Format

Send to

Choose Destination
Appl Environ Microbiol. 2003 Jan;69(1):233-40.

Synthesis of GDP-mannose and mannosylglycerate from labeled mannose by genetically engineered Escherichia coli without loss of specific isotopic enrichment.

Author information

1
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal.

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.

PMID:
12514000
PMCID:
PMC152461
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center