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
Abstract
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.