Effect of redox potential on the gene expression of Saccharomyces cerevisiae during very-high-gravity ethanol fermentation

该提案的目标之一是研究氧化还原电位对酿酒酵母在极高浓度（VHG）乙醇发酵过程中基因表达的影响。为了实现上述目标，将开发一种连续的氧化还原电位控制发酵工艺，使VHG乙醇发酵可以在特定的氧化还原电位水平下进行。一旦建立稳态，收获酵母细胞并提取RNA。然后应用微阵列技术获得在特定氧化还原电位水平下的全基因表达谱。结果，建立了基因表达模式和氧化还原电位之间的相关性。将确定由氧化还原电位调节的基因，并阐明它们在VHG条件下与乙醇生产相关的代谢功能。众所周知，NADH/NAD+是细胞内关键的还原等价物对。它调节细胞内氧化还原电位平衡。为了维持平衡，特定的代谢途径在乙醇发酵期间被重新路由朝向某些代谢产物的产生，例如乙醇、甘油。到目前为止，细胞外氧化还原电位和细胞内还原当量对之间的关系缺乏。该提案的第二个目的是利用上述发酵过程与微阵列技术相结合，以建立两者之间缺失的联系。短期目标包括（1）开发氧化还原电位控制的VHG乙醇发酵过程，和（2）阐明和建立细胞外氧化还原电位与酵母基因表达模式之间的相关性。在短期目标研究的基础上，提出了长期目标：（1）在分子水平上开发新的VHG乙醇发酵策略;（2）设计基因工程策略以提高S.H.酿酒酵母在乙醇生产过程中。尽管拟议项目针对的是酵母属。对于乙醇生产，当使用天然或遗传修饰的梭菌属时，也可以采用和实施相同的方法来研究丁醇生产。

One of the objectives of the proposal is to study the effect of redox potential on the gene expression of Saccharomyces cerevisiae during very-high-gravity (VHG) ethanol fermentation. To achieve the stated objective, a continuous redox potential-controlled fermentation process will be developed, so that VHG ethanol fermentation can be operated at a specified redox potential level. Once steady state is established, yeast cells are harvested and RNAs are extracted. Microarray technology is then applied to obtain whole gene expression profile under a specified redox potential level. As a result, correlation between gene expression pattern and redox potential is established. The genes modulated by redox potential will be identified and their metabolic functions relating to ethanol production under VHG conditions will be elucidated. It is known that NADH/NAD+ is a key reducing equivalent pair inside a cell. It modulates intracellular redox potential balance. To maintain the balance, specific metabolic pathway is re-routed towards the production of certain metabolite, such as ethanol, glycerol during ethanol fermentation. Until now, a relationship between extracellular redox potential and intracellular reducing equivalent pairs is lacking. The second objective of the proposal is to utilize the above-noted fermentation process in conjunction with microarray technology to establish the missing linkage between the two. The short-term objectives include (1) to develop redox potential-controlled VHG ethanol fermentation process, and (2) to elucidate and to establish correlations between extracellular redox potential and yeast gene expression patterns. Based on findings obtained from short-term objectives, the long-term objectives include (1) to develop novel VHG ethanol fermentation strategies at molecular level, and (2) to design genetic engineering strategies to improve performance of S. cerevisiae during ethanol production. Although the proposed project is targeting at Saccharomyces spp. for ethanol production, the same approach can also be adopted and implemented to investigate butanol production when using Clostridium spp, either native or genetically modified.