Development of an enzymatic reaction cascade for the production of KDG from seaweed carbohydrates

Abstract

In this paper development of a cell-free enzymatic cascade is described for conversion of brown seaweed carbohydrates (alginate and glucose from laminarin) to 2-keto-3-deoxygluconate (KDG), a precursor in bioplastics production. The process involves five enzymatic steps in two parallel pathways (alginate to KDG and glucose to KDG) including alginate depolymerization, cofactor recycling (NADH/NAD+), and conversion of intermediates to KDG, all integrated in a one-pot reaction system. Conversion of alginate to KDG was achieved in a three-step enzymatic process, starting with depolymerization of alginate by one endo- (RmAly3) and one exo-acting alginate lyase (RmAly4), followed by reduction of spontaneously formed 4-deoxy-L-erythro-5-hexoseulose uronate (DEH) to KDG by a novel reductase (Adh51 from Rhodothermus marinus). Cofactor recycling and conversion of glucose to gluconate were integrated by introduction of a novel glucose dehydrogenase (GDH16 from Thermobaculum strain MAT1704[E1] ) to oxidize glucose to gluconate while simultaneously regenerating NADH from NAD⁺. Generated gluconate was converted to KDG via dehydration catalyzed by the dihydroxyacid dehydratase DhadSs. The KDG productivity of the enzymatic cascade was 1 g/Lh from alginate and glucose, supplying a proof of concept of the cell free system for KDG production, but with DhadSs identified as the rate-limiting factor, resulting in conversion of 35% of the combined alginate and glucose addition to the system.