Saccharomyces cerevisiae is considered one of the most promising organisms for ethanol production from lignocellulosic feedstock. Unfortunately, pentose sugars, which make up to 30% of lignocellulose, cannot be utilized by S. cerevisiae. Pentoses can only enter yeast cells through hexose transporters, which have two orders of magnitude lower affinities for pentose sugars. Additionally, inefficient pentose uptake has been shown to be the limiting step for some D-xylose metabolizing yeast strains.In this thesis,we report the discovery of seven active pentose transporters from pentose assimilating fungal species Pichia stipitis and Neurospora crassa based on sequence homology with the glucose/xylose/H+ symporter (GXS1) in Candida intermedia. These transporters were cloned and heterologously expressed in S. cerevisiae and their sugar uptake activities were studied by analysis of intracellular sugar accumulation using HPLC.Among the seven active transporters, one L-arabinose-specific and two D-xylose-specific transporters were identified. These transporters were functionally expressed and properly localized in S. cerevisiae as indicated by HPLC analysis and immunofluorescence microscopy, respectively. Kinetic parameters of the transporters were determined using a 14C-labeled sugar uptake assay. Sugar uptake assay in un-buffered cell suspension indicated the sugar uptake through these three transporters was not coupled with proton uptake, revealing that these three sugar transporters are facilitators.Introduction of these pentose-specific transporters may facilitate pentose sugar utilization in S. cerevisiae by improving pentose uptake. More efficient utilization of pentose sugars will lower the cost for lignocellulosic ethanol production.
【 预 览 】
附件列表
Files
Size
Format
View
Discovery and characterization of pentose-specific transporters in Saccharomyces cerevisiae
PDF
download
878987797
028-85220240
