一种新型人工蛋白骨架技术的建立及其在微生物制造中的应用

Natural biocatalytic systems usually form physically and spatially organized multi-enzyme complexes, protein scaffolds or reaction in microdomains in cells. The high organization of this multi-enzyme reaction pathway can lead to efficient catalytic performance. However, the artificial synthesis system has spatial effects and imbalanced metabolic fluxes, which greatly reduces the catalytic efficiency of the artificial pathway and greatly limits the bio-manufacturing potential. At present, the known multi-enzyme assembly technologies are very limited, especially multi-enzyme assembly in space. This study proposes the RIAD-RIDD artificial protein scaffold technology, which can spatially perform multi-enzyme 1:2 ratio assembly by protein interaction. This technology has the characteristics of low molecular weight, strong affinity, high specificity, et al. This technology has realized the key enzyme assembly of vitamin K synthesis in vitro, and has improved the efficiency of the synthesis. This project is proposed to carry out key enzymes assembly in the artificial synthesis system of two common chassises—Escherichia coli and Saccharomyces cerevisiae. The studies of the enzyme assemblies using of RIAD-RIDD system in vivo, and the analysis of target metabolic flux, can solve the problems of substrate transfer channel, the improvement of target metabolic flux and the inhibition of intermediate metabolites, to achieve the innovative high yield of the target products.

天然的生物催化体系通常在细胞内会形成物理上、空间上组织有序的多酶复合体、酶分子脚手架或者反应微区，这种多酶反应通路的高度组织性能够带来高效的催化能力。然而，人工合成体系多存在空间效应以及代谢流不平衡的问题，大大降低了人工改造途径的催化效率，很大程度上限制了人工合成体系的生物制造潜力。目前，已知能够有效进行底物传递的多酶组装蛋白骨架技术非常有限，且缺少在空间上有机组织酶分子的手段。本研究提出的RIAD-RIDD人工蛋白骨架技术，具有低分子量、强亲和力、高特异性等特点，能够通过蛋白-蛋白相互作用在空间上进行多酶1:2比例组装，该技术已经在体外实现了维生素K合成的关键酶组装，并提高了反应效率。现拟将该技术在体内分别对大肠杆菌和酿酒酵母两个常用底盘的人工合成体系进行关键酶组装，通过体内组装、目标代谢流通量的研究，解决底物传递通道、代谢流通量及中间代谢产物的抑制反馈等问题，实现目标产物的创新高产。

天然的生物催化体系通常在细胞内会形成物理上、空间上组织有序的多酶复合体、酶分子脚手架或者反应微区，这种多酶反应通路的高度组织性能够带来高效的催化能力。然而，人工合成体系多存在空间效应以及代谢流不平衡的问题，大大降低了人工改造途径的催化效率，很大程度上限制了人工合成体系的生物制造潜力。目前，已知能够有效进行底物传递的多酶组装蛋白骨架技术非常有限，且缺少在空间上有机组织酶分子的手段。本研究开发了一种人工蛋白骨架技术RIAD-RIDD，具有低分子量、强亲和力、高特异性等特点，能够通过蛋白-蛋白相互作用在空间上进行多酶1:2比例组装。将该技术在体内分别对大肠杆菌和酿酒酵母两个常用底盘的人工合成体系进行类胡萝卜素关键酶组装，大肠杆菌工程菌虾青素产量提高了2.7倍，类胡萝卜素产量提高了5.7倍，酿酒酵母工程菌番茄红素产量提高了58%。通过本研究构建多酶复合物的方式链接生物合成的关键代谢节点来实现目标途径产量的提升，不仅解决了底物传递问题，也减轻了人工代谢体系的不平衡问题，对于实现目标产物的创新高产具有重要意义。