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

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.

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。

胞苷是核苷类保健食品与功能性食品的重要原料，市场需求量很大。目前，微生物发酵法是生产胞苷的主要方法，获得能够高产胞苷的优良菌株是发酵的前提，通过研究胞苷的相关合成途径，利用现代分子生物技术手段对菌株进行定向改造是研究的热点。本研究以解淀粉芽孢杆菌NXBG-11为出发菌株，重点研究了UMP与PRPP代谢对胞苷过量合成的协同作用模式及调控机理。通过切断胞苷降解为尿苷途径，弱化丙酮酸生成乙酸途径，初步提高了胞苷产量，为进一步提高胞苷产量对重组菌株NXBG-11进行转录组测序分析，确定改造胞苷降解途径中的显著变化基因udk和rihB，并采用CRISPR/Cas9基因编辑成功构建了udk基因缺失重组菌NXBG-12和rihB缺失重组菌NXBG-13。之后将突变菌解淀粉芽孢杆菌NXBG-13在优化前及优化后的培养基里同时发酵，结果表明菌株在优化后培养基里耗糖量增加了1.09倍，但是胞苷含量提高了1.3倍，糖苷转化率提高了1.2倍。在此基础上继续对产胞苷重组菌解淀粉芽孢杆菌NXBG-13进行改造，采用相同的方法成功构建了pgi基因缺失重组菌NXBG-14和edd缺失重组菌NXBG-15。为了进一步明确解淀粉芽孢杆菌产胞苷的机制，在这项研究中，应用13C-MFA来阐明以葡萄糖为碳源的工程菌株的碳代谢通量分布。以EMP途径、PPP、TCA途径和UMP合成途径作为主要的网络途径，结合KEGG中胞苷合成代谢网络图以及有关解淀粉芽孢杆菌合成胞苷途径的参考文献，构建出胞苷生物合成代谢网络。最后基于转录组学，分析菌种基因表达的变化动态并解析基因改造影响胞苷合成过程的机制，提出了UMP途径中多基因改造与PRPP代谢中多基因改造对胞苷合成的协同调控作用模式。