本项目拟在大肠杆菌中分别引入以乳酸和丙二酸单酰辅酶A为中间体的3-羟基丙酸合成途径，构建适合于微氧发酵生产3-羟基丙酸的代谢工程菌株。首先，通过对乳酸中间体途径中酶的动力学解析，优选相关酶基因进行组合表达，通过最优组合表达将该途径引入高产D-乳酸底盘细胞，随后通过基因表达微调、微氧水平调控等方法实现3-HP的高效生产。另外，在引入丙二酸单酰辅酶A中间体途径的基础上，通过辅因子工程、副产物合成途径阻断、微氧水平调控等方法平衡还原力和强化3-羟基丙酸的合成。最后在系统水平上分析溶氧扰动对两类工程菌株代谢生理的影响。本项目研究将加深相关酶动力学特征和催化机理的认识，并从系统水平上深入了解微氧发酵生产3-羟基丙酸工程菌株的代谢特征，这具有重要的科学意义。同时构建微氧条件下可由葡萄糖高效生产3-羟基丙酸新型代谢工程菌株，也具有很大的应用价值。

In this project, metabolic engineering of E.coli for 3-hydroxypropionic acid production under microaerobic condition through two different pathways, the lactate pathway and malonyl-CoA pathway will be studied. Firstly, enzymes in the lactate pathway will be selected based on the kinetics analysis, then the combinational expression of the relating genes will be optimized. The lactate pathway will be introduced into D-lactate-overproducing chassis cell for 3-hydroxypropionic acid production by expressing the optimized gene combination. Subsequently, efficient 3-hydroxypropionic acid production will be achieved by fine-tuning gene expression and controling dissoved oxygen at a proper low level. On the other hand, based on that the malonyl-CoA pathway is introduced in E.coli, we will balance the reduction power and enhance 3-hydroxypropionic acid production by cofactor engineering, blocking pathways of byproduct and controling dissoved oxygen at a proper low level. Finally, we will analyze the effect of DO disturbance on metabolic physiology of the engineered strains at a system level. This project will build a deep understanding for the enzyme kinetics, catalytic mechanisms and the strains' metabolic characteristic under microaerobic condition, which is of important scientific significance. Meanwhile, the construction of excellent and novel metabolic engineering strains for efficient bioconversion from glucose to 3-hydroxypropionic acid with breakthrough titer, productivity and yield is also very important for practical applications.