酿酒酵母酚类抑制物耐受性脂质组学研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-1338

摘要/Abstract

摘要：

通过脂质组学分析方法从细胞膜磷脂分布方面探究适应进化酿酒酵母酚酸耐受性机制。主要利用高效液相色谱-质谱（LC-MS）对酚酸胁迫下适应进化菌株和原始菌株脂质成分检测并进行统计学比较分析。检测出565种脂质代谢物,包含细胞膜磷脂185种。相比初始菌株,适应进化菌株细胞膜中磷脂酰胆碱（PC）、磷脂酰乙醇胺（PE）和磷脂酰肌醇（PI）类磷脂分子相对含量增加,含有长链（C32-C36）和双不饱和脂酰链的磷脂分子含量增加。统计学分析表明显著性差异磷脂分子主要为含有长链不饱和脂酰链的PC和PE类磷脂分子。推测适应进化菌株通过膜磷脂重塑提高细胞膜完整性,对酚类抑制物起到选择性屏障作用,从而保持细胞活性。

关键词: 脂质组学, 酚类抑制物, 酿酒酵母, 膜磷脂重塑, 木质纤维素

Abstract:

The mechanism of the adapted Saccharomyces cerevisiae tolerating to phenolic inhibitors was analyzed by lipidomics from the phospholipid profiles on cell membrane. The lipids of S. cerevisiae wild-type（WT）and adapted strains cultured under the phenolic compound stress were detected using liquid chromatography mass spectrometry（LC-MS）and systematically compared and analyzed by statistical method. The results showed that 565 lipid metabolites were identified,including 185 cell membrane phospholipids. The adapted strain presented the increase in the relative abundance of phosphatidylcholine（PC）,phosphatidylethanolamine（PE）,phosphatidylinositol（PI）,and the phospholipids with long-chain（C32-C36）and diunsaturated fatty acyl chain,while compared with the WT strain. The result of statistical analysis showed that most significant difference in the phospholipids of the WT and the adapted strain were PC and PE with long-chain and unsaturated fatty acyl chain. The mechanism of S. cerevisiae tolerating to phenolics was speculated that the plasma membrane integrity was improved via membrane phospholipids remodeling,i.e.,selectively shielded to phenolic compounds,thus the cells remained high activity.

Key words: lipidomics, phenolic inhibitor, Saccharomyces cerevisiae, membrane phospholipids remodeling, lignocellulose

顾翰琦, 邵玲智, 刘冉, 刘晓光, 李玲, 刘倩, 李洁, 张雅丽. 酿酒酵母酚类抑制物耐受性脂质组学研究[J]. 生物技术通报, 2021, 37(1): 15-23.

GU Han-qi, SHAO Ling-zhi, LIU Ran, LIU Xiao-guang, LI Ling, LIU Qian, LI Jie, ZHANG Ya-li. Lipidomics Analysis of Saccharomyces cerevisiae with Tolerance to Phenolic Inhibitors[J]. Biotechnology Bulletin, 2021, 37(1): 15-23.

图/表 4

图1 酚酸胁迫下酿酒酵母脂质成分LC-MS分析总离子流图（TIC） a、b：正、负离子模式下OPLS-DA模型的得分图;c、d：正、负离子模式下Permutation检验图;e、f：正、负离子模式下S-Plot图

图2 酚酸抑制物作用下酿酒酵母脂质种类分布

图3 酚酸胁迫下酵母菌株细胞膜磷脂相对含量分布 a：磷脂种类分布;b：不同饱和度磷脂分布;c：不同脂酰链长度磷脂分布

图4 酚酸适应进化菌株脂质正负离子模式下OPLS-DA模型分析

参考文献 31

[1]	曹运齐, 刘云云, 胡南江, 等. 燃料乙醇的发展现状分析及前景展望[J]. 生物技术通报, 2019,35(4):169-175.
	Cao Y, Liu Y, Hu N, et al. Current status and prospects of fuel ethanol production[J]. Biotechnology Bulletin, 2019,35(4):169-175.
[2]	李洪兴, 张笑然, 沈煜, 等. 纤维素乙醇生物加工过程中的抑制物对酿酒酵母的影响及应对措施[J]. 生物工程学报, 2009(9):1321-1328.
	Li H, Zhang X, Shen Y, et al. Inhibitors and their effects on Saccharomyces cerevisiae and relevant countermeasures in bioprocess of ethanol production from lignocellulose-a review[J]. Chinese Journal of Biotechnology, 2009(9):1321-1328.
[3]	Gu H, Zhang J, Bao J. Inhibitor analysis and adaptive evolution of Saccharomyces cerevisiae for simultaneous saccharification and ethanol fermentation from industrial waste corncob residues[J]. Bioresour Technol, 2014,157:6-13. URL pmid: 24518544
[4]	Yi X, Gu H, Gao Q, et al. Transcriptome analysis of Zymomonas mobilis ZM4 reveals mechanisms of tolerance and detoxification of phenolic aldehyde inhibitors from lignocellulose pretreatment[J]. Biotechnol Biofuels, 2015,8:153.
[5]	Liu ZL. Molecular mechanisms of yeast tolerance and in situ detoxification of lignocellulose hydrolysates[J]. Appl Microbiol Biotechnol, 2011,90(3):809-825.
[6]	Aulitto M, Fusco S, Nickel D, et al. Seed culture pre-adaptation of Bacillus coagulans MA-13 improves lactic acid production in simultaneous saccharification and fermentation[J]. Biotechnol Biofuels, 2019,12:45.
[7]	Gu H, Zhu Y, Peng Y, et al. Physiological mechanism of improved tolerance of Saccharomyces cerevisiae to lignin-derived phenolic acids in lignocellulosic ethanol fermentation by short-term adaptation[J]. Biotechnol Biofuels, 2019,12:268. URL pmid: 31755875
[8]	张苗苗, 陆栋, 剡倩, 等. 细胞膜对酿酒酵母乙醇耐受性影响的研究进展[J]. 中国酿造, 2016,35(9):16-19.
	Zhang M, Lu D, Yan Q, et al. Research progress on effect of cell membrane on ethanol tolerance of Saccharomyces cerevisiae[J]. China Brewing, 2016,35(9):16-19.
[9]	Qi Y, Liu H, Chen X, et al. Engineering microbial membranes to increase stress tolerance of industrial strains[J]. Metab Eng, 2019,53:24-34.
[10]	Gu H, Zhang J, Bao J. High tolerance and physiological mechanism of Zymomonas mobilis to phenolic inhibitors in ethanol fermentation of corncob residue[J]. Biotechnol Bioeng, 2015,112(9):1770-1782.
[11]	巩林林, 杨波, 杨光, 等. 有机溶剂乙腈对酵母菌细胞膜透性的影响[J]. 工业微生物, 2019,49(6):44-49.
	Gong L, Yang B, Yang G, et al. Effects of organic solvent acetonitrile on cell membrane permeability of yeast[J]. Industrial Microbiology, 2019,49(6):44-49.
[12]	郭红, 邱月, 魏建平, 等. 酿酒酵母细胞壁和细胞膜应对高渗胁迫机制研究[J]. 农业机械学报, 2020,51(6):353-359.
	Guo H, Qiu Y, Wei J, et al. Hyperosmotic stress response of Saccharomyces cerevisiae cell wall and cell membrane[J]. Transactions of the Chinese Society for Agricultural Machinery, 2020,51(6):353-359.
[13]	Tian H, Zhou J, Qiao B, et al. Lipidome profiling of Saccharomyces cerevisiae reveals pitching rate-dependent fermentative performance[J]. Appl Microbiol Biotechnol, 2010,87(4):1507-1516.
[14]	Xia J, Jones A, Lau M, et al. Comparative lipidomic profiling of xylose-metabolizing S. cerevisiae and its parental strain in different media reveals correlations between membrane lipids and fermentation capacity[J]. Biotechnol Bioeng, 2011,108(1):12-21.
[15]	Jeucken A, Brouwers J. High-throughput screening of lipidomic adaptations in cultured cells[J]. Biomolecules, 2019,9(2):42.
[16]	Kodedová M, Valachovič M, Csáky Z, et al. Variations in yeast plasma-membrane lipid composition affect killing activity of three families of insect antifungal peptides[J]. Cell Microbiol, 2019,21(12):e13093. doi: 10.1111/cmi.13093 URL pmid: 31376220
[17]	De Kroon A, Rijken P, De Smet C. Checks and balances in membrane phospholipid class and acyl chain homeostasis, the yeast perspective[J]. Prog Lipid Res, 2013,52(4):374-394. doi: 10.1016/j.plipres.2013.04.006 URL pmid: 23631861
[18]	Wang X, Jiang S, Zhang W, et al. Study on reproductive toxicity of fine particulate matter by metabolomics[J]. Chinese J of Anal Chem, 2017,45(5):633-640.
[19]	Dong Y, Hu J, Fan L, et al. RNA-Seq-based transcriptomic and metabolomic analysis reveal stress responses and programmed cell death induced by acetic acid in Saccharomyces cerevisiae[J]. Sci Rep, 2017,7:42659. doi: 10.1038/srep42659 URL pmid: 28209995
[20]	Yang J, Ding M, Li B, et al. Integrated phospholipidomics and transcriptomics analysis of Saccharomyces cerevisiae with enhanced tolerance to a mixture of acetic acid, furfural, and phenol[J]. OMICS, 2012,16(7-8):374-386. doi: 10.1089/omi.2011.0127 URL pmid: 22734833
[21]	Renne M, De Kroon A. The role of phospholipid molecular species in determining the physical properties of yeast membranes[J]. FEBS Letters, 2018,592(8):1330-1345. URL pmid: 29265372
[22]	Dawaliby R, Trubbia C, Delporte C, et al. Phosphatidylethanolamine is a key regulator of membrane fluidity in eukaryotic cells[J]. J Biol Chem, 2016,291(7):3658-3667. doi: 10.1074/jbc.M115.706523 URL pmid: 26663081
[23]	Muthukumar K, Nachiappan V. Phosphatidylethanolamine from phosphatidylserine decarboxylase2 is essential for autophagy under cadmium stress in Saccharomyces cerevisiae[J]. Cell Biochem and Biophys, 2013,67(3):1353-1363.
[24]	Koynova R, Caffrey M. Phases and phase transitions of the hydrated phosphatidylethanolamines[J]. Chem and Phys Lipids, 1994,69(1):1-34.
[25]	Yin N, Zhu G, Luo Q, et al. Engineering of membrane phospholipid component enhances salt stress tolerance in Saccharomyces cerevisiae[J]. Biotechnol Bioeng, 2020,117(3):710-720. doi: 10.1002/bit.27244 URL pmid: 31814106
[26]	Wang X, Bai X, Chen D, et al. Increasing proline and myo-inositol improves tolerance of Saccharomyces cerevisiae to the mixture of multiple lignocellulose-derived inhibitors[J]. Biotechnol Biofuels, 2015,8:142. URL pmid: 26379774
[27]	Besada-Lombana P, Fernandez-Moya R, Fenster J, et al. Engineering Saccharomyces cerevisiae fatty acid composition for increased tolerance to octanoic acid[J]. Biotechnol Bioeng, 2017,114(7):1531-1538.
[28]	Ballweg S, Sezgin E, Doktorova M, et al. Regulation of lipid saturation without sensing membrane fluidity[J]. Nat Commun, 2020,11(1):756. URL pmid: 32029718
[29]	Liu P, Chernyshov A, Najdi T, et al. Membrane stress caused by octanoic acid in Saccharomyces cerevisiae[J]. Appl Microbiol Biotechnol, 2013,97(7):3239-3251. URL pmid: 23435986
[30]	Fang Z, Chen Z, Wang S, et al. Overexpression of OLE1 enhances cytoplasmic membrane stability and confers resistance to cadmium in Saccharomyces cerevisiae[J]. Appl Environ Microb, 2016,83(1):e02319-16.
[31]	Kim H, Kim N, Choi W. Total fatty acid content of the plasma membrane of Saccharomyces cerevisiae is more responsible for ethanol tolerance than the degree of unsaturation[J]. Biotechnol Lett, 2011,33(3):509-515.