Probing Metabolic Pathways using an RccR Based Genetically Encoded Biosensor

Abstract

The study of metabolic pathways is of utmost importance to contemporary biotechnology. However, mapping metabolic pathways is a burdensome process due to the entangled and complex nature of biological systems. Due to the direct involvement of transcription factors in metabolism, biosensors based on them are a powerful tool for researching metabolism. In this study, we used a biosensor vector where the RccR transcription factor regulates the expression of eGFP in response to the metabolite KDPG to investigate the central metabolism of two bacterial species of high relevancy to biotechnology: Pseudomonas Putida and Escherichia Coli, with an experimental focus on the first. Our research demonstrates that acetate, aromatic acids, and fatty acids enter the central metabolism of P. Putida through the TCA cycle. We also show among the aromatic acid, PCA and benzoic acid flux into central metabolism through one metabolic intermediate through a pathway with moderately low metabolic leakage. On E. Coli’s end, we show that acetate and the fatty acid C18:2 enter metabolism through the TCA cycle as well. We also employ bioinformatic databases to show that HexR is closely related to RccR and that the differences between the HexR proteins in P. Putida and E. Coli might show differing interference with RccR’s function. Our results also indicate that P. Putida preferentially consumes glucose, then acetate, and then aromatic acids when presented with mixtures of them. Finally, we saw strong evidence of the presence of a metabolic pathway that leads from acetate into the central metabolism of E. Coli. through acetyl-CoA.