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.

Carotenoids: Distribution, Function in Nature, and Analysis Using LC-Photodiode Array Detector (DAD)-MS and MS/MS System.

Carotenoids are tetraterpene pigments that are present in photosynthetic bacteria, some species of archaea and fungi, algae, plants, and animals. Carotenoids are essential pigments in photosynthetic organs along with chlorophylls. Carotenoids also act as photo-protectors, antioxidants, color attractants, and precursors of plant hormones in plants. Carotenoids in animals play important roles, such as precursors of vitamin A, photo-protectors, antioxidants, enhancers of immunity, and contributors to reproduction. More than 850 kinds of carotenoids are present in nature. The structures are similar and all of them are labile. Analysis of natural carotenoids requires the establishment of reliable methods for analyzing them. Liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry/mass spectrometry (MS/MS) coupled with photodiode array detector (DAD) is an important tool for analysis of natural carotenoids. Electrospray ionization and atmospheric pressure chemical ionization are commonly used for ionization of LC-MS of carotenoids. MS and MS/MS provide not only molecular weight information but also some structural information on carotenoids. Ultraviolet-visible spectra from DAD provide information on chromophore systems, which cannot be provided by MS spectral data. In the present review, I report the structural diversity and function of natural carotenoids, and also describe the techniques for analysis of natural carotenoids using the LC-DAD-MS and MS/MS system.

Identification of Desiccation Stress-Inducible Antioxidative and Antiglycative Ultraviolet-Absorbing Oxylipins, Saclipin A and Saclipin B, in an Edible Cyanobacterium Aphanothece sacrum.

Aphanothece sacrum, a freshwater cyanobacterium, is an edible cyanobacterial strain. We identified two compounds belonging to the oxylipin family that possess UV-absorbing abilities and accumulate in the dried sample of A. sacrum. The compounds, named saclipin A and saclipin B, exhibited strong UV-absorption properties with the absorption maxima at 316 and 319 nm, respectively, and the molar extinction coefficients of 26,454 and 30,555 M-1 cm-1, respectively. The chemical structures of saclipins A and B have been elucidated, revealing that they have an all-E and a 12Z isomeric relationship within the triene structure. The saclipins could be isomerized by photoirradiation, with the cis-form saclipin B proving to be more stable in methanol, ethanol, or acetonitrile. Under drought stress conditions, the accumulation of saclipins A and B in A. sacrum was found to be increased 20- and 10-fold, respectively. Purified saclipins from A. sacrum showed biocompatibility and valuable bioactivities. Specifically, saclipins exhibited radical scavenging activity, maintaining their activity even 40 min after the reaction began. Additionally, they demonstrated inhibitory activity against glycation of elastin and collagen, which are constituents of dermal tissue. Notably, saclipins showed higher activity than the well-known glycation inhibitor aminoguanidine against collagen glycation.

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.

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.

Reconstruction of the Native Biosynthetic System of Carotenoids in E. coli─Biosynthesis of a Series of Carotenoids Specific to Paprika Fruit.

Capsanthin, capsorubin, cucurbitaxanthin A, and capsanthin 3,6-epoxide, a series of carotenoids specific to the red fruit of paprika (Capsicum annuum), were produced in pathway-engineered Escherichia coli cells. These cells functionally expressed multiple genes for eight carotenogenic enzymes, two of which, paprika capsanthin/capsorubin synthase (CaCCS) and zeaxanthin epoxidase (CaZEP), were designed to be located adjacently. The biosynthesis of these carotenoids, except for capsanthin, was the first successful attempt in E. coli. In a previous study, the levels of capsanthin synthesized were low despite the high expression of the CaCCS gene, which may have been due to the dual activity of CaCCS as a lycopene ß-cyclase and CCS. An enhanced interaction between CaCCS and CaZEP that supplies antheraxanthin and violaxanthin, substrates for CaCCS, was considered to be crucial for an efficient reaction. To achieve this, we adapted S·tag and S-protein binding. The S·tag Thrombin Purification Kit (Novagen) is merchandized for in vitro affinity purification, and S·tag-fused proteins in the E. coli lysate are specifically trapped by S-proteins fixed on the agarose carrier. Furthermore, S-proteins have been reported to oligomerize via C-terminal swapping. In the present study, CaCCS and CaZEP were individually fused to the S·tag and designed to interact on oligomerized S-protein scaffolds in E. coli, which led to the biosynthesis of not only capsanthin and capsorubin but also cucurbitaxanthin A and capsanthin 3,6-epoxide. The latter reaction by CaCCS was assigned for the first time. This approach reinforces the scaffold's importance for multienzyme pathways when native biosynthetic systems are reconstructed in microorganisms.

Identification and tissue distribution of fucoxanthinol and amarouciaxanthin A fatty acid esters in fucoxanthin-fed mice.

Administered carotenoid fatty acid esters are thought to be hydrolyzed to their free forms and absorbed into the body, and information on the tissue distribution of carotenoid fatty acid esters has been limited. Fucoxanthin, a marine carotenoid, exhibits various health benefits, including anti-diabetic and anti-obesity effects. However, fucoxanthin metabolism in mammals remains unclear. Herein, we investigated the fatty acid esters of fucoxanthin metabolites, fucoxanthinol and amarouciaxanthin A, in the tissues of male C57BL/6J mice fed a fucoxanthin-containing diet for one week. Fucoxanthinol and amarouciaxanthin A-3-esters accumulated abundantly in the liver and epididymal white adipose tissue, respectively. These esters were less detectable in the serum and other tissues. Therefore, it is suggested that fucoxanthinol and amarouciaxanthin A are partially acylated in the liver and epididymal white adipose tissue after being transported through the body as their free forms. This study presents a novel carotenoid metabolic pathway in mammals.

Synthetic-biological approach for production of neoxanthin in Escherichia coli.

Carotenoids are isoprenoid pigments produced typically in plants, algae, and part of bacteria and fungi. Violaxanthin, neoxanthin, and lutein are xanthophylls biosynthesized specifically in land plants and part of algae. Nowadays, it is feasible to produce violaxanthin and lutein in Escherichia coli by pathway engineering, whereas there is no report to synthesize neoxanthin in E. coli. So far, several genes have been reported to code for neoxanthin synthases, e.g., NSY (NXS), ABA4 and VDL, which were assigned to catalyze a reaction for forming neoxanthin from violaxanthin. However, neither gene of these was common in plants or algae that biosynthesize neoxanthin, nor was confirmed by the E. coli complementation system. This study showed that the algal VDL gene (PtVDL1) was functional in recombinant E. coli cells accumulating violaxanthin to produce neoxanthin, whereas the E. coli cells failed to generate neoxanthin, when the NSY or ABA4 gene was introduced there instead of VDL. This result notes that VDL is one of veritable neoxanthin synthase 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.

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.

Preparation of Apoastaxanthinals and Evaluation of Their Anti-inflammatory Action against Lipopolysaccharide-Stimulated Macrophages and Adipocytes.

Apocarotenoids are carotenoid derivatives in which the polyene chain is cleaved via enzymatic or nonenzymatic action. They are found in animal tissues and carotenoid-containing foods. However, limited information on the biological functions of apocarotenoids is available. Here, we prepared apocarotenoids from astaxanthin via chemical oxidation and evaluated their anti-inflammatory action against macrophages and adipocytes. A series of astaxanthin-derived apoastaxanthinals, apo-11-, apo-15-, apo-14'-, apo-12'-, apo-10'-, and apo-8'-astaxanthinals, were successfully characterized by chromatography and spectroscopic analysis. The apoastaxanthinals inhibited inflammatory cytokine production and mRNA expression against lipopolysaccharide-stimulated RAW 264.7 macrophages. Apoastaxanthinals suppressed interleukin-6 overexpression in an in vitro model with macrophages and adipocytes in the following cultures: (1) contact coculture of 3T3-L1 adipocytes and RAW264.7 macrophages and (2) 3T3-L1 adipocytes in a RAW264.7-derived conditioned media. These results indicate that the apoastaxanthinals have the potential for regulation of adipose tissue inflammation observed in obesity.

Carotenoids: Carotenoid and apocarotenoid analysis-Use of E. coli to produce carotenoid standards.

Authentic samples of many carotenoids are commercially available, but are often too expensive and of insufficient purity. As an alternative, a wide range of carotenoids can be biosynthesized in recombinant Escherichia coli strains. E. coli cells naturally produce the carotenoid biosynthetic precursors and provide a blank slate for manufacturing different target carotenoids, depending on the introduced genes encoding carotenoid biosynthetic enzymes. This chapter presents a series of plasmids that carry genes encoding carotenoid biosynthetic enzymes and methods to produce in E. coli 21 different carotenoids and two apocarotenoids, which can be used as authentic standards. For the structural confirmation of these carotenoids or apocarotenoids, we show their individual spectral data containing MS/MS and UV-visible, along with their biosynthetic routes in the E. coli transformants.

Promiscuous activity of ß-carotene hydroxylase CrtZ on epoxycarotenoids leads to the formation of rare carotenoids with 6-hydroxy-3-keto-ε-ends.

Carotenoids with rare 6-hydroxy-3-keto-ε-end groups, such as piprixanthin, vitixanthin, or cochloxanthin, found in manakin birds or plants, are rare carotenoids with high antioxidant activity. The same chemical structure is found in abscisic acid or blumenol, apocarotenoids found in plants or fungi. In this study, we serendipitously discovered that the promiscuous activity of the ß-carotene hydroxylase CrtZ, a diiron-containing membrane protein, can catalyze the formation of 6-hydroxy-3-keto-ε-end by using epoxycarotenoids antheraxanthin or violaxanthin as substrate. We suggest that the reaction mechanism is similar to that of a rhodoxanthin biosynthetic enzyme. Our results provide a further understanding of the reaction mechanism of diiron-containing ß-carotene hydroxylases, as well as insight into the biosynthesis of natural compounds with 6-hydroxy-3-keto-ε-end carotenoid derivatives.

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.

Multivariate Analysis Reveals That Unsubstituted ß-Ring and C8-Keto Structures Are Important Factors for Anti-Inflammatory Activity of Carotenoids.

Carotenoids are natural lipophilic pigments with substantial health benefits. Numerous studies have demonstrated the anti-inflammatory activities of carotenoids, especially toward lipopolysaccharide-induced inflammatory responses. As such, there are few reports on the evaluation and comparison of the anti-inflammatory activities of carotenoids against inflammation induced by other stimuli. In this study, we used pathogen-associated molecular patterns, proinflammatory cytokines, degenerated proteins, and chemical irritants as inflammatory inducers to evaluate the anti-inflammatory activities of eight different carotenoids. Each carotenoid showed characteristic anti-inflammatory activities; thus, we conducted a multivariate analysis to clarify the differences among them. Unsubstituted ß-ring (i.e., provitamin A) and C8-keto structures of carotenoids were found to be crucial for their inhibitory effects on the activation of nuclear factor-kappa B and interferon regulatory factors, respectively. Furthermore, we found that ß-carotene and echinenone treatment increased intracellular retinoid levels in monocytes and that the retinoids showed the similar activities to ß-carotene and echinenone. Taken together, the intake of both provitamin A and C8-keto carotenoids (e.g., siphonaxanthin and fucoxanthin) might be effective in improving the inflammatory status of individuals. A multivariate analysis of anti-inflammatory activities is a useful method for characterizing anti-inflammatory compounds.

Construction of transgenic Ipomoea obscura that exhibits new reddish leaf and flower colors due to introduction of ß-carotene ketolase and hydroxylase genes.

Ipomoea obscura, small white morning glory, is an ornamental plant belonging to the family Convolvulaceae, and cultivated worldwide. I. obscura generates white petals including a pale-yellow colored star-shaped center (flower vein). Its fully opened flowers were known to accumulate trace amounts of carotenoids such as ß-carotene. In the present study, the embryogenic calli of I. obscura, were successfully produced through its immature embryo culture, and co-cultured with Agrobacterium tumefaciens carrying the ß-carotene 4,4'-ketolase (crtW) and ß-carotene 3,3'-hydroxylase (crtZ) genes for astaxanthin biosynthesis in addition to the isopentenyl diphosphate isomerase (idi) and hygromycin resistance genes. Transgenic plants, in which these four genes were introduced, were regenerated from the infected calli. They generated bronze (reddish green) leaves and novel petals that exhibited a color change from pale-yellow to pale-orange in the star-shaped center part. Especially, the color of their withered leaves changed drastically. HPLC-PDA-MS analysis showed that the expanded leaves of a transgenic line (T0) produced astaxanthin (5.2% of total carotenoids), adonirubin (3.9%), canthaxanthin (3.8%), and 3-hydroxyechinenone (3.6%), which indicated that these ketocarotenoids corresponded to 16.5% of the total carotenoids produced there (530 µg g-1 fresh weight). Furthermore, the altered traits of the transgenic plants were found to be inherited to their progenies by self-crossing.

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.

Capsanthin Production in Escherichia coli by Overexpression of Capsanthin/Capsorubin Synthase from Capsicum annuum.

Capsanthin, a characteristic red carotenoid found in the fruits of red pepper (Capsicum annuum), is widely consumed as a food and a functional coloring additive. An enzyme catalyzing capsanthin synthesis was identified as capsanthin/capsorubin synthase (CCS) in the 1990s, but no microbial production of capsanthin has been reported. We report here the first successful attempt to biosynthesize capsanthin in Escherichia coli by carotenoid-pathway engineering. Our initial attempt to coexpress eight enzyme genes required for capsanthin biosynthesis did not detect the desired product. The dual activity of CCS as a lycopene ß-cyclase as well as a capsanthin/capsorubin synthase likely complicated the task. We demonstrated that a particularly high expression level of the CCS gene and the minimization of byproducts by regulating the seven upstream carotenogenic genes were crucial for capsanthin formation in E. coli. Our results provide a platform for further study of CCS activity and capsanthin production in microorganisms.

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.