Sugar kinases play a central role in carbohydrate metabolism, since they phosphorylate sugars after uptake in order to trap them inside the cell and prepare them for further catabolism. The pyrenomycetous ascomycete Hypocrea jecorina features amongst other sugar kinases one galactokinase (gal1), one hexokinase (hxk1) and one glucokinase (glk1). The role of these enzymes in carbohydrate metabolism and signalling was investigated. The single galactokinase of H. jecorina is responsible for the first step of the Leloir pathway, the phosphorylation of D-galactose to D-galactose 1-phosphate. Induction of the other genes of the Leloir pathway is independent from the H.
jecorina GAL1, which is in contrast to the situation in yeasts such as Saccharomyces cerevisiae or Kluyveromyces lactis, where the galactokinase acts as a regulatory protein and activates the transcription of the GAL pathway in the presence of D-galactose. We could show that galactokinase activity, but not the GAL1 protein itself is essential for induction of cellulases on lactose by replacing the H.
jecorina GAL1 with the Escherichia coli galactokinase. Additionally we present evidence for the existence of a second D-galactose degrading pathway which proceeds via reduction ofD-galactose to galactitol. The enzyme responsible for the second step in this pathway was identified as the L-arabinitol 4-dehydrogenase (lad1), since a [Delta]gal1[Delta]lad1 strain was unable to grow on galactitol or D-galactose as carbon source.
To facilitate successive gene knock-outs in H. jecorina we applied a blaster cassette approach. This cassette consisting of the H. jecorina pyr4 gene flanked by two direct repeats was used to delete the single hexokinase and glucokinase in H. jecorina. The cassette was removed after deletion of the glucokinase encoding gene to construct a strain deleted in both hexo- and glucokinase. Strains deleted in both genes were unable to grow on D-glucose and also showed a defect on a number of other carbon source. Using three genetic systems, diagnostic for carbon catabolite (de)repression (cellulase formation, cbh1; xylanase formation, xyn1; [beta]-galactosidase formation, bga1), we demonstrated, that only a deletion of both kinases, hxk1 and glk1, leads to derepression, suggesting a catalytic effect to be responsible.
Interestingly the induction of cbh1 on the cellulase inducer sophorose and bga1 on D-galactose were impaired in the [Delta]glk1[Delta]hxk1 strain, while the transcript levels of xyn1 on D-xylose were increased in both single and the double deletion strains.