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Dec 2016

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Sterol Analysis in Kluyveromyces lactis
乳酸克鲁维酵母中的甾醇分析   

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Abstract

Sterols are essential lipids of most eukaryotic cells with multiple functions (structural, regulatory and developmental). Sterol profile of yeast cells is often determined during the studies of ergosterol synthesis mutants used to uncover a number of functions for various sterols in yeast cells. Molecular studies of ergosterol biosynthesis have been also employed to identify essential steps in the pathway against which antifungals might be developed. We present here a protocol for the isolation of non-saponifiable lipids (sterols) from Kluyveromyces lactis yeast cells and a chromatographic method for quantitative analysis of sterols in lipid extracts (HPLC) that can be performed in laboratories with standard equipment.

Keywords: Ergosterol biosynthesis (麦角甾醇生物合成), Kluyveromyces lactis (乳酸克鲁维酵母), Lipid extraction (脂质提取), High-performance liquid chromatography (高效液相色谱法)

Background

Ergosterol, the primary membrane sterol found in yeast cells, serves a structural role in cellular membranes similar to that of cholesterol in mammalian systems. Sterols have been shown to be responsible for a number of important physical characteristics of membranes by affecting rigidity, fluidity and permeability of membranes. Through their interactions with phospholipids and sphingolipids, sterols are proposed to maintain the lateral heterogeneity of the protein and lipid distribution in the plasma membrane because of their putative role in inducing microdomains called lipid rafts (Dupont et al., 2011; Souza et al., 2011). Sterol biosynthesis in yeast is an energy-expensive, multistep aerobic process, requiring heme and molecular oxygen. Ergosterol and its biosynthetic steps are the major targets for antifungal compounds which have minor effects on cholesterol synthesis of the host organism (Daum et al., 1998). Both ergosterol and some of its biosynthetic intermediates (squalene, 7-dehydrocholesterol) belong to chemicals with a direct positive appeal to people. Therefore a simple and reliable method for sterol isolation is highly rewarding (Valachovic and Hapala, 2017). In this protocol, we describe the analytical method for sterol isolation used for determination of sterol profile in yeast cells.

Materials and Reagents

  1. Pipette tips (Eppendorf, GBO)
  2. Inoculation loop
  3. 15-ml polypropylene centrifuge tubes (Corning, catalog number: 430791 )
  4. Acid-washed glass beads, diameter 0.45 mm (Sigma-Aldrich, catalog number: G8772 ) (see Note 1)
  5. Glass Pasteur pipette (Sigma-Aldrich, catalog number: Z628018 )
  6. 20 ml Pyrex® glass tubes with Teflon cups (Corning) (Sigma-Aldrich, catalog number: Z653527) (see Note 1)
    Manufacturer: Pyrex, catalog number: 1622/10M .
  7. Kluyveromyces lactis yeast strain to be analysed
  8. n-Hexane (anhydrous 95%) (Sigma-Aldrich, catalog number: 439177 ) (see Note 1)
  9. HPLC standard ergosterol (purity ≥ 95%) (Sigma-Aldrich, catalog number: 45480 ); 0.3 mg/ml stock solution in methanol
  10. Yeast extract (Biolife Italiana, catalog number: 4122202 )
  11. Bacto peptone (Biolife Italiana, catalog number: 4122592 )
  12. D-Glucose (Biolife Italiana, catalog number: 4125012 )
  13. Potassium hydroxide (KOH) pellets (Merck, catalog number: 1050331000 )
  14. Methanol (CHROMASOLVTM for HPLC, ≥ 99.9%) (Honeywell, catalog number: 24229 )
  15. Water (CHROMASOLV® Plus, for HPLC) (Sigma-Aldrich, catalog number: V270733 )
    Note: This product has been discontinued.
  16. YEPD rich growth medium (see Recipes)
  17. Methanolic KOH solution (60% KOH, 50% methanol) (see Note 2 and Recipes)

Equipment

  1. Pipettes (Eppendorf, HTL)
  2. Incubation shaker Multitron Standard (Infors HT, Bottmingen, Switzerland)
  3. Haemocytometer
  4. Centrifuge 5804R (Eppendorf, model: 5804 R )
  5. High speed Vortex-Genie 2 (Scientific Industries, model: Digital Vortex-Genie 2 , catalog number: SI-A246)
  6. Automatic sampler (Shimadzu Scientific Instruments, model: SIL-20AC )
  7. HPLC instrument (Shimadzu Scientific Instruments, model: Prominence 20A ) equipped with reversed phase C18 column (Ascentis® Express C18, particle size 5 μm, column size 3 x 150 mm, Supelco), UV-Vis detector (Shimadzu Scientific Instruments, model: SPD-20A )
  8. FastPrep® cell homogenize (MP Biomedicals, model: FastPrep®-24, catalog number: 116004500 )

Procedure

  1. Use an inoculation loop to transfer a tip of a single colony from an agar plate into 100 ml of sterile liquid YEPD medium (see Recipes). Incubate the culture in a shaker at 150 rpm at 28 °C to reach the exponential phase (1 x 107 cells/ml). Monitor the cell density by counting the cell number in a haemocytometer.
  2. Harvest the cells by centrifugation at 3,000 x g for 5 min at room temperature (RT) and discard the supernatant.
  3. Wash the cell pellets with deionized water (RT) and harvest the cells by centrifugation (3,000 x g, 5 min, RT).
  4. Resuspend the cell pellets in deionized water (RT) to a concentration of 1 x 109 cells in 1 ml of water. Transfer 1 ml of cell suspension into a new polypropylene 15 ml tube.
  5. Add 1 ml of sterile glass beads to the tube and cool 3 min on ice. 
  6. Break the pre-cooled cells in vortex 6 x 60 sec at 2,500 rpm with 1 min cooling on ice between the breaking runs (see Note 2).
  7. Transfer broken cell suspension (without any glass beads) with glass Pasteur pipette to 20 ml Pyrex glass tube with Teflon cup.
  8. Add 3 ml of methanolic KOH solution and incubate for 2 h at 70 °C (see Notes 3 and 4).
  9. Cool the mixture, add 3 ml of n-hexane and mix well (10 sec) on a vortex mixer.
  10. Separate the organic and water phases at RT by centrifugation for 5 min at 3,000 x g.
  11. Transfer the upper organic phase with glass Pasteur pipette to a clean Pyrex glass tube with Teflon cup.
  12. Re-extract the water phase with 3 ml of n-hexane, collect the upper phase separated by centrifugation (5 min, 3,000 x g, RT) and join both organic phases.
  13. Evaporate n-hexane from the joined organic phases under the stream of nitrogen going into the tube for 10 min at RT, 3 bar.
  14. For HPLC analysis of non-saponifiable lipids (sterols), dissolve dry lipid extract equivalent of 1 x 109 cells in 1 ml of solvent (e.g., n-hexane or acetone) (see Note 5).
  15. Load 10 μl aliquot on the C18 column using automatic sampler. Ergosterol is separated with 95% methanol and 5% water as the mobile phase, flow 0.8 ml/min. Column temperature is set to 25 °C (see Note 6).
  16. Ergosterol is detected using UV-Vis detector at 280 nm.

Data analysis

The peak identity was determined from the retention times of ergosterol standard and from characteristic spectra (Figure 1). The quantity of sterols could be calculated from calibration curves constructed for individual standards used.


Figure 1. Chromatogram of sterols (A) Standard ergosterol (0.8 mmol/L) and (B) Sterol profile of K. lactis cells (ergosterol represents more than 80% of total amount of yeast sterols)

Notes

  1. All organic solvents used in lipid extraction should be of highest purity. Plastic material must not be used during the extraction procedure (with exception of the cell breaking step). Organic solvents release additives from the plastics which might interfere with subsequent analysis. Glass and Teflon® materials should be used throughout the procedure.
  2. Due to a rigid cell wall it is essential to break yeast cells for efficient extraction of lipids. FastPrep® cell homogenizer is recommended (e.g., MP Biomedicals). 2 x 45 sec breaking intervals with 5 min cooling on ice between the subsequent runs is recommended.
  3. To prepare 10 ml of methanolic KOH solution it is recommended to dissolve 6 g of KOH pellets in 5 ml in methanol mixed with 2.5 ml water. When KOH is fully dissolved, final volume should be adjusted to 10 ml with water. As the reaction is strongly exothermic special care should be taken during preparation of the solution.
  4. Efficiency of neutral lipid hydrolysis depends on the quality of methanolic KOH mixture. It is strongly recommended to use freshly prepared solution.
  5. Due to high volatility of the solvents, lipid extracts are always dried under the stream of nitrogen and dissolved in an exact volume of the solvent immediately before chromatographic analysis.
  6. Sterols present in the non-saponifiable lipid extracts originate from the free sterol fraction and from hydrolysed steryl ester fraction. If HPLC analysis is not used for preparative purification, column temperature can be increased to 40 °C to speed up the separation process.
  7. The HPLC-UV method proposed here may be useful for the sterol analysis in various yeasts and possibly for other types of sample (Valachovic and Hapala, 2017). Using standards of other sterols e.g., lanosterol, squalene, zymosterol, it is possible to determine their quantity in the sample.

Recipes

  1. YEPD rich growth medium
    2% D-glucose
    1% yeast extract
    2% Bacto peptone
  2. Methanolic KOH solution (60% KOH, 50% methanol)
    6 g KOH pellets
    5 ml methanol
    H2O (distilled H2O) up to 10 ml

Acknowledgments

This protocol was adapted from Valachovic and Hapala (2017) and used in our previous studies (Goffa et al., 2014; Konecna et al., 2016). This work was supported by the Slovak Research and Development Agency grant APVV-0282-10 and VEGA 2/0111/15.

References

  1. Daum, G., Lees, N. D., Bard, M. and Dickson, R. (1998). Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast 14(16): 1471-1510.
  2. Dupont, S., Beney, L., Ferreira, T. and Gervais, P. (2011). Nature of sterols affects plasma membrane behavior and yeast survival during dehydration. Biochim Biophys Acta 1808(6): 1520-1528.
  3. Goffa, E., Balazfyova, Z., Toth Hervay, N., Simova, Z., Balazova, M., Griac, P. and Gbelska, Y. (2014). Isolation and functional analysis of the KlPDR16 gene. FEMS Yeast Res 14(2): 337-345.
  4. Konecna, A., Toth Hervay, N., Valachovic, M. and Gbelska, Y. (2016). ERG6 gene deletion modifies Kluyveromyces lactis susceptibility to various growth inhibitors. Yeast 33(12): 621-632.
  5. Souza, C. M., Schwabe, T. M., Pichler, H., Ploier, B., Leitner, E., Guan, X. L., Wenk, M. R., Riezman, I. and Riezman, H. (2011). A stable yeast strain efficiently producing cholesterol instead of ergosterol is functional for tryptophan uptake, but not weak organic acid resistance. Metab Eng 13(5): 555-569.
  6. Valachovic, M. and Hapala, I. (2017). Biosynthetic approaches to squalene production: The case of yeast. Methods Mol Biol 1494: 95-106.

简介

甾醇是具有多种功能(结构,调节和发育)的大多数真核细胞的必需脂质。 在用于揭示酵母细胞中各种甾醇的许多功能的麦角甾醇合成突变体的研究期间,酵母细胞的甾醇分布通常被确定。 麦角甾醇生物合成的分子研究也被用于鉴定可能开发抗真菌剂途径的基本步骤。 我们在这里介绍从乳酸克鲁维酵母酵母细胞中分离不皂化脂质(甾醇)的方案和用于在实验室中用标准设备进行的脂质提取物(HPLC)中固醇的定量分析的色谱法。
【背景】在酵母细胞中发现的主要膜固醇的麦角固醇在哺乳动物系统中与胆固醇相似的细胞膜中起着结构的作用。通过影响膜的刚性,流动性和渗透性,甾醇已被证明对膜的许多重要物理特征负责。通过它们与磷脂和鞘脂的相互作用,提出甾醇以维持蛋白质的侧向异质性和质膜中的脂质分布,因为它们在诱导称为脂筏的微区域中的推定作用(Dupont等人,2011; Souza等, 2011)。酵母中的甾醇生物合成是一种能量昂贵的多步需氧过程,需要血红素和分子氧。谷歌甾醇及其生物合成步骤是对宿主生物体胆固醇合成影响较小的抗真菌化合物的主要目标(Daum等,1998)。麦角甾醇及其一些生物合成中间体(角鲨烯,7-脱氢胆固醇)均属于直接吸引人的化学物质。因此,用于甾醇分离的简单可靠的方法是非常有益的(Valachovic和Hapala,2017)。在该方案中,我们描述了用于测定酵母细胞中甾醇分布的固醇分离的分析方法。

关键字:麦角甾醇生物合成, 乳酸克鲁维酵母, 脂质提取, 高效液相色谱法

材料和试剂

  1. 移液器提示(Eppendorf,GBO)
  2. 接种环
  3. 15-ml聚丙烯离心管(Corning,目录号:430791)
  4. 酸洗玻璃珠,直径0.45毫米(Sigma-Aldrich,目录号:G8772)(见注1)
  5. 玻璃巴斯德移液器(Sigma-Aldrich,目录号:Z628018)
  6. 20微米具有特氟龙杯(Corning)(Sigma-Aldrich,目录号:Z653527)的Pyrex 玻璃管(见注1)
    制造商:Pyrex,目录号:1622 / 10M。
  7. 待分析的乳酸克鲁维酵母酵母菌株
  8. - 无水(无水95%)(Sigma-Aldrich,目录号:439177)(参见注1)
  9. HPLC标准麦角甾醇(纯度≥95%)(Sigma-Aldrich,目录号:45480); 0.3毫克/毫升甲醇储备溶液
  10. 酵母提取物(Biolife Italiana,目录号:4122202)
  11. Bacto蛋白胨(Biolife Italiana,目录号:4122592)
  12. D-葡萄糖(Biolife Italiana,目录号:4125012)
  13. 氢氧化钾(KOH)颗粒(Merck,目录号:1050331000)
  14. 甲醇(CHROMASOLV TM,用于HPLC,≥99.9%)(Honeywell,目录号:24229)
  15. 水(CHROMASOLV Plus,用于HPLC)(Sigma-Aldrich,目录号:V270733)
    注意:本产品已停产。
  16. YEPD丰富的生长培养基(见食谱)
  17. 甲醇KOH溶液(60%KOH,50%甲醇)(见注2和配方)

设备

  1. 移液器(Eppendorf,HTL)
  2. 孵化器Multitron Standard(Infors HT,Bottmingen,Switzerland)
  3. 血细胞计数器
  4. 离心机5804R(Eppendorf,型号:5804 R)
  5. 高速Vortex-Genie 2(Scientific Industries,型号:Digital Vortex-Genie 2,目录号:SI-A246)
  6. 自动取样器(Shimadzu Scientific Instruments,型号:SIL-20AC)
  7. 装备有反相C18柱(Ascentis,Express C18,粒径5μm,柱尺寸3×150mm,Supelco)的HPLC仪(Shimadzu Scientific Instruments,型号:Prominence 20A),UV-Vis检测器(岛津科学仪器,型号:SPD-20A)
  8. FastPrep ®细胞匀浆(MP Biomedicals,型号:FastPrep -24,目录号:116004500)

程序

  1. 使用接种环将单个菌落的尖端从琼脂平板转移到100ml无菌液体YEPD培养基中(参见食谱)。在振荡器中在28℃下以150rpm孵育培养物达到指数期(1×10 7个细胞/ ml)。通过计数血细胞计数器中的细胞数来监测细胞密度。
  2. 通过在室温(RT)下以3,000×g离心5分钟收获细胞,并弃去上清液。
  3. 用去离子水(RT)洗涤细胞沉淀,并通过离心(3,000×g,5分钟,RT)收获细胞。
  4. 将细胞沉淀物在去离子水(RT)中悬浮至浓度为1×10 9细胞的1ml水中。将1ml细胞悬浮液转移到新的聚丙烯15ml管中
  5. 将1ml无菌玻璃珠加入管中,并在冰上冷却3分钟
  6. 在2,500 rpm转速下以6 x 60秒的涡旋方式将预先冷却的电池分开,并在断路之间用冰冷却1分钟(见注2)。
  7. 将破碎的细胞悬浮液(没有玻璃珠)用玻璃巴斯德移液管转移到具有特氟隆杯的20毫升派雷克斯玻璃管中。
  8. 加入3毫升甲醇的KOH溶液,并在70℃下孵育2小时(见注释3和4)
  9. 冷却混合物,加入3ml正己烷,并在涡旋混合器上充分混合(10秒)。
  10. 在室温下通过在3,000×g离心5分钟分离有机相和水相。/ / em>
  11. 将带有玻璃巴斯德移液器的上部有机相转移到带特氟龙杯的干净的派雷克斯玻璃管上
  12. 用3ml正己烷重新萃取水相,收集通过离心(5分钟,3000×g,RT)分离的上部相,并加入两个有机相。
  13. 在氮气流下从连接的有机相中蒸发正己烷,在室温下进入管中10分钟,3巴。
  14. 对于不皂化脂质(甾醇)的HPLC分析,将相当于1×10 9细胞的干脂质提取物溶解在1ml溶剂(例如,n - 己烷或丙酮)(见注5)
  15. 使用自动进样器在C18柱上加载10μl等分试样。用95%甲醇和5%水作为流动相分离出麦角甾醇,流速为0.8ml / min。色谱柱温度设置为25°C(见注6)
  16. 使用280nm的UV-Vis检测器检测到麦角甾醇

数据分析

峰值同一性来自麦角固醇标准品的保留时间和特征谱(图1)。甾醇的数量可以从为所使用的各个标准构建的校准曲线计算。


图1.甾醇的色谱图(A)标准麦角甾醇(0.8mmol / L)和(B)乳酸克鲁维酵母细胞的甾醇分布(麦角甾醇占酵母总量的80%以上)甾醇)

笔记

  1. 脂质提取中使用的所有有机溶剂应具有最高的纯度。在提取过程中不得使用塑料材料(细胞破碎步骤除外)。有机溶剂从塑料中释放出可能会干扰后续分析的添加剂。在整个程序中应使用玻璃和特氟隆®材料。
  2. 由于刚性细胞壁,破坏酵母细胞以有效提取脂质是至关重要的。推荐使用FastPrep ®细胞匀浆器(例如,MP Biomedicals)。推荐使用2 x 45秒的断开间隔,并在后续运行中在冰上冷却5分钟。
  3. 为了制备10ml甲醇的KOH溶液,推荐将6g KOH颗粒溶解在5ml与2.5ml水混合的甲醇中。当KOH完全溶解时,最终体积应用水调节至10ml。由于反应强烈放热,因此在制备溶液时应特别小心
  4. 中性脂质水解的效率取决于甲醇KOH混合物的质量。强烈建议您使用新鲜的溶液。
  5. 由于溶剂的高挥发性,脂质提取物总是在氮气流下干燥,并在色谱分析之前立即溶于精确体积的溶剂中。
  6. 存在于非皂化脂质提取物中的甾醇源自游离甾醇级分和来自水解的甾醇酯级分。如果HPLC分析不用于制备纯化,则柱温可以提高到40℃,以加速分离过程。
  7. 这里提出的HPLC-UV方法可用于各种酵母和可能用于其他类型样品的固醇分析(Valachovic和Hapala,2017)。使用其他固醇的标准,例如,羊毛甾醇,角鲨烯,季戊四醇,有可能确定样品中的数量。

食谱

  1. YEPD丰富的生长培养基
    2%D-葡萄糖
    1%酵母提取物
    2%Bacto蛋白胨
  2. 甲醇KOH溶液(60%KOH,50%甲醇)
    6克KOH颗粒 5毫升甲醇
    H 2 O(蒸馏H 2 O 2)至多10毫升

致谢

该协议由Valachovic和Hapala(2017)进行了改编,并用于我们以前的研究(Goffa等人,2014; Konecna等人,2016年)。这项工作得到了斯洛伐克研究与发展局的批准APVV-0282-10和VEGA 2/0111/15的支持。

参考

  1. Daum,G.,Lees,N.D.,Bard,M。和Dickson,R。(1998)。 酿酒酵母脂质的生物化学,细胞生物学和分子生物学。 / a>  酵母 14(16):1471-1510。
  2. Dupont,S.,Beney,L.,Ferreira,T。和Gervais,P。(2011)。 甾醇的性质影响脱水过程中的质膜行为和酵母存活。 / a>  Biochim Biophys Acta 1808(6):1520-1528。
  3. Goffa,E.,Balazfyova,Z.,Toth Hervay,N.,Simova,Z.,Balazova,M.,Griac,P。和Gbelska,Y。(2014)。 "KlPDR16"的隔离和功能分析基因。   FEMS酵母研究所 14(2):337-345。
  4. Konecna,A.,Toth Hervay,N.,Valachovic,M.and Gbelska,Y。(2016)。 ERG6 基因删除修改乳酸克鲁维酵母对各种生长抑制剂的易感性。  酵母 33(12):621-632。
  5. Souza,C.M.,Schwabe,T.M.,Pichler,H.,Ploier,B.,Leitner,E.,Guan,X.L.,Wenk,M.R.,Riezman,I.and Riezman,H。(2011)。 有效生产胆固醇而不是麦角固醇的稳定酵母菌对色氨酸有作用吸收,但不是弱有机酸抗性。  Metab Eng 13(5):555-569。
  6. Valachovic,M.和Hapala,I.(2017)。 角鲨烯生产的生物合成方法:酵母的情况。  Methods Mol Biol 1494:95-106。
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引用:Gbelska, Y., Toth Hervay, N., Morvova, M. and Konecna, A. (2017). Sterol Analysis in Kluyveromyces lactis. Bio-protocol 7(17): e2527. DOI: 10.21769/BioProtoc.2527.
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