Multi-enzyme Cascades for the In Vitro Synthesis of Guanosine Diphosphate L-Fucose

Abstract

Recombinant Leloir glycosyltransferases can be exploited to synthesize a wide range of HMOs using in vitro biocatalytic reactions. However, high costs and unavailability of bulk amounts of most nucleotide sugars, such as guanosine diphosphate L-fucose (GDP-Fuc), are major obstacles for the efficient large-scale synthesis. Here, we report two novel multi-enzyme cascades for the synthesis of GDP-Fuc from readily available and low cost precursors. The first cascade was developed to produce GDP-Fuc from guanosine (Guo), fucose (Fuc), polyphosphate (PolyPn) and catalytic amounts of adenine triphosphate (ATP). GDP-Fuc was produced with a final concentration of 7 mM (4.1 g/L) and a reaction yield of 68 % from Guo and Fuc within 48 h with a biocatalyst load of 0.34 genzyme/gproduct. A second cascade, consisting of ten enzymes and eleven reactions was developed to carry out the synthesis from mannose (Man), Guo, PolyPn, L-glutamine (L-Glu) and catalytic amounts of ATP, and nicotinamide adenine dinucleotide phosphate (NADPH). Utilizing this cascade, GDP-Fuc was produced with a final concentration of 7.6 mM (4.5 g/L) and a reaction yield of 72 % in a reaction time of 48 h with a biocatalyst load of 0.97 genzyme/gproduct. Finally, a method for chromatographic purification of GDP-Fuc was established achieving product purities of 90.5 %.