Energy security, environmental pollution, and economic development drive the development of alternatives to fossil fuels as an urgent global priority. Lignocellulosic biomass has the potential to contribute to meeting the demand for biofuel production via hydrolysis and fermentation of released sugars, such as glucose, xylose, and arabinose. Construction of robust cell factories requires introducing and rewiring of their metabolism to efficiently use all these sugars. Here, we review recent advances in re-constructing pathways for metabolism of pentoses, with special focus on xylose metabolism in the most widely used cell factories Saccharomyces cerevisiae and Escherichia coli. We also highlight engineering advanced biofuels-synthesis pathways and describes progress toward overcoming the challenges facing adoption of large-scale biofuel production.