酿酒酵母蛋白激酶A催化亚基抑制环腺苷酸合成的调控机制研究

Protein kinase A,composed of Bcy1 regulatory subunit and Tpks catalytic subunits, displays a rigorous feedback regulation on cAMP synthesis in the yeast Saccharomyces cerevisiae. In this work, we will uncover the feedback regulation mechanism of Protein kinase A catalytic subunits on cAMP synthesis in W303 background via the following three points: Firstly, we will study the effect of Protein kinase A catalytic subunits in regulating transcription, translation and protein activity of adenylate cyclase, to find the distinct biological functions of catalytic subunits Tpks on negative regulation of adenylate cyclase; Secondly, through further study on Tpks protein level and the negative regulation capacity on adenylate cyclase of catalytic subunits,as well as the protein interaction of adenylate cyclase and Tpk catalytic subunits, elaborating the feedback regulation mechanism of catalytic subunits Tpks on adenylate cyclase; Finally, a random mutant library of Tpk3 catalytic subunit will be constructed using error-prone polymerase chain reaction (PCR) of the open reading frame of the TPK3 gene for identifying the important functional sites and regulatory motifs in cAMP synthesis and relieving its feedback regulation on adenylate cyclase through genome integration,constructing a cell model of Protein Kinase A catalytic subunit Tpk3 feedback regulation on cAMP synthesis. The results will not only be helpful to understand the feedback regulation mechanism of Protein Kinase A catalytic subunits in Saccharomyces cerevisiae, but also with theoretical importance for the breeding of industrial yeast strains with improved cAMP synthesis.

酿酒酵母蛋白激酶A由调节亚基和催化亚基Tpk1/2/3组成，对环腺苷酸合成有严格抑制作用。本项目以实验室菌株W303为背景，拟从三方面阐述催化亚基抑制环腺苷酸合成的调控机制：首先，通过研究各催化亚基对环腺苷酸积累、腺苷酸环化酶转录、翻译、酶活的特异性抑制作用，揭示催化亚基对环腺苷酸合成的反馈抑制作用；其次，深入研究各催化亚基的蛋白表达量，对腺苷酸环化酶酶活的抑制功能，以及各催化亚基与腺苷酸环化酶之间的蛋白质相互作用，阐述各催化亚基对腺苷酸环化酶的抑制机制；最后，利用易错PCR技术构建催化亚基随机突变文库，确定催化亚基对腺苷酸环化酶的抑制作用位点，并将突变位点整合到酵母基因组消除催化亚基对腺苷酸环化酶抑制作用，建立酿酒酵母细胞环腺苷酸合成反馈抑制的调控模型。研究结果既有助于揭示酿酒酵母蛋白激酶A催化亚基反馈抑制环腺苷酸信号通路的调控机制，同时为酵母发酵生产环腺苷酸技术研究与开发奠定理论基础。

环腺苷酸(cAMP)对生物体的生命活动有着重要的作用。本研究通过调控环腺苷酸信号通路中编码蛋白激酶A（PKA）调节亚基的BCY1基因和编码PKA催化亚基的TPK（TPK1，TPK2，TPK3）基因的表达水平来调控胞内cAMP的积累，同时研究其耐受性，旨在研究环腺苷酸信号通路调控对酿酒酵母耐受性及胞内cAMP水平的影响。基于酿酒酵母多变的发酵环境及蛋白激酶A（PKA）信号通路对其耐受性的调控为背景，研究酿酒酵母PKA改造对其耐受性的影响，并通过弱化蛋白激酶A活性构建了一系列重组菌株，并检测其生长和发酵耐受性。通过改造环腺苷酸信号通路以及旁支通路中的关键基因，来获得具有良好生长发酵性能且耐受性提高的酿酒酵母改造菌株。