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′)-β,β-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.


Introduction
Carotenoids are isoprenoids, and these yellow to orange-red pigments are widely distributed in nature [1]. They are predominantly synthesized by phototrophic organisms and by some non-phototrophic fungi, bacteria, and archaea [1,2]. Carotenoids are used commercially, especially astaxanthin, as a color enhancer for marine aquaculture resources such as salmon, and for dietary supplements, cosmetics, and pharmaceuticals because of its high antioxidant activity [3,4]. Industrial demand for carotenoids has recently increased, which has resulted in an increase in studies of carotenoid production, especially using marine organisms [5][6][7][8].
In this study, we isolated a microorganism from deep sea collected from Suruga Bay, Shizuoka, Japan (Table 1) and we iden ture of the main carotenoid produced by the isolated microo showed high nostoxanthin composition in total carotenoids, and rial strain. Furthermore, we characterized the salt tolerance and ity by supplemention with artificial sea salt.

Isolation of a Yellow Microorganism from Deep Sea Sediment
Firstly, colored microorganisms were isolated from deep se strain SG73, from the sampling Spot 2 in Table 1, showed accu carotenoid at a retention time of approximately 3.8 min and thre tion time of 4 to 5 min (Figure 2A). The three minor peaks were tography for HPLC and LC/MS (Supplemental Figure S1). The p noid was 97% (area%). These carotenoids did not correspond t standards of astaxanthin, α-carotene, or β-carotene ( Figure 2B from strain SG73 was expected to have higher hydrophilicity tha its faster retention time. In this study, we isolated a microorganism from deep sea sediment that had been collected from Suruga Bay, Shizuoka, Japan (Table 1) 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 nostoxanthin productivity by supplementation with artificial sea salt.

Isolation of a Yellow Microorganism from Deep Sea Sediment
Firstly, colored microorganisms were isolated from deep sea sediment. One isolate, strain SG73, from the sampling Spot 2 in Table 1, showed accumulation of highly pure carotenoid at a retention time of approximately 3.8 min and three minor peaks at a retention time of 4 to 5 min (Figure 2A). The three minor peaks were detected in the chromatography for HPLC and LC/MS (Supplemental Figure S1). The purity of the main carotenoid 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.

Identification of the Hydrophilic Carotenoid-Producing Strain SG73
The colony of strain SG73 exhibited a strong yellow color and a smooth surface (Figure 2C). The 16S rRNA gene sequence (1412 bp, accession no. LC618681) amplified from strain SG73 had 99.8% homology to that of Sphingomonas sanguinis NBRC 13937. Strain SG73 was clustered with S. sanguinis NBRC 13937 in the phylogenetic tree, but the bootstrap value was low (76%) ( Figure 3). However, these results indicate that there may be only a small genetic distance between these strains. Therefore, strain SG73 was identified as Sphingomonas sp. SG73.
Sphingomonas is a group of Gram-negative, rod-shaped, chemoheterotrophic, aerobic bacteria. Sphingomonas species are known to contain glycosphingolipids [17], and are widely distributed in nature, having been isolated from many different air, land, and water habitats, including seawater and marine soils. Furthermore, Sphingomonas species have been reported to produce the carotenoids lycopene, β-carotene, zeaxanthin, caloxanthin, and nostoxanthin [9,13,17].

Identification of the Hydrophilic Carotenoid-Producing Strain SG73
The colony of strain SG73 exhibited a strong yellow color and a smooth surface ( Figure 2C). The 16S rRNA gene sequence (1412 bp, accession no. LC618681) amplified from strain SG73 had 99.8% homology to that of Sphingomonas sanguinis NBRC 13937. Strain SG73 was clustered with S. sanguinis NBRC 13937 in the phylogenetic tree, but the bootstrap value was low (76%) ( Figure 3). However, these results indicate that there may be only a small genetic distance between these strains. Therefore, strain SG73 was identified as Sphingomonas sp. SG73.
Sphingomonas is a group of Gram-negative, rod-shaped, chemoheterotrophic, aerobic bacteria. Sphingomonas species are known to contain glycosphingolipids [17], and are widely distributed in nature, having been isolated from many different air, land, and water habitats, including seawater and marine soils. Furthermore, Sphingomonas species have been reported to produce the carotenoids lycopene, β-carotene, zeaxanthin, caloxanthin, and nostoxanthin [9,13,17].

Identification of the Main Carotenoid Produced by Sphingomonas sp. SG73
Identification of the main carotenoid produced by Sphingomonas sp. SG73 was con-  Figure 4B). These product ion patterns were identical with nostaoxanthin [18], and major product ions from carotenoids [19], m/z 508.  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 toluene 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

Sphingomonas panni C52 T (AJ575818)
Novosphingobium capsulatum NBRC 12533 T (AB680290) 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 other Sphingomonas strains has been reported [9,13]; however, the major carotenoids produced 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. Sphingomonas sp. SG73, found in this study, produced nostoxanthin with high purity of approximately 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. Caloxanthin and zeaxanthin, which are the main carotenoids in S. elodea ATCC 31461, are precursors of nostoxanthin, and they are synthesized from β-carotene through β-cryptoxanthin. gomonas sp. SG73, found in this study, produced nostoxanthin with high purity of approximately 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. Caloxanthin and zeaxanthin, which are the main carotenoids in S. elodea ATCC 31461, are precursors of nostoxanthin, and they are synthesized from β-carotene through β-cryptoxanthin.

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

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 increased 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 concentration was analyzed by HPLC analysis. Nostoxanthin production by Sphingomonas sp. 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 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. 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.

Materials
The following products were used in the experiments described below: Trypton (Thermo Fisher Scientific, Waltham, MA, USA); yeast extract and malt extract (Becton, Dickinson and Company, Franklin Lakes, NJ, USA); D-glucose (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); and Daigo's artificial seawater (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). All solvents for HPLC analysis were purchased from Kanto Kagaku, Tokyo, Japan.

Strains and Media
The bacteria in this study were isolated from deep sea sediment of Suruga Bay, Shizuoka, Japan (Table 1). Rich yeast malt (YM) medium was used as base medium, containing 5 g/L tryptone, 3 g/L yeast extract, 3 g/L malt extract, and 10 g/L D-glucose. Deep sea microorganisms in the sediment were cultivated on YM agar medium containing 0.36% (w/v) Daigo's artificial seawater at 22 °C for 3 to 7 days, and bacteria that grew as colored colonies were isolated.

Materials
The following products were used in the experiments described below: Tryptone (Thermo Fisher Scientific, Waltham, MA, USA); yeast extract and malt extract (Becton, Dickinson and Company, Franklin Lakes, NJ, USA); D-glucose (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); and Daigo's artificial seawater (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). All solvents for HPLC analysis were purchased from Kanto Kagaku, Tokyo, Japan.

Strains and Media
The bacteria in this study were isolated from deep sea sediment of Suruga Bay, Shizuoka, Japan (Table 1). Rich yeast malt (YM) medium was used as base medium, containing 5 g/L tryptone, 3 g/L yeast extract, 3 g/L malt extract, and 10 g/L D-glucose. Deep sea microorganisms in the sediment were cultivated on YM agar medium containing 0.36% (w/v) Daigo's artificial seawater at 22 • C for 3 to 7 days, and bacteria that grew as colored colonies were isolated.

Cultivation 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 OD 660 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.

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