Cell-free synthesis of 2-keto-3-deoxy gluconate from seaweed polysaccharides

Abstract

Macroalgae biomass has a high potential as a feedstock for biotechnological production of fuels or platform chemicals due to their high productivity and high carbohydrate content. Brown macroalgae are especially interesting as they are abundant along coasts and some species are relatively easily cultivated offshore. The carbohydrates are mainly in the form of the polysaccharides laminarin and alginate that are not used much as feedstocks in industrial biotechnology today.Laminarin is the glucose-containing beta-glucan polysaccharide that can be used as a carbon source for production of valuable compounds rather easily with high efficiency by conventional industrial production microbes, but alginate is a polyuronate and cannot be used as a carbon source for such species. Enzymatic cell free synthesis of platform chemicals from alginate is therefore an interesting and challenging option.The platform chemical, 2-keto-3-deoxy-D-gluconate (KDG), is an intermediate in microbial utilization of alginate and has an industrial potential in the manufacture of building blocks of bioplastics and various pharmaceuticals. In this study, robust thermophilic enzymes were used to develop and optimize a multi-step cell-free enzymatic process for the conversion of alginate to KDG. Alginate lyases were used to degrade alginate to unsaturated mono-uronates that were subsequently reduced by a specific NADH dependent oxidoreductase to KDG. NADH was regenerated by oxidation of glucose by a glucose dehydrogenase and the product, gluconic acid, converted to KDG by gluconic acid dehydratase. Necessary enzymes were cloned and expressed, and optimum reaction conditions determined.