Understanding metabolism and stress conditions of recombinant E. coli

在我们之前的工作中，我们确定了大肠杆菌B比大肠杆菌k更活跃的乙醛酸分流、TCA循环和乙酰辅酶a合成酶的乙酸摄取。通过系统生物学方法，我们发现了其他代谢途径：糖原生成、sfcA分流、ppc分流、糖原生物合成和脂肪酸降解在两个菌株中运作不同。结果表明，在大肠杆菌K中，丙酮酸氧化酶（poxB）以丙酮酸为底物产生乙酸酯，而不是以乙酰辅酶a为底物的磷酸转乙酰酶-乙酸激酶（Pta-AckA）体系产生乙酸酯。糖原酶磷酸烯醇丙酮酸合成酶（ppsA）失活、sfcA分流激活以及低而稳定的丙酮酸脱氢酶（aceE, aceF）导致丙酮酸积累，并被丙酮酸氧化酶b转化为醋酸盐。我们假设Cra蛋白是控制ppsA aceBAK和acs活性的调节分子，是导致两种菌株差异的原因。

In our previous work, we established that the glyoxylate shunt, the TCA cycle and acetate uptake by acetyl-CoA synthetase are more active in E.coli B than in E.coli K. By implementing system biology approach, we showed that, other metabolic pathways: the glucoenogensis, sfcA shunt, ppc shunt, glycogen biosynthesis and fatty acid degradation are operate differently in the two strains. It was found that in E.coli K, acetate is produced by pyruvate oxidase (poxB) using pyruvate as a substrate rather than by phosphotransacetylase-acetate kinase (Pta-AckA) system which uses acetyl-CoA. The inactivation of the gluconegensis enzyme phosphoenolpyruvate synthase (ppsA), the activation of the anaplerotic sfcA shunt, and the low and stable pyruvate dehydrogenase (aceE, aceF), cause pyruvate accumulation which is converted to acetate by pyruvate oxidase B. We hypothesized that the Cra protein, a regulatory molecule that controls the activity of ppsA aceBAK and acs and is responsible for the difference between the two strains. To further understand this phenomenon we investigated the effect of Cra on the growth, acetate production and gene expression in E. coli B and K. The deletion of the Cra gene in E. coli B minimally affected the growth and acetate accumulation, while the deletion of the same gene in E.coli K caused the cells to stop growing as soon as acetate concentration reached 6.6 g/L and the media conductivity reached 21 mS/cm. It was found that the lower growth of E. coli K-12 (JM109) strain was the result of transcription inhibition of the osmoprotectant producing bet operon (betABT). The conclusions are that the transcriptional changes caused by the deletion of Cra gene did not affect the activity of the central carbon metabolism instead Cra deletion caused transcription inhibition of the bet operon in E. coli K but did not affect this operon transcription in E. coli B. This property, together with the insensitivity to high glucose concentrations, makes E. coli B strain more resistant to environmental changes and better equipped for high density growth and recombinant protein production. During the last two years we expanded our research towards understanding the effect of stress condition on E. coli growth and especially the role of small regulatory RNAs that believed to be expressed when E. coli is exposed to stress conditions. Our assumption is that by manipulating the expression of small RNAs it will be possible to minimize the environmental effect on the bacterial growth and recombinant protein production. We concentrated on the following stress conditions: high glucose concentration, high dissolved oxygen concentration and lower pH. The results concerning the high glucose concentration and the involvement of the small RNA SgrS were summarized in part in last year report. We observed that in E. coli B, which is resistant to high glucose concentration, the small RNA SgrS is over expressed when the bacteria is exposed to high glucose concentration and reduces the glucose transport into the cells by reducing the translation of the glucose transporter ptsG. In E. coli K which is sensitive to high glucose concentration, SgrS was not expressed. By over-expressing SgrS in E. coli K it was possible to reduce the stress effect caused by the high glucose concentration and to allow this strain to grow as well as E. coli B strain. This observation opens a new approach towards controlling bacterial metabolism utilizing non-coding RNA. Since using oxygen-enriched air is a common strategy to support high density growth of E. coli, another possible stress factor is the creation of regions in the bioreactor containing high dissolved oxygen concentration. These areas may promote oxidative stress on the cells through the formation of reactive oxygen species such as hydrogen peroxide (H2O2) and superoxide anion (O2.-) that can cause irreversible damage to the cells. We observed that no significant difference in the growth parameters was found when the cells were transferred from 30% to 300% dissolved oxygen saturation. Transcriptional analysis and enzyme activity indicated that E. coli was able to protect itself from the poisoning effects of pure oxygen by activating the superoxide dismutase system and soxS which is part of the soxRS regulon. It was also found that the OxyR defend system was not activated an indication that H2O2 did not increase to stressing levels. The significance of these findings is that although very high concentrations of ROS affect bacterial growth and viability these conditions are currently do not exist in high density bacterial fermentations where pure oxygen is supplying to the growing culture. Research work on the stress effect of low pH is currently on going.