Synthesis of GDP-mannose and mannosylglycerate from labeled mannose by genetically engineered Escherichia coli without loss of specific isotopic enrichment.
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.
Sampaio
Maria-Manuel
MM
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal.
Santos
Helena
H
Boos
Winfried
W
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
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