroism (CD) spectra of peak-2 and -3 pigments were measured and found to be compatible with those of T. elongatus (Iwai et al. 2008). The structure of the peak-2 component was analyzed by ¹H-nuclear magnetic resonance (NMR) after acetylation in order to dissolve CDCl₃. Its ¹H-NMR signals of the aglycone moiety were compatible with those of myxol (Iwai et al. 2008). The signals of the glycoside moiety indicated that this component consisted of two kinds of glycosides. ¹H–¹H signal connections of individual glycosides were analyzed by ¹H-¹H COSY spectroscopy (Supplementary Fig. S1). One glycoside was compatible with triacetyl-α-fucoside, and the other was triacetyl-α-chinovoside (Table 2). Furthermore, the ratio of fucoside and chinovoside was assigned to be approximately 1 : 4 by ¹H signal area. Consequently, peak-1, -2, -3, -4, -7 and -8 pigments were identified as oscillol 2,2′-dimethylpentoside, (3R,2′S)-myxol 2′-methylpentoside, (3R,3′R)-zeaxanthin, 3′-hydroxyechinenone, β-cryptoxanthin and β-carotene, respectively.

The carotenoids in A. platensis NIES-39 were identified (see Table 1). Major carotenoids were β-carotene, zeaxanthin and myxol 2′-methylpentoside. Minor carotenoids (Fig. 2) were also found (Table 3). The composition was very similar to that of Spirulina (Arthrospira) platensis green NIVA-CYA 136/2, in which the methylpentoside moiety was also found to be a mixture of fucoside and chinovoside with an approximate ratio of 2 : 7 (Aakermann et al. 1992). To our knowledge, A. platensis is the only species to have this mixture of the methylpentoside moiety of myxol 2′-methylpentoside. Since the identification of glycoside moieties is difficult, most have not yet been identified. As trace amounts of the ketocarotenoid, 3′-hydroxyechinenone, were found, β-carotene ketolase (CrtO) activity should be present but its activity may be low.

Table 3

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Carotenoid composition of Arthrospira platensis NIES-39

Carotenoid .	mol % of total carotenoids .
.	Arthrospira platensis NIES-39 .	Spirulina (Arthrospira) platensis green NIVA-CYA 136/2a .
β-Carotene	44	35
β-Cryptoxanthin	3	2
(3R,3′R)-Zeaxanthin	25	31
Echinenone		2
3′-Hydroxyechinenone	1	1
β-Caroten-5,8-epoxideb		2
(3R,2′S)-Myxol-glycoside	23c	24d
Oscillol-diglycoside	4	3
Carotenoid content (mg g–1 DW)	5.9	1.3

Carotenoid .	mol % of total carotenoids .
.	Arthrospira platensis NIES-39 .	Spirulina (Arthrospira) platensis green NIVA-CYA 136/2a .
β-Carotene	44	35
β-Cryptoxanthin	3	2
(3R,3′R)-Zeaxanthin	25	31
Echinenone		2
3′-Hydroxyechinenone	1	1
β-Caroten-5,8-epoxideb		2
(3R,2′S)-Myxol-glycoside	23c	24d
Oscillol-diglycoside	4	3
Carotenoid content (mg g–1 DW)	5.9	1.3

^aAakermann et al. (1992)

^b This might be misidentification, since the epoxy group has not been found in prokaryotes.

^c A mixture of fucoside and chinovoside with the ratio of approximately 1 : 4.

^d A mixture of fucoside and chinovoside with the ratio of approximately 2 : 7.

Table 3

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Carotenoid composition of Arthrospira platensis NIES-39

Carotenoid .	mol % of total carotenoids .
.	Arthrospira platensis NIES-39 .	Spirulina (Arthrospira) platensis green NIVA-CYA 136/2a .
β-Carotene	44	35
β-Cryptoxanthin	3	2
(3R,3′R)-Zeaxanthin	25	31
Echinenone		2
3′-Hydroxyechinenone	1	1
β-Caroten-5,8-epoxideb		2
(3R,2′S)-Myxol-glycoside	23c	24d
Oscillol-diglycoside	4	3
Carotenoid content (mg g–1 DW)	5.9	1.3

Carotenoid .	mol % of total carotenoids .
.	Arthrospira platensis NIES-39 .	Spirulina (Arthrospira) platensis green NIVA-CYA 136/2a .
β-Carotene	44	35
β-Cryptoxanthin	3	2
(3R,3′R)-Zeaxanthin	25	31
Echinenone		2
3′-Hydroxyechinenone	1	1
β-Caroten-5,8-epoxideb		2
(3R,2′S)-Myxol-glycoside	23c	24d
Oscillol-diglycoside	4	3
Carotenoid content (mg g–1 DW)	5.9	1.3

^aAakermann et al. (1992)

^b This might be misidentification, since the epoxy group has not been found in prokaryotes.

^c A mixture of fucoside and chinovoside with the ratio of approximately 1 : 4.

^d A mixture of fucoside and chinovoside with the ratio of approximately 2 : 7.

Fig. 2

Structure of carotenoids in Arthrospira platensis NIES-39. Numbers indicate the peak numbers in Fig. 1.

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Cloning of the lycopene cyclase and β-carotene hydroxylase genes

From the genome sequence of A. platensis NIES-39 (Fujisawa et al. 2010), we found two lycopene cyclase genes, cruA and cruP, and one β-carotene hydroxylase gene, crtR, by homology search. However, the crtL-like gene from Synechococcus elongatus PCC 7942 and the crtY-like gene from Pantoea were not found. The three genes we found were amplified by PCR and cloned into the expression vector pET21a for E. coli. The deduced amino acid sequences of the Arthrospira CruA and CruP were found to have 65% and 53% identities with functional Synechococcus sp. PCC 7002 CruA and CruP, respectively (Fig. 3A; Supplementary Fig. S2). The deduced amino acid sequence of Arthrospira CrtR was found to have 71% identity with functional Synechocystis sp. PCC 6803 CrtR (Fig. 4A).

Fig. 3

Comparison of Arthrospira CruA with other lycopene cyclases. (A) Alignment of deduced amino acid sequences of CruA from A. platensis and Synechococcus sp. PCC 7002. The alignment was performed by ClustalW. Identical and similar residues are shown in shades of black and gray, respectively, by BOXSHADE. Dashes indicate gaps introduced to maximize sequence similarity. Numbering of amino acid residues for each polypeptide is indicated at the right side. (B) Phylogenetic tree generated based on the deduced amino acid sequences of four classes of lycopene cyclase, CruA, CruP, CrtL and CrtY. The tree was constructed on the basis of the Neighbor–Joining method. The bootstrap values on the nodes indicate the number of times that each group occurred with 1,000 replicates. Accession numbers of protein sequences are in parentheses.

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Fig. 4

Comparison of Arthrospira CrtR with other members of β-carotene hydroxylase. (A) Alignment of deduced amino acid sequences of CrtR from A. platensis and Synechocystis sp. PCC 6803. The alignment was performed by ClustalW. Identical and similar residues are shown in shades of black and gray, respectively, by BOXSHADE. Dashes indicate gaps introduced to maximize sequence similarity. Numbering of amino acid residues for each polypeptide is indicated at the right side. (B) Phylogenetic tree generated based on the alignment of deduced amino acid sequences of three classes of β-carotene hydroxylase, CrtR, CrtZ and BCH. The tree was constructed on the basis of the Neighbor–Joining method. The bootstrap values on the nodes indicate the number of times that each group occurred with 1,000 replicates. Accession numbers of protein sequences are in parentheses.

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The relationship between Arthrospira CruA/CruP and the functional lycopene cyclases of other bacterial species was investigated by generating a phylogenetic tree (Fig. 3B). As a result, the different clades for CruA/CruP and CrtL/CrtY were formed. Arthrospira CruA and CruP were grouped with Synechococcus CruA and CruP, respectively. In contrast, Arthrospira CrtR was grouped with CrtR in other cyanobacteria, and formed different clades from the CrtZ-type β-carotene hydroxylase in bacteria and the CrtR and BCH-type in plants (Fig. 4B). Among β-carotene hydroxylases, bacterial CrtZ and eukaryotic CrtR and BCH were more closely related to one another than either was related to the cyanobacterial CrtR.

Functional analysis of Arthrospira cruA and cruP

To investigate the functions of the Arthrospira lycopene cyclase, we used a heterologous E. coli expression system (Harada and Misawa 2009). The E. coli strain carrying the plasmid pACCRT-EIB that contains the crtE (coding GGPP synthase), crtB and crtI genes from the soil bacterium Pantoea ananatis (previous name, Erwinia uredovora) accumulates lycopene (Misawa et al. 1995). When pET21-CruA was introduced into the lycopene-accumulating E. coli, the yellow color of the carotenoid was observed. Production of β-carotene was confirmed with HPLC (Fig. 5A, peak-1). In contrast, neither the empty vector pET21a nor pET21-CruP influenced the composition of carotenoids (Fig. 5B; Supplementary Fig. S3), although a band corresponding to Arthrospira CruP was detected by SDS–PAGE in the E. coli strain carrying the plasmid pET21-CruP (Supplementary Fig. S4). These results indicated that the Arthrospira CruA could catalyze the cyclization reaction from lycopene to β-carotene, whereas CruP could not. Currently, the role of Arthrospira CruP in the biosynthesis of carotenoids remains unknown.

Fig. 5

LC/MS analysis of the carotenoids produced in E. coli cells harboring different combinations of plasmids. (A) pACCRT-EIB and pET21-CruA, which encodes A. platensis cruA; (B) pACCRT-EIB and pET21a, the empty vector; (C) pACCAR16ΔcrtX and pET21-CrtR, which encodes A. platensis crtR; (D) pACCAR16ΔcrtX and pET21a, the empty vector. The UV–visible and MS spectral data for numbered peaks are shown on the right. Peak 1 is β-carotene, peak 2 is lycopene and peak 3 is zeaxanthin.

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The E. coli strain carrying the plasmid pACCRT-EB-RcI that contains the crtE and crtB genes from P. ananatis and the crtI gene from Rhodobacter capsulatus accumulates neurosporene (Takaichi et al. 1996). When pET21-CruA or pET21-CruP was introduced into the E. coli, no cyclase activity was observed (data not shown). In contrast, the lycopene cyclases from P. ananatis (CrtY), the higher plant Capsicum annuum (LCY) and fungus Xanthophyllomyces dendrorhous (CrtYB) are known to convert neurosporene into a bicyclic carotenoid of 7,8-dihydro-β-carotene (Takaichi et al. 1996, Verwaal et al. 2007).

We demonstrated that cruA from A. platensis NIES-39 had lycopene cyclase activity, whereas cruP did not. This is the third functional analysis of CruA as a lycopene cyclase in a cyanobacterial species, and the fourth phototroph identified. CrtL was absent in the genome. CruA from A. platensis NIES-39 and CruP from Synechococcus sp. PCC 7002 (Maresca et al. 2007) have activity expressed in E. coli, whereas CruA from Synechococcus sp. PCC 7002 has been reported to be inactive in E. coli. However, the activity of CruA was shown from the phenotype of a cruA mutant in Synechococcus sp. PCC 7002 (Maresca et al. 2007). On the other hand, CruA from Synechocystis sp. PCC 6803 requires bound Chl a for activity (Xiong et al. 2016). Further studies are needed to elucidate the different characteristics. With regard to evolution, it is interesting that cyanobacteria obtained CruA/CruP instead of CrtY/CrtL. From cyanobacteria to chloroplasts, why it changed to CrtL from CruA/CruP is unknown. In conclusion, for lycopene cyclases among cyanobacteria, three species have CruA/CruP, two species have CrtL and others are unknown.

Functional analysis of Arthrospira crtR

To identify the catalytic activity of the β-carotene hydroxylase, pET21-CrtR was transformed into β-carotene-accumulating E. coli, which contained the plasmid pACCR16ΔcrtX with crtE, crtB, crtI and crtY from P. ananatis (Misawa et al. 1995). The respective E. coli transformants produced zeaxanthin (Fig. 5C, peak-3), which was not detectable in the β-carotene-accumulating E. coli carrying the empty vector pET21a (Fig. 5D). These results indicated that CrtR of Arthrospira may be involved in the hydroxylation of β-carotene to zeaxanthin.

In Synechocystis sp. PCC 6803, CrtR has been found to catalyze not only β-carotene to zeaxanthin but also deoxymyxol 2′-dimethyl-fucoside to myxol 2′-dimethyl-fucoside and echinenone to 3′-hydroxyechienone (Takaichi and Mochimaru 2007). Since A. platensis NIES-39 contains (3R,2′S)-myxol 2′-methylpentoside and 3′-hydroxyechienone (see Table 3), CrtR from Arthrospira may also play a role in these synthesis pathways.

Candidates of other carotenoid biosynthetic genes

The identification of the carotenoids in this study (Table 3; Fig. 2) and the completion of the entire nucleotide sequence of the genome in A. platensis NIES-39 (Fujisawa et al. 2010) will help facilitate the elucidation of the biosynthetic pathways and the corresponding enzymes and genes involved. Fig. 6 presents our proposed biosynthetic pathway of the carotenoids in A. platensis NIES-39. We have summarized the genes in A. platensis NIES-39 (Fujisawa et al. 2010) on the basis of sequence homology, in which only functionally confirmed genes were chosen for query sequences. This suggests the presence of CrtE, CrtB, CrtP, CrtQ, CrtH, CrtO and CruF (carotenoid 1,2-hydratase), in addition to functional CruA and CrtR (Table 4).

Table 4

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Candidates of other carotenoid biosynthesis genes in Arthrospira platensis NIES-39 based on carotenoids present

Gene .	Enzyme .	Query sequence for BLASTPa,b .	Accession number .		Identity (%)/ e-value .
.	.	.	CyanoBase .	NCBI .
crtE	Geranylgeranyl diphosphate synthase	Thermosynechococcus elongatus BP-1, tll0020	NIES39_J00820	BAI91134	74/3e-162
crtB	Phytoene synthase	Synechocystis sp. PCC 6803, slr1255	NIES39_C00950	BAI88965	70/8e-156
crtP	Phytoene desaturase	Synechocystis sp. PCC 6803, slr1254	NIES39_C00940	BAI88964	80/0.0
crtQ	ζ-Carotene desaturase	Synechocystis sp. PCC 6803, slr0940	NIES39_B00980	BAI88855	75/0.0
crtH	cis-Carotene isomerase	Synechocystis sp. PCC 6803, sll0033	NIES39_C05040	BAI89370	76/0.0
cruA	Lycopene cyclase	Synechococcus sp. PCC 7002, SYNPCC7002_A2153	NIES39_A06350	BAI88473	65/0.0
cruP	Lycopene cyclase	Synechococcus sp. PCC 7002, SYNPCC7002_A0043	NIES39_K00660	BAI91715	53/0.0
crtR	β-Carotene hydroxylase	Synechocystis sp. PCC 6803, sll1468	NIES39_R00430	BAI94352	71/8e-165
crtO	β-Carotene ketolase	Synechocystis sp. PCC 6803, slr0088	NIES39_L03850	BAI92542	24/2e-26
cruF	Carotenoid 1,2-hydratase	Synechococcus sp. PCC 7002, SYNPCC7002_A2032	NIES39_B01080	BAI88865	51/7e-88

Gene .	Enzyme .	Query sequence for BLASTPa,b .	Accession number .		Identity (%)/ e-value .
.	.	.	CyanoBase .	NCBI .
crtE	Geranylgeranyl diphosphate synthase	Thermosynechococcus elongatus BP-1, tll0020	NIES39_J00820	BAI91134	74/3e-162
crtB	Phytoene synthase	Synechocystis sp. PCC 6803, slr1255	NIES39_C00950	BAI88965	70/8e-156
crtP	Phytoene desaturase	Synechocystis sp. PCC 6803, slr1254	NIES39_C00940	BAI88964	80/0.0
crtQ	ζ-Carotene desaturase	Synechocystis sp. PCC 6803, slr0940	NIES39_B00980	BAI88855	75/0.0
crtH	cis-Carotene isomerase	Synechocystis sp. PCC 6803, sll0033	NIES39_C05040	BAI89370	76/0.0
cruA	Lycopene cyclase	Synechococcus sp. PCC 7002, SYNPCC7002_A2153	NIES39_A06350	BAI88473	65/0.0
cruP	Lycopene cyclase	Synechococcus sp. PCC 7002, SYNPCC7002_A0043	NIES39_K00660	BAI91715	53/0.0
crtR	β-Carotene hydroxylase	Synechocystis sp. PCC 6803, sll1468	NIES39_R00430	BAI94352	71/8e-165
crtO	β-Carotene ketolase	Synechocystis sp. PCC 6803, slr0088	NIES39_L03850	BAI92542	24/2e-26
cruF	Carotenoid 1,2-hydratase	Synechococcus sp. PCC 7002, SYNPCC7002_A2032	NIES39_B01080	BAI88865	51/7e-88

^aTakaichi and Mochimaru (2007)

^bMaresca et al. (2007)

Table 4

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Candidates of other carotenoid biosynthesis genes in Arthrospira platensis NIES-39 based on carotenoids present

Gene .	Enzyme .	Query sequence for BLASTPa,b .	Accession number .		Identity (%)/ e-value .
.	.	.	CyanoBase .	NCBI .
crtE	Geranylgeranyl diphosphate synthase	Thermosynechococcus elongatus BP-1, tll0020	NIES39_J00820	BAI91134	74/3e-162
crtB	Phytoene synthase	Synechocystis sp. PCC 6803, slr1255	NIES39_C00950	BAI88965	70/8e-156
crtP	Phytoene desaturase	Synechocystis sp. PCC 6803, slr1254	NIES39_C00940	BAI88964	80/0.0
crtQ	ζ-Carotene desaturase	Synechocystis sp. PCC 6803, slr0940	NIES39_B00980	BAI88855	75/0.0
crtH	cis-Carotene isomerase	Synechocystis sp. PCC 6803, sll0033	NIES39_C05040	BAI89370	76/0.0
cruA	Lycopene cyclase	Synechococcus sp. PCC 7002, SYNPCC7002_A2153	NIES39_A06350	BAI88473	65/0.0
cruP	Lycopene cyclase	Synechococcus sp. PCC 7002, SYNPCC7002_A0043	NIES39_K00660	BAI91715	53/0.0
crtR	β-Carotene hydroxylase	Synechocystis sp. PCC 6803, sll1468	NIES39_R00430	BAI94352	71/8e-165
crtO	β-Carotene ketolase	Synechocystis sp. PCC 6803, slr0088	NIES39_L03850	BAI92542	24/2e-26
cruF	Carotenoid 1,2-hydratase	Synechococcus sp. PCC 7002, SYNPCC7002_A2032	NIES39_B01080	BAI88865	51/7e-88

Gene .	Enzyme .	Query sequence for BLASTPa,b .	Accession number .		Identity (%)/ e-value .
.	.	.	CyanoBase .	NCBI .
crtE	Geranylgeranyl diphosphate synthase	Thermosynechococcus elongatus BP-1, tll0020	NIES39_J00820	BAI91134	74/3e-162
crtB	Phytoene synthase	Synechocystis sp. PCC 6803, slr1255	NIES39_C00950	BAI88965	70/8e-156
crtP	Phytoene desaturase	Synechocystis sp. PCC 6803, slr1254	NIES39_C00940	BAI88964	80/0.0
crtQ	ζ-Carotene desaturase	Synechocystis sp. PCC 6803, slr0940	NIES39_B00980	BAI88855	75/0.0
crtH	cis-Carotene isomerase	Synechocystis sp. PCC 6803, sll0033	NIES39_C05040	BAI89370	76/0.0
cruA	Lycopene cyclase	Synechococcus sp. PCC 7002, SYNPCC7002_A2153	NIES39_A06350	BAI88473	65/0.0
cruP	Lycopene cyclase	Synechococcus sp. PCC 7002, SYNPCC7002_A0043	NIES39_K00660	BAI91715	53/0.0
crtR	β-Carotene hydroxylase	Synechocystis sp. PCC 6803, sll1468	NIES39_R00430	BAI94352	71/8e-165
crtO	β-Carotene ketolase	Synechocystis sp. PCC 6803, slr0088	NIES39_L03850	BAI92542	24/2e-26
cruF	Carotenoid 1,2-hydratase	Synechococcus sp. PCC 7002, SYNPCC7002_A2032	NIES39_B01080	BAI88865	51/7e-88

^aTakaichi and Mochimaru (2007)

^bMaresca et al. (2007)

Fig. 6

Proposed biosynthetic pathways of carotenoids and their enzymes in Arthrospira platensis NIES-39. CrtE, geranylgeranyl diphosphate synthase; CrtB, phytoene synthase; CrtP, phytoene desaturase; CrtQ, ζ-carotene desaturase; CrtH, cis-carotene isomerase; CruA, lycopene cyclase; CrtR, β-carotene hydroxylase; CrtO, β-carotene ketolase; CruF, carotenoid 1,2-hydratase.

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In the desaturation from phytoene to lycopene, two enzymes, CrtP (phytoene desaturase) and CrtQ (ζ-carotene desaturase), are required, and CrtH (cis-carotene isomerase) is needed in darkness. The substrates of CrtR (β-carotene hydroxylase) have been reported to be β-carotene, echinenone and deoxymyxol in Synechocystis sp. PCC 6803 (Masamoto et al. 1998). Myxol-synthesizing enzymes for the right half of myxol are still unknown, but CrtR and CruF should be included (see Figs. 2, 6). As a ketocarotenoid, trace amounts of 3′-hydroxyechinenone were found. Although two distinct β-carotene ketolases, CrtO and CrtW, have been functionally confirmed in some cyanobacteria (Takaichi and Mochimaru 2007), a crtO-like gene with low homology was present and crtW-like genes were not found in the genome of A. platensis NIES-39 (Table 4).

Among the genus of Arthrospira, the genome DNA sequences of six species or strains have been published. All of these genomes have the same set of carotenoid synthesis genes, and their sequences are very similar to those of A. platensis NIES-39 (Supplementary Table S1). Synechoxanthin was absent in A. platensis NIES-39 (Fig. 1; Table 3), whereas homologous genes of cruE and cruH for synechoxanthin synthesis in Synechococcus sp. PCC 7002 (Graham and Bryant 2008) were present in the genome. It was also absent in A. maxima, and homologous genes are present (Maresca et al. 2008). There remain some possibilities that synechoxanthin is present in other culture conditions, and that cruE and/or cruH in these strains are inactive. Further investigations are needed.

Materials and Methods

Bacterial strain and culture conditions

Arthrospira platensis NIES-39 (= IAM M-135) was obtained from the Institute of Applied Microbiology of the University of Tokyo and cultured in SOT medium at 25°C under continuous illumination at 16 µmol photon m^–2 s^–1 with aeration (Hirano et al. 1998).

Purification and identification of pigments

The carotenoids were isolated and purified as follows: the pigments were extracted with acetone/methanol (7 : 2, v/v) using an ultrasonicator, and the solvent was evaporated. Each carotenoid was purified using a DEAE-Toyopearl 650 M column (Tosoh), silica gel 60 HP-TLC (Merck) and, finally, a C₁₈-HPLC column described below (Iwai et al. 2008).

Spectroscopic analysis

The HPLC system was equipped with a µBondapak C₁₈ column (8 × 100 mm, RCM type; Waters). The elution (1.8 ml min^–1) was started with a linear gradient from methanol/water (9 : 1, v/v) to 100% methanol at 20 min, and then continued with 100% methanol. The absorption spectra of the pigments were measured using an MCPD-3600 photodiode array detector (Otsuka Electronics) attached to the HPLC apparatus. For roughly quantitative analysis, the molar extinction coefficients at the maximum wavelength of each carotenoid were assumed to be the same. The CD spectra of the purified carotenoids were measured using a J-820 spectropolarimeter (JASCO) in diethyl ether/2-pentane/ethanol (5 : 5 : 2, by vol.) at room temperature. The relative molecular masses of the purified carotenoids were measured using an FD-MS; M-2500 double-focusing gas chromatograph–mass spectrometer (Hitachi) equipped with a field-desorption apparatus. The ¹H-NMR (500 MHz) spectra of the carotenoids in CDCl₃ at 24°C were measured using the UNITY INOVA-500 system (Varian) (Iwai et al. 2008).

Extraction of genomic DNA from A. platensis NIES-39

Genomic DNA was extracted from A. platensis NIES-39 cells using NucleoSpin^® Tissue (TAKARA) according to the manufacturer’s instructions.

Cloning of cruA, cruP and crtR from A. platensis NIES-39

To find sequences homologous to the known lycopene cyclase and β-carotene hydroxylase genes, homology searches of genome sequences were performed and primers were designed based on the homologous sequences obtained. The cruA, cruP and crtR genes were amplified by PCR from A. platensis NIES-39 genomic DNA by using KOD-Plus polymerase (Toyobo). The following primers were used: 5′-TCGCGGATCCGAATTCATGAAAGAGATTCTATATC-3′ as a forward primer and 5′-GTGCGGCCGCAAGCTTTTAGGAAGTCTTGGGTTGT-3′ as a reverse primer for cruA; 5′-TCGCGGATCCGAATTCATGTCTCTGACTCAACAAATTCTT-3′ as a forward primer and 5′-GTGCGGCCGCAAGCTTTTAACTATGATAATCTCCCCCGGA-3′ as a reverse primer for cruP; and 5′-TCGCGGATCCGAATTCATGTCGGAGGGCCAGAAG-3′ as a forward primer and 5′-GTGCGGCCGCAAGCTTTCAGTCCTCTGATTTAGATAACTT-3′ as a reverse primer for crtR. PCR products were cloned into the plasmid vector and sequenced. These PCR products were cloned into the pET21a vector (Merk Millipore) by an infusion cloning reaction following the manufacturer’s protocol (TAKARA). Sequencing of these clones was carried out using dideoxy chain-termination methods.

Sequence analysis of Arthrospira cruA, cruP and crtR

Homology search was performed by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and CyanoBase (http://genome.microbedb.jp/cyanobase/). The alignment of amino acids and a phylogenetic tree were constructed using CLUSTAL W (http://clustalw.ddbj.nig.ac.jp/).

Functional analysis of Arthrospira cruA, cruP and crtR in E. coli cells

The Arthrospira cruA, cruP and crtR genes were cloned into pET21a independently. These plasmids were designated pET21-CruA, pET21-CruP and pET21-CrtR, respectively. Escherichia coli strain BL21 (DE3) (Thermo Fisher Scientific) carrying the plasmid pACCRT-EIB, which contained the three carotenoid biosynthesis genes, crtE, crtB and crtI from Pantoea ananatis, and the plasmid pACCR16ΔcrtX with crtE, crtB, crtI and crtY from P. ananatis (Misawa et al. 1995), were used as the host for synthesizing lycopene and β-carotene. The plasmids pET21-CruA and pET21-CruP were transformed into the lycopene-accumulating E. coli, and the pET21-CrtR plasmid was transformed into the β-carotene-accumulating E. coli. The transformants were pre-cultured at 37°C until the OD₆₀₀ reached 0.4–0.6 in Luria–Bertani broth containing chloramphenicol (30 mg l^–1) and ampicillin (50 mg l^–1). Transformants were then treated with 0.5 mM isopropyl-β-d-thiogalactopyranoside, followed by culture for 24 h at 28°C. Two plasmids, pACCRT-EIB and pACCR16ΔcrtX, were gifts from Professor Misawa. The plasmid pACCRT-EB-RcI, which contained the crtE and crtB genes from P. ananatis and the crtI gene from R. capsulatus was also used (Takaichi et al. 1996).

Extraction and analysis of carotenoids from E. coli cells

Escherichia coli cultures were centrifuged, and pigments from cell pellets were extracted with cold acetone using a mixer for 5 min. After centrifugation, the supernatant fraction was collected and dried under N₂ flow. The dried residues were dissolved in methyl tert-butyl ether/methanol (7 : 3, v/v). HPLC analysis was performed on an Agilent 1200 Series (Agilent Technologies). A C₃₀ YMC column (250 × 4.6 mm, 5 µm) (YMC) was employed for the separation. The extract was eluted at a rate of 0.8 ml min^–1 with solvent A (water/methanol, 5 : 95, v/v) for 2 min, followed by a linear gradient from solvent A to solvent B (methyl tert-butyl ether/methanol, 7 : 3, v/v) for 23 min and solvent B alone for 15 min. For mass analysis, an LTQ Orbitrap hybrid ion-trap/Fourier transform mass spectrometer (Thermo Fisher Scientific) with an atmospheric pressure chemical ionization was used. The capillary temperature was set to 250°C, the APCI vaporizer temperature was held at 375°C and the capillary voltage was optimized to 9 V. Screening was performed in full scan, covering the range from m/z 200 up to 1,200. Carotenoids were identified by their retention time, characteristic absorption spectra and mass spectra, and compared with those of authentic lycopene, β-carotene and zeaxanthin.

Supplementary data

Supplementary data are available at PCP online.

Funding

This research was carried out without funding.

Abbreviations

Abbreviations
 
• CD

  circular dichroism

 
• GGPP

  geranylgeranyl diphosphate

 
• NMR

  nuclear magnetic resonance

Acknowledgments

We thank Professor Norihiko Misawa (Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Japan) for providing the pACCRT-EIB, pACCR16ΔcrtX and pACCRT-EB-RcI plasmids.

Disclosures

The authors have no conflicts of interest to declare.

References