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Applied and Environmental Microbiology, January 2003, p. 233-240, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.233-240.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Synthesis of GDP-Mannose and Mannosylglycerate from Labeled Mannose by Genetically Engineered Escherichia coli without Loss of Specific Isotopic Enrichment

Maria-Manuel Sampaio,1 Helena Santos,1 and Winfried Boos2*

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal,1 Department of Biology, University of Konstanz, 78457 Konstanz, Germany2

Received 23 July 2002/ Accepted 22 October 2002

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.

* Corresponding author. Mailing address: Department of Biology, University of Konstanz, 78457 Konstanz, Germany. Phone: 49-7531-882658. Fax: 49-7531-883356. E-mail: Winfried.Boos{at}Uni-Konstanz.de.

Applied and Environmental Microbiology, January 2003, p. 233-240, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.233-240.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Empadinhas, N., Albuquerque, L., Costa, J., Zinder, S. H., Santos, M. A. S., Santos, H., da Costa, M. S. (2004). A Gene from the Mesophilic Bacterium Dehalococcoides ethenogenes Encodes a Novel Mannosylglycerate Synthase. J. Bacteriol. 186: 4075-4084 [Abstract] [Full Text]  


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