Cell-free enzymatic synthesis of GDP-L-fucose from mannose

  • AMB Express. 2019 May 27;9(1):74. doi: 10.1186/s13568-019-0798-1.
Weiyang Wang  1 ,  Fan Zhang  1 ,  Yanyun Wen  1 ,  Yanbo Hu  1 ,  Ye Yuan  1 ,  Min Wei  2 ,  Yifa Zhou  3
Affiliations
  • 1. Jilin Province Key Laboratory on Chemistry and Biology of Changbai Mountain Natural Drugs, School of Life Sciences, Northeast Normal University, Changchun, 130024, People's Republic of China.
  • 2. Jilin Province Key Laboratory on Chemistry and Biology of Changbai Mountain Natural Drugs, School of Life Sciences, Northeast Normal University, Changchun, 130024, People's Republic of China. [email protected].
  • 3. Jilin Province Key Laboratory on Chemistry and Biology of Changbai Mountain Natural Drugs, School of Life Sciences, Northeast Normal University, Changchun, 130024, People's Republic of China. [email protected].
PMID: 31134391 DOI: 10.1186/s13568-019-0798-1
Abstract

GDP-L-fucose, the key substrate for fucosyloligosaccharide biosynthesis, has been synthesized via a de novo pathway in bacteria. In the present study, genes for GDP-L-fucose biosynthesis were cloned into the expression vector pET-28a (+) to construct five E. coli strains, with recombinant Enzymes being purified by using Ni-NTA chromatography. Following optimization of the 3-step reaction, Glk, ManB and ManC were added to the reaction mixture, after which Gmd and WcaG were added to overcome feedback inhibition from the end-product to produce GDP-L-fucose at 178.6 mg/l, with a yield of 14.1%. Our studies provide the basis for using cell-free enzyme production of GDP-L-fucose.

Keywords
Enzymatic synthesis; GDP-D-mannose; GDP-L-fucose; Mannose-6-phosphate.
Products