Gram-Scale Production of GDP-β-l-fucose with Multi-Enzyme Cascades in a Repetitive-Batch Mode

Abstract

Guanosine 5’-diphospho-β-l-fucose (GDP-Fuc) is an important and expensive nucleotide sugar only available in low quantities. We established two processes for the gram-scale production of GDP-Fuc with multi-enzyme cascades. We used the bifunctional enzyme fucokinase/GDP-fucose pyrophosphorylase from Bacteroides fragilis either with an ATP-regeneration system using polyphosphate or an excess of ATP. The optimized enzyme modules were scaled up to a 20 mL repetitive-batch production with 15 and 12 repetitive batches for ATP-regeneration and ATP excess, respectively. With ATP-regeneration a final product amount of 1.1 g (31 % yield) GDP-Fuc was obtained. The production process with ATP excess reached 1.6 g (57 % yield). Exceptional high total turnover numbers of 27 gP*gE−1 and 31 gP*gE−1, as well as space-time-yields of 0.083 gp*L−1*h−1 and 0.217 gp*L−1*h−1 were reached for each process respectively. Furthermore, we established a product purification protocol that enables a 95 % removal of free phosphates from the product solution, to ease further purification.