Phosphomannose isomerase and phosphomannomutase gene disruptions in Streptomyces nodosus: impact on amphotericin biosynthesis and implications for glycosylation engineering.
Streptomycetes synthesise several bioactive natural products that are modified with sugar residues derived from GDP-mannose. These include the antifungal polyenes, the antibacterial antibiotics hygromycin A and mannopeptimycins, and the anticancer agent bleomycin. Three enzymes function in biosynthesis of GDP-mannose from the glycolytic intermediate fructose 6-phosphate: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMPP). Synthesis of GDP-mannose from exogenous mannose requires hexokinase or phosphotransferase enzymes together with PMM and GMPP. In this study, a region containing genes for PMI, PMM and GMPP was cloned from Streptomyces nodosus, producer of the polyenes amphotericins A and B. Inactivation of the manA gene for PMI resulted in production of amphotericins and their aglycones, 8-deoxyamphoteronolides. A double mutant lacking the PMI and PMM genes produced 8-deoxyamphoteronolides in good yields along with trace levels of glycosylated amphotericins. With further genetic engineering these mutants may activate alternative hexoses as GDP-sugars for transfer to aglycones in vivo.
Nic Lochlainn
Laura
L
School of Biomolecular and Biomedical Science and Centre for Synthesis and Chemical Biology, University College Dublin, Ardmore House, Belfield, Dublin 4, Ireland.
Caffrey
Patrick
P
D000666:Amphotericin B / Q000096:biosynthesis*; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D006031:Glycosylation; D008359:Mannose-6-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D017875:Phosphotransferases (Phosphomutases) / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D000900:Anti-Bacterial Agents; D000666:Amphotericin B; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase; D008359:Mannose-6-Phosphate Isomerase; D017875:Phosphotransferases (Phosphomutases); C052799:phosphomannomutase