Alterations in the transcription factors GntR1 and RamA enhance the growth and central metabolism of Corynebacterium glutamicum

Evolution, i.e. the change in heritable characteristics of biological populations over successive generations, has created the diversity of life that exists today. In this study we have harnessed evolution to create faster growing mutants of Corynebacterium glutamicum, i.e. to debottleneck growth rate of this highly important industrial workhorse. After approximately 1500 generations of Adaptive Laboratory Evolution (ALE) in defined minimal medium with glucose, we obtained faster growing mutants with specific growth rate as high as 0.64h-1 as compared with 0.45h-1 for the wild type, and this 42% improvement is the highest reported for C. glutamicum to date. By genome resequencing and inverse metabolic engineering, we were able to pinpoint two mutations contributing to most of the growth improvement, and these resided in the transcriptional regulators GntR1 (gntR1-E70K) and RamA (ramA-A52V). We confirmed that the two mutations lead to alteration rather than elimination of function, and their introduction in the wild-type background resulted in a specific growth rate of 0.62h-1. The glycolytic and pentose phosphate pathway fluxes had both increased significantly, and a transcriptomic analyses supported this to be associated with increased capacity. Interestingly, the observed fast growth phenotype was not restricted to glucose but was also observed on fructose, sucrose and xylose, however, the effect of the mutations could only be seen in minimal medium, and not rich BHI medium, where growth was already fast. We also found that the mutations could improve the performance of resting cells, under oxygen-deprived conditions, where an increase in sugar consumption rate of around 30% could be achieved. In conclusion, we have demonstrated that it is feasible to reprogram C. glutamicum into growing faster and thus enhance its industrial potential.