Systems and Synthetic Biology Program, Centro Nacional de Biotecnología CNB-CSIC, 28049 Madrid, Spain.
Environ Microbiol. 2013 Jun;15(6):1772-85. doi: 10.1111/1462-2920.12069. Epub 2013 Jan 10.
Glucose catabolism of Pseudomonas putida is carried out exclusively through the Entner-Doudoroff (ED) pathway due to the absence of 6-phosphofructokinase. In order to activate the Embden-Meyerhof-Parnas (EMP) route we transferred the pfkA gene from Escherichia coli to a P. putida wild-type strain as well as to an eda mutant, i.e. lacking 2-keto-3-deoxy-6-phosphogluconate aldolase. PfkA(E. coli) failed to redirect the carbon flow from the ED route towards the EMP pathway, suggesting that ED was essential for sugar catabolism. The presence of PfkA(E. coli) was detrimental for growth, which could be traced to the reduction of ATP and NAD(P)H pools along with alteration of the NAD(P)H/NADP(+) ratio. Pseudomonas putida cells carrying PfkA(E. coli) became highly sensitive to diamide and hydrogen peroxide, the response to which is very demanding of NADPH. The inhibitory effect of PfkA(E. coli) could in part be relieved by methionine, the synthesis of which relies much on NADPH. These results expose the role of the ED pathway for generating the redox currency (NADPH) that is required for counteracting oxidative stress. It is thus likely that environmental bacteria that favour the ED pathway over the EMP pathway do so in order to gear their aerobic metabolism to endure oxidative-related insults.
由于缺乏 6-磷酸果糖激酶，恶臭假单胞菌的葡萄糖分解代谢完全通过 Entner-Doudoroff（ED）途径进行。为了激活 Embden-Meyerhof-Parnas（EMP）途径，我们将 pfkA 基因从大肠杆菌转移到恶臭假单胞菌野生型菌株和 eda 突变体（缺乏 2-酮-3-脱氧-6-磷酸葡萄糖醛酸醛缩酶）中。PfkA（大肠杆菌）未能将碳流从 ED 途径重新引导到 EMP 途径，这表明 ED 对糖分解代谢至关重要。PfkA（大肠杆菌）的存在不利于生长，这可以追溯到 ATP 和 NAD(P)H 池的减少以及 NAD(P)H/NADP(+) 比的改变。携带 PfkA（大肠杆菌）的恶臭假单胞菌细胞对二酰胺和过氧化氢高度敏感，对此的反应非常需要 NADPH。PfkA（大肠杆菌）的抑制作用在一定程度上可以通过蛋氨酸缓解，蛋氨酸的合成非常依赖 NADPH。这些结果揭示了 ED 途径在产生氧化还原货币（NADPH）方面的作用，这是抵御氧化应激所必需的。因此，那些有利于 ED 途径而不是 EMP 途径的环境细菌很可能是为了使它们的有氧代谢适应氧化相关的损伤。
