Osmotic Stress Response: Quantification of Cell Maintenance and Metabolic Fluxes in a Lysine-Overproducing Strain of Corynebacterium glutamicum

Abstract

Osmotic stress diminishes cell productivity and may cause cell inactivation in industrial fermentations. The quantification of metabolic changes under such conditions is fundamental for understanding and describing microbial behavior during bioprocesses. We quantified the gradual changes that take place when a lysineoverproducing strain of Corynebacterium glutamicum is grown in continuous culture with saline gradients at different dilution rates. The use of compatible solutes depended on environmental conditions; certain osmolites predominated at different dilution rates and extracellular osmolalities. A metabolic flux analysis showed that at high dilution rates C. glutamicum redistributed its metabolic fluxes, favoring energy formation over growth. At low dilution rates, cell metabolism accelerated as the osmolality was steadily increased. Flexibility in the oxaloacetate node proved to be key for the energetic redistribution that occurred when cells were grown at high dilution rates. Substrate and ATP maintenance coefficients increased 30-and 5-fold, respectively, when the osmolality increased, which demonstrates that energy pool management is fundamental for sustaining viability.In industrial fermentations, increases in osmotic pressure, mostly a direct result of product accumulation, disrupt bacterial growth and the production of desirable compounds and may inactivate cells in many bioprocesses (26).One strategy that microbes adopt to counteract higher osmotic pressure and to improve the likelihood of surviving such conditions is making use of organic intracellular solutes. Compatible solutes enable the cell to achieve osmotic balance without altering the cell's metabolic functions (35). The regulation of intracellular solute concentrations in response to the salinity of the medium provides the cell significant scope for adapting to changes in osmolality in the cell's environment (19).Aside from altering compatible solute concentrations, the higher energy requirements for cell maintenance under osmotic stress also disrupt energy pool management (18). "Maintenance" covers every cellular reaction involving the consumption of ATP that does not contribute to the net synthesis of biomass. Such reactions include the need to build up and maintain steep ion concentration gradients across the membrane and macromolecular turnover (27).Microbial growth and cellular productivity are driven by the flux balance generated between anabolic and catabolic reactions, as a higher osmotic pressure provokes numerous changes in the cell's metabolism. These fluxes vary considerably in response to changing environmental conditions (4, 34). Hence, a means for evaluating osmo-induced changes is necessary for the assessment and quantification of significant shifts within the metabolic network. Metabolic flux analysis is a powerful tool for quantifying such changes (22,(31)(32)(33).Corynebacterium glutamicum is particularly appropriate for studying responses to osmotic stress, as this microorganism adapts efficiently to changes in osmoti...

Phenotypic characterization of Corynebacterium glutamicum under osmotic stress conditions using elementary mode analysis

“…On the other hand, OaA node exhibited relative inflexibility as evident from no substantial change in the flux partitioning. The last observation is in agreement with the results reported by Varela et al [32], who reported that OaA shows rigidity up to 1,000 mosmol/kg [32,33].…”

“…Varela et al [32] for the first time quantified metabolic response in terms of metabolic flux redistribution in C. glutamicum grown under discrete and gradient continuous culture to gradually increasing osmolarity (between 270 and 1,880 mosmol/kg). Varela et al [33] also estimated the alteration in the maintenance coefficients of substrate and ATP under osmotic stress conditions using flux balance analysis. In the current study, we aim to quantify the metabolic fluxes across the network using the elementary mode approach.…”

Role of Central Metabolism in the Osmoadaptation of the Halophilic Bacterium Chromohalobacter salexigens

“…Therefore, the excess glucose is diverted toward overflow metabolites. The high ratio of anaplerotic to catabolic fluxes is a striking fact, especially if we consider that thriving under osmotic stress is usually energetically expensive, especially in microorganisms that use the organicsolutes-in strategy (60,61).…”

Physiological response of Corynebacterium glutamicum to oxidative stress induced by deletion of the transcriptional repressor McbR

“…This included Y X/ATP 510.5 g mol 21 (de Hollander, 1994). For the P : O ratio, earlier estimates range between 1.5 and 2.0, so that here a mean value of 1.75 was assumed (de Hollander, 1994;Varela et al, 2003Varela et al, , 2004.…”