Abstract
BackgroundBaker's yeast (Saccharomyces cerevisiae) has been engineered for xylose utilization to enable production of fuel ethanol from lignocellulose raw material. One unresolved challenge is that S. cerevisiae lacks a dedicated transport system for pentose sugars, which means that xylose is transported by non-specific Hxt transporters with comparatively low transport rate and affinity for xylose.ResultsIn this study, we compared three heterologous xylose transporters that have recently been shown to improve xylose uptake under different experimental conditions. The transporters Gxf1, Sut1 and At5g59250 from Candida intermedia, Pichia stipitis and Arabidopsis thaliana, respectively, were expressed in isogenic strains of S. cerevisiae and the transport kinetics and utilization of xylose was evaluated. Expression of the Gxf1 and Sut1 transporters led to significantly increased affinity and transport rates of xylose. In batch cultivation at 4 g/L xylose concentration, improved transport kinetics led to a corresponding increase in xylose utilization, whereas no correlation could be demonstrated at xylose concentrations greater than 15 g/L. The relative contribution of native sugar transporters to the overall xylose transport capacity was also estimated during growth on glucose and xylose.ConclusionsKinetic characterization and aerobic batch cultivation of strains expressing the Gxf1, Sut1 and At5g59250 transporters showed a direct relationship between transport kinetics and xylose growth. The Gxf1 transporter had the highest transport capacity and the highest xylose growth rate, followed by the Sut1 transporter. The range in which transport controlled the growth rate was determined to between 0 and 15 g/L xylose. The role of catabolite repression in regulation of native transporters was also confirmed by the observation that xylose transport by native S. cerevisiae transporters increased significantly during cultivation in xylose and at low glucose concentration.
Highlights
Baker’s yeast (Saccharomyces cerevisiae) has been engineered for xylose utilization to enable production of fuel ethanol from lignocellulose raw material
Xylose transport kinetics Xylose transport kinetics were determined in the xyloseutilizing S. cerevisiae strain TMB 3662 [19] expressing the heterologous transporters Gxf1, Sut1 and At5g59250 from C. intermedia, P. stipitis and A. thaliana, respectively
Cells were grown in 20 g/L glucose for two cell divisions, after which xylose transport was assayed as initial rate of D-[U-14C] xylose uptake
Summary
Baker’s yeast (Saccharomyces cerevisiae) has been engineered for xylose utilization to enable production of fuel ethanol from lignocellulose raw material. One unresolved challenge is that S. cerevisiae lacks a dedicated transport system for pentose sugars, which means that xylose is transported by non-specific Hxt transporters with comparatively low transport rate and affinity for xylose. Baker’s yeast (Saccharomyces cerevisiae) is used for industrial production of fuel ethanol from starch (corn) and sucrose (sugar cane). In recombinant xylose-utilizing S. cerevisiae, xylose is transported by non-specific hexose transporters with poor affinity for xylose [6,7]. The first constructed xylose-utilizing S. cerevisiae strains were not constrained by transport because of their slow xylose catabolism [10], whereas current recombinant S. cerevisiae strains are transport-constrained, at least at low substrate concentrations [9]. There have been recent reports of improved transport and utilization of xylose in S. cerevisiae by independent expression of the heterologous xylose transporters Gxf1 [9], Sut1 [11] and
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