体内连续进化新体系提高D-阿洛酮糖3-差向异构酶转化率的分子机制

D-阿洛酮糖因热量低且口感与蔗糖类似等优势备受瞩目，其制造用酶D-阿洛酮糖3-差向异构酶(DPEase)对原料D-果糖转化率却一直未能突破33%，致使成本居高不下。前期研究中，我们挖掘出该制造壁垒的理论根源在于DPEase结合D-果糖这种非生理性底物时呈现“葫芦”形构象，大尺度偏离经典酶锁钥模型，不仅直接导致转化率低，亦对分子改造提升酶功能提出极大挑战。为攻克上述难题，我们跳出实验室定向进化局限，创制基因突变和酶性能筛选偶联于同一生物体的新体系。本项目拟阐明“靶向可控触发突变【变得多】、信号转导承接偶联【联得紧】、程序死亡驱动筛选【选得准】”的DPEase体内连续进化分子基础，揭示DPEase底物转化率提升的酶学机制并获得转化率显著提升的突变体，攻克D-阿洛酮糖制备壁垒。本项目不仅对推动D-阿洛酮糖降低成本面向大众消费具有积极意义，而且对于发展酶分子改造新理论新技术亦具有重要参考价值。

D-psicose has attracted great attention due to its low calorie and similar taste to sucrose, the conversion rate of raw material D-fructose by its manufacturing enzyme, D-psicose 3-epimerase (DPEase), has not been broken through 33%, resulting in the high cost of D-psicose. In previous studies, we found that the theoretical root of this manufacturing barrier was that DPEase exhibited a "gourd"-shaped conformation when bounded with non-physiological substrate D-fructose, and deviated from the classic enzyme lock-and-key model on a large scale, which not only directly leads to extremely low conversion rate, but also poses great challenges to molecular modification for enzyme function enhancement. In order to overcome the above-mentioned problems, we have broken away from the limitation of laboratory-directed evolution and created a new system coupling gene mutation and enzyme performance screening in vivo. This project aims to clarify the molecular basis of DPEase continuous evolution in vivo: controllable targeting-triggered mutation for broad diversity, signal transduction-undertaken coupling for tight linkage, programmed death-driven screening for accurate selection, reveal the enzymatic mechanism for enhancement of DPEase substrate conversion, and obtain mutants with significantly increased conversion rate, so as to overcome the barrier of D-psicose manufacturing. This project not only has positive significance for reducing the cost for mass consumption, but also has important reference value for the development of new theory and technology of enzyme molecular modification.