51. One-pot synthesis of GDP-l-fucose by a four-enzyme cascade expressed in Lactococcus lactis.
- Author
-
Li L, Kim SA, Heo JE, Kim TJ, Seo JH, and Han NS
- Subjects
- Bacterial Proteins genetics, Escherichia coli genetics, Lactococcus lactis genetics, Mannose-6-Phosphate Isomerase genetics, Mannose-6-Phosphate Isomerase metabolism, Mannosyltransferases genetics, Metabolic Networks and Pathways genetics, Oxidoreductases genetics, Oxidoreductases metabolism, Plasmids, Recombinant Proteins genetics, Recombinant Proteins metabolism, Bacterial Proteins metabolism, Guanosine Diphosphate Fucose metabolism, Lactococcus lactis metabolism, Mannosyltransferases metabolism, Metabolic Engineering methods
- Abstract
GDP-l-fucose is an l-fucose donor to synthesize fucosylated compounds such as human milk oligosaccharides or Lewis antigen. In this study, we used Lactococcus lactis subsp. cremoris NZ9000 to express 4 enzymes, ManB, ManC, Gmd, and WcaG and produced GDP-l-fucose by using one-pot synthesis method with mannose-6-phosphate as substrate and the enzymes as biocatalyst. For preparation of enzyme mixture, 4 genes (manB, manC, gmd, and wcaG) cloned from Escherichia coli were transformed into L. lactis strains using pNZ8008 and the recombinant cell lysates were obtained after cultivation. When mannose-6-phosphate was used as the substrate, the consecutive reactions with ManB, ManC, Gmd, and WcaG resulted in the successful production of GDP-l-fucose (0.13mM). When GDP-d-mannose was used as the substrate, it was entirely converted to GDP-l-fucose (0.2mM; 0.12g/L) via 2 enzymatic reactions mediated by Gmd and WcaG. This is the first report of GDP-l-fucose production by using multiple enzymes expressed in lactic acid bacteria., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF