a. In our previous work, by using system biology approaches and by 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 are known to be 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 initially showed the small RNA effect in E. coli K; which does not expresses the small RNA SgrS. By over-expressing this molecule it was possible to reduce the stress effect caused by high glucose concentration and to allow this strain to grow as well as the B strain. This observation opened new approach towards controlling bacterial metabolism by utilizing non-coding RNA; we continued this line of work by identifying the small RNA Gady as a potential molecule that can increase the bacteria resistance to acid conditions. b. It was reported that at mild acidic conditions (pH 5.8), the Hfq-associated sRNA GadY is activated, also it was reported that the two decarboxylase systems - the lysine decarboxylase system (LDS) and the glutamate decarboxylase system (GDS) - are activated to maintain intracellular balance of protons. Based on the above GadY was constitutively expressed in E. coli and its effect on the bacterial growth was evaluated. We found that both the parental E. coli K-12 and the constitutively expressing GadY strains grew at pH 7.0 to similar cell densities of 43 OD, but the constitutively expressing GadY strain produced around 6 g/L acetate compared with 10 g/L by the parental strain. At pH 6.0, the parental strain grew to an OD of 20 and produced 10 g/L of acetate while the GadY strain grew to an average OD of 31 and produced 4 g/L acetate. After analyzing 17 genes associated with acid stress, it was found that at pH 7.0 LDS was expressed in the early exponential phase and GDS was expressed in the late exponential phase in both strains. c. 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). By exposing the cells to high oxygen concentration and by performing transcriptional analysis, we determined that E. coli responds to oxidative stress by activating the SoxRS regulon which causes the stimulation of the superoxide dismutase system. This enables the E. coli to protect itself from the poisoning effects of oxygen. 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. We therefore cloned the soxS promoter 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. By 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. d. This direction of research is currently being expanded to Bacillus another bacteria being used t for recombinant protein expression. So far we established that the bacteria harboring plasmid, is growing different than the parental bacterial strain. We are now in the process of comparing the gene expression pattern of the non-producing train with those of the producing strain.
