ME: Metabolic Engineering of PABA-Free Folic Acid Production

该项目的目标是通过代谢工程细菌直接从葡萄糖中产生高水平的叶酸，而不是转化更昂贵的原料，如对氨基苯甲酸（PABA）。磷酸烯醇丙酮酸（PEP）、磷酸红-4-磷酸（E4P）、谷氨酸和GTP是葡萄糖衍生的叶酸前体。因此，从浓度和地层通量的角度来看，这四种前驱体需要充分利用。代谢工程方法最初将集中于进一步增加choris酸的形成，这是叶酸生物合成中PEP和e4p衍生的中间体。建议的任务是：(1)改变调控以增加对choris酸的通量，同时限制从choris酸到氨基酸的副反应；(2)利用研究者软件和核磁共振分析建立枯草芽孢杆菌产叶酸候选菌株的通量模型和图谱；(3)对菌株进行批量和连续培养测试；(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chorisate侧取得成功后，后续的工作可以探索其他前体（例如GTP）是否已经成为限制。

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.