Ethanol produced from lignocellulose is an environment-friendly alternative to fossil fuels. The filamentous fungus Fusarium oxysporum is known for its ability to ferment hexoses and pentoses directly to ethanol. During xylose fermentation an accumulation of sedoheptulose-7-phosphate was observed, which might indicate a limitation in the transaldolase reaction or a competition of glyceraldehyde-3-phosphate between pentose phosphate pathway and glycolysis, resulting in acetate production and reduced xylose consumption. Moreover, accumulation of glucose-1,6-diphosphate may indicate reduced activity of phosphoglucomutase, which is involved in the conversion of glucose-6-phosphate to glucose-1-phosphate via glucose-1,6-diphosphate, and hence potential difficulties of glucose to be channelled towards cell wall biosynthesis. Purification and characterization of transaldolase and phosphoglucomutase was studied. In the case of transaldolase, LC-MS/MS analysis provided peptide mass and sequence information, allowing us to identify the enzyme from F. oxysporum proteome. As long as phosphoglucomutase is concerned, the effect of different metals in enzyme’s activity was also studied. The information of purification and characterization of the two enzymes aimed at the construction of a genetically modified strain of F. oxysporum which would constitutively express both phosphoglucomutase and transaldolase. Three strains were constructed; ANPGM1 and ANPgM2 strains emerged from the heterologous expression of phosphoglucomutase of F. oxysporum while FF11 strain emerged from the homologous expression of phosphoglucomutase and transaldolase of F. oxysporum. Conversion of glucose, xylose, galactose and their mixtures to ethanol by the wild type strain (F3) and the genetically modified strains of F. oxysporum was investigated in flasks while glucose and xylose metabolism by F3 and FF11 strains was studied in batch fermentations. The cultivations were performed in two phases; an aerobic growth phase and a production phase (anaerobic/oxygen-limited). Growth characteristics were evaluated in both phases of F. oxysporum strains while intracellular metabolites were determined under batch bioreactors. As a result, the constitutive expression of the two enzymes increased ethanol and reduced acetate production in glucose. In the case of xylose the specific xylose uptake rate was increased while increased yields of acetate and xylitol were detected.