Efficient Bioconversion of Sucrose to High‐Value‐Added Glucaric Acid by In Vitro Metabolic Engineering

Abstract Glucaric acid (GA) is a major value‐added chemicals feedstock and additive, especially in the food, cosmetics, and pharmaceutical industries. The increasing demand for GA is driving the search for a more efficient and less costly production pathway. In this study, a new in vitro multi‐enzyme cascade system was developed, which converts sucrose efficiently to GA in a single vessel. The in vitro system, which does not require adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD+) supplementation, contains seven enzymes. All enzymes were chosen from the BRENDA and NCBI databases and were expressed efficiently in Escherichia coli BL21(DE3). All seven enzymes were combined in an in vitro cascade system, and the reaction conditions were optimized. Under the optimized conditions, the in vitro seven‐enzyme cascade system converted 50 mm sucrose to 34.8 mm GA with high efficiency (75 % of the theoretical yield). This system represents an alternative pathway for more efficient and less costly production of GA, which could be adapted for the synthesis of other value‐added chemicals.