title: Unraveling the kinetic diversity of microbial 3-dehydroquinate dehydratases of shikimate pathway.
abstract: 3-Dehydroquinate dehydratase (DHQase) catalyzes the conversion of 3-dehydroquinic acid to 3-dehydroshikimic acid of the shikimate pathway. In this study, 3180 prokaryotic genomes were examined and 459 DHQase sequences were retrieved. Based on sequence analysis and their original hosts, 38 DHQase genes were selected for chemical synthesis. The selected DHQases were translated into new DNA sequences according to the genetic codon usage bias by both Escherichia coli and Corynebacterium glutamicum. The new DNA sequences were customized for synthetic biological applications by adding Biobrick adapters at both ends and by removal of any related restriction endonuclease sites. The customized DHQase genes were successfully expressed in E. coli, and functional DHQases were obtained. Kinetic parameters of Km, kcat, and Vmax of DHQases were determined with a newly established high-throughput method for DHQase activity assay. Results showed that DHQases possessed broad strength of substrate affinities and catalytic capacities. In addition to the DHQase kinetic diversities, this study generated a DHQase library with known catalytic constants that could be applied to design artificial modules of shikimate pathway for metabolic engineering and synthetic biology.
authors: State Key Laboratory of Microbial Resources, Chinese Academy of Sciences, Beijing, 100101 China ; Environmental Microbiology Research Center, Chinese Academy of Sciences, Beijing, 100101 China.
authors: Chang
authors: C
authors: Liu
authors: State Key Laboratory of Microbial Resources, Chinese Academy of Sciences, Beijing, 100101 China ; Environmental Microbiology Research Center, Chinese Academy of Sciences, Beijing, 100101 China.
authors: Yi-Ming
authors: YM
authors: Liu
authors: Bioinformatic Center at Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101 China.
authors: Qing-Lan
authors: QL
authors: Sun
authors: State Key Laboratory of Microbial Resources, Chinese Academy of Sciences, Beijing, 100101 China ; Environmental Microbiology Research Center, Chinese Academy of Sciences, Beijing, 100101 China.
authors: Cheng-Ying
authors: CY
authors: Jiang
authors: State Key Laboratory of Microbial Resources, Chinese Academy of Sciences, Beijing, 100101 China ; Environmental Microbiology Research Center, Chinese Academy of Sciences, Beijing, 100101 China.
authors: Shuang-Jiang
authors: SJ
authors: Liu
keywords: 3-Dehydroquinate dehydratase (DHQase); Biobrick; Kinetic constants; Shikimate pathway; Synthetic biology