The energetic conversion competence of Escherichia coli during aerobic respiration studied by 31P NMR using a circulating fermentation system

被引:25
作者
Noguchi, Y [1 ]
Nakai, Y
Shimba, N
Toyosaki, H
Kawahara, Y
Sugimoto, S
Suzuki, E
机构
[1] Ajinomoto Co Inc, Fermentat & Biotechnol Labs, Kawasaki, Kanagawa 2108681, Japan
[2] Ajinomoto Co Inc, Inst Life Sci, Kawasaki, Kanagawa 2108681, Japan
关键词
energy metabolism; in vivo P-31 NMR; P/O ratio; saturation transfer;
D O I
10.1093/jb/mvh147
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
To determine the actual potential of the energetic conversion efficiency of Escherichia coli during aerobic respiration, apparent P/O ratios (P/O-app) under either limited or standard glucose-feeding conditions were estimated. The previously reported circulating fermentation system (CFS) was used, and P-31 NMR saturation-transfer (ST) techniques were employed. By coupling with on-line NAIR observations, CFS allowed us to evaluate cellular energetics directly, with both the dissolved oxygen tension and glucose feeding precisely controlled to prevent the effect of substrate-level phosphorylation based on aerobic or anaerobic acidogenesis in E. coli cells. Phosphate consumption rates under standard and limited glucose-conditions were estimated as 4.62 +/- 0.46 and 1.99 +/- 0.11 mumol/s g of dry cell weight (DCW), respectively. Using simultaneously assessed O-2 consumption rates, the P/O-app values under these two conditions were estimated as 1.4 +/- 0.3 and 1.5 +/- 0.1, respectively. To correlate the obtained P/O-app values with the potential efficiency of respiratory enzymes, we determined the activities of two NADH dehydrogenases (NDH 1 and 2) and two ubiquinol oxidases (bo- and bd-type) during the periods when ST was performed. NDH-1 activities in standard or limited glucose cultures were maintained at 57% or 58% of the total NADH oxidizing activity. The percentages of bo-type oxidase activity in relation to the total ubiqinol oxidizing activity under the standard and limited glucose conditions were 32% and 36%, respectively. These percentages of enzymatic activities represent the respiratory competence of E. coli cells, suggesting that, during the NAIR observatory period, the enzymatic activity was not at a maximum, which could also explain the estimated P/O-app values. If this is the case, enhancing the expression of the bo-type oxidase or disrupting of the bd-type oxidase gene could be effective approach to increasing both the P/O ratio and cellular yields.
引用
收藏
页码:509 / 515
页数:7
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