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

研究了参与能量代谢的H+-ATPase活性的调控对工业重要细菌特性的影响。以下研究结果表明，能量代谢的调控使我们能够以不同于普通方式的方式改善微生物的功能。大肠埃希氏菌：利用在微量培养基中连续培养的生理定义细胞，分析了大肠杆菌F1-ATPase缺陷突变株的中心代谢。突变株的糖酵解酶、呼吸链NADH脱氢酶活性升高，TCA循环酶活性降低。在基因组信息的基础上，进行蛋白质组分析。结果1.揭示了突变体细胞蛋白质组成的变化。棒状谷氨酸产生菌：对一株H+-ATPase活性缺陷的谷氨酸棒杆菌突变株的发酵特性进行了研究。在突变体中，每个细胞的葡萄糖消耗率被发现增加。谷氨酸的产生几乎没有观察到，丙酮酸、丙氨酸、乳酸的产生增加。克隆了H+-ATPase操纵子的全部基因。将这些基因插入到大肠杆菌-棒状杆菌穿梭载体中，转化谷氨酸杆菌野生型菌株。转化子的H+-ATPase活性是野生型的2.7倍。乳酸菌：乳酸菌中H+-ATPase活性的降低导致了乳酸菌对酸的敏感性，从而证明了该酶活性对乳酸菌pH动态平衡的重要性。克隆了乳酸乳杆菌H+-ATPase操纵子的全部基因。

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.