基于乙酰辅酶A和NADPH调控优化代谢流分配强化乙酸合成3-羟基丙酸的研究

3-Hydroxypropionic acid (3-HP) is an important platform chemical. The Chinese biobased 3-HP production is limited by substrate cost and thus lack competition. The ample and cheap acetic acid is considered as a competitive substrate for 3-HP production. Moreover, biological production of 3-HP from acetic acid is economy, efficient in carbon yield recovery and environmental friendly (fix one carbon into CO2). In our study, we construct a metabolic engineered Pseudomonas denitrificans (P. denitrificans) for 3-HP production through acetic acid metabolism. Acetyl-CoA is the key precursor of 3-HP biosynthesis pathway as well as cell growth, well balance of acetyl-CoA distribution and avoid of unnecessary carbon lost is important to improve 3-HP yield. To this end, wild range of dynamic 3-HP responsive promoter library will be created and engineered to regulate 3-HP biosynthesis pathway genes (acc and msr). The flux distribution of acetyl-CoA will be optimized based on the relative gene expression, protein expression and metabolites levels; and regulation relationship between cell growth and 3-HP production is identified. On the other hand, NADPH is the enzyme cofactor of MSR and its regeneration is crucial to high level of 3-HP accumulation. Overexpression of NADPH regeneration pathways, however, down-regulation of NADPH consumption shunt will increase NADPH level and hence enhancing carbon conversion of acetyl-CoA to 3-HP. Through above, construct the novel recombinant strain P. denitrificans with high 3-HP yield from acetate is significant, and provides deep insight into bioconversion of acetic acid into value-added chemicals.

3-羟基丙酸（3-Hydroxypropionic acid，3-HP）是重要的平台化合物，目前中国生物基3-HP生产的竞争力受到原料来源和成本的限制。乙酸来源丰富、价格低廉，利用乙酸生物合成3-HP可以降低原料成本、实现CO2固定、提高碳收率和原子经济性。本项目拟通过构建重组脱氮假单胞菌代谢乙酸生产3-HP，针对代谢网络中乙酰辅酶A碳流的无效流失，提出利用不同强度的3-HP响应启动子调控3-HP合成路径中相关基因的表达的思路，从基因、蛋白质和代谢物水平研究3-HP合成路径过表达过程中乙酰辅酶A代谢流调控的机理、明晰3-HP合成和细胞生长的相互制约作用。其次针对NADPH的再生问题，提出利用辅酶代谢工程导入辅因子再生途径、削弱竞争途径的策略调控脱氮假单胞菌胞内辅酶存在形式及浓度，迫使代谢流发生迁移以满足3-HP合成路径关键酶对NADPH的需求，最终构建能实现细胞高效累积3-HP的代谢网络。

3-羟基丙酸（3-Hydroxypropionic acid，3-HP）是重要的C3平台化合物，利用乙酸作为碳原料可以提高当前生物生产的可持续性和经济性。本项目研究了乙酸在脱氮假单胞菌工程菌生产3-羟基丙酸中的应用潜力。通过在脱氮假单胞菌中引入嗜热光合绿曲菌来源的异源mcr（编码丙二酰辅酶a还原酶）和过表达内源性accABCD（编码乙酰辅酶a羧化酶），构建了代谢乙酸合成3-HP的重组菌株。为了研究3-HP合成和细胞生长在乙酰辅酶A代谢节点上的相互作用关系，项目合成了不同强度启动子和5’UTR非编码区序列，并以此调控3-HP合成路径关键基因mcr和acc，研究发现增强3-HP合成路径活性可以增加3-HP的产量。乙酰辅酶A是TCA循环与3-HP合成途径的竞争节点，通气量的强弱也会影响乙酰辅酶A的碳流分布，为了进一步了解乙酰辅酶A代谢流的分布，静息细胞模式下研究表明通气不足限制了乙酸钠的消耗和3-HP的生产，乙酰辅酶A向3-HP的转化可能受到NAD（P）H和ATP供应的限制。乙醛酸分流和/或TCA循环在异柠檬酸代谢节点上的碳通量分布也会影响3-HP合成依赖所需的NAD(P)H和A(G)TP当量。研究表明iclR基因敲除可以加强乙醛酸循环活性进而增强乙酸利用，而aceK基因敲除可以推动更多的碳流通量进入TCA循环进而提高NAD(P)H和A(G)TP当量。此外，研究发现通过抑制脂肪酸合成路径（添加浅蓝菌素和缺失脂肪酸合成酶基因fabF）提高丙二酰辅酶A节点处进入3-HP合成的碳通量提升了3-HP产量和得率。