解淀粉芽孢杆菌UMP与PRPP代谢对胞苷过量合成的协同调控机理研究

胞苷作为合成药物的中间体，市场需求量大，主要通过微生物发酵获得。多途径协同调节是胞苷生物合成的调控方式之一，UMP与PRPP代谢途径影响最大，但其协同调控机理尚不清楚。本项目运用胞苷代谢理论与代谢工程方法，研究解淀粉芽孢杆菌中UMP与PRPP代谢对胞苷过量合成的协同作用模式及调控机理。拟采用基因编辑、流量组学和代谢组学技术，重点研究PRPP合成关键酶(G6PDH、6PGDH和PRS)、UMP合成关键酶(CPS、OPRTase)、辅酶代谢(NADPH、NADH)与上游代谢物(α-KG、天冬氨酸)等调节因子在胞苷合成中的协同作用，分析调节因子对细胞生长及代谢通量的影响，提出UMP与PRPP代谢对胞苷合成的协同作用模式，诠释胞苷合成的多途径协同调节方式及调控代谢稳态的分子途径，阐明菌株内调节因子间的关系及其对胞苷合成的协同作用，揭示其分子调控机理，为从分子水平上优化胞苷最大生产能力提供理论依据。

Cytidine is an anti-tumor, antiviral good intermediates, and its market demand is growing fast. It is mainly obtained by microbial fermentation. Multiple pathway coordinated regulation of cytidine synthesis is one of the main approaches to control overproduction in Bacillus amyloliquefaciens. UMP and PRPP synthesis pathway play the most important role, however, its molecular mechanism of coordinated regulation is still unclear. Using the research methods of cytidine metabolic theory and metabolic engineering, Bacillus amyloliquefaciens BG-13 will be used as initial strain to investigate the effects and molecular regulation mechanism of the key enzyme of synthesis of PRPP (G6PDH, 6PGDH and PRS), the key enzyme of synthesis of UMP (CPS, OPRTase), coenzyme metabolism(NADPH、NADH), and upstream metabolites (α-KG, Aspartic acid) on synthesis system of cytidine by using the methods of metabolic engineering, gene editing technology, fluxomics and metabonomics technology. These metabolic regulators as the research object, influence of regulating factor on the biosynthesis of cytidine, cell growth, metabolic flux during cytidine synthesis process will be analyzed. And, a multiple pathway synergy regulation modes of cytidine synthesis by UMP and PRPP will be established. The theoretical maximal cytidine synthesis capability and molecular regulation pathway of cytidine metabolic homeostasis will be interpreted. The interactions between the various metabolic regulation factors and coordination regulation effect on cytidine secretion will be expounded. The molecular mechanism of cytidine regulation system will be revealed. Therefore, it is very important to academic significance and application value in optimization of cytidine maximum capacity on molecular level, molecular genetics, breeding research of producing-cytidine strain.