In Vitro Cascade Oxidization of D-Glucose to D-Glucaric Acid by Three Coenzyme-Free Oxidoreductases

D-Glucaric acid (GA) is one of the top 12 value-added biochemicals with broad applications in the detergent, polymer, pharmaceutical and food industries. The conversion of D-glucose to GA requires full oxidization for both C6 and C1 termini but these highly-selective oxidation of D-glucose remains challenging. Previous in vitro enzymatic pathways have obvious weakness, for example, up to eight enzymes used and the addition of costly coenzyme NAD. Here we develop an in vitro coenzyme-free minimal enzymatic pathway through six-electron oxidation catalyzed by three coenzyme-free oxidoreductases for the conversion of D-glucose to GA. This new-to-nature enzymatic pathway comprises an engineered galactose oxidase (GAO), glucose oxidase (GOX), and a newly discovered high-activity pyrroloquinoline quinone (PQQ)-dependent aldehyde dehydrogenase (ALDH), together with catalase for hydrogen peroxide (H2O2) removal. Structure-guided engineering of GAO improved its catalytic activity on its non-natural product D-glucose by a factor of thousands relative to that of GAO variant A3.E7, achieving the complete oxidation of C6 terminus of D-glucose. Sequence-guided mining identified ALDH from Devosia limi (DelALDH), which showed approximately five-fold higher activity toward L-guluronic acid than the best reported ALDH. Three-step oxidization of D-glucose was implemented by stepwise enzyme addition of GAO, GOX and ALDH for its respective oxidation of C6, C1 and C6 terminus of D-glucose. This four-enzyme cocktail achieves a molar yield of 98.3% GA from D-glucose with a productivity of 0.89 ± 0.01 g L-1 h-1. This in vitro coenzyme-free minimal-enzyme pathway could open a better door to biomanufacturing GA from D-glucose instead of myo-inositol.