枯草芽胞杆菌嘌呤核苷通用底盘菌的从头构建及应用

Purine nucleosides are of great importance in pharmaceutical, food, and health care industry due to their important physiological roles in cellular metabolism. Although they have been produced in bulk by Bacillus for decades, the majority of industrial Bacillus strains were screened by conventional mutagenesis approaches, which have introduced undesirable secondary mutations and thus limit the further improvements of their production capacity. To break through the bottleneck of purine nucleosides fermentation, systematic metabolic engineering is necessary to construct the genetically engineered bacteria. To date, the metabolic engineering for purine nucleosides production has developed slowly because it is hindered by two major problems: (i) the low metabolic flux of purine nucleosides synthesis carried by pentose phosphate pathway (PPP), and (ii) the lack of efficiently seamless genetic manipulation system in Bacillus. In the proposed project, we plan to use the model strain Bacillus subtilis W168 to construct a universal chassis for the purine nucleosides biosynthesis through the seamless genetic manipulation system, which has been previously constructed and will be further optimized in this project. Firstly, an efficient nucleoside synthesis module will be constructed by protein scaffold and related technologies to precisely control the cell metabolic flux of the central carbon metabolism. The effective metabolic flux of PPP will be increased by introducing spatially recruited metabolic enzymes in a designable manner. Then, in an attempt to increase the accumulation of the precursors PRPP and IMP for purine nucleosides synthesis, several key targets for the pentose phosphate and purine biosynthetic pathway will be genetically modified and optimized. A universal chassis will be subsequently constructed and applied to optimize the inosine, adenosine, and guanosine biosynthesis pathways. Finally, the universal chassis with known genetic background will be successfully obtained when we can achieve efficient synthesis of 2-3 purine nucleosides in this project. Taken together, the results of this project will provide substantial scientific foundation to clarify the regulatory mechanisms of purine nucleoside biosynthesis, and will further provide a new theoretical and practical basis for breeding industrial microorganisms.

嘌呤核苷在细胞代谢中具有重要的生理作用，广泛应用于医药、食品和保健等领域。目前嘌呤核苷多采用传统诱变选育的芽胞杆菌进行发酵法生产, 但多轮诱变积累的负效应限制了菌株发酵性能的提高。因此，亟需开展嘌呤核苷的系统代谢工程研究，突破嘌呤核苷发酵生产的瓶颈。而构建工程菌目前主要面临两大问题：①嘌呤核苷合成载流途径为戊糖磷酸途径(PPP)，流量低；②缺乏高效的无痕操作系统。本项目以模式菌株枯草芽胞杆菌W168为研究对象，完善前期工作中构建的无痕操作系统；利用蛋白支架等技术定向改造中心碳代谢途径的代谢流量分配，精确调控PPP的代谢通量；进一步优化PPP和嘌呤合成途径，增强嘌呤核苷合成前体的积累，最终获得一株遗传背景清楚的嘌呤核苷通用底盘菌；在此基础上改造不同的嘌呤核苷终端代谢途径，实现2-3种嘌呤核苷的高效合成。本项目的完成将有助于阐明嘌呤核苷生物合成的调控机制，为工业细菌育种提供新的理论和实践基础。

嘌呤核苷作为生物体内重要的代谢产物，参与核酸合成、能量供给和蛋白质合成等许多重要的细胞代谢过程，在维持细胞的生理代谢功能中发挥着重要作用，在医药、保健和食品等领域中具有非常广泛的应用价值。目前嘌呤核苷代谢工程改造只局限在产物合成相关途径基因的局部改造，缺乏对目标微生物代谢网络的全局调控，难以实现嘌呤核苷的高效积累。本项目首先以模式菌株枯草芽胞杆菌W168为研究对象，通过重构毒素-抗毒素系统构建抗毒素开关调控的毒素反筛模块（TCCRAS），建立了芽胞杆菌高效无痕的遗传操作系统；在此基础上，进一步利用限制性修饰系统建立了一套简单、高效且应用范围广的基因组编辑新技术（RMGE），该技术能够广泛适用于大肠杆菌、枯草芽胞杆菌和酿酒酵母的基因组编辑。在此基础上，以基因组代谢网络模型为基础，在细胞水平对代谢网络进行分析和预测，运用局部搜索启发式算法分析预测最佳的敲除靶点。运用最小开关调整算法对预测的靶点进行分析筛选，发现了有利于嘌呤核苷合成的改造靶点转醛醇编码基因。根据预测结果，构建的工程菌肌苷产量分别显著提高了9.6%和10.6%；进一步在染色体上过表达zwf基因使肌苷产量提高的同时菌株生产强度显著提高了15.7%，在此基础上对pgi靶点进行敲除或弱化，发现弱化策略更有利于肌苷的积累，使肌苷产量显著提高了21.1%。同时，利用支架蛋白对磷酸戊糖途径关键酶进行线性组装表达，发酵罐结果显示肌苷积累提高10.7%，通过回补编码腺苷酸琥珀酸合成酶基因获得了嘌呤核苷通用底盘菌。在通用底盘菌的基础上，通过阻断分支代谢途径和分解途径，同时过表达关键酶基因，构建了肌苷和腺苷基因工程菌，产量分别为22.81±0.82 g/L 和3.58±0.17g/L，获得文献报道以来枯草芽胞杆菌从头构建肌苷工程菌的最高产量。本项目在基因组代谢网络模型指导下，对枯草芽胞杆菌嘌呤核苷的合成进行理性代谢工程研究，从细胞整体水平上分析枯草芽胞杆菌嘌呤核苷合成的代谢机理，为嘌呤核苷生产菌株的选育提供理论基础和实践经验。

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