Metabolic impact of increased NADH availability in Saccharomyces cerevisiae

Abstract

Engineering the level of metabolic cofactors to manipulate metabolic flux is emerging as an attractive strategy for bioprocess applications. We present the metabolic consequences of increasing NADH in the cytosol and the mitochondria of Saccharomyces cerevisiae. In a strain that was disabled in formate metabolism, we either overexpressed the native NAD(+)-dependent formate dehydrogenase in the cytosol or directed it into the mitochondria by fusing it with the mitochondrial signal sequence encoded by the CYB2 gene. Upon exposure to formate, the mutant strains readily consumed formate and induced fermentative metabolism even under conditions of glucose derepression. Cytosolic overexpression of formate dehydrogenase resulted in the production of glycerol, while when this enzyme was directed into the mitochondria, we observed glycerol and ethanol production. Clearly, these results point toward different patterns of compartmental regulation of redox homeostasis. When pulsed with formate, S. cerevisiae cells growing in a steady state on glucose immediately consumed formate. However, formate consumption ceased after 20 min. Our analysis revealed that metabolites at key branch points of metabolic pathways were affected the most by the genetic perturbations and that the intracellular concentrations of sugar phosphates were specifically affected by time. In conclusion, the results have implications for the design of metabolic networks in yeast for industrial applications.

FIG. 1.

Confirmation of the localization of GFP-tagged formate dehydrogenase fused with the mitochondrial signal sequence encoded by the CYB2 gene. The corresponding strain was designated mFDH-GFP. As a control, the cytosolic localization of unmodified GFP is also shown. Overlaying of the bright-field image with that of GFP clearly indicates the localization of the Cyb2-Fdh1-GFP construct in the mitochondria.

FIG. 2.

Metabolic changes to formate supplementation. (A) Steady-state biomass yields as a function of the molar ratio of formate to glucose (F:G) in the feed medium. (B) Specific glucose uptake rates (r_G) as a function of the specific formate consumption rates (r_F) for F-REF (circles), cFDH (squares), and mFDH (triangles). The rates of consumption of glucose and formate were calculated from the data obtained from the chemostat cultures. gDW, grams (dry weight).

FIG. 3.

Specific rates of ethanol production (r_eth) (A) and specific rates of glycerol production (r_gly) (B) for F-REF (circles), cFDH (squares), and mFDH (triangles) at different concentrations of formate in the inlet feed in glucose-limited chemostat cultures. The carbon content in the inlet feed is shown as the ratio of formate to glucose (F:G).

FIG. 4.

Specific rates of CO₂ evolution (r_CO2) (A) and RQs (B) for F-REF (circles), cFDH (squares), and mFDH (triangles) at different concentrations of formate in the inlet feed in glucose-limited chemostat cultures. The carbon content in the inlet feed is shown as the ratio of formate to glucose (F:G).

FIG. 5.

Consumption of formate by F-REF (circles), cFDH (squares), and mFDH (triangles) after pulsing of steady-state, glucose-limited cultures with formate. The inset shows the raw data on the residual concentration of formate. The specific formate uptake rate was calculated from the raw data.

FIG. 6.

(A) Hierarchical clustering of the strains and the 23 intracellular metabolites that exhibited significantly different profiles (P < 0.05) as determined by ANOVA. Also shown are the time points (in minutes) at which the different samples were quantified. Numbers at the bottom are amounts of change (n-fold). AKG, α-ketoglutarate; acetyl-CoA, acetyl coenzyme A; F6P, fructose-6-phosphate; G6P, glucose-6-phosphate; cAco, cis-aconitate; E4P, erythrose-4-phosphate; 2-/3-PG, 2-phosphoglycerate/3-phosphoglycerate; PEP, phosphoenolpyruvate; S7P, sedoheptulose-7-phosphate; R5P, ribose-5-phosphate; 6PG, 6-phosphogluconate; Ru-/Xy5P, ribulose-5-phosphate/xylulose-5-phosphate. (B) The data presented in panel A were used to illustrate the effect of time using PC analysis for F-REF (data points clustered together), cFDH (dark gray data points), and mFDH (light gray data points). The inset shows the amount of variance that is explained by the different PCs. The data are projected only on the first two PCs, which captured 83% of the variance.

FIG. 7.

Variation in the NADH/NAD⁺ (A), NADPH/NADP⁺ (B), and ATP/ADP (C) ratios in F-REF (open circles), cFDH (squares), and mFDH (triangles) after pulsing of steady-state, glucose-limited cultures with formate.