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The most prevalent xylose-assimilating pathways in recombinant Saccharomyces cerevisiae , i.e. the xylose isomerase (XI) and the xylose reductase/xylitol dehydrogenase (XR/XDH) pathways, channel the carbon flux through the pentose phosphate pathway and further into glycolysis. In contrast, the oxidative and non-phosphorylative bacterial Weimberg pathway channels the xylose carbon through five steps into the metabolic node α-ketoglutarate (αKG) that can be utilized for growth or diverted into production of various metabolites. In the present study, steps preventing the establishment of a functional Weimberg pathway in S. cerevisiae were identified. Using an original design where a S. cerevisiae strain was expressing the essential four genes of the Caulobacter crescentus pathway (xylB, xylD, xylX, xylA) together with a deletion of FRA2 gene to upregulate the iron-sulfur metabolism, it was shown that the C. crescentus αKG semialdehyde dehydrogenase, XylA was not functional in S. cerevisiae. When replaced by the recently described analog from Corynebacterium glutamicum, KsaD, significantly higher in vitro activity was observed but the strain did not grow on xylose. Adaptive laboratory evolution (ALE) on a xylose/glucose medium on this strain led to a loss of XylB, the first step of the Weimberg pathway, suggesting that ALE favored minimizing the inhibiting xylonate accumulation by restricting the upper part of the pathway. Therefore three additional gene copies of the lower Weimberg pathway (XylD, XylX and KsaD) were introduced. The resulting S. cerevisiae strain (ΔΔfra2, xylB, 4x (xylD-xylX-ksaD)) was able to generate biomass from xylose and Weimberg pathway intermediates were detected. To our knowledge this is the first report of a functional complete Weimberg pathway expressed in fungi. When optimized this pathway has the potential to channel xylose towards value-added specialty chemicals such as dicarboxylic acids and diols. • Growth on xylose via the Weimberg pathway established in S. cerevisiae. • >50% of the xylose carbon channeled through the Weimberg pathway. • XylA identified as a bottleneck when using the pathway gene from C. crescentus. • C. glutamicum ksaD expression gives higher enzyme activity than C. crescentus xylA. • S. cerevisiae growth on xylose requires fine-tuning of Weimberg pathway enzymes. [ABSTRACT FROM AUTHOR]