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A high-efficiency optically pure L-lactic acid producing strain was engineered from a thermophilic anaerobic bacterium, Thermoanaerobacterium aotearoense SCUT27 isolated by us, which is able to efficiently utilize sugar mixtures derived from lignocellulosic biomass in the absence of other microorganisms or enzymes under non-sterile fermentation conditions.This mutant strain, designated as △pta-ack, was constructed by chromosomal inactivation of genes encoding acetate kinase (ack) and phosphate acetyltransferase (pta) to eliminate the competing acetic acid pathway.During the batch fermentations using glucose and xylose as sole carbon source, the substrate conversions of the mutant strain were increased by 2.0 and 2.5 folds compared with the wild type strain, respectively.The largest yields of lactic acid are up to 0.98 g/g glucose and 0.93 g/g xylose, respectively.On the contrary, the △pta-ack strain showed decreasing volumetric ethanol productivities, which are less than 40% of that produced by the initial strain.In 5-liter batch fermentation with non-sterilized medium containing 50 g/L glucose and xylose mixture (1∶1, g∶g), 44.8 g/L of L-lactic acid was obtained with the yield of 89.6%.The optical purity of L-lactic acid exceeded 99.5%.Results indicated that a new strain was identified and constructed by metabolic engineering, which could produce optically pure L-lactic acid effectively from renewable cellulosic resources.