Rapid selection of glucose-utilizing variants of the polyhydroxyalkanoate producerRalstonia eutrophaH16 by incubation with high substrate levels

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Aims:The application of Ralstonia eutropha H16 for producing polyhydroxyalkanoates as bioplastics is limited by the incapability of the bacterium to utilize glucose as a growth substrate. This study aims in characterizing glucose-utilizing strains that arose after incubation with high glucose levels, in comparison with previously published mutants, generated either by mutagenesis or by metabolic engineering.Methods and Results:Cultivations on solid and liquid media showed that the application of high substrate concentrations rapidly induced a glucose-positive phenotype. The time span until the onset of growth and the frequency of glucose-utilizing colonies were correlated to the initial glucose concentration. All mutants exhibited elevated activities of glucose-6-phosphate dehydrogenase. The glucose-positive phenotype was abolished after deleting genes for the N-acetylglucosamine phosphotransferase system.Conclusions:A procedure is provided for selecting glucose-utilizing R. eutropha H16 in an unprecedented short time period and without any mutagenic treatment. An altered N-acetylglucosamine phosphotransferase system appears to be a common motif in all glucose-utilizing mutants examined so far.Significance and Impact of the Study:The correlation of the applied glucose concentration and the appearance of glucose-utilizing mutants poses questions about the randomness or the specificity of adaptive mutations in general. Furthermore, glucose-adapted strains of R. eutropha H16 could be useful for the production of bioplastics.

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