In-depth analysis of the purine biosynthetic pathway of Corynebacterium glutamicum : from local pathway analysis to global phenotype profiling

摘要

This thesis focused on the purine biosynthetic pathway of C. glutamicum in order to increase the general understanding of this essential pathway. In a first step, a suitable approach for the reliable quantitation of intracellular purine pools was established and validated, thus allowing targeted metabolite profiling of genetically engineered C. glutamicum strains. Based on a metabolic engineering strategy, site-directed mutants were generated and comprised modifications of the purine precursor supply (pgi), deletions of degrading reactions (purA and guaB2) and a point mutation (purFK348Q) targeting at a deregulation of the feedback inhibitory control. The individual modifications were combined in C. glutamicum ΔpurA ΔguaB2 purFK348Q Δpgi. Conducting a systems-level approach, a metabolic shift of the purine intermediate distribution was revealed, promoting a tremendous increase of the intracellular concentration of the degradation product hypoxanthine at the expense of IMP. Furthermore, a global phenotypic adaptation, expressed in transient growth stagnation, was observed. These adverse effects were attributed to a decline in the ATP generating capacity and imbalances of the NADPH metabolism caused by the deletion of the first glycolytic enzyme, glucose 6-phosphate isomerase (pgi). Cultivations applied on complex substrates showed a release of these adverse effects, temporarily delaying the growth stagnation phenomenon.