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Novel insights into amino-acid catabolism and the characterisation of a ncRNA regulating BCAA biosynthesis in bacteria

Novel insights into amino-acid catabolism and the characterisation of a ncRNA regulating BCAA biosynthesis in bacteria

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KNORR, Sebastian, 2020. Novel insights into amino-acid catabolism and the characterisation of a ncRNA regulating BCAA biosynthesis in bacteria [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Knorr2020Novel-50505, title={Novel insights into amino-acid catabolism and the characterisation of a ncRNA regulating BCAA biosynthesis in bacteria}, year={2020}, author={Knorr, Sebastian}, address={Konstanz}, school={Universität Konstanz} }

2020-08-14T12:28:26Z Amino acid metabolism is not only of interest because of its impact on protein biosynthesis as the motor for catalytic processes in the cell, but also catabolism of amino acids themselves presents a very important biochemical process in bacterial metabolism. Bacteria can exploit amino acids as alternative carbon sources by directly utilizing the carbon backbone for energy metabolism. Furthermore, amino acid degradation presents an important defense strategy against various stress factors. However, white spots on the map still exist for many bacterial degradation pathways of amino acids. For example, complete catabolic pathways of the basic amino acid lysine, the branched chain amino acids and most of the aromatic amino acids are still missing for E. coli, the best studied organism. This thesis presents the complete characterisation of a novel lysine degradation pathway in E. coli which can also be found in other bacterial species. We discovered a putative αketoglutarate dependent dioxygenase, CsiD, that hydroxylates glutarate producing succinate thus connecting lysine degradation to central carbon metabolism. Furthermore, we showed that the reaction product of CsiD, L-2-hydroxyglutarate, is further metabolised via LhgO to channel electrons into the membrane for respiratory energy metabolism. We present evidence that lysine is degraded, in E. coli, under nutrient starvation conditions contributing to bacterial adaptation to stationary phase conditions. Furthermore, because of the association of L-2-hyroxyglutarate to human diseases such as cancer and organic acidurias, we discuss a potential relation between the human gut microbiome as a potential source of the oncometabolite. To shed light on other possibly unknown amino acid degradation pathways in E. coli, this thesis contains the implementation of a screening assay for the identification of novel enzyme functions involved in amino acid catabolism. The screening results offer new insights into what processes influence the utilization of amino acids and derived metabolites. Different RNA based regulatory elements, like transcription attenuators and riboswitches, can be found in bacteria regulating important processes in amino acid metabolism.<br />This thesis presents the characterisation of the putative non-coding RNA motif ilvH as a cis-regulatory acting RNA in branched-chain-amino-acid (BCAA) biosynthesis. We were able to show that the ilvH-motif RNA regulates downstream gene expression in response to isoleucine and α-ketobutyrate, an intermediate of isoleucine biosynthesis. However, we could not validate the motif RNA as a classical riboswitch. Mutational analysis of a conserved tetraloop of the ilvH-motif indicated that this sub-motif is involved in the regulation mechanism of the small ncRNA. It seems likely that the ilvH-motif RNA presents a novel regulation mechanism sensed by BCAA biosynthesis metabolites. However, further investigations are needed to fully understand the regulatory mechanism underlying the ilvH-motif RNA. 2020-08-14T12:28:26Z eng 2020 terms-of-use Knorr, Sebastian Knorr, Sebastian Novel insights into amino-acid catabolism and the characterisation of a ncRNA regulating BCAA biosynthesis in bacteria

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