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.

Engineering of a thermophilic dihydroxy-acid dehydratase to enhance its dehydration ability on glycerate to pyruvate and its application in in vitro synthetic enzymatic biosystems

The low activity of dihydroxy-acid dehydratase (DHAD) on dehydration of glycerate to pyruvate hampers its applications in the biosystems. Protein engineering of a thermophilic DHAD from Sulfolobus solfataricus (SsDHAD) was performed to increase its dehydratation activity. A novel high-throughput method was established. A triple-mutant (I161M/Y145S/G205K) with a 10-fold higher activity on glycerate dehydration was obtained after three rounds of iterative saturation mutagenesis (ISM) based on computational analysis. The shrunk substrate-binding pocket and newly formed hydrogen bonds were the reason for the activity improvement of the mutant. For the in vitro synthetic enzymatic biosystems of converting glucose or glycerol to L-lactate, the biosystems with the mutant SsDHAD showed 3.32- and 2.34-times of the reaction rate than that of wild type, respectively. This study demonstrates the potential of protein engineering to improve the efficiency of in vitro synthetic enzymatic biosystems by enhancing the enzyme activity of rate-limited enzymes.

Synthesis of Modified Aldonic Acids and Studies of Their Substrate Efficiency for Dihydroxy Acid Dehydratase (DHAD)

Modified aldopentonic and aldohexonic acids were synthesized in order to study the electronic requirements for a successful enzymatic conversion into their corresponding 2-keto 3-deoxy analogues by dihydroxy acid dehydratase (DHAD), an enzyme from the biosynthetic pathway of branched chain amino acids. Analytical tests with the novel artificial substrates (18)-(21) and (27) provided evidence that the amount of conversion could be enhanced by replacement of the hydroxy group at C4 of L-arabinonic acid (21) with less electron-withdrawing, ambivalent or electron-donating substituents. Modified aldohexonic acids were no substrates for DHAD, perhaps due to less perfect binding to the active site presumably for steric reasons. For 4-deoxy-L-threo-pentonic acid (18) the enzymatic conversion into 3,4-dideoxy-2-ketopentonic acid (29) by DHAD could be achieved on a preparative scale.