One-pot synthesis of GDP-l-fucose by a four-enzyme cascade expressed in Lactococcus lactis

• Published in: Journal of biotechnology 2017-12, Vol.264, p.1-7
• Main Authors: Li, Ling, Kim, Seul-Ah, Heo, Ji Eun, Kim, Tae-Jip, Seo, Jin-Ho, Han, Nam Soo
• Format: Article
• Language: English
• Subjects: 5-epimerase-4-reductase; 6-dehydratase; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Escherichia coli - genetics; GDP-4-keto-6-deoxymannose-3; GDP-d-mannose-4; GDP-l-fucose; Guanosine Diphosphate Fucose - metabolism; Lactococcus lactis; Lactococcus lactis - genetics; Lactococcus lactis - metabolism; Mannose-1-phosphate guanylyltransferase; Mannose-6-Phosphate Isomerase - genetics; Mannose-6-Phosphate Isomerase - metabolism; Mannosyltransferases - genetics; Mannosyltransferases - metabolism; Metabolic Engineering - methods; Metabolic Networks and Pathways - genetics; Oxidoreductases - genetics; Oxidoreductases - metabolism; Plasmids; Recombinant Proteins - genetics; Recombinant Proteins - metabolism
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2017.10.009

•Four genes (manB, manC, gmd, and wcaG) cloned from E. coli were expressed in Lactococcus lactis.•The one-pot reaction of ManB, ManC, Gmd, and WcaG with mannose-6-P resulted in the successful production of GDP-l-fucose.•This is the first report of GDP-l-fucose production by using multiple enzymes expressed in lactic acid bacteria.•GRAS LAB has potentials to be used for production of bioactive compounds to be used for human. 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.