Recombinant Hypocrea strains were generated using protoplast transformation of H. jecorina QM9414. In the derived strains, the two Hypocrea codon-optimized genes (without GST-tag) were placed under the control of either the H. jecorina pyruvate kinase (pki) promoter, which is a strong constitutive promoter, or the H. jecorina xylanase 1 (xyn1) promoter, which is a strict shut-off system if an inducer (e.g. D-xylose) is missing. Such a system was used to avoid interference of the introduced recombinant pathway with cell wall biosynthesis and consequently, biomass formation. However, when comparing both promoter systems the strong pki promoter did not lead to decreased growth, diminished cell integrity or other adverse effects (data not shown). Therefore, we used strains in which both genes were under the control of the pki promoter for further studies as we observed a remarkably higher Neu-NAc formation.