To enhance ethanol production in Thermoanaerobacterium aotearoense, the lactate dehydrogenase (10) gene, which is responsible for lactic acid production in a key branch pathway, was successfully disrupted via homologous recombination. ldh-up and ldh-down were designed and amplified based on JW/SL-YS485-AY 278026, and they were subsequently used as homologous fragments with an inserted erythromycin resistance gene to construct the targeted vector based on pBLUESCRIPT II SK(+). Southern hybridization and PCR-based assay definitely confirmed that the ldh gene in the Delta ldh mutant was disrupted by the insertion of the erythromycin resistance gene. Compared with the wild type, the Delta ldh mutant exhibited increases of 31.0% and 31.4% in cell yield under glucose and xylose cultivation, respectively, probably because knocking out the ldh gene results in increased acetate and ATP levels. Knockout of lactate dehydrogenase produced 2.37- and 2.1-fold increases in the yield of ethanol (mole/mole substrate) under glucose and xylose cultivation, respectively. Moreover, no lactic acid was detected in Delta ldh mutant fermentation mixtures (detection limit of HPLC: 0.5 mM), but lactic acid was readily detected for growth of the wild-type strain on both glucose and xylose, with final concentrations up to 59.24 mM and 56.06 mM, respectively. The success of this process thoroughly demonstrates the methodological possibility of gene knockout through homologous recombination in Thermoanaerobacterium. (C) 2010 Elsevier Inc. All rights reserved.