Xylose and glucose from lignocellulose are sustainable sources for production of pyruvate, which is the starting material for the synthesis of many drugs and agrochemicals. In this study, the pyruvate decarboxylase gene (KmPDC1) and glycerol-3-phosphate dehydrogenase gene (KmGPD1) of Kluyveromyces marxianus YZJ051 were disrupted to prevent ethanol and glycerol accumulation. The deficient growth of PDC disruption was rescued by overexpressing mutant KmMTH1-Delta T. Then pentose phosphate pathway and xylitol dehydrogenase SsXYL2-ARS genes were overexpressed to obtain strain YZB053 which produced pyruvate with xylose other than glucose. It produced 24.62 g/L pyruvate from 80 g/L xylose with productivity of 0.51 g/L/ h at 42 degrees C. Then, xylose-specific transporter ScGAL2-N376F was overexpressed to obtain strain YZB058, which simultaneously consumed 40g/L glucose and 20 g/L xylose and produced 29.21 g/L pyruvate with productivity of 0.81 g/L/h at 42 degrees C. Therefore, a platform for pyruvate production from glucose and xylose at elevated temperature was developed. (C) 2016 Elsevier Ltd. All rights reserved.