Alginate biosynthesis in mucoid recombinants of Pseudomonas aeruginosa overproducing GDP-mannose dehydrogenase.
The Pseudomonas aeruginosa algD gene, encoding GDP-mannose dehydrogenase (GMD) and cloned at Chakrabarty's Laboratory in the expression vector pMMB24 (plasmid pVD211), was mobilized into P.aeruginosa strains 8821 and 8821M. Strain 8821M was a high-alginate-producing variant, spontaneously obtained from mucoid strain 8821, with derepressed levels of GMD, a key enzyme in the regulation of alginate biosynthesis, leading to the irreversible oxidation of GDP-mannose to GDP-mannuronic acid. A slight increase in the level of GMD, in both strains harboring the plasmid pVD211 and batch-grown at 37 degrees C without IPTG induction, led to the increase of production rate and the final concentration of alginate produced by control strains harboring the cloning vector. However, the viscosity of the aqueous solutions prepared with the alginate (3 g l-1) produced by mucoid strains harboring pVD211 was lower than those with the alginate produced by the controls (shear rates in the range 0.6-12 s-1). The specific activity of GMD assayed in crude extracts from cells harboring pVD211 and subjected to IPTG induction (0.5 and 3 mM) presented the highest values. However, either the rate of biosynthesis and final concentration of alginate or the viscosity of solutions prepared with the alginate produced by recombinants grown with IPTG were lower than that possible without overproduction. Therefore, the stimulation of the alginate pathway only by manipulating the rate of the step catalysed by GMD, although possible within certain levels, was at the expense of the final exopolysaccharide quality.
Laboratório de Engenharia Bioquímica, Instituto Superior Técnico, Lisboa, Portugal.
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Martins
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Sá-Correia
D000464:Alginates / Q000378:metabolism*; D002237:Carbohydrate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D007700:Kinetics; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D011994:Recombinant Proteins / Q000378:metabolism
D000464:Alginates; D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases; C020562:GDPmannose dehydrogenase