Investigation of benzoic acid and ferulic acid related genes in Escherichia coli with metabolomic and genomic approaches

Phenolic compounds are secondary metabolites whose biosynthesis takes place in different living groups, especially plants. It is noteworthy that these compounds are produced in response to environmental stresses such as protection of plants against UV, defense against pathogens, and have many important activities including antibacterial, antioxidant, anticancer, antiviral. Due to the bioactive properties of phenolic compounds and their positive effects on human health even with dietary intake, these compounds are used as natural compounds in many sectors, including the health sector. However, it is not known exactly which genes and metabolites they interact with in the cell. In order for these compounds to be used more effectively, it is necessary to expard the knowledge about their potential roles on living things and human health, to detail the studies at the gene/genome level in the cell, and to determine the pathways in which they interact within the cell. In this thesis, it was aimed to determine the target genes and metabolites associated with phenolic compounds benzoic acid and ferulic acid in Escherichia coli bacteria. In addition to genomic, gene expression and metabolomic analyzes, various microbiology experiments and statistical analysis methods were applied in the studies. In the first part of the thesis, genomic studies were carried out with the ASKA plasmid genome-wide library, which was created by combining 4123 clones in the ASKA clone set. The ASKA plasmid genome-wide library was exposed to different concentrations of benzoic acid and ferulic acid stress using gradient cultivation method. 91 strains from benzoic acid gradient cultivation and 132 strains with relatively high tolerance level were determined from ferulic acid gradient selection. Streaking method was used to confirm the tolerance of these strains and to perform an additional selection. Colony PCR was performed by selecting strains that were confirmed to be tolerant as a result of selection studies with these compounds and insert sizes were examined. Plasmid isolation and sequence analysis were performed by selecting 6 strains from benzoic acid and ferulic acid. Tolerant clones, whose insert information was obtained after sequence analysis, were found to carry yjtD, yabQ, ydfB, yegP, osmE, hisS (benzoic acid) and prpE, sbmC and fliS (ferulic acid) genes. In another part of the thesis study, gene expression analysis was performed in wild type Escherichia coli by selecting from tolerant clones for each compound. In Real-Time PCR experiments, an increase in the expression levels of osmE and prpE genes was observed. In the last part of the thesis studies, 1H-NMR spectroscopy analysis was used to determine the metabolites related to benzoic acid and ferulic acid stress. Metabolite extracts for 1H-NMR analysis were prepared in 4 concentrations, 3 replicates separately for each compound using E.coli BW25113 strain. As a result of 1H-NMR analysis of benzoic acid with E.coli BW25113 strain, 80 metabolites were obtained. In the statistical analyzes performed with 80 metabolites, a significant change was observed in the concentrations of 24 metabolites compared to the control group, in at least one benzoic acid concentration application. Sucrose, IMP, Pantothenate, Creatine, Mannitol, Uridine, Glycine, Adenosine, Cytidine, Histamine, Putrescine, N6-Acetyllysine, 2-Aminobutyrate, dTTP, 2-Aminobutyrate, Oanithine, in statistical analysis after benzoic acid and E.coli BW25113 strain NMR analysis There were significant changes in metabolite concentrations of Acetamide, Inosine, Hydroxyacetone, Glycolate, Guanosine, Creatine phosphate, Betaine and Malate compared to the control group. In NMR analysis of E. coli BW25113 strain with benzoic acid, "Galactose (Galactose)", "Starch and sucrose (Starch and sucrose)", "Purine (Purine)", "Pyrimidine (Pyrimidine)" and "Glycine, serine and threonine (Glycine)" , serine and threonine)" metabolic pathways were found to be affected. As a result of NMR analysis with ferulic acid, 71 metabolite profiles were obtained. As a result of the statistical analyzes performed with 71 metabolites, it was observed that there was a change in the concentration of 41 metabolites in at least one ferulic acid application compared to the control group. Metabolites with significant changes, Acetamide, Fumarate, Uracil, Oxypurinol, N-Acetylornithine, Glycolate, Erythritol, Succinate, Trimethylamine N-oxide, Hydroxyacetone, N-Acetylglucoseamine, Pyroglutamate, Betaine, Isocitrate, Malateyl glycolamide, Erythyl glycoseamine, , Fructose, Adenosine, AMP, Creatine phosphate, Inosine, Histamine, O-phosphocholine, Cytidine, Cytosine, Maleate, Pyridoxine, Methionine, Glutamate, Malonate, Cadaverine, Guanosine, Alanine, Tartrate and Sebacate. Metabolic pathways affected in NMR analysis of ferulic acid and E. coli BW25113 strain, "Tricarboxylic acid cycle (TCA cycle)", "Glutathione (Glutathione)", "Propanoate (Propanoate)", "Sulfur (Sulfur)", "Methane (Methane) ", "Butanoate (Butanoate)", "Arginine biosynthesis (Arginine biosynthesis)", "Nicotinate and nicotinamide (Nicotinate and nicotinamide)", "Lysin degradation", "Alanine, aspartate and glutamate (Alanin, aspartate and glutamate) )", "Pyruvate (Pyruvate)", "Glyoxylate and dicarboxylate (Glyoxylate and dicarboxylate)", "Glycolysis/gluconeogenesis (Glycolysis/gluconeogenesis)", "Purine (Purine)", "Glycine, serine and threonine (Glycin, serine and threonine)", "Pyrimidine (Pyrimidine)", "Fructose and mannose (Fructose and mannose)" and "Starch and sucrose (Starch and sucrose)". In this thesis, the genes and metabolites that may be associated with benzoic acid and ferulic acid in E. coli bacteria were determined. Keywords: Benzoic acid, Ferulic acid, ASKA, Escherichia coli, gene, metabolite.