Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase.

Edward King, Sarah Maxel, Yulai Zhang, Karissa C Kenney, Youtian Cui, Emma Luu, Justin B Siegel, Gregory A Weiss, Ray Luo, Han Li

Journal: Nature communications 2022;13(1):7282

PMID: 36435948

Free Full Text on Nature Publishing Group View on PubMed

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 >10 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.

Address: Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine, CA, 92697, USA.; Department Chemical and Biomolecular Engineering University of California Irvine, Irvine, CA, 92697, USA.; Department of Chemistry, University of California Irvine, Irvine, CA, 92697, USA.; Genome Center, University of California Davis, Davis, CA, 95616, USA.; Department of Chemistry, Molecular Medicine University of California, Davis, Davis, CA, USA.; Department of Biochemistry and Molecular Medicine University of California, Davis, Davis, CA, USA.; Department of Pharmaceutical Sciences, University of California Irvine, Irvine, CA, 92697, USA.; Department Materials Science and Engineering, University of California Irvine, Irvine, CA, 92697, USA.; Department of Biomedical Engineering, University of California Irvine, Irvine, CA, 92697, USA.; Department Chemical and Biomolecular Engineering University of California Irvine, Irvine, CA, 92697, USA. [email protected].; Department of Biomedical Engineering, University of California Irvine, Irvine, CA, 92697, USA. [email protected].

DOI: 10.1038/s41467-022-35021-x

Link outs

Free resources

Full Text Sources:

Other Literature Sources:

https://www.lens.org/lens/search/patent/list?q=ref...

MeSH Terms: Oxidoreductases, Nicotinamide Mononucleotide, Escherichia coli, Models, Molecular, Molecular Conformation