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

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 %. Multi‐enzyme cascade reactions: In vitro enzyme cascades for the one‐pot synthesis of guanosine diphosphate fucose (GDP‐Fuc) from low cost and readily available substrates. In situ co‐factor regeneration was implemented and a robust and scalable ion exchange chromatography protocol for the purification of GDP‐Fuc from the reaction was established. The developed cascades and the purification protocol can be employed for the efficient biocatalytic production of GDP‐Fuc.