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 lysine-overproducing 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.

FIG. 1.

Effect of medium osmolality on intracellular content at different dilution rates. The graphs show the intracellular accumulation of trehalose (A) and proline (B) at 0.09 h⁻¹ (circles), 0.13 h⁻¹ (squares), 0.17 h⁻¹ (triangles), and 0.21 h⁻¹ (diamonds).

FIG. 2.

Map of flux distributions at 0.09 h⁻¹ (black numbers) and 0.21 h⁻¹ (white numbers in black columns) at osmolalities of 280, 1,000, and 1,800 mosmol kg⁻¹ (from top to bottom). The fluxes are expressed in millimoles per gDCW per hour and are normalized with regard to biomass formation. Abbreviations: TRE, trehalose; RIBU5P, ribulose-5-phosphate; F6P, fructose-6-phosphate; XYL5P, xylulose-5-phosphate; XYL6P, xylulose-6-phosphate; E4P, erythrose-4-phosphate; RIB5P, ribose-5-phosphate; SED7P, sedoheptulose-7-phosphate; GAP, glyceraldehyde-3-phosphate; G3P, glyceraldehyde-6-phosphate; ACCOA, acetyl-CoA; ISOCIT, isocitrate; AKG, α-ketoglutarate; SUCCOA, succinyl-CoA; IN, intracellular metabolite; EX, extracellular metabolite.

FIG. 3.

Effect of rises in medium osmolality on the ratios between the main exit branches of the principal nodes at different dilution rates. (A) Glycolysis (EMP)/pentose phosphate pathway (PPP) ratio (G6P node). (B) Tricarboxylic acid cycle (TCA)/aspartate synthesis (ASP) ratio (OAA node). The graphs show data for 0.09 h⁻¹ (circles), 0.13 h⁻¹ (squares), 0.17 h⁻¹ (triangles), and 0.21 h⁻¹ (diamonds).

FIG. 4.

Effect of medium osmolality on cell maintenance. The bars indicate the specific maintenance coefficients for cellular integrity (m_s [black]) and for cellular productivity (m_sp [white]).

FIG. 5.

Effect of dilution rate on the amount of carbon flux employed for maintenance at the following osmolalities: 280 (circles), 700 (squares), 1,200 (triangles), and 1,800 (diamonds) mosmol kg⁻¹.