构建人工多酶体是提高级联催化效率的有效手段。申请人创建了在胞内通过自组装形成具有高度有序结构及高效耦合催化功能的超分子多酶体技术。在此基础上，本项目拟在大肠杆菌中将合成重要生物质平台化合物-衣康酸的关键三个异源酶，通过与不同相互作用元件的串联组合，利用元件的相互作用及寡聚酶的自聚作用，使三酶按一定比例和排列进行自组装，形成结构紧密有序、具有区室化构造的人工多酶体，提高三酶级联催化效率。通过研究多酶体的构成、结构特征及其催化特性，揭示区室化多酶体的形成过程与分子机制，阐明其高效级联催化反应机理。同时，对大肠杆菌柠檬酸代谢途径进行改造与优化，实现TCA循环与衣康酸合成的最佳平衡，达到衣康酸合成路径的最优化，最终实现在大肠杆菌中高产衣康酸。本研究将首次在胞内通过无支架自组装构建具有区室化结构及高效级联催化功能的异源人工多酶体。该技术具有一定的普适性，可为胞内构建新的人工多酶反应体系提供有效途径。

Itaconic acid is one of the most important platform chemical source of biomass. Based on the previous research, itaconic acid will be biosynthesized by the combination of the self-assembly of three key enzymes and metabolic engineering in E. coli in this study. .The artificial multienzyme complexes with highly catalytic efficiency, which are compartmentalized of three key heterologous enzymes: citrate synthase, aconitase and cis-aconitatedecarboxylase for biosynthesis of itaconic acid, will be formed in the in E. coli cell by self-assembled via two different host-guest protein interactions in series. The enzymes are fused with the host-guest protein using alpha-helical linker, respectively. The self-assembled process of the complexes will be controlled by regulating enzymes’ expressed process, their expression in the living cells and the combination modes of the host-guest proteins. The molecule fluorescence complementation approach will be used to investigate the influence of the type of alpha-helical linker and their length, the site of the enzyme which fuses with the linker, and the orientation of the enzyme in the complexes’ architectural characteristic and catalytic properties. The formation mechanism of the multienzyme complexes will also be investigated using this approach. Meanwhile, the metabolic pathways of itaconic acid will be reformed and optimized using Crispr/Cas9 technology for itaconic acid production with high yield. .The artificial compartmentalized multienzyme complexes with highly catalytic efficiency will be realized by self-assembled for itaconic acid production with high yield in E. coli cell for the first time in this project. This facile approach is also a convenient platform technique for designing and construction of other artificial multienzyme systems in living cell.