分段式微流控生物被膜催化天然产物糖基定向改造研究

Miniaturizing biocatalysis is an effective way for the process to intensify directional glycosyl-modification of natural products. However, the industrial implementation of this process is not cost-effective, mainly due to the high costs of enzymes (free or immobilized) and suspended cell catalysts catalyzed in the traditional way. The products yield is also impeded by low solubility of natural glycoside substrates, and the inhibition of thermally unstable product. Therefore, we propose a novel concept of “Microfluidic Biofilms Biocatalysis”, which is a self-designed and integrated microchannel microreactor developed to produce a novel biofilms catalyst based on the surface display of recombinant Escherichia coli with glycosidase gene, and the docking of enzyme and substrate occurred outside the cells and inside the biofilms for the first time. The aim of this project is to comprehensively answer the fundamental scientific questions of microscale biofilms-catalyzed glycosyl-modification of natural products. The proposed schemes include: (1) Analyze the life cycle, physicochemical properties, morphology, stress effects and catalytic properties of the biofilms, to reveal the mechanism of biofilm formation and regulatory on a molecular level; (2) Illustrate the catalytic mechanism and the interaction mechanism of biofilm-enzyme-substrate by studing the factors of catalytic system, dynamics, affinity between biofilms and substrate, and the mass transfer process model; (3) Design a multi-inlet looped-shape micro-channel reactor to achieve continue feeding of substrates with “buffer-ionic liquid/solvent-complexing agent” based on the novel concept of the microfluidic integrated reaction and separation, to reveal the intensification mechanism of biofilms-catalytized process. Finally, the catalytic mechanism of microfluidic biofilms will be eventually clarified. This could be favorable to enrich the theoretical perspectives of industrial biocatalysis, and provide some new methodologies and approaches for the biosynthesis of rare natural products.

微型生物催化是天然产物糖基定向改造过程增效的有效途径，但由于传统催化剂应用成本高、糖苷底物溶解度低、产物抑制严重且热不稳定，限制了应用。由此，我们提出“微流控生物被膜（Biofilms）催化”的设想，设计分段式微通道集成反应器，基于重组糖苷酶基因的表面展示大肠杆菌制备新型生物被膜催化剂，首次实现了底物与酶在菌膜内、细胞外的对接，剖析微尺度催化基础问题：（1）分析生命周期、理化性质、形态结构、胁迫效应及催化性能，揭示生物被膜的形成、驯化及调控规律；（2）研究生物被膜催化体系相关因素、动力学及其与底物的亲和力，建立传质介导的反应限速模型，揭示生物被膜-酶-底物的相互作用机理；（3）设计多入口回路添加底物，基于“缓冲液-离子液体/溶剂-络合剂”构建反应分离耦合的新工艺，揭示生物被膜催化的过程强化机制。从而阐明微流控生物被膜催化的机理，丰富工业生物催化的基础理论，为生物制造珍稀天然产物提供新思路。

微型生物催化是天然产物糖基定向改造过程增效的有效途径，但由于传统催化剂应用成本高、糖苷底物溶解度低、产物抑制严重且热不稳定，限制了应用。由此，我们提出了“微流控生物被膜（Biofilms）催化”的设想，设计了分段式微通道集成反应器，率先开展了生物被膜新型催化剂的分段式微流控制备与催化应用基础研究。以α-L-鼠李糖苷酶为模式糖苷酶，构建了异源表达糖苷酶的系列重组工程菌，在硅烷化修饰的微通道中采用“空气/培养液（含适量石墨烯）”的分段式微流控技术实现了生物被膜催化剂的快速可控制备，系统分析了生命周期、理化性质、形态结构、胁迫效应及催化性能，解析了微流体作用力限制和胞外多糖等物质粘附对微流控生物被膜的形成、驯化及调控的作用机理。通过研究生物被膜催化体系相关因素、动力学及其与底物的亲和力，构建了微流控生物被膜催化反应的高效体系，建立了传质介导的反应限速模型，发现生物被膜对pH、温度及底物浓度等反应条件具有相对更广的耐受范围，其中的细胞催化活性相对稳定，形成的三维立体结构使得微通道表面粗糙度增加，流体发生界面分离或形成回流区频率增大，进而增强传质和传热，从而提高了生物催化效率。其中，酶、流速与介质是影响催化反应体系的关键要素，如：密码子优化可提高酶的表达量和酶活，截短突变可增强酶的催化性能和稳定性，双启动子和融合表达可获得可视化的全细胞催化剂，表面展示酶的生物被膜首次实现了底物与酶在菌膜内、细胞外的对接；调节微流的流速可改变停留时间和流体形态来强化传质传热，添加适量的低共熔剂等介质可改善底物溶解性和细胞膜通透性。在此基础上，设计了多入口回路型微通道反应器添加底物，优化了液液两相体系，构建了微流控反应分离耦合的新工艺，揭示了生物被膜催化的过程强化机制。研究结果阐明了微流控生物被膜催化的机理，进一步丰富了工业生物催化的基础理论，为生物制造珍稀天然产物提供了新的方法和技术。