As the need for fuels increases, value of ethanol as an alternative fuel for automobiles has recently received much attention. One of the key steps in improving ethanol production is to efficiently convert hemicellulosic biomass to ethanol. However, none of the wild-type microbial organisms currently known, can produce ethanol as a homo-fermentation product by fermenting all sugars present in hemicellulosic biomass. In this study, a non-recombinant E.coli, strain SE2378, was obtained that can convert all the monomeric sugars present in hemicellulose to ethanol with higher than 80% yield. Strain SE2378 was derived from strain AH242 which lacks both lactate dehydrogenase (ldhA) and pyruvate-formate lyase (pflB). Strain AH242 shows growth deficiency under anaerobic conditions without external electron acceptors due to the inability to reoxidize NADH generated during glycolysis. Strain SE2378 was isolated under anaerobic conditions after strain AH242 was mutagenized. Strain SE2378 produced ethanol as a major fermentation product at a yield of 85% (mol/mol) from glucose and 80 % (mol/mol) from xylose. One mutation that is responsible for anaerobic growth of strain SE2378 and ethanol production was mapped in lpdA encoding dihydrolipoamide dehydrogenase (LPD) that is a component of pyruvate dehydrogenase complex (PDHc). Unlike native LPD known to be highly sensitive to high NADH/NAD ratio, the mutated LPD is insensitive to higher NADH/NAD ratio. Therefore, the mutated PDHc is active under anaerobic conditions. Apparently, mutated PDHc supports the conversion of pyruvate to acetyl-CoA under anaerobic condition and the resultant acetyl-CoA is reduced to ethanol by alcohol dehydrogenase encoded by adhE.