Production of recombinant proteins from different microorganism such as bacteria and yeast are an efficient process for acquiring large amounts of proteins needed for clinical and structural studies. To obtain the proper amount of the producing microorganisms, elaborated growth strategies that yield higher densities of the microorganism (e.g. 100 g/l) are used routinely. However, high-producing strains do not always respond well to these growth strategies and, in several cases, it is impossible to grow them efficiently and to obtain the needed amount of the required protein. Understanding the reasons for such growth behavior can improve the production process and can lead to construction of bacterial and yeast strains that are resistant to stress and produce recombinant proteins more efficiently. To overcome this obstacle, we are investigating the bacterial and yeast metabolism and their response to external physiological conditions especially during high density growth process. We report here on our work with E. coli, Bacillus and pichia pastoris Regarding E. coli: we concentrated on understanding the resistant mechanism of the organism to high oxygen concentration that is routinely supplied to the growing culture. 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. As for Bacillus: Protective antigen (PA) of Bacillus anthracis is being considered as a vaccine candidate against anthrax and its production has been explored in several heterologous host systems. Since the systems tested introduced adverse issues such as inclusion body formation and endotoxin contamination, the production from B. anthracis is considered as a preferred method. We examined the effect of PA expression on the metabolism of B. anthracis producing strain, BH500, by comparing it with a control strain carrying an empty plasmid. The strains were grown in a bioreactor and RNA-seq analysis of the producing and non-producing strain was conducted. Among the observed differences, the strain expressing rPA had increased transcription of sigL, the gene encoding RNA polymerase 54, sigB, the general stress transcription factor gene and its regulators rsbW and rsbV, as well as the global regulatory repressor ctsR. There were also decreased expression of intracellular heat stress related genes such as groL, groES, hslO, dnaJ, and dnaK and increased expression of extracellular chaperons csaA and prsA2. Also, major central metabolism genes belonging to TCA, glycolysis, PPP, and amino acids biosynthesis were up-regulated in the PA-producing strain during the lag phase and down-regulated in the log and late-log phases, which was associated with decreased specific growth rates. The information obtained from this study may guide genetic modification of B. anthracis to improve PA production. Concerning Pichia pastoris: Work is being done on expression of P-glycoprotein (Pgp) from pichia pastoris which is needed for structural studies. The expression of this protein is poor likely because of stress on the producing cells. We established that expression of this protein using the conventional growth strategy causes the cells to produce high concentrations of formaldehyde and hydrogen peroxide, and lower activity of FLD, FDH, IDH and -KGDHs. By developing a feeding strategy of methanol containing fresh media components it was possible to reduce the production of formaldehyde and hydrogen peroxide and to increase the activity of FLD, FDH, IDH and -KGDHs.
