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

自克隆酵母温度诱导基因表达和代谢组调节的生物技术评价

基本信息

批准号：
269148392
负责人：
Professor Dr.-Ing. Thomas Becker
金额：
--
依托单位：
Hochschulpräsidium
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2015
资助国家：
德国
起止时间：
2014-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/269148392?language=en
关键词：
Biotechnological evaluation temperature induced gene

项目摘要

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.

饮料加工酵母菌是几种应激源的基础，如温度、渗透压、氧气和营养饥饿。所有这些因素都调节应激反应及其相关代谢基因的诱导。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表达。本研究利用天然启动子对巴斯德酵母的胁迫诱导基因进行了启动子研究。将通过不同的热冲击和冷冲击对卡尔酵母菌株TUM 34/70和酿酒酵母菌株TUM 68进行评价。通过绿色荧光蛋白（eGFP）的表达水平及其荧光检测来分析启动子的诱导能力。将基于工业上可实现的工艺参数，评价不同强制降解条件下表达强度的诱导性以及与温度、底物可用性和酵母生理条件相关的生物加工相容性，以合理使用工艺优化的酵母。选定的基因（ATF 1和GPD 1）在挥发性酯和相关代谢物的生物合成方面具有产品改进特性，将受到评估的启动子的控制，以创建自克隆酵母菌株。这些表达系统的温度诱导的活化有助于酵母发酵饮料的效率，以优化风味特征。此外，将通过差分离线LC/NMR分析法结合已建立的HILIC-MS/MS方法，对酵母代谢的关键代谢物进行定量和定性测定。此外，还将分析酶活性和蛋白质（Atf 1 p和Gpd 1 p）的稳定性，特别是在发酵过程结束时。此外，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将被研究。