Novel insights into characteristics, relevance and regulation of corynebacterial aconitase

Abstract

Strains of the Gram-positive soil bacterium $\textit{Corynebacterium glutamicum}$ are widely used in industrial biotechnology for the production of amino acids, predominantly L-glutamate and L-lysine. In this context, the tricarboxylic acid cycle (TCA cycle) is of particular interest, as it provides energy and the biosynthetic precursors 2-oxoglutarate and oxaloacetate. A detailed understanding of the enzymes and of the regulation of this metabolic pathway is crucial for rational improvement of production strains. In this dissertation, the properties, physiological relevance and regulation of aconitase, a [4Fe-4S]-cluster containing enzyme catalysing the second step of the TCA cycle, have been investigated. In the first part of this thesis, the biochemical properties of aconitase were studied. The comparison of different enzyme assays revealed the highest activity for the hydration of $\textit{cis}$-aconitate (0.433 ± 0.054 U mg$^{-1}$ protein in cell-free extracts) and the lowest activity for the physiological reaction from citrate to isocitrate (0.134 ± 0.026 U mg$^{-1}$). Aconitase was heterologously overproduced, purified, reactivated and used for the determination of kinetic constants. The K$_{m}$ values for citrate (480 ± 200 μM) and isocitrate (552 ± 302 μM) were much higher than the one for $\textit{cis}$-aconitate (18.5 ± 3.4 μM), a feature reported also for aconitases from other organisms. The pH and temperature optima were found to be between 7.5 and 7.75 and at 50°C. These results are useful for mechanistic in $\textit{silico}$ models of the central metabolism of $\textit{C. glutamicum}$. The second aim of this thesis was to study the impact of an aconitase deletion on growth and metabolism of $\textit{C. glutamicum}$. A $\Delta \textit{acn}$ strain was found to be glutamate-auxotrophic and severely inhibited in its growth. It secreted large amounts of acetate into the medium and showed strongly elevated mRNA and protein levels of enzymes involved in acetate metabolism. When searching for the reason of acetate production, we discovered that the $\Delta \textit{acn}$ strain was lacking citrate synthase activity due to a single point mutation in the corresponding $\textit{gltA}$ gene. Subsequent analysis of 28 independent $\Delta \textit{acn}$ mutant strains revealed that 16 of them contained none or only inactive citrate synthase due to 16 different kinds of mutations (point mutations, insertions, deletions, transposon insertions). This result clearly showed that in $\Delta \textit{acn}$ mutants there is a strong selection pressure for secondary mutations in the $\textit{gltA}$ gene, probably caused by the toxicity of high cytoplasmic citrate concentrations that accumulate in the absence of aconitase. Similar results were obtained for $\textit{C. glutamicum}$ mutants lacking the $\textit{icd}$ gene encoding isocitrate dehydrogenase. In this case, 8 out of 25 $[...