One of the challenges in strain improvement by evolutionary engineering is to subsequently determine the molecular basis of the improved properties that were enriched from the natural genetic variation during the selective conditions. This study focuses on Saccharomyces cerevisiae IMS0002 which, after metabolic and evolutionary engineering, ferments the pentose sugar arabinose. Glucose- and arabinose-limited anaerobic chemostat cultures of IMS0002 and its non-evolved ancestor were subjected to transcriptome analysis, intracellular metabolite measurements and metabolic flux analysis. Increased expression of the GAL-regulon and deletion of GAL2 in IMS0002 confirmed that the galactose transporter is essential for growth on arabinose. Elevated intracellular concentrations of pentose-phosphate-pathway intermediates and upregulation of TKL2 and YGR043c (encoding transketolase and transaldolase isoenzymes) suggested an involvement of these genes in flux-controlling reactions in arabinose fermentation. Indeed, deletion of these genes in IMS0002 caused a 21% reduction of the maximum specific growth rate on arabinose.