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Accumulation of Zymosterol in Yeast Grown in the Presence of Ethionine

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Accumulation of Zymosterol in Yeast Grown in the Presence of Ethionine J. Biochem. 83, 1109-1116 (1978) Accumulation of Zymosterol in Yeast Grown in the Presence of Ethionine Nakao ARIGA,* Hiroshi HATANAKA ,' Jun NAGAI,2 and Hirohiko KATSUKI Department of Chemistry, Faculty of Science , Kyoto University, Sakyo-ku, Kyoto 606, and *Department of Chemistry , Faculty of General Education, Gifu University, Nagara, Gifu 502 Received for publication, September 17, 1977 In order to identify the methyl acceptor for the methylation of sterol side-chains in ergosterol biosynthesis, Saccharolnyces cerevisiae (wild type) was grown in the presence and absence of ethionine which was expected to be an inhibitor of the methylation. Gas-liquid chromato graphic analyses of the sterols in the cells grown in the absence of ethionine showed that ergosterol was the most abundant sterol. On the other hand, a sterol, named sterol Z, accounted for more than 50% of the total sterols in the cells grown in the presence of ethionine. As a result of experiments to raise the yield of sterol Z, the best concentration of DL-ethionine for the production was found to be 1.0 mm. The use of the methionine-less mutant was less effective for the production of sterol Z. Sterol Z was isolated by repeated TLC and was identified as zymosterol from its melting point, GLC and mass spectrometry. The role of zymosterol and other sterols as the methyl-acceptor sterol in ergosterol bio synthesis is also discussed. Although it is well known that ergosterol is syn a methylated sterol intermediate in yeast, 4a thesized via lanosterol in yeast as in the case of methyl-5ƒ¿-ergosta-8,24-dien-3 ƒÀ-ol which has a cholesterol biosynthesis in animals, much remains methyl group at position 4. Akhtar et al. (2) unsolved on the synthesis of ergosterol from synthesized 3H-labeled 4,4,14ƒ¿-trimethyl-5 ƒ¿-ergo lanosterol, especially on the methylation reaction sta-8,24(28)-dien-3 ƒÀ-ol and demonstrated that the of the sterol side-chain. Two theories have been compound was converted into ergosterol in yeast. proposed concerning the problem whether the They claimed that the methylation occurs before reaction occurs before or after complete elimination complete elimination of the three methyl groups. of the three methyl groups attached to the nucleus On the other hand, Katsuki and Bloch (3) examined of the lanosterol molecule. Barton et al. (1) found the methylation reaction with cell-free extracts of yeast. According to their studies, most of the methylated sterols had a carbon skeleton of ergo 1 Present address: Mitsubishi-Kasei Institute of Life Sciences, Tokyo. stane, suggesting that Cg,-sterol is the methyl 2 Present address: Department of Biochemistry, Fa acceptor in the methylation reaction. Moore and culty of Medicine, Mie University, Tsu. Gaylor (4, 5) investigated the substrate specificity Vol. 83, No. 4, 1978 1109 1110 N. ARIGA, H. HATANAKA, J . NAGAI, and H. KATSUKI of S-adenosylmethionine: ƒ¢21 sterol methyltrans flask was, after sterilization, inoculated with 0.5 ml ferase with a partially purified enzyme preparation . of an overnight culture of the yeast. The cultiva They claimed that the methylation occurs at the tion was carried out with shaking at 30•Ž for the stage of C27-sterol, since zymosterol showed the indicated periods. Aliquots of the culture were highest activity as methyl acceptor so far examined withdrawn aseptically and diluted with an appro-. and 4ƒ¿-methyl-5ƒ¿-cholesta-8 ,24-lien-3ƒÀ-o1 and priate volume of saline. The absorbance at 660 4,4-dimethyl-5a-cholesta-8,24-dien-3ƒÀ-ol scarcely nm was measured in a Hitachi Model 101 Spectro showed any activity. Recently, we studied the photometer. The dry weight of the cells was methylation reaction with cell-free extracts of yeast estimated from the absorbance on the basis of the and reported that homocysteine inhibited the predetermined standard curve. methyltransfer reaction from S-adenosylmethionine Respiratory adaptation of yeast was carried to sterols, giving rise to an accumulation of 5 out in the following way: 800 ml of the growth ƒ¿ cholesta-7,24-dien-3ƒÀ-ol (6). Parks et al. (7) also medium in a 1 liter Erlenmeyer flask was, after reported the occurrence of this sterol in yeast. In sterilization, inoculated with 50 ml of an overnight spite of the studies reported, it has not been culture of the yeast. After bubbling nitrogen gas established yet whether the methylation of the through the growth medium under sterile conditions side-chain occurs before or after the demethylation for 1 h, the outlet of the glass tubing was sealed of lanosterol in intact yeast cells and, even if the with a mercury trap. The yeast was grown under latter possibility is valid, whether one or both of anaerobic conditions for 38 h at 28•Ž with stirring. the two C27-sterols is the methyl acceptor. In The harvested cells were washed twice with 0.1 mt connection with this, Fryberg et al. (8) proposed potassium phosphate buffer, pH 7.0, resuspended multiple pathways for ergosterol biosynthesis and in the same buffer containing 3 % glucose (0.4 mg they considered that the methylation reaction of dry weight of cells per ml). The cells were occurs at the stage of zymosterol or a 4-methyl incubated with shaking for 4 h at 28•Ž for respira derivative of it and that episterol is an important tory adaptation. The absorbance at 660 nm of intermediate in the biosynthesis. We have found the culture increased by 25 % during the incubation . that ethionine is an effective inhibitor of the Analysis of Sterols-The cells harvested by methylation of sterol side-chains in intact yeast centrifugation, were saponified at 80•Ž for 2 .5 h cells. This paper describes the accumulation of with 10% KOH in 50% methanol in a nitrogen zymosterol in yeast grown in the presence of atmosphere. Non-saponifiable lipids were ex ethionine under various conditions. The role of tracted by the usual method and analyzed with a zymosterol in the methylation reaction of the Shimadzu GC-5A or Hitachi K-53 gas-liquid side-chain is also discussed. chromatograph equipped with a flame ionization detector. A glass column (4 mm •~ 1 .5 m) was MATERIALS AND METHODS used. The liquid phase was SE-30 (1 .5•“) ad sorbed on acid-washed Chromosorb W (Shimadzu Growth of Yeast-The yeast strains used were Co., Ltd.). Column temperature was 230•Ž and Saccharomyces cerevisiae ATCC 12341 (wild strain) flow-rate of nitrogen carrier gas was 50 ml per and a methionine-less mutant (strain No . 16) minute, unless otherwise indicated . For the iden obtained by UV-irradiation of the haploid-type of tification of sterol Z, GLC with the following three Saccharomyces cerevisiae (mating type a, galactose columns was carried out , using cholestane as an fermentable, pantothenate-non-requiring). The internal standard: 5 % DEGS , 1.5% OV-17 basal growth medium for the wild strain was the (Shimadzu Co., Ltd.) and Diasolid ZS (Nihon same as described previously (3), except for the Chromato Works, Ltd.). The determination of addition of DL-ethionine, and that for the mutant non-saponifiable lipids was carried out as follows: strain was the same as described by Naiki and Squalene, sterol Z and ergosterol were determined Iwata (9), except for the addition of the indicated by measuring the height of each peak . Total concentrations of DL-ethionine and 100 PM DL sterols were determined by weighing tracings of all methionine. For most of the growth experiments , areas of sterol peaks, using ƒÀ-cholestanol as a 50 ml of the growth medium in a 250-m1 Erlenmeyer standard. The amount of other sterols including J. Biochem. ACCUMULATION OF ZYMOSTEROL IN YEAST 1111 lanosterol, fecosterol, and episterol, etc. was ob yeast cells, the effects of various compounds on the tained by subtracting the amounts of sterol Z and incorporation of radioactivity from L-[methyl-14C-] ergosterol from that of total sterols. methionine into sterols were examined. Among Mass Spectrometry-For the analysis of sterols the compounds tested, only ethionine inhibited the by mass spectrometry, a Hitachi RMU-6D was methylation and other compounds such as betaine, used. The electron energy and temperature of sarcosine, and norleucine were ineffective (data the sample heater were 70 ell and 120•Ž, respec not shown). tively. Injection of the sample heater was carried The yeasts were grown in the presence and out by the direct injection method. absence of ethionine and were harvested at the Thin-Layer Chromatography-For the isolation late logarithmic growth phase, and the non of sterol Z, TLC was carried out. Kieselguhr G saponifiable lipids extracted from the cells were (Merck, Art. 8129) was used as the adsorbent. analyzed by GLC as described in " MATERIALS The solvent system used was cyclohexane-ethyl AND METHODS. " The chromatograms in Fig. 1 acetate (99.5 : 0.5, v/v). A part of the chromato show a typical example of gas-liquid chromato plate, after development, was sprayed with 20 grams of non-saponifiable lipids obtained from the SbCl5 solution in chloroform. wild strain of yeast grown under both conditions. Cleavage of Sterol Side-Chain-The cleavage of Three major peaks representing squalene, unidenti the sterol side-chain was carried out as described fied sterol Z and ergosterol, and three minor peaks previously (3) with some modifications. Thesterol representing lanosterol, fecosterol, and episterol are sample was allowed to react with osmium tetroxide seen in the yeast grown in the absence of ethionine in ether. The osmium complex with the sterol was (Fig. IA). In the yeast grown in the presence of degraded with sodium sulfite and the resulting ethionine, in contrast, sterol Z is the most abundant steroid compound was extracted with ether.
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