In our previous report, we concentrated on the effect of acidic conditions on growth of recombinant E. coli and its protection against this condition by expression small RNAs. 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. In that report we also demonstrated that the E. coli mechanism to resist high oxygen concentration can be utilized for expression of recombinant proteins. By cloning the soxS promoter into the pGFPmut3.1 plasmid creating pAB49, an expression vector that can be induced by increasing oxygen concentration was created. 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. This research work was continued by better understanding the resistant mechanism of E. coli to high oxygen concentration and by investigating recombinant protein expression from bacillus. a. To obtain large quantities of E. coli needed for recombinant protein production molecular oxygen is used often, but high oxygen concentration is known to have a damaging effect on the bacteria. Therefore, it is important to study the effect of over-oxygenation on E. coli metabolism and the bacteria protecting mechanism. Following change in the dissolved oxygen (DO) from 30% to 300% air saturation the expression of sodC, the periplasmic CuZn-containing SOD, and sodA, the cytosolic Mn-containing SOD, was higher, while the expression of the sodB, the cytosolic Fe-containing SOD, was lower. The down-regulation of the sodB was found to be related to the activation of the small RNA RyhB. It was revealed that iron homeostasis was involved in the RyhB activation and in sodB regulation but not in sodA. Supplementation of amino acids to the culture medium reduced the intracellular ROS accumulation and reduced the activation of both SodA and SodC following the increase in the oxygen concentration. The study provides evidence that at conditions of over-oxygenation, sodA and sodC are strongly regulated by the amount of ROS, in particular superoxide; and sodB is regulated by iron availability through the small RNA RyhB. In addition, information on the impact of NADH, presence of amino acids and type of iron on SOD regulation, and consequently, on the ROS concentration is provided. b. Protective antigen (PA) expressed by Bacillus anthracis has been used as a vaccine against anthrax infections in humans. Since its expression in the Bacillus anthracis is low, E.coli has been used for higher expression of recombinant PA (rPA), but the expressed protein was counteracted by issues related to the host strain endotoxin contamination. As a result, improved expression of rPA was investigated in its natural producer Bacillus anthracis (non-virulent) ames strain. The work is focused on understanding the effect of over expression the protein on the bacillus metabolism and genes expression with the purpose of improving the production of this recombinant toxin and recombinant proteins in general. Therefore, controlled growth of the B. anthracis ames with and without the plasmid encoding the protective antigen were performed and protein production, bacterial growth characteristics and gene transcription pattern were analyzed. Comparative RNAseq analysis showed that the stress response global regulator ctsR (fc 4.9) was up-regulated along with other stress related gene transcripts like sigB (fc 3), rsbW & rsbV. In addition, htrA, dps, GBAA_1113, GBAA_0326, GBAA_5290 and GBAA_4875 were predominantly expressed in PA producing culture. Contrary to the general observation of increase in chaperone transcripts post recombinant protein induction in E.coli, we observed that groL, groES, hslO, narJ, hscC, dnaJ, dnaK, grpE, clpB, clpP were down-regulated in log and late log phase except csaA which was up-regulated 2.2-fold throughout the cultivation time. Most of the genes belonging to transport, TCA, glycolysis, PPP and amino acid biosynthesis were up-regulated in the lag phase samples which help the cell coping with the increased requirements of nutrient and energy for PA expression. These pathways are down-regulated in the log and late log phase as cellular stress response onsets, which is reflected in decreased specific growth rate. The information obtained from this study would be further verified by qRT-PCR, which will be used for genetic modification of B. anthracis to improve PA production.
