New and Rare Carotenoids Isolated from Marine Bacteria and Their Antioxidant Activities

Marine bacteria have not been examined as extensively as land bacteria. We screened carotenoids from orange or red pigments-producing marine bacteria belonging to rare or novel species. The new acyclic carotenoids with a C30 aglycone, diapolycopenedioc acid xylosylesters A–C and methyl 5-glucosyl-5,6-dihydro-apo-4,4′-lycopenoate, were isolated from the novel Gram-negative bacterium Rubritalea squalenifaciens, which belongs to phylum Verrucomicrobia, as well as the low-GC Gram-positive bacterium Planococcus maritimus strain iso-3 belonging to the class Bacilli, phylum Firmicutes, respectively. The rare monocyclic C40 carotenoids, (3R)-saproxanthin and (3R,2′S)-myxol, were isolated from novel species of Gram-negative bacteria belonging to the family Flavobacteriaceae, phylum Bacteroidetes. In this review, we report the structures and antioxidant activities of these carotenoids, and consider relationships between bacterial phyla and carotenoid structures.


Introduction
Some species of bacteria, yeast, and fungi, as well as algae and higher plants, synthesize a large number of carotenoids with different molecular structures, and more than 750 carotenoids with different structures have been isolated from natural sources [1]. Many beneficial pharmaceutical effects of carotenoids have recently been reported. Therefore, evaluating the pharmaceutical potentials of various carotenoids may represent an interesting field in medical research. However, the number of carotenoid species that have been examined for this purpose has been limited, and has included C 40 carotenoids possessing skeletons composed of 40 carbon atoms, such as dicyclic carotenoids, e.g., β-carotene, α-carotene, β-cryptoxanthin, zeaxanthin, lutein, canthaxanthin, astaxanthin, and fucoxanthin, and the acyclic carotenoid lycopene [2][3][4][5][6][7][8]. Difficulties have been associated with identifying natural sources to supply sufficient amounts of new or rare carotenoids, with the exception of carotenoids that can be isolated from a species of higher plants or algae or chemically synthesized. It has therefore been desirable to find cultivable bacteria that produce new or rare carotenoids, since they can easily be reproduced.
The generation of free radicals has been suggested to play a major role in the progression of a wide range of pathological disturbances, including myocardial and cerebral ischemia [20], atherosclerosis [21], renal failure [22], inflammation [23], and rheumatoid arthritis [24]. The subsequent peroxidative disintegration of cells and organelle membranes has also been implicated in various pathological processes [25]. Carotenoid pigments, which have been shown to possess strong antioxidant activities, have been attracting increasing attention due to their beneficial effects on human health, e.g., their potential to prevent diseases such as cancer and cardiovascular diseases [26].
We have attempted to identify novel or rare types of carotenoids from yellow or red pigment-producing marine bacteria that were classified to belong to rare or novel species by 16S rRNA analyses since 2002. The results of this screening led to the isolation of diapolycopenedioc acids xylosylesters A-C (new carotenoids) from Rubritalea squalenifaciens [27,28], methyl 5-glucosyl-5,6-dihydro-apo-4,4′-lycopenoate (a new carotenoid) from Planococcus maritimus [29], and (3R)-saproxanthin and (3R,2′S)-myxol (rare carotenoids) from a novel species belonging to the family Flavobacteriaceae [30]. In this review, we report the structures and antioxidant activities of these carotenoids, and consider relationships between bacterial phyla and carotenoid structures. [27,28] A yellow pigment-producing bacterium (strain HOact23 T ) that was found to produce squalene was isolated from the homogenate of the marine sponge Halicondria okadai, which had been collected from the Miura peninsula (Kanagawa, Japan), and was subsequently classified as a novel species in the genus Rubritalea, belonging to phylum Verrucomicrobia, based on 16S rRNA gene sequence data. The name proposed for the new taxon was Rubritalea squalenifaciens [31], with the type strain HOact23 T (=MBIC08254 T = DSM 18772 T ).
The antioxidant activity of compound 1 was evaluated using 1 O 2 suppression activity. Its IC 50 was 5.1 μM (the IC 50 values of astaxanthin and β-carotene were 8.9 μM and >100 μM, respectively).

Methyl 5-Glucosyl-5,6-Dihydro-Apo-4,4′-Lycopenoate from Planococcus Maritimus [29]
A yellow pigment-producing bacterium (strain iso-3), which was found to be solvent-tolerant, was isolated as an orange-pigmented colony from the microbial analysis of a sample derived from an intertidal sediment from the Clyde estuary, UK. The 16S rRNA gene sequence of strain iso-3 was the most similar to that of type strain Planococcus maritimus (99.5 as a similarity score, and 96.4 as an s_ab score, from the Sequence match analysis of RDP), which belongs to the class Bacilli, phylum Firmicutes, and was identified as Planococcus maritimus strain iso-3.

(3R)-Saproxanthin and (3R,2′S)-Myxol [30]
Strain 04OKA-13-27 (MBIC08261) was isolated from the dense mats of filamentous algae from within the territory of damselfish (Stegastes nigricans). Strain YM6-073 (MBIC06409) was isolated from a sediment sample collected 0.1 m below the surface of the sea by cultivating for 30 days on an HSV medium. The two marine bacteria, which had been collected off the coast of Okinawa Prefecture, were classified on the basis of this 16S rRNA gene sequences. A similarity search in the databases of the DNA Data Bank of Japan (DDBJ) and RNA Database Project II (RDPII) showed the 16S rRNA gene sequences of the both strains (04OKA-13-27 and YM6-073) to be 96.5% (1408 bp/1459 bp) similar to Stanierella latercula ATCC 23177 T , 95.5% (1324 bp/1386 bp) similar to Gaetbulimicrobium brevivitae strain SMK-19 T , and 94.2% (1306 bp/1386 bp) similar to Robiginitalea biformata strain HTCC2501 T . The phylogenetic relationship between these strains was deduced with already known species in the family Flavobacteriaceae. The result obtained revealed that the two bacterial strains should be classified as novel species of the family Flavobacteriaceae.
We found two-type new C 30 carotenoids diapolyconedioc acid xylosylesters (compound 1-3) from HOact23 T and methyl 5-glucosyl-5,6-dihydro-apo-4,4′-lycopenoate (compound 4) from iso-3 through the isolation and structural analyses of carotenoids produced by these strains. Acyclic C 30 carotenoids were previously shown to be contained in land bacteria including Staphylococcus aureus, belonging to the class Bacilli, and the methanotrophs Methylobacterium rhodium (formerly Pseudomonas rhodos), belonging to the class α-Proteobacteria, and Methylomonas sp., belonging to the class γ-Proteobacteria [17,34]. Thus, acyclic C 30 carotenoids are likely to widely exist in domain bacteria (prokaryotes), i.e., they are present not only in some low-GC Gram-positive bacteria, but also in some phyla in Gram-negative bacteria. The strong singlet-oxygen-quenching activities of our C 30 carotenoids also indicated that such C 30 carotenoids are promising as functional carotenoids, although these in vivo functional analyses have not yet been conducted.
The carotenoids produced by the six other strains isolated were all zeaxanthin, which is a common carotenoid in domain bacteria. Our study may be effective for identifying rare and new carotenoids based on its ratio (4/10). In addition, all the rare and new carotenoids (1-6) isolated possessed potent antioxidant activities.

Conclusions
Marine bacteria are likely to produce carotenoids to protect themselves from activated oxygen produced by sunlight (mainly 1 O 2 ); therefore, their potent antioxidant activities were expected and reasonable. Therefore, the techniques performed in our study effectively identified new antioxidant carotenoids.

Author Contributions
Kazutoshi Shindo performed the experiments and wrote the text; Norihiko Misawa supervised the project and corrected the manuscript.