marine drugs

Article Carotenoid Nostoxanthin Production by 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 () by 16S rRNA gene sequence analysis; members of this 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 , 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 Location 34°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◦3994 34°91520 E′ N, 138°6534 691 m ′ E 1523 m Spot 3Spot 5 34◦91490 N, 138◦6507 34°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◦4220 34°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◦6345 34°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◦4803 34°98670 E′ N, 138°6967 3372 m ′ E 1299 m ◦ 0 ◦ 0 Spot 10Spot 9 34 7835 N, 138 6171 34°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.

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FigureFigure 2.2. Chromatograph of carotenoids. (A) Carotenoids extracted from strain SG73; (B) authentic standards: astaxanthin, αα-carotene,-carotene, andand ββ-carotene;-carotene; ((CC)) yellowyellow wetwet coloniescolonies ofof strainstrain SG73.SG73. TheThe coloniescolonies werewere growngrown onon YMYM agarager mediummedium atat 2222 ◦°CC forfor 33 days.days. The separation separation was was performed performed at at 35 35 °C,◦C, with with methanol/tetrahydrofuran methanol/tetrahydrofuran = 8/2 = 8/2 (v/v ()v as/v the) as mobile the mobile phase phase at a flow at a rate of 1.0 mL/min, and the detection was performed at 470 nm with an SPD-20AV detector. flow rate of 1.0 mL/min, and the detection was performed at 470 nm with an SPD-20AV detector.

2.2. IdentificationIdentification of the Hydrophilic Carotenoid-ProducingCarotenoid-Producing StrainStrain SG73SG73 The colony of of strain strain SG73 SG73 exhibited exhibited a strong a strong yellow yellow color color and anda smooth a smooth surface surface (Fig- (ureFigure 2C).2 CThe). The16S 16SrRNA rRNA gene gene sequence sequence (1412 (1412 bp, accession bp, accession no. LC618681) no. LC618681) amplified amplified from fromstrain strainSG73 SG73had 99.8% had 99.8%homology homology to that to of that Sphingomonas of Sphingomonas sanguinis sanguinis NBRC NBRC13937. 13937.Strain StrainSG73 was SG73 clustered was clustered with S. with sanguinisS. sanguinis NBRCNBRC 13937 13937in the inphylogenetic the phylogenetic tree, but tree, the but boot- the bootstrapstrap value value was was low low (76%) (76%) (Figure (Figure 3).3 ).However, However, these these results results indicate indicate that there maymay bebe only a small genetic distance between thesethese strains.strains. Therefore, strain SG73 waswas identifiedidentified as Sphingomonas sp.sp. SG73. Sphingomonas isis a group of Gram-negative, rod-shaped,rod-shaped, chemoheterotrophic, aerobicaerobic bacteria. SphingomonasSphingomonas speciesspecies are are known to contain glycosphingolipidsglycosphingolipids [[17],17], andand areare widelywidely distributeddistributed inin nature,nature, having having been been isolated isolated from from many many different different air, air, land, land, and and water wa- habitats,ter habitats, including including seawater seawater and and marine marine soils. soils. Furthermore, Furthermore,Sphingomonas Sphingomonasspecies species havehave been reported to produceproduce thethe carotenoidscarotenoids lycopene,lycopene, ββ-carotene,-carotene, zeaxanthin,zeaxanthin, caloxanthin,caloxanthin, and nostoxanthin [[9,13,17].9,13,17].

2.3. Identification of the Main Carotenoid Produced by Sphingomonas sp. SG73 Identification of the main carotenoid produced by Sphingomonas sp. SG73 was con- ducted by high-performance liquid chromatography (HPLC) equipped with an ultraviolet- visible absorption spectrometer (UV-Vis), an electro-spray ionization (ESI) a time-of-flight (TOF) mass spectrometer (MS), and MS/MS systems. The molecular formula of this carotenoid was determined to be C40H56O4 by ESI MS spectra data of m/z 601.4243 [M + H]+ and m/z 623.4085 [M + Na]+ (Figure4A). The MS/MS spectra that used pre- cursor ion at m/z 600.41 [M+] showed characteristic product ion patterns of tetrahydroxy carotenoid (Figure4B). These product ion patterns were identical with nostaoxanthin [ 18], and major product ions from carotenoids [19], m/z 508.3515 [M-92] generated by elim- ination of toluene moiety from the polyene chain and m/z 494.3283 [M-106] generated

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Mar. Drugs 2021, 19, x by elimination of xylene moiety from the polyene chain, were observed. The UV-Vis4 of 10

spectrum [λmax values (427, 452, and 480 nm)] indicated the presence of β,β-carotene type chronopher (Figure4C) [20].

76 SG73 strain 65 Sphingomonas sanguinis NBRC 13937T (AB680528) 0.01 59 Sphingomonas pseudosanguinis G1-2T (AM412238) Sphingomonas yabuuchiae GTC868T (AB071955) 99 84 Sphingomonas parapaucimobilis NBRC 15100T (BBPI01000114)

89 Sphingomonas paucimobilis ATCC 29837T (U37337) 88 Sphingomonas zeae JM-791T (KP999966) 54 Sphingomonas carotinifaciens L9-754T (JQ659512) Sphingomonas yunnanensis YIM 003T (AY894691)

19 T 84 GIFU11458 (D16146) 100 Sphingomonas ginsenosidimutans Gsoil 1429T (HM204925) Sphingomonas desiccabilis CP1DT (AJ871435) Sphingomonas spermidinifaciens DSM 27571T (JQ608324) 15 98 71 Sphingomonas yantingensis 1007T (JX566547) 61 Sphingomonas carri PR0302T (KP185150) Sphingomonas mucosissima CP173-2T (AM229669)

28 Sphingomonas aracearum WZY27T (MH636880) 3 24 Sphingomonas panacis DCY99T (KM819014) Sphingomonas naasensis KIS18-15T (KC735149)

9 JSS-26T (AF131296) Sphingomonas oligophenolica S213T (AB018439) 35 Sphingomonas asaccharolytica IFO 10564T (Y09639) 70 T 91 Sphingomonas mali NBRC 15500 (AB680884) 96 Sphingomonas pruni IFO 15498T (Y09637)

100 Sphingomonas aquatilis JSS-7T (AF131295) DAPP-PG 224T (AB055863)

33 Sphingomonas cynarae SPC-1T (HQ439186)

T 60 Sphingomonas hankookensis ODN7 (FJ194436) 94 Sphingomonas panni C52T (AJ575818) capsulatum NBRC 12533T (AB680290)

Figure 3. Phylogenetic tree of strain SG73 and related microorganisms created with the neighbor-joining method. Numbers indicate bootstrap values. T indicates type strain of a species. Figure 3. Phylogenetic tree of strain SG73 and related microorganisms created with the neighbor-joining method. Num- bers indicate bootstrap values. T indicatesThese results type strain indicate of a thatspecies. the main carotenoid produced by Sphingomonas sp. SG73 is nostoxanthin ((2,3,20,30)-β,β-carotene-2,3,20,30-tetrol). Production of nostoxanthin by other 2.3.Sphingomonas Identificationstrains of the has Main been Caro reportedtenoid [Produced9,13]; however, by Sphingomonas the major carotenoids sp. SG73 produced by thisIdentification strain were caloxanthin of the main and carotenoid zeaxanthin, produced and the ratioby Sphingomonas of nostoxanthin sp. wasSG73 estimated was con- to be about 11% (area%) and less than half of the two main carotenoids. Sphingomonas sp. ducted by high-performance liquid chromatography (HPLC) equipped with an ultravio- SG73, found in this study, produced nostoxanthin with high purity of approximately 97% of let-visible absorption spectrometer (UV-Vis), an electro-spray ionization (ESI) a time-of- the total carotenoid production. A biosynthesis pathway for nostoxanthin by Sphingomonas flight (TOF) mass spectrometer (MS), and MS/MS systems. The molecular formula of this species has been reported and is shown in Figure5[ 9]. Nostoxanthin is synthesized from carotenoidβ-carotene was via fourdetermined hydroxylation to be C steps40H56O performed4 by ESI MS by spectra two enzymes. data of Caloxanthinm/z 601.4243and [M + + + H]zeaxanthin, and m/z 623.4085 which are [M the + Na] main (Figure carotenoids 4A). The in S. MS/MS elodea ATCC spectra 31461, that used are precursors precursor ofion + atnostoxanthin, m/z 600.41 [M and] showed they are characteristic synthesized from productβ-carotene ion patterns through ofβ tetrahydroxy-cryptoxanthin. carotenoid (Figure 4B). These product ion patterns were identical with nostaoxanthin [18], and major product ions from carotenoids [19], m/z 508.3515 [M-92] generated by elimination of tolu- ene moiety from the polyene chain and m/z 494.3283 [M-106] generated by elimination of xylene moiety from the polyene chain, were observed. The UV-Vis spectrum 9λmax values (427, 452, and 480 nm) indicated the presence of β,β-carotene type chronopher (Figure 4C) [20]. These results indicate that the main carotenoid produced by Sphingomonas sp. SG73 is nostoxanthin ((2,3,2′,3′)-β,β-carotene-2,3,2′,3′-tetrol). Production of nostoxanthin by

Mar. Drugs 2021, 19, x 5 of 10

other Sphingomonas strains has been reported [9,13]; however, the major carotenoids pro- duced by this strain were caloxanthin and zeaxanthin, and the ratio of nostoxanthin was estimated to be about 11% (area%) and less than half of the two main carotenoids. Sphin- gomonas sp. SG73, found in this study, produced nostoxanthin with high purity of approx- imately 97% of the total carotenoid production. A biosynthesis pathway for nostoxanthin by Sphingomonas species has been reported and is shown in Figure 5 [9]. Nostoxanthin is synthesized from β-carotene via four hydroxylation steps performed by two enzymes. Mar. Drugs 2021, 19, 274 Caloxanthin and zeaxanthin, which are the main carotenoids in S. elodea ATCC 31461, are5 of 10 precursors of nostoxanthin, and they are synthesized from β-carotene through β-crypto- xanthin.

Mar. Drugs 2021, 19, x 6 of 10

FigureFigure 4. Spectra 4. Spectra of of mass mass spectroscopy spectroscopy (MS) ( (AA),), MS/MS MS/MS (B), ( Band), and ultraviolet ultraviolet visible visible absorption absorption spectroscopy spectroscopy (UV-Vis) (UV-Vis) (C) (C) ofof thethe main carotenoid carotenoid from from SphingomonasSphingomonas sp.sp. SG73. SG73. * Main * Main product product ions ionsderived derived from carotenoids. from carotenoids.

β-Carotene

β-Cryptoxanthin

Zeaxanthin

Caloxanthin

Nostoxanthin

Figure 5. Presumed pathway of nostoxanthin biosynthesis in Sphingomonas sp. SG73.

2.4. Effect of Sea Salt on Growth and Nostoxanthin Production To confirm that Sphingomonas sp. SG73 is derived from the sea and is tolerant to sea salt, Sphingomonas sp. SG73 was cultured in YM liquid medium containing from 0% to 7.2% artificial sea salt. As shown in Figure 6A, the addition of 1.8% and 3.6% sea salt in- creased growth of Sphingomonas sp. SG73, and the strain grew best with 1.8% of sea salt (1.6-fold). When grown in 5.4% sea salt concentration, growth of the strain was decreased, and the strain did not grow in the medium containing 7.2% sea salt. Nostoxanthin production in Sphingomonas sp. SG73 cultured with each salt concen- tration was analyzed by HPLC analysis. Nostoxanthin production by Sphingomonas sp.

Mar. Drugs 2021, 19, x 6 of 10

Mar. Drugs 2021, 19, 274 6 of 10 Figure 4. Spectra of mass spectroscopy (MS) (A), MS/MS (B), and ultraviolet visible absorption spectroscopy (UV-Vis) (C) of the main carotenoid from Sphingomonas sp. SG73. * Main product ions derived from carotenoids.

β-Carotene

β-Cryptoxanthin

Zeaxanthin

Caloxanthin

Nostoxanthin

FigureFigure 5. Presumed 5. Presumed pathway pathway of nostoxanthin of nostoxanthin biosynthesis biosynthesis in Sphingomonas in sp.Sphingomonas SG73. sp. SG73. 2.4. Effect of Sea Salt on Growth and Nostoxanthin Production 2.4.To Effect confirm of Sea that SaltSphingomonas on Growthsp. and SG73 Nostoxanthin is derived from Production the sea and is tolerant to sea salt, SphingomonasTo confirmsp. that SG73 Sphingomonas was cultured in YM sp. liquid SG73 medium is derived containing from from the 0%sea to and 7.2% is tolerant to sea artificial sea salt. As shown in Figure6A, the addition of 1.8% and 3.6% sea salt increased growthsalt, Sphingomonas of Sphingomonas sp.sp. SG73, SG73 and was the cultured strain grew in best YM with liquid 1.8% ofmedium sea salt (1.6-fold). containing from 0% to When7.2% grown artificial in 5.4% sea sea salt. salt As concentration, shown in growthFigure of 6A, the the strain addition was decreased, of 1.8% and and the 3.6% sea salt in- straincreased did not growth grow in of the Sphingomonas medium containing sp. SG73, 7.2% sea and salt. the strain grew best with 1.8% of sea salt (1.6-fold).Nostoxanthin When production grown in Sphingomonas5.4% sea saltsp. concentration, SG73 cultured with growth each salt of the concentra- strain was decreased, Sphingomonas tionand was the analyzed strain bydid HPLC not analysis.grow in Nostoxanthin the medium production containing by 7.2% sea salt.sp. SG73 was highest when grown with 1.8% of sea salt and was 2.5-fold higher as compared with the amountNostoxanthin produced without production sea salt (Figurein Sphingomonas6B). Carotenoid sp. production SG73 cultured per cell densitywith each salt concen- wastration higher was when analyzed cultured withby HPLC 5.4% sea analysis. salt than withNostoxanthin 1.8% sea salt. production When the growth by Sphingomonas sp. of Sphingomonas sp. SG73 was suppressed at higher salt concentrations, it suggested that metabolic, proteomic, and energetic flows to cell growth might be held down and their excess flows were diverted to the carotenoid biosynthetic pathway. Growth of some Sphingomonas relative species with 3.6% sea salt were surveyed (data not shown). Similar to Sphingomonas sp. SG73, the addition of 3.6% sea salt increased growth of Sphingomonas sp. NBRC 101068 and NBRC 101704, which were isolated from seawater of Tokyo Bay and Pacific Ocean, respectively, whereas Sphingomonas sp. JCM 11416 and NBRC 13937, which were isolated from air and human blood, showed no growth. Since 3.6% sea salt is the concentration in seawater, the Sphingomonas strains showed favorable growth and higher nostoxanthin production under 3.6% sea salt. However, the sea salt concentration of the deep sea of Suruga Bay has been reported to be approximately 3.4% [21]. Further optimization of sea salt concentration between 1.8% and 3.6% and other metabolic and culture conditions may improve growth and nostoxanthin production of Sphingomonas sp. SG73. Mar. Drugs 2021, 19, x 7 of 10

SG73 was highest when grown with 1.8% of sea salt and was 2.5-fold higher as compared with the amount produced without sea salt (Figure 6B). Carotenoid production per cell density was higher when cultured with 5.4% sea salt than with 1.8% sea salt. When the growth of Sphingomonas sp. SG73 was suppressed at higher salt concentrations, it sug- gested that metabolic, proteomic, and energetic flows to cell growth might be held down and their excess flows were diverted to the carotenoid biosynthetic pathway. Growth of some Sphingomonas relative species with 3.6% sea salt were surveyed (data not shown). Similar to Sphingomonas sp. SG73, the addition of 3.6% sea salt increased growth of Sphingomonas sp. NBRC 101068 and NBRC 101704, which were isolated from seawater of Tokyo Bay and Pacific Ocean, respectively, whereas Sphingomonas sp. JCM 11416 and NBRC 13937, which were isolated from air and human blood, showed no growth. Since 3.6% sea salt is the concentration in seawater, the Sphingomonas strains showed favorable growth and higher nostoxanthin production under 3.6% sea salt. How- ever, the sea salt concentration of the deep sea of Suruga Bay has been reported to be Mar. Drugs 2021, 19, 274 approximately 3.4% [21]. Further optimization of sea salt concentration between 1.8%7 of 10and 3.6% and other metabolic and culture conditions may improve growth and nostoxanthin production of Sphingomonas sp. SG73.

A B 20 ** 3.0 3.0 (fold) 2.5 2.5 660 ) 15 660 2.0 ** 2.0

10 1.5 1.5

1.0 1.0

Cell density (OD Cell density 5

Relative(fold) production 0.5 0.5 RelativeODper production 0 0 0 0 1.8 3.6 5.4 7.2 0 1.8 3.6 5.4 7.2 Sea salt concentration (%) Sea salt concentration (%)

FigureFigure 6. Effect 6. Effect of ofsea sea salt salt on on growth growth and and nostoxanthin nostoxanthin production bybySphingomonas Sphingomonassp. sp. SG73. SG73. (A ()A Cell) Cell density density (OD (OD660)660 of) of SphingomonasSphingomonas sp.sp. SG73 SG73 cultured cultured with with sea sea salt; salt; ( (BB)) relative Relative production production of nostoxanthin by bySphingomonas Sphingomonassp. sp. SG73. SG73. Black Black bars bars indicateindicate the the relative relative production production level, level, an andd gray barsbars indicate indicate the the relative relative value value of productionof production per cellper density.cell density. All values All values are aremeans means and and standard standard deviations deviations for for triplicate triplicate experiments. experiments. ** p **< 0.01.p < 0.01.

3. 3.Materials Materials and and Methods Methods 3.1.3.1. Materials Materials TheThe following following products products were usedused inin the the experiments experiments described described below: below: Tryptone Trypton (Thermo Fisher Scientific, Waltham, MA, USA); yeast extract and malt extract (Becton, (Thermo Fisher Scientific, Waltham, MA, USA); yeast extract and malt extract (Becton, Dickinson and Company, Franklin Lakes, NJ, USA); D-glucose (FUJIFILM Wako Pure Dickinson and Company, Franklin Lakes, NJ, USA); D-glucose (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); and Daigo’s artificial seawater (FUJIFILM Wako ChemicalPure Chemical Corporation, Corporation, Osaka, Osaka, Japan); Japan). and All Daigo’s solvents artificial for HPLC seawater analysis (FUJIFILM were purchased Wako Purefrom Chemical Kanto Kagaku, Corporation, Tokyo, Japan. Osaka, Japan). All solvents for HPLC analysis were pur- chased from Kanto Kagaku, Tokyo, Japan. 3.2. Strains and Media 3.2. StrainsThe bacteriaand Media in this study were isolated from deep sea sediment of Suruga Bay, Shizuoka,The bacteria Japan in (Table this 1study). Rich were yeast isolated malt (YM)from mediumdeep sea wassediment used asof baseSuruga medium, Bay, Shi- zuoka,containing Japan 5 (Table g/L tryptone, 1). Rich 3yeast g/L yeastmalt (YM) extract, medium 3 g/L malt was extract,used as and base 10 medium, g/L D-glucose. contain- ingDeep 5 g/L sea tryptone, microorganisms 3 g/L yeast in the extract, sediment 3 g/L were malt cultivated extract, on and YM 10 agar g/L medium D-glucose. containing Deep sea ◦ microorganisms0.36% (w/v) Daigo’s in the artificial sediment seawater were cultivated at 22 C for on 3 toYM 7 days,agar andmedium bacteria containing that grew 0.36% as (wcolored/v) Daigo’s colonies artificial were seawater isolated. at 22 °C for 3 to 7 days, and bacteria that grew as colored colonies3.3. Cultivation were isolated. of Isolated Strains

The isolated strains were pre-cultivated in 10 mL YM liquid medium in a 50 mL baffled Erlenmeyer flask at 22 ◦C with agitation at 200 rpm for 24 h. Each culture was inoculated into 10 mL of fresh medium in a 50 mL baffled Erlenmeyer flask to achieve ◦ initial OD660 values of 0.15. Then, cells were grown at 22 C with agitation at 200 rpm for 72 h. To evaluate the salt tolerance of isolated strains and the effect of salt on carotenoid production in the isolated strains, Daigo’s artificial seawater was supplemented into the YM liquid medium at concentrations from 0 to 7.2% (w/v); 3.6% artificial seawater is equivalent to real seawater.

3.4. Carotenoid Analysis To measure the intracellular carotenoid content of the isolated strains, harvested cells were suspended in 1 mL acetone and broken using a bead shaker (Shake Master NEO, BMS, Tokyo, Japan) with 0.6 mm diameter zirconia beads. The resulting cell extract was centrifuged at 8000× g for 10 min at 4 ◦C. Carotenoid analysis was performed using a HPLC system (Shimadzu, Kyoto, Japan) equipped with a COSMOSIL packed column Cholester (NACALAI TESQUE, Kyoto, Japan), as described previously [22]. The separation was performed at 35 ◦C, with Mar. Drugs 2021, 19, 274 8 of 10

methanol/tetrahydrofuran = 8/2 (v/v) as the mobile phase at a flow rate of 1.0 mL/min, and the detection was performed at 470 nm with an SPD-20AV detector (Shimadzu). Twenty microliters of each sample solution was injected.

3.5. Identification of Carotenoid Structure The LC/MS analysis of carotenoids was carried out using a Waters Xevo G2S Q TOF mass spectrometer (Waters Corporation, Milford, CT, USA) equipped with an Acquity UPLC system. The ESI-TOF/MS spectra were acquired by scanning from m/z 100 to 1500 with a capillary voltage of 3.2 kV, cone voltage of 20 eV, and source temperature of 120 ◦C. Nitrogen was used as a nebulizing gas at a flow rate of 30 L/h. The MS/MS spectra were measured with a quadrupole-TOF MS/MS instrument with argon as a collision gas at a collision energy of 20 V. The UV-Vis absorption spectra were recorded from 200 to 600 nm using a photodiode-array detector (PDA) (Acquity UPLC PDA eλ Detector, Waters, Milford, MA, USA). An Acquity 1.7 µm BEH UPLC C18 (2.1 id X 100 mm) column (Waters Corporation, Milford, CT, USA) was used as a stationary phase and MeCN/H2O (85:15)– MeCN/MeOH (65:35) (linear gradient 0 to 15 min) as a mobile phase, at a flow rate of 0.4 mL/min for the HPLC system.

3.6. Genetic Analysis The 16S rRNA gene sequence of the isolated strain was determined by TechnoSuruga Laboratory Co. Ltd., Shizuoka, Japan. The 16S rRNA gene sequence was subjected to ho- mology searching using ENKI v3.2 (TechnoSuruga Laboratory, Shizuoka, Japan) against the Microbial Identification Database DB-BA15.0 (TechnoSuruga Laboratory, Shizuoka, Japan) and the International Nucleotide Sequence Databases (DDBJ, ENA, and GenBank). Multi- ple alignments were analyzed using the CLUSTAL W algorithm [23], and a phylogenetic tree was constructed using MEGA v7.0 [24] by the neighbor-joining method. The 16S rRNA gene sequences of the isolated strain and its closest relatives were identified as follows: Sphingomonas sanguinis NBRC 13937T (AB680528), S. pseudosanguinis G1-2T (AM412238), S. yabuuchiae GTC868T (AB071955), S. parapaucimobilis NBRC 15100T (BBPI01000114), S. paucimobilis ATCC 29837T (U37337), S. zeae JM-791T (KP999966), S. carotinifaciens L9-754T (JQ659512), S. yunnanensis YIM 003T (AY894691), S. adhaesiva GIFU11458T (D16146), S. gin- senosidimutans Gsoil 1429T (HM204925), S. desiccabilis CP1DT (AJ871435), S. spermidinifaciens DSM 27571T (JQ608324), S. yantingensis 1007T (JX566547), S. carri PR0302T (KP185150), S. mucosissima CP173-2T (AM229669), S. aracearum WZY27T (MH636880), S. panacis DCY99T (KM819014), S. naasensis KIS18-15T (KC735149), S. koreensis JSS-26T (AF131296), S. oligophe- nolica S213T (AB018439), S. asaccharolytica IFO 10564T (Y09639), S. mali NBRC 15500T (AB680884), S. pruni IFO 15498T (Y09637), S. aquatilis JSS-7T (AF131295), S. melonis DAPP- PG 224T (AB055863), S. cynarae SPC-1T (HQ439186), S. hankookensis ODN7T (FJ194436), S. panni C52T (AJ575818), and Novosphingobium capsulatum NBRC 12533T (AB680290).

4. Conclusions In this study, a novel nostoxanthin producer Sphingomonas sp. SG73 was isolated from deep sea sediment that had been collected from Suruga Bay, Shizuoka, Japan. Nostoxanthin is expected to have high antioxidative activity. The strain was halophilic, and the growth and nostoxanthin production was stimulated with 1.8–3.6% sea salt. In the future, a com- mercial supply of nostoxanthin can be achieved by improvement of this pure nostoxanthin producer Sphingomonas sp. SG73.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.339 0/md19050274/s1, Figure S1: Chromatogram of LC/MS/MS analysis. The main peak is nostoxanthin. The small peak 1, 2, and 3 is presumed as caloxanthin, zeaxanthin, and β-cryptoxanthin, respectively. Author Contributions: Conceptualization, K.Y.H.; methodology, K.Y.H., T.M., and M.M.; validation, H.K. and T.O.; formal analysis, H.K., T.O., and T.M.; investigation, H.K. and T.O.; resources, M.M., K.M.; data curation, H.K.; writing—original draft preparation, H.K.; writing—review and editing, Mar. Drugs 2021, 19, 274 9 of 10

K.Y.H., Y.H.-H., K.M., and T.K.; visualization, H.K.; supervision, K.M., M.K., and S.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Seeds Creation Research centered on Marine Biotechnology Program of Marine Open Innovation Institute. This work was also supported by the JST-Mirai Program (no. JPMJMI17EJ) and Grant-in-Aid for Scientific Research(B) from Japan Society for the Promotion of Science(JSPS) (no. JP21H03645). Acknowledgments: The samples of deep sea sediment, which were used in this study, were obtained from the Japan Agency for Marine-Earth Science and Technology (JAMSTEC). We would like to thank TechnoSuruga Laboratory Co., Ltd. for their support in the identification of microorganisms. We thank Tara Penner for editing a draft of this manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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