ME: Metabolic Engineering of PABA-Free Folic Acid Production

该项目的目标是通过代谢工程使细菌直接从葡萄糖中生产高水平的叶酸，而不是转化更昂贵的原料，如对氨基苯甲酸(PABA)。磷酸烯醇式丙酮酸(PEP)、4-磷酸红细胞(E4P)、谷氨酸和GTP是叶酸的葡萄糖前体。因此，从浓度和地层通量的角度来看，这四种前体需要经过改造才能充分获得。代谢工程方法最初将专注于进一步增加分支酸的形成，分支酸是叶酸生物合成中的一种PEP和E4P衍生中间体。建议的任务是：(1)改变调节以增加分支酸的通量，同时限制从分支酸到氨基酸的副反应；(2)使用调查软件和核磁共振分析对枯草杆菌产生叶酸的候选菌株的通量进行建模和映射；(3)对分批和连续培养的菌株进行测试；(4)根据步骤1-3的结果，进一步微调途径。在叶酸生物合成途径的分节酸侧获得成功后，后续的工作可以探索其他前体(例如GTP)是否已经变得有限。

The objective of this project is to metabolically engineer bacteria to produce high levels of folic acid directly from glucose as opposed to transforming a more expensive raw material such as para amino benzoic acid (PABA). Phosphoenolpyruvate (PEP), erythrose-4-phosphate (E4P), glutamate, and GTP are the glucose-derived precursors of folic acid. Thus, from the concentration and formation flux standpoints, these four precursors need to be engineered to be amply available. The metabolic engineering approach will initially focus on increasing further the formation of chorismate, a PEP and E4P-derived intermediate in folic acid biosynthesis. The proposed tasks are: (1) alter regulation to increase the flux to chorismate while limiting the side reactions from chorismate to amino acids, (2) model and map the fluxes in the folic acid-producing candidate strain of B. subtilis using the investigators software and NMR analyses, (3) test the strains in batch and continuous cultivation, and (4) based on the results of steps 1-3, further fine tune the pathways. After achieving success on the chorismate-side of the folic acid biosynthetic pathway, subsequent work can explore whether other precursors (e.g. GTP) have become limiting.