解淀粉芽孢杆菌嘧啶操纵子对胞苷过量合成的转录调控机制研究

胞苷是抗肿瘤、抗病毒药物良好的中间体，也是核酸类保健食品的原料。随着胞苷市场需求的日益增大，采用微生物发酵法大规模生产胞苷已成为解决这一问题的首选途径。在提高发酵单位高效生产胞苷过程中，嘧啶操纵子的调控对胞苷的过量合成至关重要，然而其转录调控效应及关联机制尚不清楚。本项目以解淀粉芽孢杆菌为出发菌株，拟采用基因重组对嘧啶操纵子负转录调控系统进行重构来实现胞苷合成途径的改造，以弱化子与调节蛋白PyrR为研究对象，分析其对胞苷生物合成转录调控的影响；运用RT-PCR、二维电泳技术、代谢控制分析等方法系统地研究操纵子与胞苷合成酶基因的转录、表达及代谢流分布的关系，揭示嘧啶操纵子对胞苷过量合成的转录调控机制，以实现解除负调控因素、增加胞苷合成酶表达量的目标，为构建胞苷合成的理想载流途径奠定研究基础。

Cytidine is an anti-tumor, antiviral good intermediates, also can be used as health food ingredients. With the increasing of cytidine market, the large-scale production of cytidine by microbial fermentation method has become a major way to solve this problem. While improving cytidine production, the regulation of pyr operon is essential to the excessive synthesis cytidine, however, its transcriptional regulation mechanism is unclear. Bacillus amyloliquefaciens will be used as initial strain to investigate reconstruction of negative transcriptional regulation system to achieve the modification of cytidine synthesis pathway. Attenuator and regulatory protein PyrR as the research object, effects of transcriptional regulation on cytidine biosynthesis will be analyzed. Relationship between pyr operon and cytidine synthase gene transcription, expression and metabolic flux distribution will be studied using RT-PCR, two-dimensional electrophoresis, metabolic control analysis. Ttranscriptional regulation mechanisms of pyr operon to cytidine excessive synthesis will be revealed. A goal will be achieved by removing the limiting factors，expanding metabolic pathways in the cytidine biosynthesis and increasing the yield of cytidine. Therefore, it is very important to academic significance and application value in molecular genetics and breeding research of producing-cytidine strain.