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

Abstract

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 0.64 h as compared with 0.45 h 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 0.62 h. 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.

Efficient Production of the Dicarboxylic Acid Glutarate by Corynebacterium glutamicum via a Novel Synthetic Pathway

“…Coupling production to reactions or pathways important for growth bears the potential to select improved strains by laboratory evolution. This strategy has been successful for selecting faster growing C. glutamicum strains ( Pfeifer et al, 2017 ; Radek et al, 2017 ; Wang Z. et al, 2018 ) or strains more tolerant to methanol ( Leßmeier and Wendisch, 2015 ), lignocellulose derived inhibitors ( Wang X. et al, 2018 ) or thermal stress ( Oide et al, 2015 ).…”

Evolutionary engineering of Corynebacterium glutamicum

“…The time needed for biomass production represents a key factor in the economic success of biotechnological processes. While ALE approaches have been intensively explored to accelerate the growth of E. coli , so far, only two recent studies have reported on the evolution of C. glutamicum towards higher growth rates on glucose minimal medium (Table ) . Interestingly, these studies revealed similar key targets for enhancing metabolic flux and increasing substrate uptake rates for both species, indicating that microorganisms follow similar principles to adapt to fast growth.…”

“…In another recent ALE experiment, C. glutamicum cultures were evolved for about 1500 generations in defined media with glucose . The evolved cell line was found to lack large parts of the CGP3 element.…”

“…Besides the lack of the prophage element, the authors found mutations in the gntR1 gene, encoding a repressor of gluconate catabolism and an activator of ptsG , and in ramA , encoding a LuxR‐type regulator, which inherits a global role in the coordination of the central metabolism . Re‐introduction into the wild‐type background revealed that both mutations significantly increased the growth rate and glucose consumption rate (Table ) . Metabolomic flux and transcriptome analysis showed an increased flux through the pentose phosphate pathway but did not indicate significant rewiring of the central metabolic network, which is in agreement with similar studies on E. coli…”

“…Overview of different objectives in C. glutamicum ALE studies. ALE has successfully been applied to increase the growth rate, substrate utilization, stress tolerance, and small molecule production . Growth coupling strategies have been used to increase precursor supply and small molecule production …”