Strain Improvement and Analysis of Industrial Microorganisms by the Manipulation of Energy Metabolism

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Effect of the manipulation of H+-ATPase activity, involved in the energy metabolism, on the characteristics of the industrially important bacteria were investigated. The following results obtained suggest that the manipulation of energy metabolism allow us to improve microbial function differently from ordinary way.1. Escherichia coli : Central metabolism of the F1-ATPase-defective mutant of E.coli was analyzed using the physiologically defined cells cultured continuously in minimal medium. In the mutant increases in the activities of glycolytic enzymes, NADH dehydrogenase of respiratory chain, decreases in the TCA cycle enzyme activities were observed. Based on the genome information, proteome analysis was conducted. The results revealed the changes in the cell protein composition in the mutant.2. Coryneform-glutantic acid producer : Fermentation profiles of a mutant of Corynebacterium glutamicum defective in H+-ATPase activity was investigated. In the mutant the rate of glucose consumption per cell was found to be enhanced. Glutamic acid production was hardly observed, while the production of pyruvic acid, alanine, lactic acid was increased. All the genes of the H+-ATPase operon were cloned. These genes were inserted into E.coli-Corynebacterium shuttle vector, and wild type strain of C.glutamicum was transformed with this plasmid. The transformant showed 2.7-fold activity of H+-ATPase as that of the wild type.3. Lactic acid bacteria : A decrease in the H+-ATPase activity in Lactococcus lactis, a starter strain for cheese making, resulted in the acid sensitivity, thereby demonstrating the importance of this enzyme activity on the pH homeostasis of L.lactis. All the genes of H+-ATPase operon of L.lactis were cloned.