Abstract
Engineering of the yeast Saccharomyces cerevisiae towards efficient D-xylose assimilation has been a major focus over the last decades since D-xylose is the second most abundant sugar in nature, and its conversion into products could significantly improve process economy in biomass-based processes. Up to now, two different metabolic routes have been introduced via genetic engineering, consisting of either the isomerization or the oxido-reduction of D-xylose to D-xylulose that is further connected to the pentose phosphate pathway and glycolysis. In the present study, cytosolic D-xylose oxidation was investigated instead, through the introduction of the Weimberg pathway from Caulobacter crescentus in S. cerevisiae. This pathway consists of five reaction steps that connect D-xylose to the TCA cycle intermediate α-ketoglutarate. The corresponding genes could be expressed in S. cerevisiae, but no growth was observed on D-xylose indicating that not all the enzymes were functionally active. The accumulation of the Weimberg intermediate D-xylonate suggested that the dehydration step(s) might be limiting, blocking further conversion into α-ketoglutarate. Although four alternative dehydratases both of bacterial and archaeon origins were evaluated, D-xylonate accumulation still occurred. A better understanding of the mechanisms associated with the activity of dehydratases, both at a bacterial and yeast level, appears essential to obtain a fully functional Weimberg pathway in S. cerevisiae.