From Semirational to Rational Design: Developing a Substrate-Coupled System of Glucose Dehydrogenase for Asymmetric Synthesis

Glucose dehydrogenase (GDH) generally functions as an expensive cofactor NAD­(P)H regenerator in an enzyme-coupled cofactor regeneration system, as in the production of fine chemicals. However, whether GDH can accept substrates other than glucose remains to be explored. Based on a known mutant of GDH with high thermostability, DN46 (E170K–K252L), we employed semirational design-based directed evolution to expand its substrate scope and promote its application in the asymmetric synthesis of methyl 2-hydroxyl carboxylates. After one round of saturation mutagenesis and two rounds of iterative saturation mutagenesis, an enantioselectivity-enhanced mutant DN46-E96Q-H147V (>99% ee, R-preference) and enantioselectivity-reversed mutant DN46-E96Q-I150A-W152L with high enantioselectivity (>97% ee, S-preference) evolved. With glucose as a cosubstrate, we developed a dual-function GDH-based substrate-coupled cofactor regeneration system for asymmetric synthesis. Furthermore, in light of a deeper understanding of the catalytic mechanism, a rational design was successfully performed to create a mutant DN46-W152G for the upscaled synthesis of (R)-2-chloromandelic acid methyl ester, the precursor of the medicine (S)-clopidogrel. This work expands the utilization of GDH, provides a design method for rational design of this enzyme, and will support future work regarding its application toward achieving even broader substrate scope.