基于GlcNAc6P特异性磷酸酶定向筛选与进化的N-乙酰氨糖生物合成研究

N-acetylglusosamine (GlcNAc) finds a variety of applications in healthcare food and pharmaceutical fields. In previous work, the applicant achieved the initial accumulation of GlcNAc in Bacillus subtilis by rational design of GlcNAc synthesis pathway and modular assemble of metabolic networks, and further identified the dephosphorylation reaction of N-acetylglucosamine-6-phosphate (GlcNAc6P) as a rate-limiting step by 13C labeling based dynamics analysis of GlcNAc synthesis pathway. However, GlcNAc6P specific phosphatase is unknown and never reported. In this project, with the glucose phosphatase YigL from E. coli as the initial target protein, the applicant attempts to accelerate the dephosphorylation reaction of GlcNAc6P and improve GlcNAc synthesis rate by construction of GlcNAc6P specific phosphatase screening system and mutant library generated by directed evolution of YigL, and analysis of catalytic dynamics and structure-function mechanism, and overexpression of the obtained GlcNAc6P specific phosphatase via chemical induced chromosome engineering. The research strategies and idea used in this project are not only useful for the screening and molecular engineering of the other substrates specific phosphatase, but also are of academic value for the studies of the unknown pathway enzymes in the metabolic engineering.

N-乙酰氨基葡萄糖(GlcNAc)在健康保健领域具有广泛应用。前期研究中，申请人通过GlcNAc合成途径理性设计与模块组装策略构建了一株具有较高GlcNAc合成效率的B.subtilis菌株，并利用基于13C同位素标记的反应动力学分析确定GlcNAc6P的去磷酸反应为GlcNAc合成的限速步骤，而GlcNAc6P特异性磷酸酶未见报道。由此，本项目以来自E. coli的葡萄糖-6-磷酸酶YigL为出发蛋白，通过GlcNAc6P特异性磷酸酶高效筛选系统设计、定向进化构建突变体文库和目标蛋白筛选、体外催化动力学分析和构效关系的分子机制解析、目标蛋白多拷贝整合表达等策略提高GlcNAc6P去磷酸反应速率，促进GlcNAc高效合成。所采用的研究思路和方法对其他底物特异性酶的高效筛选和构效关系解析具有一定参考价值，同时对代谢工程中未知途径限速酶的调控研究亦具有一定借鉴意义。