氧化还原电位调控高浓度乙醇发酵中酵母生物分子交互网络的构建

Very high gravity (VHG) fermentation can significantly improve ethanol titer, which consequently saves energy consumption for ethaol distillation, and reduce the discharge of the stillage. However, yeast cells are sufferred with environmental stresses under VHG conditions, resulting in stuck fermentation with prolonged fermenttaion time and more sugars unconverted. It was observed that oxidoreduction potential (ORP) control significantly improved viability of yeast cells, and consequently enhanced ethanol production under VHG fermentation conditions. However, the understanding of this phenomenon is still very limited, particularly at molecular levels, which is the prerequisite for process optimization. . In this project, impact of ORP control on the distribution of the intracellular metabolic fluxes of yeast cells will be investigated, with a focus on important intermediate metabolites such as pyruvate and glycerol related to ethanol production to detect key metabolic nodes and develop a debottleneck strategy for the process optimization. Meanwhile, protein analysis and differential gene expression will be explored at global levels for an insight of key enzymes that regulate the metabolic pathways. At the end, the biomolecular interaction network of yeast cells will be constructed by combining the experimental data of metabolites, enzymes and genes as well as the information from public databases for an understanding of their metabolic responses to the ORP control under VHG ethanol fermentation conditions, which may lay a foundation for developing innovative fermentation processes.

高浓度（VHG）底物发酵可以提高发酵液中乙醇浓度， 减少乙醇精馏能耗和废糟液量，但相应的环境胁迫常常使酵母细胞活性降低，发酵过程延缓。 实验证明氧化还原电位（ORP）调控可以显著改善酵母细胞活性和乙醇发酵性能。. 本项目拟首先考查ORP对酵母细胞代谢流分布的影响， 重点分析丙酮酸和甘油等与乙醇发酵相关的代谢物合成与分解过程能量和通量的变化，寻找关键节点。在此基础上，进一步研究酵母细胞蛋白组特征和基因的差异表达，寻找调控代谢途径的关键酶及相应的基因。利用代谢物、蛋白和基因表达的数据， 以及共享的组学信息，构建酵母细胞ORP控制条件下VHG 乙醇发酵过程生物分子交互网络， 揭示ORP对酵母细胞的多水平调节以及提高细胞活性和乙醇发酵性能的分子机理。预期取得的研究进展， 不仅可以加深对ORP影响酵母细胞生理代谢的理解，而且有助于优化VHG乙醇发酵过程，为创新技术开发奠定基础。

在燃料乙醇生产中，高浓度（VHG）发酵技术可以提高产物浓度，减少精馏能耗和废糟液排放，但其带来的环境胁迫常常使酵母细胞活性降低，发酵过程显著延缓。实验证明氧化还原电位（ORP）调控明显改善了细胞活性和乙醇发酵性能，但其内在机制尚不清晰。本项目首先考查不同发酵条件下ORP变化特点，发现高浓度乙醇发酵中ORP控制在-150 mV时，发酵效果最好，因为该条件兼顾了细胞的有氧呼吸和乙醇发酵两条通路。通过代谢组分析了72种胞内代谢物，主要对糖类、有机酸、核酸、氨基酸及其各衍生物进行了分类分析，发现了起重要作用的TCA和呼吸电子传递链中的代谢流改变。随后进行细胞转录组和蛋白组分析，发现蛋白合成和胁迫响应方面的基因表达和蛋白含量出现上调，从多个角度说明了ORP控制的作用机理。因此进一步全面研究了ORP 对胁迫耐受性的影响。实验结果表明ORP调控细胞对糠醛的脱毒效果最好，糠醛被100%转化为糠醇。此外在探究机理的过程中，研究开发了木质纤维素乙醇生产工艺，为ORP控制在第二代燃料乙醇生产的研究奠定了基础。取得的研究进展，不仅可以加深对ORP影响细胞生理代谢机制的理解，而且有助于优化乙醇发酵过程，为创新技术开发奠定基础。