大肠杆菌微氧产3-羟基丙酸代谢工程研究

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.

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。

本项目在大肠杆菌底盘中引入了以乳酸为中间体的3-羟基丙酸（3-HP）合成途径，首先通过动力学解析或体内表达的表征优选了丙酰辅酶A转移酶、乳酰辅酶A脱水酶、3-羟基丙酰辅酶A脱水酶和3-羟基丙酰水解酶，通过四种酶的组合表达在大肠杆菌中成功的引入了该途径。同时，为了给3-HP的生产提供充足的乳酸前体，通过进化工程和乳酸合成途径优化得到高产乳酸的底盘,并解析了进化菌的优良表型和突变基因的关系及相关机理。随后将3-HP合成途径导入该底盘中，并通过密码子替换、不同启动子组合等策略协调各基因的表达水平，使3-HP的产量从0.6g/L增加到2.5 g/L，但是有大量乳酸积累。微氧发酵表明乳酰辅酶A脱水酶的活性和稳定性受到氧的抑制而难于推动乳酸向3-HP的转化，而好氧生长和厌氧诱导结合的双阶段发酵显著提高了3-HP的合成效率，其产量从2.5 g/L提高到约9.6g/L（同时积累38.7g/L乳酸副产物）。研究结果表明该途径虽然可以实现3-羟基丙酸的合成，但乳酸向3-羟基丙酸的高效转化仍然是一个很大的挑战。经过研究内容的调整，本项目还开展了以谷氨酸棒杆菌为底盘构建高效利用乙酸生产3-HP代谢工程菌株的工作，首先通过蛋白标签融合的策略使以丙二酰辅酶A为中间体的3-HP合成途径中的关键酶——丙二酰辅酶A还原酶的表达水平提高了4倍，3-HP产量从0.14g/L提高到0.66g/L；随后，通过乙酸代谢的强化和TCA循环弱化策略使其产量提高到2.4 g/L。在此基础上，对一系列工程菌株进行了代谢组分析，解析了相关代谢途径中代谢物水平、还原力和能荷水平的变化规律，根据该规律针对性的采用了脂肪酸合成途径的弱化、中间代谢物丙二酸半醛分支途径的阻断、增加NADPH供应等策略，使工程菌在以乙酸为单一碳源时的3-HP摇瓶产量达到3.7 g/L，得率为0.42 g/g 乙酸；在发酵罐中流加发酵的产量达到 26.4 g/L 3-HP，是目前报道的以乙酸为单一碳源时的最高发酵产量，表明谷氨酸棒杆菌具有良好的利用乙酸合成生物基化学品的潜力。