In yeast strains engineered for pentose fermentation, D-xylose utilization rates were unaffected in D-xylose-maltose-cofermentations. Even in hexo-/glucokinase deletion strains that accumulate intracellular glucose no negative effect on D-xylose utilization could be seen. Extracellular glucose on the other hand had an inhibitory effect increasing with increasing D-glucose concentration. This inhibition could be alleviated but not abolished by overexpression of transporters that can transport D-xylose.
A specific D-xylose transporter which is not inhibited by D-glucose is therefore a necessity for efficient co-fermentation in S. cerevisiae. We have developed a system to screen for transporters with enhanced D-xylose specificity, which can also be used for evolutionary engineering approaches. This system is based on a D-xylose-fermenting yeast strain that has deletions of all hexose-transporters and all hexo-/glucokinases. D-glucose cannot itself be utilized as a carbon source even with expression of non-specific pentose-transporting permeases. However, it strongly inhibits D-xylose utilization and growth at transport level. As a result, mutations which alleviate D-xylose uptake and utilization in the presence of increasing concentrations of glucose can easily be selected. We will show successful generation of new D-xylose-transporters with decreased inhibition by D-glucose.