Global transcription machinery engineering (gTME) is an approach for reprogramming gene transcription to elicit cellular phenotypes important for technological applications. In our study, the application of gTME to Saccharomyces cerevisiae was to improve xylose utilization and tolerance, which is a key trait for many biofuel programs. Mutation of the transcription factor spt15 was introduced by error-prone polymerase chain reaction and then screened on media using xylose as the sole carbon source. The selected out strain spt15-25 showed modest growth rates in the media containing 50, 100, and 150 g/L of xylose or glucose. Under the following fermentation condition: 30 A degrees C, rotating speed of 200 r/min, 500-mL Erlenmeyer flask containing 100-mL media, after 109 h, 93.5% of xylose was consumed in 50 g/L xylose medium. Meanwhile, 98.3% glucose can be metabolized in 50-g/L glucose medium. And the carbon source was 50 g/L glucose-xylose (w/w = 1); the utilization ratio of xylose and glucose was 90.8% and 97.3%, respectively. And all the xylitol concentration was below 2.48 g/L.