17-28: Engineering of improved D-xylose transporters to overcome impediments in mixed sugar co-fermentation in Saccharomyces cerevisiae

Tuesday, May 1, 2012
Napoleon Ballroom C-D, 3rd fl (Sheraton New Orleans)
Alexander Farwick, Thorsten Subtil, Stefan Bruder and Eckhard Boles, Institute of Molecular Biosciences, Goethe-University Frankfurt, Frankfurt, Germany
The failure of co-consumption of xylose and hexoses by S. cerevisiae is one major obstacle on the way to efficient conversion of mixed sugar hydrolysates. We could show that the reason for this deficit is the inhibitory effect of D-glucose on D-xylose uptake. Intracellular glucose or glucose metabolism is not interfering with D-xylose metabolism.

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.

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