Biotechnological evaluation of temperature induced gene expression and metabolome modulation of self-cloning Saccharomyces yeast

在饮料加工过程中，酵母菌受到几种压力因素的影响，如温度、渗透压、氧气和营养缺乏。所有这些因素调节应激反应相关基因的诱导及其相关代谢。由于不同的特定工艺参数作为胁迫诱导剂，也可以直接诱导酵母基因的表达。本项目将通过不同的冷热冲击对卡尔斯伯根酿酒酵母TUM 34/70和酿酒酵母TUM 68的胁迫诱导基因的原生启动子进行评价。启动子的诱导能力将通过绿色荧光蛋白（eGFP）的表达水平及其荧光检测来分析。在工业上可实现的工艺参数基础上，对不同胁迫条件下酵母的表达强度的诱导性以及与温度、底物利用率和生理条件的生物加工相容性进行评价，以产生工艺优化酵母的合理利用。选定的基因（ATF1和GPD1）在挥发性酯和相关代谢物的生物合成方面具有产品改善特性，将由评估的启动子控制，以创建自克隆酵母菌株。温度诱导这些表达系统的激活有助于酵母发酵饮料的效率，以优化风味特征。此外，酵母代谢的关键代谢物的定量和定性测定将通过差分离线lc /NMR-Analytic和已建立的HILIC-MS/ ms -方法进行分析。此外，将分析不同应激源下的酶活性和蛋白质（Atf1p和Gpd1p）的稳定性，特别是在发酵过程结束时。此外，还将研究不同胁迫条件下酵母菌的代谢和生理条件以及自克隆菌株的生长动力学。

The beverage processing Saccharomyces yeast underlies several stressors, such as temperature, osmotic pressure, oxygen and nutrient starvation. All these factors regulate the induction of genes involved in stress response and its related metabolism. Yeast gene expression can also be induced directly due to different specific process parameters acting as stress inducers. In this project native promoters of stress inducible genes of Saccharomyces pastorianus var. carlsbergensis strain TUM 34/70 and Saccharomyces cerevisiae strain TUM 68 will be evaluated by different heat and cold shocks. The resulting induction capacity of the promoters will be analyzed by the expression level of the green fluorescence protein (eGFP) and its fluorescence detection. The inducibility of the expression intensity as well as the bioprocessing compatibility with respect to the temperature, substrate availability and physiological conditions of the yeast during different stress conditions will be evaluated based on industrial realizable process parameters in order to generate the rational use of process optimized yeast. Selected genes (ATF1 and GPD1) which have product improving properties with respect to the biosynthesis of volatile esters and relevant metabolites will be controlled by the evaluated promoters to create self-cloning yeast strains. The temperature induced activation of these expression systems contribute to the efficiency of yeast fermented beverages in order to optimize the flavor profile. Furthermore, quantitative and qualitative determination of key-metabolites of the yeast metabolism will be analyzed by differential off-line-LC/NMR-Analytic complimented by established HILIC-MS/MS-methods. In addition the enzyme activity and the stability of the proteins (Atf1p and Gpd1p), underlying different stressors, will be analyzed especially at the end of the fermentation process. Furthermore, the metabolic and physiological conditions of the yeast as well as the growth kinetics of the self-cloning strains under different stress situations will be studied.