Understanding metabolism and stress conditions of recombinant E. coli

在我们以前的工作中，我们发现乙醛分流、TCA循环和乙酰-辅酶A合成酶对乙酸乙酯的吸收在大肠杆菌B中比在大肠杆菌K中更活跃。通过运用系统生物学的方法，我们发现，其他代谢途径：葡萄糖生成、SFCA分流、PPC分流、糖原生物合成和脂肪酸降解在两株菌中的作用是不同的。研究发现，在E.coliK中，乙酸酯是由以丙酮酸为底物的丙酮酸氧化酶(PoxB)产生的，而不是由以乙酰辅酶A为底物的磷酸转乙酰基酶-醋酸激酶(PTA-AckA)系统产生的。葡萄糖磷酸烯醇式丙酮酸合成酶(PPSA)的失活、逆转录的SFCA分流的激活以及低而稳定的丙酮酸脱氢酶(ACEE，ACEF)引起丙酮酸的积累，丙酮酸氧化酶B将丙酮酸转化为乙酸酯。我们推测，CRA蛋白是控制PPSA aceBAK和ACS活性的调节分子，是导致这两个菌株之间差异的原因。 为了进一步了解这种现象，我们研究了CRA基因对大肠杆菌B和K的生长、醋酸盐产量和基因表达的影响。在E.coliB中，crA基因的缺失对细胞的生长和醋酸盐的积累影响最小，而在E.coliK中，相同基因的缺失会导致细胞在醋酸盐浓度达到6.6g/L和介质电导率达到21ms/cm时停止生长。结果表明，大肠杆菌K-12(JM109)生长缓慢是由于产生BET操纵子(BetABT)的渗透保护剂转录抑制的结果。结论是，CRA基因缺失引起的转录变化并不影响中心碳代谢的活性，而CRA基因缺失会抑制E.coliK中的bet操纵子的转录，但不影响E.coliB的该操纵子的转录，这一特性以及对高糖的不敏感性使E.coliB菌株对环境变化的抵抗力更强，更适合于高密度生长和重组蛋白生产。 在过去的两年里，我们扩大了我们的研究，以了解胁迫条件对大肠杆菌生长的影响，特别是当大肠杆菌暴露在胁迫条件下时，据信表达的小调控RNA的作用。我们的假设是，通过控制小RNA的表达，可以最大限度地减少环境对细菌生长和重组蛋白生产的影响。 我们集中在以下胁迫条件：高葡萄糖浓度，高溶解氧浓度和较低的pH。有关高糖浓度和小RNA SGRs参与的结果在去年的报告中进行了部分总结。我们观察到，在耐高糖的大肠杆菌B中，当细菌暴露在高糖浓度下时，小RNA SGRS过表达，并通过减少葡萄糖转运体ptsG的翻译来减少葡萄糖向细胞的转运。在对高糖敏感的E.coliK中，不表达SGRs。通过在E.coliK中过表达SGRs，可以减少高糖浓度引起的应激效应，并使该菌株能够像E.coliB菌株一样生长。这一观察结果为利用非编码RNA控制细菌新陈代谢开辟了一条新的途径。 由于使用富氧空气是支持大肠杆菌高密度生长的常见策略，另一个可能的压力因素是在生物反应器中创建包含高溶解氧浓度的区域。这些区域可能通过形成活性氧物种，如过氧化氢(H_2O_2)和超氧阴离子(O_2.-)，对细胞造成不可逆转的损害，从而促进细胞的氧化应激。我们观察到，当细胞从30%的溶解氧饱和度转移到300%的溶解氧饱和度时，细胞的生长参数没有显著差异。转录分析和酶活性分析表明，大肠杆菌能够通过激活超氧化物歧化酶系统和SOXRS调节子中的SOXS来保护自己免受纯氧的毒害。研究还发现，OxyR防御系统没有被激活，这表明过氧化氢没有增加到应激水平。这些发现的意义在于，尽管非常高浓度的ROS影响细菌的生长和生存能力，但这些条件目前不存在于高密度细菌发酵中，在高密度细菌发酵中，纯氧供应给正在生长的培养物。 有关低pH胁迫效应的研究工作目前正在进行中。

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.