Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae TMB3001 has previously been engineered to utilize xylose by integrating the genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) and overexpressing the native xylulokinase (XK) gene. The resulting strain is able to metabolize xylose, but its xylose utilization rate is low compared to that of natural xylose utilizing yeasts, like Pichia stipitis or Candida shehatae. One difference between S. cerevisiae and the latter species is that these possess specific xylose transporters, while S. cerevisiae takes up xylose via the high-affinity hexose transporters. For this reason, in part, it has been suggested that xylose transport in S. cerevisiae may limit the xylose utilization. We investigated the control exercised by the transport over the specific xylose utilization rate in two recombinant S. cerevisiae strains, one with low XR activity, TMB3001, and one with high XR activity, TMB3260. The strains were grown in aerobic sugar-limited chemostat and the specific xylose uptake rate was modulated by changing the xylose concentration in the feed, which allowed determination of the flux response coefficients. Separate measurements of xylose transport kinetics allowed determination of the elasticity coefficients of transport with respect to extracellular xylose concentration. The flux control coefficient, CtranspJ, for the xylose transport was calculated from the response and elasticity coefficients. The value of CtranspJ for both strains was found to be < 0.1 at extracellular xylose concentrations > 7.5 g L-1. However, for strain TMB3260 the flux control coefficient was higher than 0.5 at xylose concentrations < 0.6 g L-1, while CtranspJ stayed below 0.2 for strain TMB3001 irrespective of xylose concentration. © 2003 Wiley Periodicals, Inc.