基于多酶区室化自组装的大肠杆菌合成衣康酸研究

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.

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。

衣康酸是重要的精细化学品，是合成可降解聚合物的重要单体。本课题在课题组前期研究基础上，设计并开发了多种全新的提高大肠杆菌生产衣康酸的技术。1. 利用合成生物学策略，在大肠杆菌Escherichia coli中整合产衣康酸多酶级联催化反应，通过多种方式对级联反应多酶进行自组装，并结合CRISPR/Cas9基因编辑技术进行代谢工程改造，探索并开发高效生产衣康酸的代谢工程菌。2. 我们在三羧酸循环（TCA）上的关键酶异柠檬酸脱氢酶（IDH）中确定光控蛋白AsLOV2的7个插入位点得到ILOVs，在胞外探索蓝光照射对其酶活的影响，并利用CRISPR/Cas9基因编辑技术将ILOVs整合入基因组中，在胞内探索其光控效应，使得在不敲除icd的基础上通过光照来控制的酶活，积累衣康酸前体，构建高产衣康酸菌株。3. 利用响应衣康酸的启动子YpItcR/Pccl，构建了在衣康酸存在下的自调控系统。利用衣康酸诱导的dCas9，构建了靶向代谢过程中必需基因icd，pykA，sucCD的三个sgRNA，积累前体物质，提高衣康酸产量。