Adaptive Laboratory Evolution (ALE) is increasingly being used as a technique for untargeted strain optimization. This work aimed at developing an automated and miniaturized ALE approach based on repetitive batch cultivations in microtiter plates. The new method is applied to the recently published strain Corynebacterium glutamicum pEKEx3-xylXABCD(Cc), which is capable of utilizing D-xylose via the Weimberg (WMB) pathway. As a result, the significantly improved strain WMB2(evo) was obtained, showing a specific growth rate of 0.26 h(-1) on D-xylose as sole carbon and energy source. WMB2(evo) grows stable during lab-scale bioreactor operation, demonstrating the high potential of this strain for future biorefinery applications. Genome sequencing of cell samples from two different ALE processes revealed potential key mutations, e.g. in the gene cg0196 (encoding for the transcriptional regulator IolR of the myo-inositol metabolism). These findings open up new perspectives for the rational engineering of C. glutamicum towards improved D-xylose utilization. (C) 2017 Elsevier Ltd. All rights reserved.