Characterization of Sterol Lipids in Kluyveromyces Lactis Strain M-16 Accumulating a High Amount of Steryl Glucoside

Characterization of Sterol Lipids in Kluyveromyces lactis Strain M-16 Accumulating a High Amount of Steryl Glucoside Michiko Sugai1, Naoya Takakuwa2, Masao Ohnishi3, Tadasu Urashima1 and Yuji Oda3＊ 1 Graduate School of Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine (Obihiro, Hokkaido 080-8555, JAPAN) 2 Memuro Research Station, National Agricultural Research Center for Hokkaido Region (Memuro, Hokkaido 082-0081, JAPAN) 3 Department of Food Science, Obihiro University of Agriculture and Veterinary Medicine (Obihiro, Hokkaido 080-8555, JAPAN)

Abstract: Kluyveromyces lactis strain M-16 isolated from raw milk accumulates a high amount of steryl glucoside in the cells. Under high temperature or in the presence of NaCl, this strain did not show better growth than other K. lactis strains that hardly accumulated steryl glucoside. Heat shock elevated the content of steryl glucoside 3.2-fold, which accounted for 27% of the total sterol lipids, and simultaneously reduced that of acyl sterol. Both strains, M-16 and NBRC 1267, contained ergosterol as a principal component, and dihydroergosterol was also included in steryl glucoside of strain M-16. Lanosterol was a major component second to ergosterol in free sterols. In acyl sterol of strain M-16, the proportion of 4,4- dimethylzymosterol was higher than that of ergosterol. Excess synthesis of steryl glucoside in strain M-16 consumes ergosterol and dihydroergosterol in the pool of free sterols, and acyl sterol may inevitably take in 4,4-dimethylzymosterol and 4-methylfecosterol, the intermediates in the biosynthetic pathway to ergosterol, as a component sterol.

Key words: Kluyveromyces lactis, steryl glucoside, heat shock, sterol glucosyltransferase

1 INTRODUCTON The yeast Saccharomyces cerevisiae synthesizes ergos- Steryl glucoside (SG), which is composed of the sterol terol as an essential component of the cell membrane to 8) ring with a glucose residue at the C3-OH group, is included maintain its integrity but does not accumulate detectable in the membrane lipids of various organisms1,2). In plants, amounts of SG9). The yeasts alternatively used to assess SG may serve as a primer for cellulose biosynthesis the function of SG are the methanol-utilizing species, because the cotton fiber membrane synthesizes sitosterol- Pichia pastoris, and the alkane-utilizing species, Yarrowia cellodextrins from SG and UDP-glucose3). Other reports on lipolytica10). The two species require SG synthesis for the the physiological functions of SG are related with the cellu- degradation of methanol-induced peroxisomes by exoge- lar response to environmental changes through morpho- nous carbon compounds and the utilization of decane, logical differentiation. Kunimoto et al.4) suggested that SG respectively11). As for P. pastoris, the SG content in the plays a role as a mediator in the early stage of stress- cells from the complete medium was much lower than that responsive signal transduction from the rapid synthesis of in those from the minimal medium and increased with the SG in the myxoamoebae of the true slime mold5) and in stress conditions, such as heat shock or high ethanol con- human fibroblasts by heat-shock treatments6). The sterol molecules of SG and the lipid compositions of a Gram-nega- Abbreviations: SG, steryl glucoside; AS, acyl sterol; FS, free tive bacterium, Helicobacter pylori, changed during mor- sterol; DMS, 4,4-dimethylsterol; MMS, 4-methylsterol; DeMS, 4- phological transition from the spiral to the coccoid form desmethylsterol; SGT, sterol glucosyltransferase; TLC, thin-layer induced by environmental stresses7). chromatography; GC-MS, gas chromatograph mass spectrometer.

＊Correspondence to: Yuji Oda, Department of Food Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, JAPAN E-mail: [email protected] Accepted October 3, 2008 (received for review August 5, 2008) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/

91 M. Sugai, N. Takakuwa, M. Ohnishi et al.

centrations9). Recently, Park et al.12) studied the interac- tions between defensins, antifungal peptides produced by plants, and cell surface glycolipids of fungi and found that the mutant strains of Neurospora crassa which are resistant to defensin expressed highly increased levels of SG, identified as ergosteryl glucoside, when cultured in the conventional potato-dextrose broth without any environmental stress. The yeast Kluyveromyces lactis included in the family Saccharomycetaceae13) possesses the gene encoding sterol glucosyltransferase (SGT) for SG synthesis but does not produce detectable amounts of SG, as observed in S. cerevisiae. We investigated 2,150 yeast strains isolated from raw milk and milk products to produce glucosylceramide from cheese whey and unexpectedly found that K. lactis 14) strain M-16 synthesized a considerable amount of SG . Fig. 1 Thin-layer Chromatograms of Sterol Lipids This strain is a wild microorganism accumulating SG even Extracted from the Cells of K. lactis M-16. under the usual culture conditions. In the present experi- Sterol lipids were separated into SG, AS and FSs ments, the chemical composition of sterol lipids was ana- containing DMS, MMS and 4-desmethylsterol by lyzed to assess the characteristics of strain M16 in SG accumulation. two experiments. Solvent systems: (a) chloroform: methanol:acetic acid:water (20:3:5:2.3:0.7, v/v); (b) n-hexane:diethyl ether:acetic acid (80:30:1). Detection: (a) orcinol-sulfuric acid reagent; (b)

2 EXPERIMENTAL H2SO4:ethanol:water (25:50:25). 2.1 Organism and culture K. lactis strain M-16 was isolated from domestic raw milk and deposited in the NITE Patent Microorganisms Depositary (NPMD), Chiba, Japan, as NITE P-228. Other Total lipids were subjected to a silicic acid column (Sep- strains classified as K. lactis (NBRC 0433, NBRC 0648, pak cartridge) and eluted by methanol to obtain the frac- NBRC 1090, NBRC 1267, NBRC 1903) were obtained from tion containing SG and glucosylceramide. After hydrolysis 16) the NITE Biological Research Center (NBRC), Chiba, by 10% Ba(OH)2-dioxane (1:1) , sterol was extracted from Japan. Yeast cells were grown in a YPD medium composed this fraction twice by hexane and once by diethylether and of 1.0% yeast extract, 2.0% polypeptone, and 2.0% glucose further purified by a silicic acid column. AS isolated from with shaking or in the same medium solidified by agar. preparative TLC was hydrolyzed by 1M KOH in methanol15) to release the component sterol. DMS, MMS and DeMS 2.2 Sterol analysis were collected from TLC and combined. FSs and compo-

The cells reached about one-half of A600 in maximal nent sterols prepared from SG and AS were analyzed with growth; 10 for strain M-16 and 30 for strain NBRC 1267 GC-MS (QP2010, Shimadzu Corp., Kyoto, Japan) equipped were transferred under stress conditions and successively with a ULBON HR-1 capillary column (50 m×0.25 mm, i.d. cultured for 6 h. After centrifugation, the collected cells 0.25 mm, Shinwa Chemical Industries, Kyoto, Japan) and an were lyophilized and used for the extraction of total lipids15) EI detector according to a method described elsewhere17). to analyze the sterol lipids by TLC. For each sample, two A typical chromatogram of these sterols and their biosyn- TLC plates were developed with chloroform:methanol: thetic pathway in the yeast cells are shown in Figs. 2 and acetic acid:water (20:3:5:2.3:0.7, v/v) and n-hexane:diethyl 3, respectively. ether:acetic acid (80:30:1) and detected by orcinol-sulfuric acid reagent and H2SO4:ethanol:water (25:50:25), respec- 2.3 Molecular biological techniques tively. TLC chromatograms resulted in the separation of A polymerase chain reaction was conducted using Z- cellular sterols into SG, acyl sterol (AS) and free sterols Taq-DNA polymerase as recommended by the supplier (FSs) containing 4,4-dimethylsterol (DMS), 4-methylsterol (Takara Bio Inc., Kyoto, Japan). The primers used were (MMS) and 4-desmethylsterol (DeMS) (Fig. 1) to determine SGT-1F (5’-CAGATGCAAAACGTTTCC-3’) and SGT-11R their contents by comparing their densities to those of (5’-TGGACGACGTTCCTATTT-3’) for the SGT gene. The authentic standards14) or to prepare samples for further fragments amplified from the genomic DNA of yeast cells analysis. were sequenced and assigned the DDBJ/EMBL/GenBank

92 J. Oleo Sci. 58, (2) 91-96 (2009) Yeast Steryl Glucosides

Accession Numbers AB426891 to AB426892.

3 RESULTS AND DISCUSSION 3.1 Effects of stress conditions on the growth and sterol compositions The relationship between the cellular SG and environmental changes9) suggested that strain M-16 may be more resistant to stress conditions than the NBRC strains hardly synthesizing SG. Then, serially diluted suspensions of yeast cells were spotted on agar plates and incubated for 2 days to compare their growth responses to external stresses (Fig. 4). Under the standard conditions at 25℃, vital Fig. 2 GC-MS Spectrum of Sterols Included in AS Isolated growth of all the strains was observed with suspensions from K. lactis Strain NBRC 1267. diluted up to 103 and more or less inhibited at 35℃ or in the Each peak of component sterol prepared by the presence of 4.0% NaCl. Strain NBRC 1267 grew exception- hydrolysis of AS was identified as follows: (a) ally well even at 35℃. Strains NBRC 0433, NBRC 1090 and zymosterol; (b) ergosterol; (c) dihydroergosterol; (d) NBRC 1903 were less sensitive to 4.0% NaCl than the other 4-methylzymosterol; (e) 5-dehydroepisterol; (f) strains. Individual strains differed in resistance to each fecosterol; (g) episterol; (h) ergosta-7-ene-3b-ol; (i) stress condition, and strain M-16 did not show the best growth. These observations mean that SG is unlikely to 4-methylfecosterol; (j) lanosterol; (k) 4,4- protect the cells from stress conditions in strain M-16. dimethylzymosterol. Under stress conditions, the syntheses of SG, AS, and FSs in the cells of strain M-16 were compared with those of strain NBRC 1267, selected as a reference (Table 1). By heat-shock treatment at 42℃, the content of SG in strain M-16 was elevated 3.2-fold, which accounted for 27% of total sterol lipids, and simultaneously reduced that of AS. Strain NBRC 1267 produced a detectable amount of SG only by a similar treatment. Ethanol stimulated the synthesis of AS in strain M-16. In the two strains, appreciable amounts of sterol lipids appeared as FSs including DMS, MMS and DeMS. The contents of DeMS, which covered most parts of FSs, were maintained at a relatively constant level compared with those of DMS and MMS. Growth at 42℃ and that in the presence of 5% ethanol caused accumulation of DMS in the two strains. Differences in the contents of sterol lipids indicate that the cellular responses by these stresses are not identical. Strains M-16 and NBRC 1267 seem to share basic stress-responding mechanisms, and the ability to synthesize SG of the former may be much higher than that of the latter.

3.2 Chemical structures of sterol lipids FSs and component sterols of SG and AS in the cells with heat-shock treatments at 42℃ were analyzed, except for SG in strain NBRC 1267 (Table 2). The two strains contained ergosterol, the characteristic sterol in fungi and yeasts, as a principal component, and dihydroergosterol was also included in SG of strain M-16. Lanosterol was a major component second to ergosterol in FSs. In AS of strain M-16, the proportion of 4,4-dimethylzymosterol was Fig. 3 Biosynthetic Pathway of Sterols in Yeast. higher than that of ergosterol. SGT generally catalyzes the

93 J. Oleo Sci. 58, (2) 91-96 (2009) M. Sugai, N. Takakuwa, M. Ohnishi et al.

Fig. 4 Growth of K. lactis Strains under Stress Conditions. Ten-fold serial dilutions of a yeast cell suspension (106 cell/ml) were spotted toward the right on YPD plates at 5 mL and incubated at 25℃ for 2 days. The growth under the standard condition was compared with those at 35℃ or in the presence of 4.0% NaCl.

Table 1 Contents of Sterol Lipids in the Cells of Kluveromyces lactis Strains under Stress Conditions. Content (mg/g as dry basis) Strain Condition FSs SG AS Total DMS MMS DeMS Standard 0.77 ± 0.03 1.81 ± 0.17 0.29 ± 0.04 0.39 ± 0.24 3.72 ± 0.64 7.83 ± 0.57 42℃ 2.50 ± 0.18 0.75 ± 0.18 1.64 ± 0.41 0.72 ± 0.35 3.19 ± 0.36 9.31 ± 0.83 M-16 +4% NaCl 0.45 ± 0.11 1.84 ± 0.64 0.24 ± 0.47 0.30 ± 0.22 3.78 ± 0.29 7.62 ± 0.36 +5% Ethanol 0.55 ± 0.04 3.85 ± 1.62 1.27 ± 0.27 0.56 ± 0.28 3.61 ± 0.21 12.10 ± 1.15 Standard <0.01 3.18 ± 0.27 0.24 ± 0.03 0.18 ± 0.02 2.92 ± 0.21 6.75 ± 0.47 42℃ 0.04 ± 0.01 3.72 ± 0.11 0.67 ± 0.07 0.15 ± 0.02 2.89 ± 0.15 7.66 ± 0.19 NBRC 1267 +4% NaCl <0.01 3.44 ± 0.08 0.55 ± 0.03 0.45 ± 0.00 3.00 ± 0.08 8.34 ± 1.01 +5% Ethanol <0.01 3.76 ± 0.05 1.06 ± 0.02 0.44 ± 0.01 2.85 ± 0.13 8.34 ± 0.28

Data are shown as the average values and standard deviations from three independent experiments.

transfer of the glucose residue to various FSs in vitro1). concentrations when supplied to rats (Japanese patent Ergosterol and dihydroergosterol, the products in the 3009017). Oral administration of choresteryl glucoside downstream of the biosynthetic pathway (Fig. 3), seem to inhibited gastric ulcers induced by cold-restraint stress in be preferentially incorporated into SG of strain M-16. rats18). SG can be used as a constituent of a hair restorer There might be some mechanisms of selective reaction by (Japanese patent 3113763) or liposomes for drug delivery19). SGT in vivo. Excess synthesis of SG in strain M-16 con- Ergosteryl glucoside, the major component in yeast SG, is sumes ergosterol and dihydroergosterol in the pool of FSs expected to show similar effects. and AS inevitably may take in 4,4-dimethylzymosterol and 4-methylfecosterol, the intermediates in the biosynthetic 3.3 Properties of SGT genes pathway to ergosterol, as a component sterol. The entire region spanning SGT genes was amplified There have been some reports on the desirable effects of from strains M-16 and NBRC 1267 based on the genome the internal and external uses of SG. A mixture of SG and database of K. lactis strain NBRC 1267. The obtained frag- its acyl derivatives from oil seeds reduced the serum lipid ments revealed a single ORF of 3,627 bp but differed in a

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Table 2 Composition of Sterols in Sterol Lipids Isolated from the Cells of K. lactis with Heat-shock Treatments. M-16 NBRC 1267 Component SG AS FSs AS FSs (%) (%) (%) (%) (%) Lanosterol 1.2 4.1 23.8 1.8 19.6 DMS 4,4-Dimethylzymosterol <0.1 38.6 4.5 12.3 6.0 4-Methylfecosterol <0.1 10.0 0.8 1.3 <0.1 MMS 4-Methylzymosterol <0.1 9.3 1.1 9.8 2.6 Fecosterol 2.4 0.4 8.6 3.3 5.4 Zymosterol <0.1 2.3 1.5 19.3 3.1 Episterol 5.3 2.1 3.5 1.8 1.1 DeMS Ergosta-7-ene-3b-ol 0.4 <0.1 2.7 <0.1 1.8 Dihydroergosterol 28.2 3.3 3.0 0.3 <0.1 5-Dehydroepisterol 0.4 <0.1 1.0 2.9 1.7 Ergosterol 62.1 29.9 49.5 47.2 58.7

single nucleotide. At position 1859, C in strain NBRC 1267 References was replaced with T in strain M-16, resulting in the con- 1. Warnecke, D.; Erdmann, R.; Fahl, A.; Hube, B.; Muller, version from threonine to methionine in the polypeptide. F.; Zank, T.; Zahringer, U.; Heinz, E. Cloning and func- This change is not specific for strain M-16 because other tional expression of UGT genes encoding sterol gluco- strains of K. lactis showed the same sequence as strain M- syltransferases from Saccharomyces cerevisiae, Can- 16. The sequences of the 5’-upstream region to -464 were dida albicans, Pichia pastoris, and Dictyostelium dis- identical in the two strains. Some mechanisms other than coideum. J. Biol. Chem. 274, 13048-13059 (1999). the nucleotide sequence of the SGT gene may regulate the 2. Takakuwa, N.; Saito, K.; Ohnishi, M.; Oda, Y. Determi- expression that causes the excess synthesis of SG in strain nation of glucosylceramide contents in crop tissues M-16. and by-products from their processing. Bioresour. Technol. 96, 1089-1092 (2005). 3. Peng, L.; Kawagoe, Y.; Hogan, P.; Delmer, D. Sitosterol- b-glucoside as primer for cellulose synthesis in plants. 4 CONCLUSIONS Science 295, 147-150 (2002). K. lactis strain M-16 accumulates a high amount of SG 4. Kunimoto, S.; Murofushi, W.; Kai, H.; Ishida, Y.; that incorporates ergosterol in the pool of FSs and conse- Uchiyama, A.; Kobayashi, T.; Kobayashi, S.; Murofushi, quently elevates the proportion of other component sterol H.; Murakami-Murofushi, K. Steryl glucoside is a lipid in AS. Although the physiological role of SG accumulated mediator in stress-responsive signal transduction. Cell in strain M-16 is not fully understood, the peculiar accumu- Struct. Funct. 27, 157-162 (2002). lation of SG will be of interest from the industrial view- 5. Murakami-Murofushi, K.; Nishikawa, K.; Hirakawa, E.; point. This strain will enable the efficient production of SG Murofushi, H. Heat stress induces a glycosylation of from agricultural by-products such as cheese whey. membrane sterol in myxoamoebae of a true slime mold, Physarum polycephalum. J. Biol. Chem. 272, 486-489 (1997). 6. Kunimoto, S.; Kobayashi, T.; Kobayashi, S.; Murakami- ACKNOWLEDGEMENTS Murofushi, K. Expression of cholesteryl glucoside by We thank Dr. K. Yunoki for his assistance with the analy- heat shock in human fibroblasts. Cell Stress Chaper- sis of sterols with a gas chromatograph mass spectrome- ones 5, 3-7 (2000). ter. This work was partly supported by the Program for the 7. Shimomura, H.; Hayashi, S.; Yokota, K.; Oguma, K.; Promotion of Basic Research Activities for Innovative Bio- Hirai, Y. Alteration in the composition of cholesteryl sciences (PROBRAIN). glucosides and other lipids in Helicobacter pylori undergoing morphological change from spiral to coc-

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