College of Light Industry and Food Engineering, Guangxi University, Nanning, Guangxi 530004, People's Republic of China; Key Laboratory for Industrial Biocatalysis, Ministry of Education of China, Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, People's Republic of China.
Key Laboratory for Industrial Biocatalysis, Ministry of Education of China, Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, People's Republic of China; Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, People's Republic of China.
Bioresour Technol. 2017 Dec;245(Pt B):1782-1789. doi: 10.1016/j.biortech.2017.05.144. Epub 2017 May 31.
The aim of this work was to demonstrate the first proof-of-concept for the use of ab initio-aided assembly strategy intensifying in vivo biosynthesis process to construct Escherichia coli cell factory overproducing highly active xanthine dehydrogenase (XDH). Three global regulator (IscS, TusA and NarJ) and four chaperone proteins (DsbA, DsbB, NifS and XdhC) were overexpressed to aid the formation and ordered assembly of three redox center cofactors of Rhodobacter capsulatus XDH in E. coli. The NifS, IscS and DsbB enhanced the specific activity of RcXDH by 30%, 94% and 49%, respectively. The combinatorial expression of NarJ and IscS synergistically increased the specific activity by 129% and enhanced the total enzyme activity by a remarkable 3.9-fold. The crude enzyme showed nearly the same coupling efficiency of electron transfer and product formation as previously purified XDHs, indicating an integrity and efficient assembly of highly active XDH.
这项工作的目的是首次证明，使用从头辅助组装策略可以强化体内生物合成过程，从而构建能够大量生产高活性黄嘌呤脱氢酶（XDH）的大肠杆菌细胞工厂。我们过表达了三个全局调控因子（IscS、TusA 和 NarJ）和四个伴侣蛋白（DsbA、DsbB、NifS 和 XdhC），以帮助形成和有序组装类球红细菌 XDH 的三个氧化还原中心辅因子。NifS、IscS 和 DsbB 分别使 RcXDH 的比活性提高了 30%、94%和 49%。NarJ 和 IscS 的组合表达协同将比活性提高了 129%，并显著将总酶活性提高了 3.9 倍。粗酶显示出与先前纯化的 XDH 几乎相同的电子转移和产物形成的偶联效率，表明高活性 XDH 具有完整性和高效的组装。
