Figure 6.

Overexpression of HUT1 and YEA4 nucleotide sugar transporters increases the rate of UDP-glucose release. (A) A wild-type copy of the YMD8 gene was cloned by PCR into a GAL1 overexpression vector to form the pYMD8 plasmid, which was then transformed into wild-type and ymd8Δ strains. Cells for the extracellular collection were grown to confluence in selective media with galactose to induce gene expression. The bioassay was used to detect UDP-glucose in extracellular collections from 1 h incubations in 1% (w/v) galactose. Overexpression of YMD8 has no impact on UDP-glucose release in WT strains but did significantly decrease the rate of release in a ymd8Δ strain (n = 3). (B) Wild-type copies of the HUT1 and YEA4 genes were cloned into the overexpression vector to form plasmids pHUT1 and pYEA4, which were transformed into wild-type strains. The bioassay was used to detect extracelluar UDP-glucose in extracellular collections. Both HUT1 and YEA4 overexpression led to significant increases in the rate of UDP-glucose release (n = 3). An asterisk indicates p < 0.05 vs vector alone.