大肠杆菌借助SecY蛋白转位通道非特异性跨内膜运输糖的研究

The main purpose of this project is to realize the non-specific transport of sugars across the inner membrane of Escherichia coli. This research includes the following key contents: Release of the SecY protein translocation channel from fully preventing the permeation of the water-soluble substances by rational mutation of its plug and pore ring residues, thus weakening the diffusion barrier of sugars across the inner membrane. Using of the mutant SecY channel, the SARS shell protein SCVE and the sugar kinase to construct a simple-diffusion transmembrane system and a phosphorylation supplementary system. The replacement of the transport system of xylose, arabinose, lactose or cellobiose with the simple-diffusion system will be tried, and the combination of the simple-diffusion system and the phosphorylation supplementary system will be employed to replace the transport system of glucose, fructose or mannose. The recombinant strains with new sugar transport systems will be used to produce xylitol in a mixed-sugar fermentation process. Moreover, the principles for designing, constructing and co-working of the simple-diffusion system and the phosphorylation supplementary system will be intensively studied. The realization mechanism of overcoming the substrate specificity, saturation phenomenon, inhibitor sensitivity, and inducer exclusion by using the simple-diffusion system will also be discussed. This project is expected to provide a new sugar transport strategy for E. coli, and the results will help to further expand the application prospect of E. coli in metabolic engineering and synthetic biology.

本项目以大肠杆菌非特异性跨内膜转运糖分子为目的，研究内容包括：利用栓塞结构域和氨基酸孔环的理性突变，解除SecY蛋白转位通道对水溶性物质的严格密封，削弱细胞内膜对糖分子的屏障作用；结合突变型SecY通道、SARS病毒外壳蛋白SCVE和糖类激酶，构建简单扩散跨膜系统及磷酸化辅助系统；以简单扩散跨膜系统替代木糖、阿拉伯糖、乳糖和纤维二糖转运系统；结合简单扩散跨膜系统和磷酸化辅助系统替代葡萄糖、果糖和甘露糖转运系统；尝试新型糖运输系统在混糖发酵制备木糖醇中的应用。此外，拟探讨简单扩散跨膜系统的设计、构建原理以及简单扩散跨膜系统和磷酸化辅助系统的适配机制，阐明简单扩散跨膜系统克服底物狭窄、“饱和现象”、抑制物敏感和“诱导物排斥”的实现机理和调控策略。本项目有望为大肠杆菌提供新的糖转运策略，研究成果有助于进一步拓展大肠杆菌在代谢工程和合成生物学中的应用前景。

糖特异性转运蛋白在糖代谢的初始阶段，即糖通过质膜进入细胞质的过程中，起决定性作用。细菌中的SecY复合物（SecYEG）是负责蛋白质易位的膜通道。本项目的研究表明，透化的SecY通道可以被用作大肠杆菌的非特异性糖转运蛋白。删除栓塞结构域的SecY允许葡萄糖、果糖、甘露糖、木糖和阿拉伯糖通过，并且通过额外的孔环突变能够促进乳糖转运，表明利用透化后的SecY通道转运糖类分子与糖的立体特异性无关。重组大肠杆菌在多种单糖上表现出快速生长特性，并有助于消除运输饱和，提高糖耐受性，减少竞争性抑制和解除碳分解代谢物抑制效应，这些现象在天然的糖转运系统中无法避免。项目组在产木糖醇的大肠杆菌JM109 (DE3)中引入了SecY (ΔP)作为木糖摄取的通道。发现SecY (ΔP)通道除了可以快速运输木糖外，它还不受 XylB 催化形成的木糖醇磷酸的抑制干扰，解决了XylFGH和XylE转运蛋白运输木糖产木糖醇的技术瓶颈。更重要的是，在SecY (ΔP)通道和碳分解代谢物抑制效应的协同作用下，大肠杆菌可以响应葡萄糖浓度自动控制木糖流向戊糖磷酸 (PP) 途径和木糖醇合成途径的通量，从而确保重组细胞能够充分利用碳源，并尽可能提高木糖醇的得率。此外，本项目提出了一种通过重组大肠杆菌JM109 (DE3)发酵，高效生产D-阿洛酮糖的方法。该菌株共表达了SecY (ΔP)通道和D-阿洛酮糖3-差向异构酶(DPEase)，确保了D-果糖能够以其非磷酸化形式转运，继而被细胞转化为D-阿洛酮糖。通过补料分批发酵，D-阿洛酮糖的产量达到≈23.3 g/L。