Investigation of trans-cinnamic acid and p-coumaric acid related genes and metabolites in Escherichia coli

Phenolic compounds are natural compounds that are widely found in nature, have a wide place in the human diet and have a broad spectrum of biological activity. Trans-cinnamic acid and p-coumaric acid, which we used in our thesis, are members of the phenolic acids class and belong to the phenolic compounds group. In the literature reviews, it has been determined that trans-cinnamic acid and p-coumaric acid have a wide variety of biological activities such as antibacterial, antibiofilm, antifungal, anticancer, but it has not been clarified yet in relation to which pathways in the cell they perform these activities. In this thesis, it was aimed to investigate the genes and metabolites related to trans-cinnamic acid and p-coumaric acid in Escherichia coli. In this thesis, first of all, first of all, tolerant clones were determined compared to the control by using the ASKA clone set plasmid library by gradient seeding method. Compared to the control, 99 strains of trans-cinnamic acid and 100 strains of p-coumaric acid were selected. Then, for the first selection, streaking experiments were carried out in nutrient media containing increasing phenolic acid concentration. Relatively tolerant strains were identified compared to the control by streaking experiments. Colony PCR experiments were carried out to determine the presence and size of the inserts in the plasmids of the selected strains. In colony PCR experiments, plasmid isolation was performed from the clones with different insert sizes. The nucleotide sequences of the inserts contained in the isolated plasmids were elucidated by the Sanger Sequencing method, and then the obtained nucleotide sequences were subjected to bioinformatic analysis. As a result of bioinformatic analysis, it was determined that trans-cinnamic acid-tolerant strains had prpE, yddL, flhD, sbmC, p-coumaric acid-tolerant strains were pgaB, prpE, dinI, bax and yadI. In the last stage of the thesis study, metabolites that play a role in the response of Escherichia coli BW25113 to trans-cinnamic acid and p-coumaric acid were investigated using NMR (Nuclear Magnetic Resonance) Technology. BW25113 strain was exposed to increasing sublethal concentrations of trans-cinnamic acid and p-coumaric acid to obtain metabolite extracts and 1H-NMR analyzes were performed. After exposure to trans-cinnamic acid, an increase in pantothenate, pyridoxine, adenine, cellobiose and nicotinate metabolites of the BW25113 strain, and a decrease in pyroglutamate, ethylene glycol, glycerol, IMP, UMP and sucrose metabolites were detected. In Pathway analysis, it was determined that Pentothenate and CoA biosynthesis, Glycerolipid Metabolism, Galactose Metabolism, Pyrimidine Metabolism, Purine Metabolism, Glyoxylate and Dicarboxylate Metabolism, Glutathione Metabolism and Vitamin B6 Metabolism were affected. After exposure to p-coumaric acid, BW25113 strain had an increase in ADP, cytosine, dTTP, fumarate, ethanol, inosine methanol, oxypurinol and UMP metabolites, and decreased acetamide, choline, cis-aconitate, hydroxyacetone, hypoxanthine, succinate and pantothenate metabolites. In the pathway analyzes, it was determined that Purine Metabolism, Pyrimidine Metabolism, Pyruvate Metabolism, Pentothenate and CoA Biosynthesis, Glycerolipid Metabolism, Beta-Alanine Metabolism, Glyoxylate and Dicarboxylate Metabolism, Glutathione Metabolism, TCA Cycle, Nicotinatabolism and Nikon's Metabolism were affected.