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- Title
Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase.
- Authors
King, Edward; Maxel, Sarah; Zhang, Yulai; Kenney, Karissa C.; Cui, Youtian; Luu, Emma; Siegel, Justin B.; Weiss, Gregory A.; Luo, Ray; Li, Han
- 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 instead of directly contacting NCBs, the mutations optimize the enzyme's global conformational dynamics to resemble the WT with the native cofactor bound. Restoring the enzyme's access to catalytically competent conformation states via deep navigation of protein sequence space with high-throughput evolution provides a universal route to engineer NCB-dependent enzymes. Engineering enzymes to accept noncanonical cofactor biomimetics is difficult. Here, the authors establish a self-sufficient growth selection method and demonstrate its application in engineering the Lactobacillus pentosus NADH oxidase to efficiently recycle reduced nicotinamide mononucleotide (NMNH).
- Subjects
WNT signal transduction; BIOMIMETIC chemicals; AMINO acid sequence; SEQUENCE spaces; ANIMAL navigation; ESCHERICHIA coli
- Publication
Nature Communications, 2022, Vol 13, Issue 1, p1
- ISSN
2041-1723
- Publication type
Article
- DOI
10.1038/s41467-022-35021-x