Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform.

ABSTRACT: Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)⁺) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN⁺). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN⁺ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN⁺ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation wi

SUBMITTER: Zhang L 

PROVIDER: S-EPMC9418148 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature