转录因子RpoD调控运动发酵单胞菌乙醇耐受性的机制研究

The ethanol stress is one of the key factors which influence ethanol fermentation efficiency of biomass. We have confirmed that the mutation of rpoD gene, encoding gene of sigma 70 factor, could remarkably improve the ethanol tolerance of Z.mobilis. However, the molecular mechanism of ethanol tolerance in mutant strain remains unclear. The relationship between transcription factor RpoD and ethanol tolerance will be studied by analyzing the character of mutant strain and the content of cell membrane component, identifying the sequence of mutant RpoD and protein structure. Moreover, to reveal the molecular mechanism how the transcription regulator factor RpoD regulate the ethanol tolerance of Z. mobilis, the gene expression difference between mutant strain and parent strain will be further compared by transcriptome analysis and the differentially expressed genes will be identified. Then the DNA-binding site of transcription factor RpoD to the promoter region of key response genes will also be analyzed by using the electrophoretic mobility shift assay technology in vitro. The results will not only improve the ethanol tolerance of Z. mobilis, but also solve the key problem of large scale production of bioethanol, and promote the development of biofuel ethanol production, so as to provide the engineering strain and technical reserve for the sustainable development of biomass energy.

乙醇胁迫是影响生物质乙醇发酵效率的关键制约因素之一。我们前期实验证明σ70因子的编码基因rpoD 的突变能够显著提高运动发酵单胞菌(Zymomonas mobilis) 的乙醇耐受性，但突变菌株中乙醇耐受性提高的机制并不清楚。本项目拟通过表征突变株的特性、细胞膜各组分含量的变化分析、鉴定RpoD突变后的DNA序列和蛋白结构，研究转录因子RpoD与乙醇耐受机制之间的关系；进一步通过转录组分析突变株和出发菌株的基因表达差异，以及通过凝胶阻滞电泳体外检测RpoD与差异表达基因启动子的相互作用，揭示转录因子RpoD调控运动发酵单胞菌乙醇耐受性的分子机制，最终阐明RpoD调控运动发酵单胞菌的乙醇耐受性的机制这一科学问题。本项目的开展既能提高运动发酵单胞菌乙醇耐受性，亦能解决运动发酵单胞菌生物乙醇规模化生产的关键问题，推动生物燃料乙醇产业的发展，为生物质能源的可持续发展提供工程菌株和技术储备。

乙醇胁迫是影响生物质乙醇发酵效率的关键制约因素之一。前期实验证明σ70因子的编码基因rpoD 的突变能够显著提高运动发酵单胞菌(Zymomonas mobilis) 的乙醇耐受性，但突变菌株中乙醇耐受性提高的机制并不清楚。本项目表征了突变株和对照菌株在乙醇胁迫下的表型特性以及相应关键酶基因的表达水平；通过Clustal W软件对突变前后RpoD基因进行了比对和分析，推测了这些突变在启动子识别和转录起始中所发挥的作用；通过转录组分析了突变株和出发菌株的基因表达差异及相应的GO分析及KEGG富集分析；并针对纤维素燃料乙醇稀酸预处理工艺中存在呋喃甲醛等抑制物质对发酵菌株的影响，从rpoD突变文库中筛选到对呋喃甲醛耐性提高的工程菌株，为进一步提高运动发酵单胞菌在木质素水解液中的发酵性能提供理论依据和技术支持。本项目的开展为推动生物燃料乙醇产业的发展，以及为生物质能源的可持续发展提供了工程菌株和技术储备。