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

通过操纵能量代谢对工业微生物进行菌株改良和分析

基本信息

批准号：
10460033
负责人：
YOKOTA Atsushi
金额：
$ 4.1万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

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.

研究了参与能量代谢的H+- atp酶活性对工业重要细菌特性的影响。以下结果表明，对能量代谢的操纵使我们能够以不同于普通方式改善微生物的功能。大肠杆菌：利用在最小培养基中连续培养的生理定义细胞，分析大肠杆菌f1 - atp酶缺陷突变体的中心代谢。突变体呼吸链中糖酵解酶NADH脱氢酶活性升高，TCA循环酶活性降低。基于基因组信息，进行蛋白质组分析。结果揭示了突变体细胞蛋白组成的变化。棒状-谷氨酸生产者：研究了H+- atp酶活性缺陷的谷状棒状杆菌突变体的发酵概况。在突变体中，发现每个细胞的葡萄糖消耗率提高了。谷氨酸的产量几乎没有变化，而丙酮酸、丙氨酸、乳酸的产量增加。H+- atp酶操纵子的所有基因均被克隆。将这些基因插入大肠杆菌-棒状杆菌穿梭载体中，利用该质粒转化谷氨酸梭菌野生型菌株。转化后的H+- atp酶活性是野生型的2.7倍。乳酸菌：乳酸乳球菌（Lactococcus lactis）是一种用于奶酪制作的发酵剂，其H+- atp酶活性降低，导致其对酸敏感，从而证明了该酶活性对乳酸乳球菌pH稳态的重要性。克隆了乳酸菌H+- atp酶操纵子的所有基因。