Comparison of heterologous xylose transporters in recombinant Saccharomyces cerevisiae

Abstract

Background: 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.

Results: In 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.

Conclusions: Kinetic 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.

Figure 1

Xylose uptake rates of S. cerevisiae strains expressing the Gxf1, Sut1 and At5g59250 transporters determined using D-[U¹⁴C] labeled sugar. Cells were grown on 20 g/L glucose. Lines represent the calculated fit according to Michaelis-Menten kinetics. Gxf1, square; Sut1, upwards pointing triangle; At5g59250, diamond; control, downwards pointing triangle. Biological replicates are designated by empty and filled version of the same symbol.

Figure 2

Aerobic batch cultivation xylose 4 g/L of recombinant S. cerevisiae strains expressing the Gxf1, Sut1 and At5g59250 transporters. Gxf1, square; Sut1, upwards pointing triangle; At5g59250, diamond; Control, downwards pointing triangle. Deviations between biological replicates are indicated by error bars.

Figure 3

Xylose uptake rates of S. cerevisiae grown under different conditions. Lines represent the calculated fit according to Michaelis-Menten kinetics. 20 g/L Glucose, square; 0.5 g/L glucose, circle; 60 g/L xylose, diamond. Biological replicates are designated by empty and filled version of the same symbol.