Gluconobacter oxydans can rapidly and incompletely oxidize glycerol to dihydroxyacetone (DHA), a versatile product extensively used in cosmetic, chemical and pharmaceutical industries. To improve DHA production, the glycerol dehydrogenase (GDH) responsible for DHA formation was overexpressed in G. oxydans M5AM, in which the gene coding for the membrane-bound alcohol dehydrogenase (ADH) was interrupted. Real-time PCR and enzyme activity assay revealed that the absence of ADH together with the overexpression of GDH gene resulted in an increased GDH activity in the resulting strain M5AM/GDH, which led to a substantially enhanced production of DHA in a resting cell system. In a batch biotransformation process. M5AM/GDH exhibited a 2.4-fold increased DHA productivity of 2.4 g/g CDW/h from 1.0 g/g CDW/h, yielding 96 g/L DHA from 100 g/L glycerol. When 140 a glycerol was supplied, a final DHA concentration of 134 g/L was accumulated within 14 h. In four repeated batch runs, 385 g DHA over a time period of 34 h was achieved from 400 g glycerol with an average productivity of 2.2 g/g CDW/h. These results indicated that this newly developed strain G. oxydans M5AM/GDH with high productivity and increased tolerance against product inhibition has potential for DHA production in an industrial bioconversion process. (C) 2010 Elsevier Ltd. All rights reserved.