It was found that Corynebacterium glutamicum ΔiolR 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 ΔiolR on defined d-xylose/d-glucose mixtures under batch and fed-batch conditions. The resulting yield matched the theoretical maximum of 1 mol mol-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 d-xylonate production in the near future.