Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase.

ABSTRACT: Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN⁺) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. Here, we establish a growth selection platform to evolve enzymes to utilize NMN⁺-based reducing power. This is based on an orthogonal, NMN⁺-dependent glycolytic pathway in Escherichia coli which can be coupled to any reciprocal enzyme to recycle the ensuing reduced NMN⁺. With a throughput of >10⁶ variants per iteration, the growth selection discovers a Lactobacillus pentosus NADH oxidase variant with ~10-fold increase in NMNH catalytic efficiency and enhanced activity for other NCBs. Molecular modeling and experimental validation suggest that ins

SUBMITTER: King E 

PROVIDER: S-EPMC9701214 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature