Multi-enzyme cascade excluding costly cofactors for pyruvate production from glucose

In vitro enzymatic cascade is a useful biomanufacturing technique, but requirement for costly exogenous cofactors restricts its economic feasibility. In this study, we designed a costly cofactor-free enzymatic cascade to transform glucose into pyruvate, an important platform compound widely used in different industries. Only five enzymes including glucose oxidase from Aspergillus niger, mutant alditol oxidase from Streptomyces coelicolor, 2-keto-3-deoxy-d-gluconate aldolase from Sulfolobus acidocaldarius, catalase from Aspergillus niger, dihydroxy acid dehydratase from Paralcaligenes ureilyticus, and Mg2+ were needed for pyruvate production from glucose. Under the optimal reaction conditions, 30.82 mM pyruvate was produced with a yield of 74.27% of the theoretical value in 8 h. In addition, acetoin, another important platform chemical, can also be produced from glucose through further introducing pyruvate decarboxylase from Zymomonas mobilis and thiamine pyrophosphate into the enzymatic cascade. Acetoin at a concentration of 17.79 mM was produced from glucose with 84.71% of the theoretical yield in 10 h.