Intracellular fluxes in a recombinant xylose-utilizingSaccharomyces cerevisiae cultivated anaerobically at different dilution rates and feed concentrations

Abstract

A metabolic flux model was constructed for the yeast Saccharomyces cerevisiae comprising the most important reactions during anaerobic metabolism of xylose and glucose. The model was used to calculate the intracellular fluxes in a recombinant, xylose‐utilizing strain of S. cerevisiae (TMB 3001) grown anaerobically in a defined medium at dilution rates of 0.03, 0.06, and 0.18 h−1. The feed concentration was varied from 0 g/L xylose and 20 g/L glucose to a mixture of 15 g/L xylose and 5 g/L glucose, so that the total concentration of carbon source was kept at 20 g/L. The specific uptake of xylose increased with the xylose concentration in the feed and with increasing dilution rate. The excreted xylitol was less than half of the xylose consumed. With increasing xylose concentration in the feed, the fluxes in the pentose phosphate pathway increased, whereas the flux through glycolysis decreased. Under all cultivation conditions, nicotinamide adenine dinucleotide (NADH) was the preferred cofactor for xylose reductase. The model showed that the flux through the reaction from ribulose 5‐phosphate to xylulose 5‐phosphate was very low under all cultivation conditions. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 289–296, 2001.

Metabolic flux analysis of xylose metabolism in recombinant Saccharomyces cerevisiae using continuous culture

“…Considering the differences in the substrate levels the results are very similar between the two studies, which demonstrates how reproducible the chemostat studies, and even these simple flux models, can be. The models used in this work and in the studies reported by Wahlbom and co-workers (Wahlbom et al, 2001;Wahlbom and Hahn-Ha¨gerdal, 2002) are very similar. Both models were derived from the work done by Nissen and co-workers, and van Gulik and Heijnen (Nissen et al, 1997;van Gulik and Heijnen, 1995).…”

“…Thus also under anaerobic conditions some carbon is lost as carbon dioxide, the carbon which will never reach ethanol and the production of which would generate NAD + required for the XDH reaction. Also Wahlbom (Wahlbom et al, 2001) have demonstrated the xylose metabolism under anaerobic conditions in a similar metabolic flux analysis study. Their results on 20 g/L glucose and 15:5 g/L xylose-glucose mixture agree well with our results on 10 g/L glucose and 27:3 g/L xylose-glucose mixture under anaerobic conditions.…”

“…This is not necessarily a good assumption, but in the case where the actual cytosolic ratio between intracellular NADPH and NADH is not known, it is not safe to assume that the in vitro determined cofactor specificity of the P. stipitis XR would be valid either. Furthermore, the ratio between NADPH-and NADHspecific XR-reaction cannot be let to settle freely by using two separate reactions in the model (Wahlbom et al, 2001), since this favors the NADH-specific reaction. However, the effect of changing the ratio of NADPH and NADH specificity was examined.…”

“…Metabolic flux analysis has been applied extensively, including the quantification of metabolic fluxes in glucose-consuming yeast (Nissen et al, 1997;van Gulik and Heijnen, 1995), and more recently also for natural xylose-utilizing yeast (Granstro¨m et al, 2000) as well as genetically engineered S. cerevisiae (Wahlbom et al, 2001;Wahlbom and Hahn-Ha¨gerdal, 2002). Metabolic flux analysis has also been used to calculate theoretical yields for ethanol production from xylose-utilizing S. cerevisiae (Lee et al, 2001).…”

Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae

“…The specific xylose utilization rate of TMB3001 in aerobic sugar-limited chemostat was strongly dependent on the extracellular xylose concentration, as has been observed earlier in anaerobic sugar-limited chemostat cultures (Eliasson et al, 2000;Wahlbom et al, 2001). Based on the sensitivity of the xylose flux to changes in the extracellular xylose concentration, i.e., the large R J X ec , it has been speculated that transport might have large control over the xylose utilization rate (Eliasson et al, 2000;Wahlbom, 2002).…”