Metabolomic and (13)C-metabolic flux analysis of a xylose-consuming Saccharomyces cerevisiae strain expressing xylose isomerase

Abstract

Over the past two decades, significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative Pentose Phosphate Pathway (PPP) is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis.

Keywords: 13C-metabolic flux analysis; Saccharomyces cerevisiae; carbon catabolite repression; cellulosic ethanol; metabolomics; xylose.

Fig. 1

Metabolite pool sizes for metabolites in Glycolysis and the PPP. All pool sizes are in units of µmol/g DCW. Error bars represent uncertainties from standard deviations of three biological replicates as well as uncertainties in OD₆₀₀-DCW correlations. GA = Glucose Aerobic, GN = Glucose Anaerobic, XA = Xylose Aerobic, XN = Xylose Anaerobic

Fig. 2

Metabolite pool sizes for metabolites in the TCA Cycle. All pool sizes are in units of µmol/g DCW. Error bars represent uncertainties from standard deviations of three biological replicates as well as uncertainties in OD₆₀₀-DCW correlations. GA = Glucose Aerobic, GN = Glucose Anaerobic, XA = Xylose Aerobic, XN = Xylose Anaerobic

Fig. 3

Metabolite pool sizes and ratios for select cofactors. All pool sizes are in units of µmol/g DCW. Pool size error bars represent uncertainties from standard deviations of three biological replicates as well as uncertainties in OD₆₀₀-DCW correlations. Metabolite ratio error bars represent uncertainties from standard deviations of three biological replicates only. GA = Glucose Aerobic, GN = Glucose Anaerobic, XA = Xylose Aerobic, XN = Xylose Anaerobic

Fig. 4

Best-fit metabolic flux distributions in xylose-consuming S. cerevisiae strain under four sets of conditions. All fluxes are given in units of mmol/g/h. For each reversible reaction the net flux is given with the exchange flux indicated inside parentheses; “nr” indicates the exchange flux could not be resolved to within one order of magnitude. Specific growth rates (shown in the bottom right of the Figure) are in units of h⁻¹. Four values are listed for each flux. These are the best-fit values for the Glucose Aerobic (top left), Glucose Anaerobic (bottom left), Xylose Aerobic (top right), and Xylose Anaerobic (bottom right) cultures

Fig. 5

Weighted Sums of Squared Residuals from flux estimations with different models. “Best-fit” = the final metabolic model used for flux estimation, “Glx shunt” = the final model with addition of the glyoxylate shunt pathway, “w/o FBPase” = the final model with removal of the gluconeogenic enzyme Fructose-1,6-Bisphosphatase, “unidirectional OAA transport” = the final model with removal of OAA transport from mitochondria to cytosol (i.e. via Malate-Aspartate or Malate-Oxaloacetate Shuttle), so that OAA can only be transported from cytosol to mitochondria. Error bars for each “Best-fit” model show the expected range for the weighted sum of squared residuals in the chi-square goodness-of-fit test. GA = Glucose Aerobic, GN = Glucose Anaerobic, XA = Xylose Aerobic, XN = Xylose Anaerobic

Fig. 6

Confidence intervals for select fluxes. All fluxes are in units of mmol/g/h. The line inside each box represents the best-fit value of the flux. Each box represents the flux 68% confidence interval and the error bars (“whiskers”) represent the flux 95% confidence interval. GA = Glucose Aerobic, GN = Glucose Anaerobic, XA = Xylose Aerobic, XN = Xylose Anaerobic. Enzyme abbreviations: PGI, Phosphoglucose isomerase; KGD, α-Ketoglutarate dehydrogenase; SDH, Succinate dehydrogenase; TPI, Triosephosphate isomerase