大肠杆菌中D-2-羟基戊二酸分解代谢机制研究

D-2-羟基戊二酸连接了L-丝氨酸合成、糖酵解与TCA循环，是一种“存在被隐藏”的代谢物，D-2-羟基戊二酸的产生并积累是合成生物学生产L-丝氨酸的瓶颈所在。已报道的D-2-羟基戊二酸分解代谢依赖于D-2-羟基戊二酸脱氢酶、电子传递黄素蛋白ETF、电子传递黄素蛋白醌氧化还原酶ETFQO。申请者基于生物信息学分析与前期实验结果，推测双结构域蛋白YdiJ为模式微生物大肠杆菌中D-2-羟基戊二酸分解代谢关键酶，结构域YdiJ-I负责催化D-2-羟基戊二酸脱氢，结构域YdiJ-II负责YdiJ-I与辅酶Q间电子传递，YdiJ具备D-2-羟基戊二酸脱氢酶、ETF、ETFQO的生物学功能。本项目拟确定YdiJ在大肠杆菌D-2-羟基戊二酸分解代谢中的作用，揭示YdiJ各结构域的功能与催化机理，阐明大肠杆菌中新的D-2-羟基戊二酸分解代谢机制，实现基于D-2-羟基戊二酸分解代谢强化的L-丝氨酸高效合成。

D-2-hydroxyglutarate is a kind of "hidden" metabolite which connects the L-serine synthesis, glycolysis and TCA cycle. The production and accumulation of D-2-hydroxyglutarate is the bottleneck of the L-serine production through synthetic biology. The reported D-2-hydroxyglutarate catabolism dependents on D-2-hydroxyglutarate dehydrogenase, electron transport flavinprotein (ETF), and electron transport flavinprotein ubiquinone oxidoreductase (ETFQO). Based on bioinformatics analysis and preliminary experiment results, we speculated that YdiJ with two domains is the key enzyme of D-2-hydroxyglutarate catabolism in Escherichia coli. The domain YdiJ-I is suspected to be responsible for the dehydrogenation of D-2-hydroxyglutarate, and domain YdiJ-II is suspected to be responsible for electron transfer between YdiJ-I and the coenzyme Q. YdiJ possesses the biological functions of D-2-hydroxyglutarate dehydrogenase, ETF and ETFQO. This project intends to identity the role of YdiJ in the catabolism of D-2-hydroxyglutarate, reveal the functions of YdiJ-I and YdiJ-II, uncover the catalytic mechanism of YdiJ, clarify the new mechanism of D-2-hydroxyglutarate catabolism in E. coli, and realize the efficient synthesis of L-serine by enhancing D-2-hydroxyglutarate catabolism.