Characterization of an fungal l-fucokinase involved in Mortierella alpina GDP-l-fucose salvage pathway

Abstract

GDP-l-fucose functions as a biological donor for fucosyltransferases, which are required for the catalysis of l-fucose to various acceptor molecules including oligosaccharides, glycoproteins and glycolipids. Mortierella alpina is one of the highest lipid-producing fungi and can biosynthesis GDP-l-fucose in the de novo pathway. Analysis of the M. alpina genome suggests that there is a gene encoding l-fucokinase (FUK) for the conversion of fucose to l-fucose-1-phosphate in the GDP-l-fucose salvage pathway, which has never been found in fungi before. This gene was characterized to explore its role in GDP-l-fucose synthesis. The yield of GDP-l-fucose is relatively higher in lipid accumulation phase (0.096 mg per g cell) than that in cell multiplication phase (0.074 mg per g cell) of M. alpina Additionally, the transcript level of FUK is up regulated by nitrogen exhaustion when M. alpina starts to accumulate lipid, highlights the functional significance of FUK in the GDP-l-fucose biosynthesis in M. alpina Gene encoding FUK was expressed heterologously in Escherichia coli and the resulting protein was purified to homogeneity. The product of FUK reaction was analyzed by liquid chromatography and mass spectrometry. Kinetic parameters and other properties of FUK were investigated. Comparative analyses between the FUK protein and other homologous proteins were performed. To our knowledge, this study is the first to report a comprehensive characterization of FUK in a fungus. Mortierella alpina could be used as an alternative source for the production of GDP-l-fucose.

Keywords: GDP-l-fucose synthesis; Mortierella alpina/salvage pathway; characterization; l-fucokinase.