It was found that Corynebacterium glutamicum Delta iolR devoid of the transcriptional regulator IolR accumulates high amounts of D-xylonate when cultivated in the presence of D-xylose. Detailed analyses of constructed deletion mutants revealed that the putative myo-inositol 2-dehydrogenase IolG also acts as D-xylose dehydrogenase and is mainly responsible for D-xylonate oxidation in this organism. Process development for D-xylonate production was initiated by cultivating C. glutamicum Delta iolR on defined D-xylose/D-glucose mixtures under batch and fed-batch conditions. The resulting yield matched the theoretical maximum of 1 mol mo1(-1) and high volumetric productivities of up to 4 g L-1 h(-1) could be achieved. Subsequently, a novel one-pot sequential hydrolysis and fermentation process based on optimized medium containing hydrolyzed sugarcane bagasse was developed. Cost-efficiency and abundance of second-generation substrates, good performance indicators, and enhanced market access using a non-recombinant strain open the perspective for a commercially viable bioprocess for Dxylonate production in the near future.