Understanding metabolism and stress conditions of recombinant E. coli

在我们以前的工作中，我们利用系统生物学的方法，通过构建不同的突变体，集中于调节E.coliK和B菌株的中心碳代谢。这项工作是为了研究各种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可能表达的小调控RNA的作用。我们假设，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。我们发现，在对高糖敏感的E.coliK中，小RNA SGRS不表达。通过过量表达该分子，有可能减少高糖浓度引起的胁迫效应，并允许K株和B株一样生长。这一观察为利用非编码RNA控制细菌代谢开辟了新的途径。我们继续这一系列的工作，以确定可以提高细菌对酸性条件的抵抗力的小RNA，但到目前为止，我们无法通过操纵在这种生长条件下差异表达的FNRs和Gady的水平来影响细菌对低pH的抵抗力。 另一个可能的压力因素是氧气。使用富氧空气是支持大肠杆菌高密度生长的常见策略。然而，高浓度的溶解氧也可能通过形成活性氧物种(ROS)来促进细胞内的氧化应激。为了确定氧气浓度升高对大肠杆菌生长特性的影响，研究了溶氧水平从30%增加到300%时，亲本菌株和一株SOD缺陷菌的特异性基因表达和酶活性。亲本菌株的生长参数没有显著差异，只是呼吸和醋酸盐积累曲线暂时下降。通过转录分析，确定亲本菌株通过激活SoxRS调节子来响应氧化胁迫。然而，在溶氧转换后，该SOD缺陷菌株同时激活了SoxRS和OxyR调节子，但无法恢复其最初的生长速度。转录分析和酶活性分析结果表明，当大肠杆菌暴露在溶氧变化中时，超氧应激调节因子SOxRS被激活，导致超氧化物歧化酶系统的刺激。这使大肠杆菌能够保护自己免受氧气的毒害。此外，由于OxyR保护系统没有被激活，这表明H_2O_2没有增加到胁迫水平。 由于SoxRS调节子的激活，SoxS基因的表达可以增加16倍。我们推测，这一特性使该基因有可能成为重组蛋白表达的候选基因。与现有的诱导方法相比，氧气诱导有几个优点：它不涉及生长因子或营养物质的添加或耗尽，不需要添加化学诱导剂或影响产生菌生长和代谢的温度变化，它不影响生长介质组成，简化了回收和纯化过程。将SoxS启动子克隆到pGFPmut3.1载体中，构建了可通过增加氧气浓度诱导表达的表达载体pAB49。通过测定氧饱和度从30%增加到300%时GFP的表达来表征SOXS启动子的效率和调控特性。重组GFP的表达水平与dO2成正比，表明pAB49是一个可控载体。氧对GFP的潜在有害影响被发现可以忽略不计，这是由蛋白质-羰基含量和比活性决定的。在高密度生长条件下，通过增加氧气浓度诱导细胞生长，在300%空气饱和度下培养3h，绿色荧光蛋白荧光强度达到109000 FU(494 mg绿色荧光蛋白/L)，占总蛋白的3.4%。

In our previous work, by using system biology approaches and the constructing of different mutants, we concentrated on the regulation of the central carbon metabolism in E. coli K and B strains. This work was expended towards the investigation of the effect of various stress condition on E. coli growth and especially the role of small regulatory RNAs that is likely expressed when E. coli is exposed to stress conditions. We hypothesized 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 showed that In E. coli K, which is sensitive to high glucose concentration; the small RNA SgrS was not expressed. By over-expressing this molecule it was possible to reduce the stress effect caused by high glucose concentration and to allow the K strain to grow as well as the B strain. This observation opens new approach towards controlling bacterial metabolism utilizing non-coding RNA. We continued this line of work to identify small RNA that can increase the bacteria resistance to acid conditions, but so far we were unable to affect the bacteria resistance to low pH by manipulating level of FnrS And Gady that were expressed differentially at this growth conditions . Another possible stress factor is oxygen. The use of oxygen-enriched air is a common strategy that supports high density growth of E. coli. However, high dissolved oxygen concentrations may also promote oxidative stress in the cells through the formation of reactive oxygen species (ROS). To determine the effect of elevated oxygen concentrations on the growth characteristics, specific genes expression and enzyme activities in parental E. coli strain and an SOD-deficient strain, were evaluated when the dissolved oxygen level was increased from 30% to 300%. No significant differences in the growth parameters were observed in the parental strain except for a temporary decrease of the respiration and acetate accumulation profile. By performing transcriptional analysis, it was determined that the parental strain responded to the oxidative stress by activating the SoxRS regulon. However, following the dissolved oxygen switch, the SOD-deficient strain activated both SoxRS and OxyR regulons but was unable to resume its initial growth rate. The transcriptional analysis and enzyme activities results indicated that when E. coli is exposed to dissolved oxygen shift, the superoxide stress regulator SoxRS is activated and causes the stimulation of the superoxide dismutase system. This enables the E. coli to protect itself from the poisoning effects of oxygen. In addition, since the OxyR protecting system was not activated it showed that H2O2 did not increase to stressing levels. As a result of the SoxRS regulon activation the expression of the soxS gene can increase by up to 16 fold. We postulated that this property makes this gene a possible candidate for recombinant protein expression. Compared with the existing induction approaches the oxygen induction offers several advantages: it does not involve addition or depletion of growth factors or nutrients, addition of chemical inducers or temperature changes that can affect growth and metabolism of the producing bacteria, it does not affect the growth-media composition simplifying the recovery and purification processes. The soxS promoter was cloned into the pGFPmut3.1 plasmid creating pAB49, an expression vector that can be induced by increasing oxygen concentration. The efficiency and the regulatory properties of soxS promoter were characterized by measuring the GFP expression when the dO2 in the culture was increased from 30% to 300% air saturation. The expression level of recombinant GFP was proportional to the dO2, demonstrating that pAB49 is a controllable vector. Potentially harmful effect of oxygen on the GFP was found negligible as determined by protein-carbonyl content and specific activity. Performing high density growth the cells were induced by increasing the dO2, after 3 hours at 300% air saturation, GFP fluorescence reached 109000 FU (494 mg of GFP/L) representing 3.4% of total protein.