<P><B>Abstract</B></P> <P>A transformant of <I>Moorella thermoacetica</I> was constructed for thermophilic ethanol production from lignocellulosic biomass by deleting two phosphotransacetylase genes, <I>pdul</I>1 and <I>pdul</I>2, and introducing the native aldehyde dehydrogenase gene (<I>aldh</I>) controlled by the promoter from glyceraldehyde-3-phosphate dehydrogenase. The transformant showed tolerance to 540mM and fermented sugars including fructose, glucose, galactose and xylose to mainly ethanol. In a mixed-sugar medium of glucose and xylose, all of the sugars were consumed to produce ethanol at the yield of 1.9mol/mol-sugar. The transformant successfully fermented sugars in hydrolysate prepared through the acid hydrolysis of lignocellulose to ethanol, suggesting that this transformant can be used to ferment the sugars in lignocellulosic biomass for ethanol production.</P> <P><B>Highlights</B></P> <P> <UL> <LI> An ethanol-producing transformant of <I>Moorella thermoacetica</I> was constructed. </LI> <LI> The transformant fermented mixed sugars of glucose and xylose simultaneously. </LI> <LI> The transformant fermented hydrolysate of lignocellulose to ethanol. </LI> <LI> The transformant is expected as ethanol producer from lignocellulosic biomass. </LI> </UL> </P>