Bioconversion of plant derived lignocellulosic materials into biofuels has drawn increasing attention as an attractive strategy for replacement of fossil fuels. Saccharomyces cerevisiae, also known as baker's yeast, is considered one of the most promising organisms for ethanol production from lignocellulosic feedstock. Unfortunately, pentose sugars, which make up to 30% of biomass hydrolysate, cannot be utilized by S. cerevisiae. Heterologous pentose utilization pathways from bacterial and fungal resources have been introduced into S. cerevisiae to enable the assimilation of pentose sugars. However, pentose utilization of recombinant S. cerevisiae strains is inefficient due to several limitations such as the low uptake rate of pentose sugars and suboptimal heterologous pentose utilization pathways. 1. Cloning and characterization of pentose specific transporters Pentoses can only enter yeast cells through hexose transporters, which have two orders of magnitude lower affinity towards pentoses than towards glucose. It has also been shown that inefficient pentose uptake is the limiting step in some xylose metabolizing yeast strains. Here we report the cloning and characterization of one arabinose-specific and two xylose-specific transporters from various pentose-utilizing species. These 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 immunofluorescence microscopy, respectively. Introduction of these pentose-specific transporters should 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. 2. Combinatorial optimization of pentose utilization pathways in Saccharomyces cerevisiae A lot of research has been carried out to overcome the limitations in pentose utilization in S. cerevisiae. However, it is challenging to come up with a single strategy which can simultaneously address all major limitations. Here we report the combinatorial optimization of a highly efficient pentose utilization pathway in S. cerevisiae. Specifically, we chose the fungal three-step xylose utilization pathway as our target pathway, and performed optimization of the pathway through shuffling of pathway enzymes or promoters. For the protein library based optimization, 10 to 20 different enzyme homologues from various fungal species with different catalytic efficiency and cofactor preference have been cloned for each pathway enzyme, and were assembled into a library of xylose utilizing pathways using our recently developed DNA assembler method. For the promoter library based optimization, promoter mutants with varied strength were created by error-prone PCR and randomly assembled into the pathway to generate a library of xylose utilizing pathways. The resultant libraries exhibited a high efficiency for correct assembly of the complete multi-gene pathway and good diversity of different homologues within the same catalytic step. Using the libraries generated, clones with better combination of enzyme homologues can be selected by faster cell growth. This method can also be applied to directly search for the pathway with a better fit in industrial yeast strains, which may have different metabolic flux patterns compared to the laboratory S. cerevisiae strains.