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In this work, we performed a comparative adaptive laboratory evolution experiment of the important biotechnological platform strain  Corynebacterium glutamicum  ATCC 13032 and its prophage-free variant MB001 towards improved growth rates on glucose minimal medium. Both strains displayed a comparable adaptation behavior and no significant differences in genomic rearrangements and mutation frequencies. Remarkably, a significant fitness leap by about 20% was observed for both strains already after 100 generations. Isolated top clones (UBw and UBm) showed an about 26% increased growth rate on glucose minimal medium. Genome sequencing of evolved clones and populations resulted in the identification of key mutations in  pyk  (pyruvate kinase),  fruK  (1-phosphofructokinase) and  corA  encoding a Mg 2+  importer. The reintegration of selected  pyk  and  fruK  mutations resulted in an increased glucose consumption rate and  ptsG  expression causative for the accelerated growth on glucose minimal medium, whereas  corA  mutations improved growth under Mg 2+  limiting conditions. Overall, this study resulted in the identification of causative key mutations improving the growth of  C. glutamicum  on glucose. These identified mutational hot spots as well as the two evolved top strains, UBw and UBm, represent promising targets for future metabolic engineering approaches.

Adaptive laboratory evolution of Corynebacterium glutamicum towards higher growth rates on glucose minimal medium