基于Cat8p转录激活模体鉴定及其与酿酒酵母2-苯乙醇合成相关基因互作

Biotransformation by Saccharomyces cerevisiae is the trend for 2-phenylethanol (2-PE) production, however, the yield in the present strains is low which are hardly applied in industry. Transcription factor Cat8p was found in our finished project that it played important role in improving 2-PE bio-synthesis and yeast tolerance to it. Aiming at the low synthesis efficiency and complicated regulatory mechanism, a series of exploring studies are designed based on the bio-information prediction of Cat8p, e.g. interactive genes, interactive proteins of functional domain. Using fluorescence microscopy and Chromatin immunoprecipitation (CHIP), Cat8p location and its interactive genes will be confirmed. Deletion of gene of interactive kinase will be used for analyzing the role of phosphorylation during Cat8p activating its target genes. The cis-acting elements in crucial genes of ARO8 and ARO10 targeted by Cat8p will be identified using promoter mutation methods. By analyzing interaction between Cat8p-Gat1p/Gln3p, and determining the change of target genes’ transcription level and synthesized 2-PE level after CAT8 expression and GAT1/GLN3 interruption, the hypothesis of transcriptional activation module will be verified, and the activation mechanism will be elucidated. The study of regulatory mechanism based on whole level is important for guiding 2-PE bio-synthesis efficiently.

酵母生物转化是2-苯乙醇(2-PE)生产的重要方向，但目前菌株产量低难以工业化应用。申请人结题项目研究发现，转录因子Cat8p在增强2-PE生物合成及提高酵母菌2-PE耐受性方面发挥重要作用。本项目针对现有菌株2-PE合成效率依然低而调控机制复杂的现状，基于Cat8p的生物信息预测(互作基因、功能域互作蛋白)，拟通过荧光显微技术、染色质免疫共沉淀技术研究Cat8p在L-苯丙氨酸响应中细胞定位及基因互作；缺失互作激酶基因，分析蛋白磷酸化在调控Cat8p转录激活功能中的作用；通过启动子突变研究鉴定Cat8p关键靶基因(ARO8、ARO10)顺式调控元件；分析Cat8p-Gat1p/Gln3p互作及CAT8表达-GAT1/GLN3阻断后靶基因mRNA及2-PE产量变化，验证基于Cat8p的转录激活模体假说、解析艾氏途径关键酶基因调控机制。整体水平调控机制研究对指导2-PE高效生物合成有重要意义。

艾氏途径是酿酒酵母合成2-苯乙醇(2-PE)的主要途径，但其调控因子及机制研究相对较少。我们前期研究发现转录因子Cat8在增强2-PE生物合成及提高酵母菌2-PE耐受性方面发挥重要作用，但其调控机制尚不清楚。本研究在对Cat8互作基因、功能域互作蛋白生物信息预测的基础上，关键因子qRT-PCR分析、模块菌构建、启动子调控研究表明，Cat8在2-PE合成中发挥重要的正调控作用。通过Snf1缺失，并对Snf1缺失株和非缺失株的Cat8荧光定位分析研究发现Snf1是影响Cat8磷酸化及细胞核定位的主要激酶；质谱分析Cat8显示，Western Blotting中三个Cat8条带具有不同的磷酸化位点。转录组学及染色质免疫共沉淀技术-测序（ChIP-seq）分析研究表明，Cat8不仅在酵母菌二次生长利用非发酵碳源方面发挥重要作用，在氮代谢调控中也发挥重要作用。ChIP-seq分析显示，Cat8在氨基酸通透酶基因AGP1/GAP1/BAP2、艾氏途径关键酶基因ARO9/ARO10/BAT2、转录激活子Gat1启动子区均有互作位点。通过共免疫沉淀（Co-IP）分析发现，Cat8与Aro80、Cat8与Gat1之间存在互作，这些转录因子协同作用共同调控艾氏途径关键酶表达，增强酵母菌2-PE生物合成。Cat8是酿酒酵母全局性调控因子，该因子对艾氏途径调控机制的解析对合理调控酵母菌2-PE代谢合成有重要意义。