The methylotrophic yeast Hansenula polymorpha has the potential to be used in the process of simultaneous saccharification and fermentation (SSF) of xylan derived xylose at elevated temperatures. To improve parameters of high-temperature resistance and high-temperature fermentation of H. polymorpha, strains carrying deletion of acid trehalase gene (ATH1) and overexpressing genes coding for heat-shock proteins Hsp16p and Hsp104p were constructed. Results indicate that the corresponding recombinant strains have up to 12-fold increased tolerance to heat-shock treatment. The deletion of ATH1 gene and constitutive expression of HSP16 and HSP104 resulted in up to 5.8-fold improvement of ethanol production from xylose at 50 degrees C. Although the maximum ethanol concentration achieved from xylose was 0.9 gL(-1), our model H. polymorpha strains with elevated thermotolerance can be further modified by metabolic engineering to construct improved high-temperature ethanol producers from this pentose. Biotechnol. Bioeng. 2009;104: 911-919. (C) 2009 Wiley Periodicals, Inc.