Discovery and characterization of novel d-xylose-specific transporters from Neurospora crassa and Pichia stipitis

Abstract

Saccharomyces cerevisiae is considered one of the most promising organisms for ethanol production from lignocellulosic feedstock. Unfortunately, pentose sugars, which comprise up to 30% of lignocellulose, cannot be utilized by wild type S. cerevisiae. Heterologous pathways were introduced into S. cerevisiae to enable utilization of D-xylose, the most abundant pentose sugar. However, the resulting recombinant S. cerevisiae strains exhibited a slow growth rate and poor sugar utilization efficiency when grown on D-xylose as the sole carbon source. D-xylose uptake is the first step of D-xylose utilization. D-xylose can only enter yeast cells through hexose transporters, which have two orders of magnitude lower affinity towards D-xylose compared to hexoses. It was also shown that inefficient pentose uptake is the limiting step in some D-xylose metabolizing yeast strains. Here we report the cloning and characterization of two novel D-xylose-specific transporters from Neurospora crassa and Pichia stipitis. These two transporters were identified from a total of 18 putative pentose transporters. They were functionally expressed and properly localized in S. cerevisiae as indicated by HPLC analysis and fluorescence confocal microscopy, respectively. Kinetic parameters of the D-xylose-specific transporters were determined using a ¹⁴C-labeled sugar uptake assay. Use of pentose-specific transporters should improve D-xylose consumption and ethanol production in fast D-xylose assimilating strains, thereby lowering the cost of lignocellulosic ethanol production.