Concerning E. coli: We looked at the effect of amino acids additions on the growth, metabolism, gene expression profile and intracellular proteins abundance, in E. coli B and K grown at a steady state in glucose minimal media. Since most microbial fermentations requires addition of amino acids which inadvertently affects microbial growth and biologics production levels, investigating their effect on cellular responses will help to design an effective process strategy. We found that adding amino acids affect significantly acetate excretion, amino acid biosynthesis, pyrimidine nucleotide degradation and fatty acid oxidation. In the presence of amino acids E. coli K, unlike E. coli B increased cell yield, biomass production rate and upregulated fatty acid biosynthesis, but down regulate expression of genes and proteins involved in amino acid biosynthesis, glucose consumption and respiration rate. In comparison E. coli B, showed constitutive synthesis of energetically demanding precursors and higher fatty acid -oxidation activity, which is the reason for its higher biomass formation. This study showed that the presence of amino acids does not affect the amount of ribosomal proteins, translation factors or related genes. As a result, we proposed that the amount of translation related proteins is associated with growth rate and not with the available nutrients. Concerning the yeast pichia pastoris: By evaluating different induction strategies, it was possible to increase the expression of Pgp more than 1000-fold. This result was obtained by inducing the expression with 20% (v/v) media containing 2.5% (v/v) methanol. By quantifying the Pichia biomass, the concentrations of methanol, formaldehyde, hydrogen peroxide, formate, and the enzymatic activities of alcohol oxidase (AOX), catalase (CAT), formaldehyde dehydrogenase (FLD), formate dehydrogenase (FDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), and -ketoglutarate dehydrogenases (-KGDHs), it was possible to establish a correlation between Pgp expression and the induction strategy. Inducing the culture by adding methanol together with fresh media was associated with decreased of formaldehyde and hydrogen peroxide and increased activities of FLD, FDH, IDH, and, -KGDHs. These results indicate that the lower Pgp expression was due to an increase in toxic formaldehyde and hydrogen peroxide and is not related to methanol oxidation. It is possible that Pgp expression is responsible for this behavior, since the increased metabolite concentrations and decreased enzymatic activities were not observed when parental Pichia was subjected to the same growth conditions, or when different proteins were expressed. The development of the optimized procedure is important for improving the production but understanding the effect of this specific protein on the host metabolism is equally important. Concerning the Bacillus anthracis: work was done to improve the expression of the recombinant protective antigen (rPA) from Bacillus anthracis, which is the current vaccine against anthrax. This was done by growing both the Bacillus anthracis expressing PA and a control strain at regulated growth conditions and comparing the profile of the expressed genes in the producing strain with those of the non-producing strain by utilizing RNAseq and LC-MS/MS proteomics. The results showed that major central metabolism genes were up-regulated in the PA-producing strain during the lag phase, and down-regulated in the log and late-log phases. The study also showed that several secretion chaperones (e.g. csaA, prsA, prsA1, prsA2 and prsA3) are responsible for limiting PA secretion. As a result, a list of candidate genes whose altered expression can potentially improve the expression was proposed. Among the main candidates were the secretion chaperones csaA, prsA, prsA1-3, sigL, sigB, groEL and the global regulatory repressors ctsR.
