A molecular transporter engineering approach to improving xylose catabolism in Saccharomyces cerevisiae

Traditional metabolic pathway engineering rarely considers the influence of molecular transport. Here, we describe the directed evolution of two heterologous transporters, Candida intermedia GXS1 and Scheffersomyces stipitis XUT3. Growth rate on xylose was improved up to 70% by mutant transporter expression. Most mutants were found to exhibit vastly improved Vmax values and display an increase in high cell density sugar consumption rates. Mixed glucose and xylose fermentations reveal that mutant transporters can alter the diauxic shift dynamics and the simultaneous sugar utilization capacity of the host strain. Analysis of mutations highlights several important residues influencing transporter function including point mutations at F40 of C. intermedia GXS1 and at E538 of S. stipitis XUT3. This work is the first to demonstrate that molecular transporter proteins can be improved for biotechnological applications through directed evolution in yeast.