Understanding metabolism and stress conditions of recombinant E. coli

在我们之前的工作中，我们利用系统生物学的方法和构建不同的突变体，重点研究了大肠杆菌K和B菌株中心碳代谢的调控。这项工作是为了研究各种应激条件对大肠杆菌生长的影响，特别是当大肠杆菌暴露于应激条件时可能表达的小调控rna的作用。我们假设，通过操纵小rna的表达，有可能将环境对细菌生长和重组蛋白生产的影响降到最低。我们发现在大肠杆菌K中，它对高葡萄糖浓度敏感；小RNA SgrS未表达。通过过表达该分子，可以减少高葡萄糖浓度引起的应激效应，并使K菌株和B菌株一样生长。这一发现为利用非编码RNA控制细菌代谢开辟了新的途径。我们继续这项工作，以确定可以增加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FnrS和Gady的水平来影响细菌对低pH值的抵抗力，FnrS和gdy在这种生长条件下的表达差异。

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.