Comparative 13C metabolic flux analysis of pyruvate dehydrogenase complex-deficient, L-valine-producing Corynebacterium glutamicum

Abstract

L-Valine can be formed successfully using C. glutamicum strains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-type C. glutamicum and four PDHC-deficient strains were compared by (13)C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP). Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand of NADPH for l-valine formation. In accordance, the introduction of the Escherichia coli transhydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into an L-valine-producing C. glutamicum strain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover, as demonstrated for L-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation via the PPP.

Fig. 1.

Selected part of the central metabolism in C. glutamicum. The l-valine pathway is marked in yellow. The enzymes inactivated or overexpressed in some of the analyzed strains are shown in green. Heterologous transhydrogenase genes (blue) are expressed in the strain C. glutamicum Δace Δpqo(pJC4ilvBNCE)(pBB1pntAB). Abbreviations: EMP, glycolysis; PPP, pentose phosphate pathway; TCA, tricarboxylic acid cycle; PTS, phosphotransferase system; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PK, pyruvate kinase; PDHC, pyruvate dehydrogenase complex; PQO, pyruvate-quinone oxidoreductase; AK, acetate kinase; PTA, phosphotransacetylase; CS, citrate synthase; PEPCx, phosphoenolpyruvate carboxylase; PEPCk, phosphoenolpyruvate carboxykinase; PCx, pyruvate carboxylase; ODx, oxaloacetate decarboxylase; ME, malic enzyme; MQO, malate-quinone oxidoreductase; MDH, malate dehydrogenase; AHAS, acetohydroxyacid synthase; AHAIR, acetohydroxyacid isomeroreductase; DHAD, dihydroxyacid dehydratase; TA, valine transaminase; THD, transhydrogenase.

Fig. 2.

Cultivation profiles of analyzed C. glutamicum strains. The arrow shows when labeled glucose was added; the dotted red line shows the time of sampling. Strains were C. glutamicum ATCC 13032 (wild type) (a), C. glutamicum ΔaceE (b), C. glutamicum ΔaceE(pJC4ilvBNCE) (c), C. glutamicum ΔaceE Δpqo(pJC4ilvBNCE) (d), and C. glutamicum ΔaceE Δpqo(pJC4ilvBNCE)(pBB1pntAB) (e). Symbols: optical density (green diamonds), glucose (blue circles), l-valine (orange squares), l-alanine (gray hexagons), ketoisovalerate (purple triangles), acetate (light blue inverted triangles) (only PDHC-deficient strains).

Fig. 3.

Quantification of intracellular fluxes in wild-type C. glutamicum and different l-valine production strains. (a to e) Estimated flux distributions. Specific values denote the molar percentage of glucose uptake. (f) Absolute flux values for NADPH-related reactions.