Metabolic Engineering Through Manipulation of Cofactors: Effects on Production of Succinate

本项目的目的是系统地研究大肠杆菌在精确的遗传扰动下调节其代谢活动的细胞反应。琥珀酸的合成将作为一个模型体系。两个关键的辅助因子NADH/NAD+和CoA的作用将被研究。此外，还将研究酶的性质对代谢通量重新分配和琥珀酸产量的影响。具体目的是验证以下假设，以通过琥珀酸合成途径增加通量：(1)降低参与产品合成途径关键步骤的酶的反馈抑制作用；(2)如果产物合成途径需要辅因子NADH，则通过操纵NADH/NAD+池可以增强通过该特定途径的总通量；(3)在一个辅因子具有多种作用的情况下，可能需要对辅因子水平进行微调以实现最佳产品形成。

The objective of this project is to systematically investigate the cellular responses of Escherichia coli in regulating its metabolic activities upon precise genetic perturbations. The synthesis of succinic acid will be used as a model system. The role of two key cofactors, NADH/NAD+ and CoA, will be investigated. In addition, the effect of enzyme properties on the redistribution of metabolic fluxes and on succinic acid productivity will also be investigated. The specific objectives are to test the following hypotheses to increase flux through the succinic acid synthesis pathway: (1) reducing the effect of feedback inhibition of an enzyme which is involved in a key step of the product synthesis pathway; (2) if the product synthesis pathway requires the cofactor NADH, the overall flux through that particular pathway will be enhanced by manipulating the NADH/NAD+ pool; and (3) in situations where a cofactor has multiple roles, fine tuning of the co-factor level may be required for optimal product formation.