An ependymin-related blue carotenoprotein decorates marine blue sponge.

Marine animals display diverse vibrant colors, but the mechanisms underlying their specific coloration remain to be clarified. Blue coloration is known to be achieved through a bathochromic shift of the orange carotenoid astaxanthin (AXT) by the crustacean protein crustacyanin, but other examples have not yet been well investigated. Here, we identified an ependymin (EPD)-related water-soluble blue carotenoprotein responsible for the specific coloration of the marine blue sponge Haliclona sp. EPD was originally identified in the fish brain as a protein involved in memory consolidation and neuronal regeneration. The purified blue protein, designated as EPD-related blue carotenoprotein-1, was identified as a secreted glycoprotein. We show that it consists of a heterodimer that binds orange AXT and mytiloxanthin and exhibits a bathochromic shift. Our crystal structure analysis of the natively purified EPD-related blue carotenoprotein-1 revealed that these two carotenoids are specifically bound to the heterodimer interface, where the polyene chains are aligned in parallel to each other like in ß-crustacyanin, although the two proteins are evolutionary and structurally unrelated. Furthermore, using reconstitution assays, we found that incomplete bathochromic shifts occurred when the protein bound to only AXT or mytiloxanthin. Taken together, we identified an EPD in a basal metazoan as a blue protein that decorates the sponge body by binding specific structurally unrelated carotenoids.

Identification of a novel monocyclic carotenoid and prediction of its biosynthetic genes in Algoriphagus sp. oki45.

Bacteria belonging to the genus Algoriphagus have been isolated from various sources, such as Antarctic sea ice, seawater, and sediment, and some strains are known to produce orange to red pigments. However, the pigment composition and biosynthetic genes have not been fully elucidated. A new red-pigmented Algoriphagus sp. strain, oki45, was isolated from the surface of seaweed collected from Senaga-Jima Island, Okinawa, Japan. Genome comparison revealed oki45's average nucleotide identity of less than 95% to its closely related species, Algoriphagus confluentis NBRC 111222 T and Algoriphagus taiwanensis JCM 19755 T. Comprehensive chemical analyses of oki45's pigments, including 1H and 13C nuclear magnetic resonance and circular dichroism spectroscopy, revealed that the pigments were mixtures of monocyclic carotenoids, (3S)-flexixanthin ((3S)-3,1'-dihydroxy-3',4'-didehydro-1',2'-dihydro-ß,ψ-caroten-4-one) and (2R,3S)-2-hydroxyflexixanthin ((2R,3S)-2,3,1'-trihydroxy-3',4'-didehydro-1',2'-dihydro-ß,ψ-caroten-4-one); in particular, the latter compound was new and not previously reported. Both monocyclic carotenoids were also found in A. confluentis NBRC 111222 T and A. taiwanensis JCM 19755 T. Further genome comparisons of carotenoid biosynthetic genes revealed the presence of eight genes (crtE, crtB, crtI, cruF, crtD, crtYcd, crtW, and crtZ) for flexixanthin biosynthesis. In addition, a crtG homolog gene encoding 2,2'-ß-hydroxylase was found in the genome of the strains oki45, A. confluentis NBRC 111222 T, and A. taiwanensis JCM 19755 T, suggesting that the gene is involved in 2-hydroxyflexixanthin synthesis via 2-hydroxylation of flexixanthin. These findings expand our knowledge of monocyclic carotenoid biosynthesis in Algoriphagus bacteria. KEY POINTS: • Algoriphagus sp. strain oki45 was isolated from seaweed collected in Okinawa, Japan. • A novel monocyclic carotenoid 2-hydroxyflexixanthin was identified from strain oki45. • Nine genes for 2-hydroxyflexixanthin biosynthesis were found in strain oki45 genome.

Astaxanthin: Past, Present, and Future.

Astaxanthin (AX), a lipid-soluble pigment belonging to the xanthophyll carotenoids family, has recently garnered significant attention due to its unique physical properties, biochemical attributes, and physiological effects. Originally recognized primarily for its role in imparting the characteristic red-pink color to various organisms, AX is currently experiencing a surge in interest and research. The growing body of literature in this field predominantly focuses on AXs distinctive bioactivities and properties. However, the potential of algae-derived AX as a solution to various global environmental and societal challenges that threaten life on our planet has not received extensive attention. Furthermore, the historical context and the role of AX in nature, as well as its significance in diverse cultures and traditional health practices, have not been comprehensively explored in previous works. This review article embarks on a comprehensive journey through the history leading up to the present, offering insights into the discovery of AX, its chemical and physical attributes, distribution in organisms, and biosynthesis. Additionally, it delves into the intricate realm of health benefits, biofunctional characteristics, and the current market status of AX. By encompassing these multifaceted aspects, this review aims to provide readers with a more profound understanding and a robust foundation for future scientific endeavors directed at addressing societal needs for sustainable nutritional and medicinal solutions. An updated summary of AXs health benefits, its present market status, and potential future applications are also included for a well-rounded perspective.

A novel carotenoid biosynthetic route via oxidosqualene.

Over 800 known carotenoids are synthesized from phytoene or 4,4'-diapophytoene (dehydrosqualene) characterized by three conjugated double bonds. In this paper, we report that carotenoid desaturase CrtN from Staphylococcus aureus and Methylomonas can accept oxidosqualene, which is the precursor for plant- or animal-type triterpenoids, yielding the yellow carotenoid pigments with 8, 9, or 10 conjugated double bonds. The resulting pathway is the second nonnatural route for carotenoid pigments and the first pathway for carotenoid pigments not biosynthesized via (diapo)phytoene.

Major Carotenoids of Meiothermus ruber Are Deinoxanthin Glucoside Esters, Not Meiothermoxanthin Glucoside Esters.

Meiothermus ruber DSMZ 1279T was isolated from a hot spring in Kamchatka and was red in color. The major carotenoid present was reported to be 1'-(ß-d-glucopyranosyloxy)-3,4,3',4'-tetradehydro-1',2'-dihydro-ß,ψ-caroten-2-one after saponification (Burgess et al. J. Nat. Prod. 1999, 62, 859-863). In this study, we purified the major carotenoids in this species without saponification. We then reidentified the major carotenoids present using spectroscopic data, including electronic circular dichroism (ECD), 1H NMR, rotating-frame nuclear Overhauser effect spectroscopy (ROESY), 13C NMR, heteronuclear single-quantum correlation spectroscopy (HSQC), heteronuclear multiple-bond correlation spectroscopy (HMBC), and MS, and enzymatic hydrolysis of fatty acid moieties and found deinoxanthin glucoside iso fatty acid esters. The bound fatty acids present included four iso types, and their composition differed from cellular lipids. Moreover, the previously identified carotenoid glucoside was a saponification artifact of deinoxanthin glucoside esters. Ketomyxocoxanthin glucoside esters and 1'-hydroxytorulene glucoside esters were also present. On the basis of the identification of carotenoids and the whole genome sequence of M. ruber, we propose a carotenoid biosynthetic pathway and note the corresponding genes.

A Novel Carotenoid with a Unique 2,6-Cyclo-ψ-End Group, Roretziaxanthin, from the Sea Squirt Halocynthia roretzi.

A novel carotenoid with a unique 2,6-cyclo-ψ-end group, named roretziaxanthin (1), was isolated from the sea squirt Halocynthia roretzi as a minor carotenoid along with (3S,3'S)-astaxanthin, alloxanthin, halocynthiaxanthin, mytiloxanthin, mytiloxanthinone, etc. This structure was determined to be 3-hydroxy-1',16'-didehydro-1',2'-dihydro-2',6'-cyclo-ß,ψ-carotene-4,4'-dione by UV-VIS, MS, and NMR spectral data. The formation mechanism of roretziaxanthin in the sea squirt was discussed.

Carotenoid Metabolism in Aquatic Animals.

Aquatic animals contain various carotenoids that exhibit structural diversity. These carotenoids originate from algae or partly from some bacteria. Herbivorous animals directly ingest carotenoids from dietary algae and metabolize them. Carnivorous animals ingest carotenoids from dietary herbivorous animals and metabolize them. Therefore, carotenoids found in these animals reflect the food chain as well as the metabolic pathways. Carotenoids in aquatic animals are described from the viewpoints of natural product chemistry, metabolism, food chain, and chemosystematics.

Carotenoid Metabolism in Terrestrial Animals.

Terrestrial animals, especially insects, contain various carotenoids that show structural diversity. These animals accumulated carotenoids derived from plants and other animals and modified them through metabolic reactions. Therefore, most of the carotenoids found in terrestrial animals originated from plants. Conversely, recent investigation revealed that some species of aphids and spider mites synthesized carotenoid themselves by carotenoid biosynthetic genes, which were horizontally transferred from fungi. In this chapter, carotenoids in terrestrial animals are described from the viewpoints of natural product chemistry, metabolism, food chain, and chemosystematics.

Fecal Microflora from Dragonflies and Its Microorganisms Producing Carotenoids.

The intestines of insects are assumed to be the niche of various microbial groups, and a unique microflora could be formed under environmental conditions different from mammalian intestinal tracts. This chapter describes the bacterial flora formed in the intestines of two dragonfly species, "akatombo" (the red dragonfly; Sympetrum frequens) and "usubaki-tombo" (Pantala flavescens), which fly over a long distance, and carotenoid-producing microorganisms isolated from this flora. C30 carotenoids, which were produced by a bacterium Kurthia gibsonii isolated from S. frequens, were structurally determined.

Carotenoid Biosynthesis in Animals: Case of Arthropods.

All the organisms that belong to the animal kingdom had been believed not to synthesize carotenoids de novo. However, several groups of arthropods, which contain aphids, spider mites, and flies belonging to the family Cecidomyiidae, have been unexpectedly shown to possess carotenoid biosynthesis genes of fungal origin since 2010. On the other hand, few reports have shown direct evidence corroborating the catalytic functions of the enzymes that the carotenogenic genes encode. In the present review, we want to overview the carotenoid biosynthetic pathway of the pea aphid (Acyrthosiphon pisum), which was elucidated through functional analysis of carotenogenic genes that exist on its genome using Escherichia coli that accumulates carotenoid substrates, in addition to carotenoid biosynthesis in the other carotenogenic arthropods.

Biological Activities of Paprika Carotenoids, Capsanthin and Capsorubin.

Paprika Capsicum annuum L. (Solanaceae) contains various carotenoids such as capsanthin, capsorubin, cryptocapsin cucurbitaxanthin A, ß-cryptoxanthin, capsanthin epoxide, zeaxanthin, and ß-carotene. Especially, capsanthin and capsorubin are characteristic carotenoid in paprika. They show strong antioxidative effect. Furthermore, these carotenoids show preventive effect of obesity-related diseases. Dietary paprika carotenoids are absorbed in blood, and they are detected in erythrocytes. It contributes to upregulate endurance performance of athletes by reducing oxygen consumption (VO2) and the heart rate.

Carotenoid Nostoxanthin Production by Sphingomonas sp. SG73 Isolated from Deep Sea Sediment.

Carotenoids are used commercially for dietary supplements, cosmetics, and pharmaceuticals because of their antioxidant activity. In this study, colored microorganisms were isolated from deep sea sediment that had been collected from Suruga Bay, Shizuoka, Japan. One strain was found to be a pure yellow carotenoid producer, and the strain was identified as Sphingomonas sp. (Proteobacteria) by 16S rRNA gene sequence analysis; members of this genus are commonly isolated from air, the human body, and marine environments. The carotenoid was identified as nostoxanthin ((2,3,2',3')-ß,ß-carotene-2,3,2',3'-tetrol) by mass spectrometry (MS), MS/MS, and ultraviolet-visible absorption spectroscopy (UV-Vis). Nostoxanthin is a poly-hydroxy yellow carotenoid isolated from some photosynthetic bacteria, including some species of Cyanobacteria. The strain Sphingomonas sp. SG73 produced highly pure nostoxanthin of approximately 97% (area%) of the total carotenoid production, and the strain was halophilic and tolerant to 1.5-fold higher salt concentration as compared with seawater. When grown in 1.8% artificial sea salt, nostoxanthin production increased by 2.5-fold as compared with production without artificial sea salt. These results indicate that Sphingomonas sp. SG73 is an efficient producer of nostoxanthin, and the strain is ideal for carotenoid production using marine water because of its compatibility with sea salt.

Protective Effect of Antioxidative Liposomes Co-encapsulating Astaxanthin and Capsaicin on CCl4-Induced Liver Injury.

Our previous study reported that co-encapsulation of potent antioxidants astaxanthin (Asx) and capsaisin (Cap) into liposomes brought about synergistically higher antioxidative activity than the calculated additive activity of each single antioxidant encapsulating liposome. Based on the previous computational chemistry analysis, the synergistic effect was revealed to be resulted from intermolecular interactions between Asx, especially 3R,3'R-form of Asx stereoisomer (Asx-R), and Cap, by which changes of electronic states of the polyene moiety of Asx-R were induced. Although liposomes co-encapsulating Asx-R and Cap (Asx-R/Cap-Lipo) at an optimal ratio clearly showed synergistic antioxidative activity in vitro, it is unclear whether the effective antioxidative activity derived from intermolecular interaction between Asx-R and Cap is also exerted in vivo. Therefore, in this study, we investigated therapeutic potential of Asx-R/Cap-Lipo as an antioxidant formulation in vivo. For this purpose, we employed carbon tetrachloride (CCl4)-induced acute liver injury rat model, since CCl4 is known to cause oxidative damage in liver. CCl4 administration significantly increased the levels of aspartate transaminase (AST) and alanine aminotransferase (ALT). Intravenous combined administration of liposomes encapsulating Asx-R (Asx-R-Lipo) and liposomes encapsulating Cap (Cap-Lipo) significantly decreased CCl4-induced increase of AST and ALT levels. Importantly, the treatment with Asx-R/Cap-Lipo tended to show higher protective effect on acute liver injury than combined treatment with Asx-R-Lipo plus Cap-Lipo. These results suggest that co-encapsulated Asx-R and Cap in liposomal membranes could exert more effective antioxidative activities in vivo, and that Asx-R/Cap-Lipo would be a hopeful antioxidant formulation for treating reactive oxygen species-related diseases.

Evaluation of Intestinal Absorption of Dietary Halocynthiaxanthin, a Carotenoid from the Sea Squirt Halocynthia roretzi.

Halocynthiaxanthin is an acetylenic carotenoid mainly found in Halocynthia roretzi. To date, several bioactivities of halocynthiaxanthin have been reported, but its mechanism of digestion and absorption in mammals has not been studied yet. In this study, we evaluated the intestinal absorption of halocynthiaxanthin in mice. The halocynthiaxanthin-rich fraction was prepared from the tunicate Halocynthia roretzi. Mice were orally administered the fraction at a dose of 5 mg/kg body weight. The halocynthiaxanthin levels in the plasma, liver, and small intestine, were quantified using HPLC-PDA, 1, 3, 6, and 9 h after ingestion. The halocynthiaxanthin-rich fraction mainly consisted of the all-trans form and a small amount of cis forms. These three isomers were detected in the plasma of mice 3 h after ingestion. Time-course changes after the ingestion of this fraction were found, with cis isomers being more abundant than the all-trans isomer in the mouse plasma and liver. In the small intestine, however, the all-trans isomer was primarily detected. The possibility that cis isomers might be absorbed rapidly from the small intestine cannot be denied, but our results suggest that dietary all-trans-halocynthiaxanthin might be isomerized to the cis isomer after intestinal absorption.

Antitumour Effects of Astaxanthin and Adonixanthin on Glioblastoma.

Several antitumour drugs have been isolated from natural products and many clinical trials are underway to evaluate their potential. There have been numerous reports about the antitumour effects of astaxanthin against several tumours but no studies into its effects against glioblastoma. Astaxanthin is a red pigment found in crustaceans and fish and is also synthesized in Haematococcus pluvialis; adonixanthin is an intermediate product of astaxanthin. It is known that both astaxanthin and adonixanthin possess radical scavenging activity and can confer a protective effect on several damages. In this study, we clarified the antitumour effects of astaxanthin and adonixanthin using glioblastoma models. Specifically, astaxanthin and adonixanthin showed an ability to suppress cell proliferation and migration in three types of glioblastoma cells. Furthermore, these compounds were confirmed to transfer to the brain in a murine model. In the murine orthotopic glioblastoma model, glioblastoma progression was suppressed by the oral administration of astaxanthin and adonixanthin at 10 and 30 mg/kg, respectively, for 10 days. These results suggest that both astaxanthin and adonixanthin have potential as treatments for glioblastoma.

Effect of astaxanthin-rich extract derived from Paracoccus carotinifaciens on the status of stress and sleep in adults.

This study investigated the effect of a dietary supplement containing astaxanthin-rich extract derived from Paracoccus carotinifaciens (astaxanthin supplement) on the status of stress and sleep in individuals aged 20-64 years. Twenty-five subjects orally administered 12 mg astaxanthin/day of astaxanthin supplement for 8 weeks (astaxanthin group) and 29 subjects given a placebo (placebo group) were evaluated with Profile of Mood States 2nd Edition for stress and Oguri-Shirakawa-Azumi Sleep Inventory for Middle-aged and Aged version for sleep. We did not observe any significant intergroup differences in the stress and sleep. A subgroup analysis was performed after dividing the subjects into two groups: those who scored >65 and those who scored ≤65 in the "Depression-Dejection" dimension of Profile of Mood States 2nd Edition. The sleep of subjects who scored >65 ("Depression-Dejection") showed significant improvement in the astaxanthin group compared with the placebo group, whereas no significant improvement was observed in stress and the other subjects. Our results indicate that people who tend to be strongly depressed may experience improved sleep after ingesting astaxanthin supplement. On the basis of the parameters tested, administration of astaxanthin supplement was not associated with any problems related to safety. Clinical registration: This study has been registered at the University Hospital Medical Information Network (https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000038619) on August 24, 2018 as "A study to evaluate the effect of intake of astaxanthin on the status of stress and sleep in adults," Identification No. UMIN000033863.

Pathway engineering for efficient biosynthesis of violaxanthin in Escherichia coli.

Carotenoids are naturally synthesized in some species of bacteria, archaea, and fungi (including yeasts) as well as all photosynthetic organisms. Escherichia coli has been the most popular bacterial host for the heterologous production of a variety of carotenoids, including even xanthophylls unique to photosynthetic eukaryotes such as lutein, antheraxanthin, and violaxanthin. However, conversion efficiency of these epoxy-xanthophylls (antheraxanthin and violaxanthin) from zeaxanthin remained substantially low. We here examined several factors affecting their productivity in E. coli. Two sorts of plasmids were introduced into the bacterial host, i.e., a plasmid to produce zeaxanthin due to the presence of the Pantoea ananatis crtE, crtB, crtI, crtY, and crtZ genes in addition to the Haematococcus pluvialis IDI gene, and one containing each of zeaxanthin epoxidase (ZEP) genes originated from nine photosynthetic eukaryotes. It was consequently found that paprika (Capsicum annuum) ZEP (CaZEP) showed the highest conversion activity. Next, using the CaZEP gene, we performed optimization experiments in relation to E. coli strains as the production hosts, expression vectors, and ribosome-binding site (RBS) sequences. As a result, the highest productivity of violaxanthin (231 µg/g dry weight) was observed, when the pUC18 vector was used with CaZEP preceded by a RBS sequence of score 5000 in strain JM101(DE3).

Structural and functional analysis of the carotenoid biosynthesis genes of a Pseudomonas strain isolated from the excrement of Autumn Darter.

There are many reports about carotenoid-producing bacteria and carotenoid biosynthesis genes. In databases for Pseudomonas genome sequences, there are genes homologous to carotenoid biosynthesis genes, but the function of these genes in Pseudomonas has not been elucidated. In this study, we cloned the carotenoid biosynthesis genes from a Pseudomonas sp. strain, named Akiakane, which was isolated from the excrement of the Autumn Darter dragonfly. Using an Escherichia coli functional expression system, we confirmed that the idi, crtE, crtB, crtI, and crtY gene products of the Akiakane strain show predictable catalytic activities. A cluster of six genes was also found, which was comparable to other carotenoid-producing bacteria that belong to the α-Proteobacteria or γ-Proteobacteria class.

Carotenoid Profiling of a Red Seaweed Pyropia yezoensis: Insights into Biosynthetic Pathways in the Order Bangiales.

Carotenoids are natural pigments that contribute to light harvesting and photo-protection in photosynthetic organisms. In this study, we analyzed the carotenoid profiles, including mono-hydroxy and epoxy-carotenoids, in the economically valuable red seaweed Pyropia yezoensis, to clarify the detailed biosynthetic and metabolic pathways in the order Bangiales. P. yezoensis contained lutein, zeaxanthin, α-carotene, and ß-carotene, as major carotenoids in both the thallus and conchocelis stages. Monohydroxy intermediate carotenoids for the synthesis of lutein with an ε-ring from α-carotene, α-cryptoxanthin (ß,ε-caroten-3'-ol), and zeinoxanthin (ß,ε-caroten-3-ol) were identified. In addition, ß-cryptoxanthin, an intermediate in zeaxanthin synthesis from ß-carotene, was also detected. We also identified lutein-5,6-epoxide and antheraxanthin, which are metabolic products of epoxy conversion from lutein and zeaxanthin, respectively, by LC-MS and ¹H-NMR. This is the first report of monohydroxy-carotenoids with an ε-ring and 5,6-epoxy-carotenoids in Bangiales. These results provide new insights into the biosynthetic and metabolic pathways of carotenoids in red seaweeds.

Effect of astaxanthin-rich extract derived from Paracoccus carotinifaciens on cognitive function in middle-aged and older individuals.

This study was conducted to investigate the effect of dietary supplement containing astaxanthin-rich extract derived from Paracoccus carotinifaciens (astaxanthin supplement) on cognitive function of subjects aged 45-64 years. Cognitive functions of 28 subjects orally administered 8 mg astaxanthin/day of astaxanthin supplement for 8 weeks (astaxanthin group) and 26 subjects given a placebo (placebo group) were compared by word memory test, verbal fluency test, and Stroop test. The astaxanthin group experienced significantly larger increase in blood astaxanthin level than the placebo group. However, there were no significant intergroup differences in the results of the tests. A subgroup analysis was performed after dividing subjects into the <55 years old and ≥55 years old age groups. The result of "words recalled after 5 minutes" in word memory test in <55 years old subjects showed significant improvement in the astaxanthin group than in the placebo group, which was not found in ≥55 years old subjects. Our results indicate that people aged 45-54 years may experience improved cognitive function after ingesting astaxanthin supplement for 8 weeks. On the basis of the parameters tested, administration of astaxanthin supplement was not associated with any problems related to safety.