title: Synthesis of GDP-mannose and mannosylglycerate from labeled mannose by genetically engineered Escherichia coli without loss of specific isotopic enrichment.
abstract: We report the construction of an Escherichia coli mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-O-alpha-D-mannosyl-D-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment. The strain carries a deletion in the manA gene, encoding phosphomannose isomerase. This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM). The strain also has a deletion of the cps gene cluster that prevents the synthesis of colanic acid, a mannose-containing polymer. Plasmid-encoded phosphomannomutase (cpsG) and mannose-1-phosphate guanylyltransferase (cpsB) ensure the formation of GDP-mannose. A second plasmid harbors msg, a gene from Rhodothermus marinus that encodes mannosylglycerate synthase, which catalyzes the formation of 2-O-alpha-D-mannosyl-D-glycerate from GDP-mannose and endogenous glycerate. The rate-limiting step in 2-O-alpha-D-mannosyl-D-glycerate formation is the transfer of GDP-mannose to glycerate. 2-O-alpha-D-mannosyl-D-glycerate can be released from cells by treatment with cold-water shock. The final product is formed in a yield exceeding 50% the initial quantity of labeled mannose, including loss during preparation and paper chromatography.
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal.
authors.forename: Maria-Manuel
authors.initials: MM
authors.lastname: Sampaio
authors.forename: Helena
authors.initials: H
authors.lastname: Santos
authors.forename: Winfried
authors.initials: W
authors.lastname: Boos
mesh_terms: D002250:Carbon Radioisotopes / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods; D005988:Glyceric Acids; D006155:Guanosine Diphosphate Mannose / Q000096:biosynthesis*; D008358:Mannose / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000378:metabolism; D008359:Mannose-6-Phosphate Isomerase / Q000235:genetics; D008364:Mannosyltransferases / Q000235:genetics; D009713:Nucleotidyltransferases / Q000235:genetics; D017875:Phosphotransferases (Phosphomutases) / Q000235:genetics; D010957:Plasmids
chemical_list: D002250:Carbon Radioisotopes; D005988:Glyceric Acids; C402688:mannosylglycerate; D006155:Guanosine Diphosphate Mannose; D008364:Mannosyltransferases; C402687:mannosylglycerate synthase; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase; D008359:Mannose-6-Phosphate Isomerase; D017875:Phosphotransferases (Phosphomutases); C052799:phosphomannomutase; D008358:Mannose