Metabolic engineering of Escherichia coli cell factory for highly active xanthine dehydrogenase production
Cheng-Hua Wang1, Chong Zhang2, Xin-Hui Xing2
1College 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.
Abstract:
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.
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