marine drugs Article Carotenoid Nostoxanthin Production by Sphingomonas sp. SG73 Isolated from Deep Sea Sediment Hiroshi Kikukawa 1,2 , Takuma Okaya 1, Takashi Maoka 3, Masayuki Miyazaki 4 , Keita Murofushi 2,5, Takanari Kato 6, Yoko Hirono-Hara 1, Masahiro Katsumata 6, Shoichi Miyahara 5 and Kiyotaka Y. Hara 1,2,* 1 Department of Environmental and Life Sciences, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan; [email protected] (H.K.); [email protected] (T.O.); [email protected] (Y.H.-H.) 2 Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan; [email protected] 3 Research Institute for Production Development, 15 Shimogamo-Morimotocho, Sakyo-ku, Kyoto 606-0805, Japan; [email protected] 4 Institute for Extra-Cutting-Edge Science and Technology Avant-Garde Research (X-Star), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan; [email protected] 5 Industrial Research Institute of Shizuoka Prefecture, 2078 Makigaya, Aoi-ku, Shizuoka 421-1298, Japan; [email protected] 6 Hagoromo Foods Corporation, 151 Shimazaki-cho, Shimizu-ku, Shizuoka 424-0823, Japan; [email protected] (T.K.); [email protected] (M.K.) * Correspondence: [email protected] Citation: Kikukawa, H.; Okaya, T.; Abstract: Carotenoids are used commercially for dietary supplements, cosmetics, and pharmaceu- Maoka, T.; Miyazaki, M.; Murofushi, ticals because of their antioxidant activity. In this study, colored microorganisms were isolated K.; Kato, T.; Hirono-Hara, Y.; from deep sea sediment that had been collected from Suruga Bay, Shizuoka, Japan. One strain was Katsumata, M.; Miyahara, S.; Hara, found to be a pure yellow carotenoid producer, and the strain was identified as Sphingomonas sp. K.Y. Carotenoid Nostoxanthin (Proteobacteria) by 16S rRNA gene sequence analysis; members of this genus are commonly isolated Production by Sphingomonas sp. SG73 from air, the human body, and marine environments. The carotenoid was identified as nostoxanthin Isolated from Deep Sea Sediment. ((2,3,20,30)-β,β-carotene-2,3,20,30-tetrol) by mass spectrometry (MS), MS/MS, and ultraviolet-visible Mar. Drugs 2021, 19, 274. https:// absorption spectroscopy (UV-Vis). Nostoxanthin is a poly-hydroxy yellow carotenoid isolated from doi.org/10.3390/md19050274 some photosynthetic bacteria, including some species of Cyanobacteria. The strain Sphingomonas sp. Academic Editor: Bill J. Baker SG73 produced highly pure nostoxanthin of approximately 97% (area%) of the total carotenoid pro- duction, and the strain was halophilic and tolerant to 1.5-fold higher salt concentration as compared Received: 8 April 2021 with seawater. When grown in 1.8% artificial sea salt, nostoxanthin production increased by 2.5-fold Accepted: 11 May 2021 as compared with production without artificial sea salt. These results indicate that Sphingomonas sp. Published: 14 May 2021 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. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: carotenoid; nostoxanthin; Sphingomonas; deep sea microorganism published maps and institutional affil- iations. 1. Introduction Carotenoids are isoprenoids, and these yellow to orange-red pigments are widely Copyright: © 2021 by the authors. distributed in nature [1]. They are predominantly synthesized by phototrophic organisms Licensee MDPI, Basel, Switzerland. and by some non-phototrophic fungi, bacteria, and archaea [1,2]. Carotenoids are used This article is an open access article commercially, especially astaxanthin, as a color enhancer for marine aquaculture resources distributed under the terms and such as salmon, and for dietary supplements, cosmetics, and pharmaceuticals because of conditions of the Creative Commons its high antioxidant activity [3,4]. Industrial demand for carotenoids has recently increased, Attribution (CC BY) license (https:// which has resulted in an increase in studies of carotenoid production, especially using creativecommons.org/licenses/by/ marine organisms [5–8]. 4.0/). Mar. Drugs 2021, 19, 274. https://doi.org/10.3390/md19050274 https://www.mdpi.com/journal/marinedrugs Mar. Drugs 2021, 19, x 2 of 10 The yellow carotenoid nostoxanthin ((2,3,2′,3′)-β,β-carotene-2,3,2′,3′-tetrol) (Figure 1) is biosynthesized from zeaxanthin by the addition of two hydroxyl groups [9]. On the basis of the chemical structure of nostoxanthin, it is expected to have a similar high anti- oxidative activity as that of zeaxanthin, but no study revealing the antioxidative activity of nostoxanthin has been reported. This carotenoid has been isolated from some prokary- otes including some species of Cyanobacteria: the bacteriochlorophyll-a containing bacte- rium Sandarakinorhabdus limnophila, photosynthetic bacteria, the non-marine Brevundimo- Mar. Drugs 2021, 19, 274 nas sp., and most Sphingomonas species [9–16]. However,2 of 10the concentration of nostoxan- thin in known nostoxanthin-producing bacteria is low [9]. TheIn yellow this carotenoid study, nostoxanthin we isolated ((2,3,2 0,3a0 )-microorganismβ,β-carotene-2,3,20,30-tetrol) from (Figure deep1 ) sea sediment that had been iscollected biosynthesized from from Suruga zeaxanthin Bay, by the Shizuoka, addition of two Japan hydroxyl (Tab groupsle 1) [9 ].and On thewe identified the chemical struc- basis of the chemical structure of nostoxanthin, it is expected to have a similar high antiox- idativeture activityof the as thatmain of zeaxanthin, carotenoid but no studyproduced revealing theby antioxidative the isolated activity microorganism. of This organism nostoxanthinshowed high has been nostoxanthin reported. This carotenoid composition has been isolated in total from some carotenoids, prokaryotes and we identified the bacte- including some species of Cyanobacteria: the bacteriochlorophyll-a containing bacterium Sandarakinorhabdusrial strain. Furthermore, limnophila, photosynthetic we characterized bacteria, the non-marine the saltBrevundimonas tolerancesp., and nostoxanthin productiv- and most Sphingomonas species [9–16]. However, the concentration of nostoxanthin in knownity by nostoxanthin-producing supplemention bacteria with isartificial low [9]. sea salt. FigureFigure 1. The 1. structureThe structure of nostoxanthin. of nostoxanthin. In this study, we isolated a microorganism from deep sea sediment that had been collectedTable 1. from Sampling Suruga Bay, spots Shizuoka, in the Japan Suruga (Table 1Bay.) and we identified the chemical structure of the main carotenoid produced by the isolated microorganism. This organism showed high nostoxanthin composition in total carotenoids, and we identified the bacterial strain. Furthermore, we characterized the salt tolerance and nostoxanthinLocation productivity by Water Depth supplementation withSpot artificial 1 sea salt. 34°8482′ N, 138°3589′ E 376 m Table 1. Sampling spotsSpot in the 2 Suruga Bay. 34°8526′ N, 138°3994′ E 691 m Spot 3 Location34°9149′ N, 138°6507 Water Depth ′ E 1548 m Spot 1 34◦84820 N, 138◦35890 E 376 m Spot 2Spot 4 34◦85260 N, 138◦399434°91520 E′ N, 138°6534 691 m ′ E 1523 m Spot 3Spot 5 34◦91490 N, 138◦650734°18720 E′ N, 138°4220 1548 m ′ E 3595 m Spot 4 34◦91520 N, 138◦65340 E 1523 m Spot 5Spot 6 34◦18720 N, 138◦422034°27360 E′ N, 138°4813 3595 m ′ E 3233 m Spot 6 34◦27360 N, 138◦48130 E 3233 m Spot 7Spot 7 34◦78470 N, 138◦634534°78470 E′ N, 138°6345 1652 m ′ E 1652 m Spot 8 34◦98670 N, 138◦69670 E 1299 m Spot 9Spot 8 34◦24090 N, 138◦480334°98670 E′ N, 138°6967 3372 m ′ E 1299 m ◦ 0 ◦ 0 Spot 10Spot 9 34 7835 N, 138 617134°2409E′ N, 138°4803 1730 m ′ E 3372 m 2. Results and DiscussionSpot 10 34°7835′ N, 138°6171′ E 1730 m 2.1. Isolation of a Yellow Microorganism from Deep Sea Sediment Firstly, colored microorganisms were isolated from deep sea sediment. One isolate, strain2. Results SG73, from and the samplingDiscussion Spot 2 in Table1, showed accumulation of highly pure carotenoid at a retention time of approximately 3.8 min and three minor peaks at a retention time2.1. of Isolation 4 to 5 min (Figure of a 2YellowA). The three Microorganism minor peaks were detectedfrom Deep in the chromatographySea Sediment for HPLC and LC/MS (Supplemental Figure S1). The purity of the main carotenoid was 97% (area%).Firstly, These colored carotenoids microorganisms did not correspond to thewere peaks isolat of authenticed from standards deep sea sediment. One isolate, ofstrain astaxanthin, SG73,α-carotene, from ortheβ-carotene sampling (Figure Spot2B). The 2 major in Tabl carotenoide 1, fromshowed strain accumulation of highly pure SG73 was expected to have higher hydrophilicity than astaxanthin because of its faster retentioncarotenoid time. at a retention time of approximately 3.8 min and three minor peaks at a reten- tion time of 4 to 5 min (Figure 2A). The three minor peaks were detected in the chroma- tography for HPLC and LC/MS (Supplemental Figure S1). The purity of the main carote- noid was 97% (area%). These carotenoids did not correspond to the peaks of authentic standards of astaxanthin, α-carotene, or β-carotene (Figure 2B). The major carotenoid from strain SG73 was expected to have higher hydrophilicity than astaxanthin because of its faster retention time.