Tandem-mass-spectrometry-driven investigation of the anaplerotic reactions in Corynebacterium glutamicum
- Tandem Massenspektrometrie getriebene Untersuchung der anaplerotischen Reaktionen in Corynebacterium glutamicum
Kappelmann, Jannick; Wiechert, Wolfgang (Thesis advisor); Takors, Ralf (Thesis advisor)
Dissertation / PhD Thesis
Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2018
The anaplerotic reactions form the basis of a circular reaction network around the metabolites phosphoenolpyruvate, pyruvate, oxaloacetate, and malate, comprising specific carboxylation and decarboxylation reactions which link glycolysis to the tricarboxylic acid cycle. The net carboxylation activity of anaplerotic reactions is essential for the replenishment of tricarboxylic acid cycle intermediates and therefore directly limits the biosynthetic flux towards amino acids derived from it, most notable L-lysine and L-glutamate. The microbial production of these amino acids is primarily accounted for by the biotechnological usage of Corynebacterium glutamicum, a major workhorse of industrial biotechnology and model organism of systems biology alike. The pivotal role of anaplerotic reactions in the biotechnological usage of C. glutamicum motivates the present thesis which is dedicated to a thorough study of these reactions in this organism. First, a theoretical analysis establishes the reason for the unsuccessfully determined anaplerotic reaction activities by 13C-Metabolic Flux Analysis (13C-MFA) in previous studies. Using a focused isotopomer network of the anaplerotic node, it is shown in full generality that for C. glutamicum only certain anaplerotic deletion mutants allow to uniquely determine the anaplerotic fluxes from stationary 13C-isotopomer data. This result prompted the investigation of a set of single and double deletion mutants whose metabolome was investigated by a protocol employing a liquid chromatography electrospray ionization triple quadrupole mass spectrometry (LC-ESI-QqQ MS) platform. By means of quadrupole-time-of-flight technology (QqTOF) a proteome data set for the investigated deletion mutants was generated and evaluated with a novel regression approach, yielding precise fold changes for almost a third of all open reading frames of C. glutamicum. A major biological finding arising from the total data set consists in the proof that the glyoxylate shunt can be active as anaplerotic reaction in C. glutamicum under solely glycolytic conditions, which is brought about by a metabolite-regulator interaction. The finding of inconsistently estimated fluxes in a 13C-Metabolic Flux Analysis of the investigated strains prompted the development of a novel LC-ESI-QqTOF MS-based 13C-profiling method, which harvests the collision induced dissociation (CID) of product anions of central metabolism as well as product cations of amino acids for the positional resolution of 13C-label enrichment. For this purpose, accurate mass spectrometry, selectively-labeled standards, and published fragmentation pathways are used to structurally annotate all dominant mass peaks of a large collection of metabolites, resulting in the most detailed map of the carbon atom fate of LC-ESI-MS/MS collisional fragments yet. Apart from providing positional resolution of 13C-enrichment, it is shown that the CID of amino acids can be harvested to separate 13C- and 15N-isotopic species of these metabolites without resolution in the m/z domain.