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

酿酒酵母在加工过程中承受着温度、渗透压、氧气和营养饥饿等多种压力。所有这些因素都调节参与应激反应及其相关代谢的基因的诱导。由于不同的特定工艺参数作为胁迫诱导剂，酵母基因的表达也可以直接被诱导。在本项目中，巴氏酵母菌胁迫诱导基因的天然启动子。卡氏酵母菌株TUM 34/70和酿酒酵母菌株TUM 68将进行不同的冷热冲击试验。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。根据工业可实现的工艺参数，将评估酵母在不同胁迫条件下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兼容性，以产生工艺优化的酵母的合理使用。选择的基因(atf1和gpd1)具有改善挥发性酯生物合成和相关代谢产物的特性，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提高酵母发酵饮料的效率，从而优化风味。此外，酵母菌代谢的关键代谢物的定量和定性测定将通过建立的HILIC-MS/MS-方法进行差示离线LC/核磁共振分析。此外，还将分析不同应激源下的蛋白质(Atf1p和Gpd1p)的酶活性和稳定性，特别是在发酵过程结束时。此外，还将研究酵母的代谢和生理条件以及自克隆菌株在不同胁迫条件下的生长动力学。

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.