Abstract
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 Mg2+ 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 Mg2+ 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.
Highlights
In recent years, adaptive laboratory evolution experiments (ALE) in combination with next-generation sequencing (NGS) became a key approach to study microbial adaptation in fundamental as well as in applied research[1,2,3,4]
Our results revealed no significant differences between the two strains in terms of mutation frequency and genomic stability and even emphasized a positive trend of MB001 to evolve to higher growth rates under the chosen conditions
We reported on the construction of a prophage-free variant of C. glutamicum strain ATCC 13032 named MB001 displaying several positive features for metabolic engineering[19]
Summary
Adaptive laboratory evolution experiments (ALE) in combination with next-generation sequencing (NGS) became a key approach to study microbial adaptation in fundamental as well as in applied research[1,2,3,4]. The activity of specialized mobile elements[16], in particular, transposable phages, genomic islands and cryptic prophages may have a considerable influence on the stability of bacterial genomes by causing genomic rearrangements e.g. integrations, deletions, disruptions or inversions[16,17,18] These elements represent prime candidates for removal in several recent genome reduction projects aiming at the construction of stable and predictable chassis strains[19,20]. We compared C. glutamicum ATCC 13032 wild type strain and its prophage-free variant MB001 in a long-term evolution experiment on glucose minimal medium. For both strains, a fast adaptation resulting in about ~20% increased growth rates was observed within the first ~100 generations. Our results revealed no significant differences between the two strains in terms of mutation frequency and genomic stability and even emphasized a positive trend of MB001 to evolve to higher growth rates under the chosen conditions
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