Glycan-modifying enzymes are increasingly sought after for in vitro glycan analysis and modification. A bottleneck limiting progress in the glycobiology research is the non-availability and/or high price of glycan-modifying enzymes on the market.

The broad aim of this project is to produce recombinant fucosyltransferases of human origin in Trichoderma reesei, a high-yielding fungal cell factory. T. reesei is a eukaryote with well-developed posttranslational protein processing machinery featuring glycosylation, phosphorylation and disulfide bond formation. In addition to a long history of safe industrial use and ability to express heterologous proteins, T. reesei is also easier and cheaper to cultivate on a large scale than mammalian or insect cells typically employed for the production of recombinant gene products of human origin.  

In the current work, cDNA encoding the C-terminal catalytic domain of the human ɑ-1,3/4 fucosyltransferase (FUT3) was synthesised according to the T. reesei codon usage and assembled into an expression vector under the strong cellobiohydrolase 1 promoter. The expression design allowed FUT3 to be produced as a fusion to the core-linker fragment of the endogenous cellobiohydrolase 1 protein to facilitate expression and secretion. DNA encoding the mCherry fluorescent protein was cloned into one of the vectors as a reporter of expression and secretion. A Strep-tag sequence was included to aid purification and detection of the recombinant protein. The constructs were transformed into a low protease-producing T. reesei strain using particle bombardment. Preliminary screening of the transformants did not only demonstrate secretion of a full-sized fusion protein but also degradation of the heterologous protein, most probably by the remaining Trichoderma proteases. The time- and pH-dependent degradation of the recombinant FUT3 makes it necessary to optimize the cultivation conditions in order to obtain sufficient amounts of the protein for purification, biochemical characterization and determination of the nature of the degradation products.