Klebsiella oxytoca strains were constructed to produce optical pure D-lactate by pH-controlled batch fermentation in mineral salts medium. The alcohol dehydrogenase gene, adhE, and the phospho-transacetylase/acetate kinase A genes. pta-ackA, were deleted from the wild type. KMS002 (Delta adhE) and KMS004 (Delta adhE Delta pta-ackA) exhibited D-lactate production as a primary pathway for the regeneration of NAD(+). Both strains produced 11-13 g/L of D-lactate in medium containing 2% (w/v) glucose with yields of 0.64-0.71 g/g glucose used. In sugarcane molasses, KMS002 and KMS004 produced 22-24 g/L of o-lactate with yields of 0.80-0.87 g/g total sugars utilized. Both strains also utilized maltodextrin derived from cassava starch and produced D-lactate at a concentration of 33-34 g/L with yields of 0.91-0.92 g/g maltodextrin utilized. These o-lactate yields are higher than those reported for engineered E. coli strains. 2012 Elsevier Ltd. All rights reserved.