Mari Mochimaru

Carotenoids and carotenogenesis in cyanobacteria: unique ketocarotenoids and carotenoid glycosides

Abstract.Cyanobacteria grow by photosynthesis, and necessarily contain chlorophyll and carotenoids, whose main functions are light harvesting and photoprotection. In this review, we discuss the carotenoids, carotenogenesis pathways, and characteristics of carotenogenesis enzymes and genes in some cyanobacteria, whose carotenogenesis enzymes have been functionally confirmed. In these cyanobacteria, various carotenoids have been identified, including the unique ketocarotenoids, echinenone and 4-ketomyxol; and the carotenoid glycosides, myxol glycosides and oscillol diglycosides. From these findings, certain carotenogenesis pathways can be proposed. The different compositions of carotenoids among these species might be due to the presence or absence of certain gene(s), or to different enzyme characteristics. For instance, two distinct β-carotene ketolases, CrtO and CrtW, are properly used in two pathways depending on the species. One β-carotene hydroxylase, CrtR, has been identified, and its substrate specificities vary across species. At present, functionally confirmed genes have been found in only a few species, and further studies are needed.

Genomic structure of an economically important cyanobacterium, Arthrospira (Spirulina) platensis NIES-39.

A filamentous non-N2-fixing cyanobacterium, Arthrospira (Spirulina) platensis, is an important organism for industrial applications and as a food supply. Almost the complete genome of A. platensis NIES-39 was determined in this study. The genome structure of A. platensis is estimated to be a single, circular chromosome of 6.8 Mb, based on optical mapping. Annotation of this 6.7 Mb sequence yielded 6630 protein-coding genes as well as two sets of rRNA genes and 40 tRNA genes. Of the protein-coding genes, 78% are similar to those of other organisms; the remaining 22% are currently unknown. A total 612 kb of the genome comprise group II introns, insertion sequences and some repetitive elements. Group I introns are located in a protein-coding region. Abundant restriction-modification systems were determined. Unique features in the gene composition were noted, particularly in a large number of genes for adenylate cyclase and haemolysin-like Ca2+-binding proteins and in chemotaxis proteins. Filament-specific genes were highlighted by comparative genomic analysis.

The cyanobacterium Anabaena sp. PCC 7120 has two distinct β-carotene ketolases: CrtO for echinenone and CrtW for ketomyxol synthesis

Two β‐carotene ketolases, CrtW and CrtO, are widely distributed in bacteria, although they show no significant sequence homology with each other. The cyanobacterium Anabaena sp. PCC 7120 was found to have two homologous genes. In the crtW deleted mutant, myxol 2′‐fucoside was present, but ketomyxol 2′‐fucoside was absent. In the crtO deleted mutant, β‐carotene was accumulated, and the amount of echinenone was decreased. Therefore, CrtW catalyzed myxol 2′‐fucoside to ketomyxol 2′‐fucoside, and CrtO catalyzed β‐carotene to echinenone. This cyanobacterium was the first species found to have both enzymes, which functioned in two distinct biosynthetic pathways.

Photobiological hydrogen production and nitrogenase activity in some heterocystous cyanobacteria

Publisher Summary This chapter discusses the activities of nitrogenase and H2 evolution in three cyanobacterial strains and some factors that affected these activities. Nitrogen-fixing heterocystous cyanobacteria are potential candidates for the development of photobiological hydrogen production systems. They produce H2 under aerobic conditions using water as an electron donor. For example A. variabilis IAM M-58 was most active in H2 production, and the amount of H2 produced was markedly higher than that of the other species. Hydrogen metabolism in these cyanobacteria involves at least three enzymes: nitrogenase, uptake hydrogenase, and bidirectional hydrogenase. Some researchers favor hydrogenase over nitrogenase as the hydrogen evolving system because of its high energy efficiency, but it was reported that continued production of H2 in air was mediated by nitrogenase in the heterocysts. To develop the hydrogen producing systems by cyanobacteria based on nitrogenase activity, it is important to find cyanobacterial strains which have a high activity of H2 production.

Three kinds of binding site for tentoxin on isolated chloroplast coupling factor 1

Tentoxin binding on chloroplast coupling factor 1 (CF1) was studied using a centrifugation column method followed by HPLC analysis. From non‐linear regression analysis of the results, the presence of three types of binding site with the following K d values was deduced: 6.9×10−8 M (first site), 1.4×10−5 M (second site), and 6.3×10−3 M (third site). The binding of one tentoxin inhibits, that of two tentoxins moderately restores, and that of three tentoxins greatly stimulates the ATPase activity of CF1. The forward rate constant of the binding of tentoxin on the first site was 6.3×103 M−1 s−1.

Survey of the Distribution of Different Types of Nitrogenases and Hydrogenases in Heterocyst-Forming Cyanobactera

As a first step toward developing the methodology for screening large numbers of heterocyst-forming freshwater cyanobacteria strains for the presence of various types of nitrogenases and hydrogenases, we surveyed the distribution of these genes and their activities in 14 strains from culture collections. The nitrogenase genes include nif1 encoding a Mo-type nitrogenase expressed in heterocysts, nif2 expressed in vegetative cells and heterocysts under anaerobic conditions, and vnf encoding a V-type nitrogenase expressed in heterocysts. Two methods proved to be valuable in surveying the distribution of nitrogenase types. The first method was Southern blot hybridization of DNA digested with two different endonucleases and hybridized with nifD1, nifD2, and vnfD probes. The second method was ethane formation from acetylene to detect the presence of active V-nitrogenase. We found that all 14 strains have nifD1 genes, and eight strains also have nifD2 genes. Four of the strains have vnfD genes, in addition to nifD2 genes. It is curious that three of these four strains had similar hybridization patterns with all of the nifD1, nifD2, and vnfD probes, suggesting that there could be some bias in strains used in the present study or in strains held in culture collections. This point will need to be assessed in the future. For surveying the distribution of hydrogenases, Southern blot hybridization was an effective method. All strains surveyed had hup genes, with the majority of them also having hox genes.

Carotenogenesis diversification in phylogenetic lineages of Rhodophyta.

Carotenoid composition is very diverse in Rhodophyta. In this study, we investigated whether this variation is related to the phylogeny of this group. Rhodophyta consists of seven classes, and they can be divided into two groups on the basis of their morphology. The unicellular group (Cyanidiophyceae, Porphyridiophyceae, Rhodellophyceae, and Stylonematophyceae) contained only β‐carotene and zeaxanthin, “ZEA‐type carotenoids.” In contrast, within the macrophytic group (Bangiophyceae, Compsopogonophyceae, and Florideophyceae), Compsopogonophyceae contained antheraxanthin in addition to ZEA‐type carotenoids, “ANT‐type carotenoids,” whereas Bangiophyceae contained α‐carotene and lutein along with ZEA‐type carotenoids, “LUT‐type carotenoids.” Florideophyceae is divided into five subclasses. Ahnfeltiophycidae, Hildenbrandiophycidae, and Nemaliophycidae contained LUT‐type carotenoids. In Corallinophycidae, Hapalidiales and Lithophylloideae in Corallinales contained LUT‐type carotenoids, whereas Corallinoideae in Corallinales contained ANT‐type carotenoids. In Rhodymeniophycidae, most orders contained LUT‐type carotenoids; however, only Gracilariales contained ANT‐type carotenoids. There is a clear relationship between carotenoid composition and phylogenetics in Rhodophyta. Furthermore, we searched open genome databases of several red algae for references to the synthetic enzymes of the carotenoid types detected in this study. β‐Carotene and zeaxanthin might be synthesized from lycopene, as in land plants. Antheraxanthin might require zeaxanthin epoxydase, whereas α‐carotene and lutein might require two additional enzymes, as in land plants. Furthermore, Glaucophyta contained ZEA‐type carotenoids, and Cryptophyta contained β‐carotene, α‐carotene, and alloxanthin, whose acetylenic group might be synthesized from zeaxanthin by an unknown enzyme. Therefore, we conclude that the presence or absence of the four enzymes is related to diversification of carotenoid composition in these three phyla.

All of α-Carotene and Its Derivatives Have a Sole Chirality?

Distribution of a-carotene and its derivatives is reported to be limited in some taxonomic groups of phototrophic organisms. In addition, C-6′ in a-carotene between e-end group and conjugated double bonds is chiral, (6′R) - and (6′S)-types. The chirality was not systematically investigated, and the reported algae and land plants contained only (6′R)-type of a-carotene and/or its derivatives. To confirm the reliability of chirality, we re-examined distribution of a-carotene and its derivatives, and analyzed their C-6′ chirality using circular dichroism or nuclear magnetic resonance spectra after purification of the carotenoids. We found a-carotene and/or its derivatives from Rhodophyceae (macrophytic type), Cryptophyceae, Euglenophyceae, Chlorarachniophyceae, Prasinophyceae, Chlorophyceae, Ulvophyceae, Charophyceae, and land plants, while we could not detected them from Glaucophyceae, Rhodophyceae (unicellular type), Chryosophyceae, Raphidophyceae, Bacillariophyceae, Phaeophyceae, Xanthophyceae, Eustigmatophyceae, Haptophyceae, and Dinophyceae. Further, loroxanthin, siphonaxanthin, and their fatty acid esters, which are synthesized from lutein, were found from Euglenophyceae, Chlorarachniophyceae, Prasinophyceae, Chlorophyceae, and Ulvophyceae. We analyzed chirality of a-carotene and/or its derivatives from around 40 species described above, and found they had only (6′R)-type.

Characterization of carotenogenesis genes in the cyanobacterium Anabaena sp. PCC 7120.

Cyanobacteria produce many kinds of carotenoids for light harvesting and light protection in photosynthesis. To elucidate the biosynthetic pathways of carotenoids in Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120), we have produced gene-disruption mutants lacking selected proposed carotenoid biosynthetic enzymes. Here we describe the construction of mutants by triparental mating. A cargo plasmid, bearing a target gene interrupted by an antibiotic-resistant cassette, is transformed to E. coli donor containing a helper plasmid, and is introduced into Anabaena cells by conjugation. Double-reciprocal recombination replaces the target genes in Anabaena genome with mutated ones on the plasmid. Carotenoids in the selected double recombinants are identified using high-performance liquid chromatography.

Functional Identification of GDP-Fucose Synthase Gene in Anabaena sp. PCC 7120

To elucidate the biosynthetic pathways of carotenoids, especially myxol 2′-fucosides in cyanobacteria, an Anabaena sp. PCC 7120 deletion mutant was analyzed that was proposed to be lacking GDP-fucose synthase (WcaG, All4826). The mutant contained polar carotenoid glycosides and also free myxol. Their glycoside moiety was identified to be rhamnose, isomer of fucose, by FD-MS and 1HNMR, instead of the usual fucose. This result suggests that in the mutant, fucose is not synthesized, and rhamnose is bound to myxol as a substrate by fucosyltransferase, so that all4826 is a functional gene for GDP-fucose synthase. We also identified a β-carotene hydroxylase, CrtR (Alr4009), in Anabaena 7120 that catalyzed deoxymyxol to myxol.