Development Of Metabolomics Method Based On GC-MS And Its Application For Studying Mechanism Of Ethanol Tolerance In Sacchromyces Cerevisiae

Metabolomics,as one of the most important means of life science research,reveals the intricate mechanism of biological regulation through qualitative and quantitative analysis of small molecular metabolites in organisms.In recent years,with the rapid expansion of industry,China has also entered the peak of resource problems and environmental pressure.At present,Greenhouse Effect ? acid rain ?ozone layer destruction and other problems are the heaviest in history.These problems have increased the demand for clean energy.Bioethanol,as a liquid fuel that can replace fuel,has attracted extensive attention.Bioethanol is mainly produced by Saccharomyces cerevisiae fermentation,and the stress of high temperature,high sugar,high pressure and increasing ethanol concentration in the fermentation process will inhibit the growth of Saccharomyces cerevisiae and lead to the decrease of fermentation performance.And then affect the final production of ethanol.Therefore,it is of great significance to explore the ethanol tolerance mechanism of Saccharomyces cerevisiae to solve the problem of energy crisis and environmental pollution.At present,the reaction mechanism of Saccharomyces cerevisiae to ethanol stress has not been fully clarified.In this paper,the sample treatment method of Saccharomyces cerevisiae S288 C was optimized.The metabolic difference of Saccharomyces cerevisiae S288 C stressed with 0% ? 2% and 5% different concentrations of ethanol and under 0% and 5% ethanol stress different time was analyzed based on GC-MS.The mechanism of ethanol tolerance of Saccharomyces cerevisiae was elucidated.The main results achieved are as follows:(1)The method of pre-treatment of saccharomyces cerevisiae S288 C was optimized and characterized.The cells were quenched with the method of cold physiological saline.This method not only can effectively inactivate the activity of the enzyme,but also can avoid the leakage of metabolites in the cell as much as possible.The effects of five extraction solvent: methanol(-20?),acetonitrile/water(1:1)(-20?),acetonitrile/methanol/water(2:2:1)(-20?),water/isopropanol/methanol(2:2:5)(-20?),and 75 % ethanol(boiling)on the extraction efficiency of metabolites wereinvestigated.The water/isopropanol/methanol(2:2:5)(-20?)was defined as the best extraction solvent.(2)Based on the metabolomics method of GC-MS,the metabolic profiles of yeast under ethanol stress were compared and analyzed.It was found that the metabolic phenotypes of yeast changed obviously under different concentrations of ethanol.Pathway analysis showed that the metabolic pathways of citrate cycle,alanine,aspartate and glutamate metabolism were changed significantly.According to the change trends of the intermediate metabolites(succinic acid,citric acid,pyruvic acid,fumaric acid,glutamic acid,aspartic acid)in these metabolic pathways,we suspect that ethanol stress hinders the citrate cycle,which may lead to insufficient energy supply,reduced utilization of carbon sources,and reduced synthesis of some amino acids(glutamic acid)required for cell growth.Furthemore,the growth of cell was slowed down.