In Vitro Biotransformation of High-Titer d-Glucarate by Stepwise-Added Enzyme Cocktails

d-Glucaric acid (GA), one of the polyhydroxy dicarboxylic acids, is known for its great potentials in the polymer, food, detergent, and pharmaceutical industries. Here, we presented a highly efficient synthesis of GA from starch by in vitro biotransformation (ivBT). This one-pot ivBT was composed of five thermophilic enzymes (i.e., isoamylase, α-glucan phosphorylase, phosphoglucomutase, inositol 1-phosphate synthase, and inositol monophosphatase) responsible for the synthesis of myo-inositol (MI) from starch, followed by three mesophilic enzymes (i.e., myo-inositol oxygenase (MIOX), uronate dehydrogenase (UDH), and H₂O-forming NADH oxidase (NOX)) responsible for the synthesis of GA from inositol. Also, a stepwise-added enzyme cocktail strategy was employed to circumvent the inhibition of GA on MIOX and mismatches in enzymes’ properties, such as temperature and pH. The three-step cascade bioprocessing in one pot resulted in the concentration of disodium glucarate of approximately 9.66 g/L (i.e., 38 mM) from 10 g/L maltodextrin with an atom efficiency of 96.6%. Up to 52.1 g/L disodium d-glucarate, the highest titer reported, was produced in a stirred bioreactor operated in two-reaction temperature ranges, featured with the stepwise addition of enzyme cocktails, and equipped with a constant pH controller and stirring for enough oxygen supplies. This study could open a new door to the industrial biomanufacturing of GA by ivBT.