Enhanced In Vitro Cascade Catalysis of Glycerol into Pyruvate and Acetoin by Integration with Dihydroxy Acid Dehydratase from Paralcaligenes ureilyticus

Recently, an in vitro enzymatic cascade was constructed to transform glycerol into the high-value platform chemical pyruvate. However, the low activity of dihydroxy acid dehydratase from Sulfolobus solfataricus (SsDHAD) limited the efficiency. In this study, the enzymatic reduction of pyruvate catalyzed by d-lactate dehydrogenase from Pseudomonas aeruginosa PAO1 was used to assay the activities of dihydroxy acid dehydratases. Dihydroxy acid dehydratase from Paralcaligenes ureilyticus (PuDHT) was identified as the most efficient candidate for glycerate dehydration. After the optimization of the catalytic temperature for the enzymatic cascade, comprising alditol oxidase from Streptomyces coelicolor A3, PuDHT, and catalase from Aspergillus niger, 20.50 ± 0.27 mM of glycerol was consumed in 4 h to produce 18.95 ± 0.97 mM of pyruvate with a productivity 12.15-fold higher than the previous report using SsDHAD. The enzymatic cascade was further coupled with the pyruvate decarboxylase from Zymomonas mobile for the production of another platform compound, acetoin. Acetoin at a concentration of 8.52 ± 0.12 mM was produced from 21.62 ± 0.19 mM of glycerol with a productivity of 1.42 ± 0.02 mM h−1.