Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform
Year: 2022
Type: article
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 without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA+), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)+.
Source: Nature Communications
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $5,890
Funders: National Science Foundation, U.S. Department of Health and Human Services, European Commission, National Institutes of Health, Advanced Research Projects Agency - Energy, University of California, Irvine, Advanced Research Projects Agency, National Institute of General Medical Sciences, National Institute of Environmental Health Sciences, H2020 European Research Council
Awards: G1308682533, G146225979, G146713361, Collaborative Research: Understanding and exploiting the structure-function link between fatty acid biosynthesis and degradation enzymes for functionalized small molecule synthesis, Biomarkers of Exposure to Hazardous Substances, G2421906508, G3611247453, G3690998425, G3874321780, G434337789, RCN-UBE: Data-to-Design Course-based Undergraduate Research Experience ? protein modeling and characterization to enhance student learning and improve computational protein design, G606658003, Engineering an unnatural redox cofactor (uRedox) system for efficient biosynthesis of medicines, CI-EN: Collaborative Research: Enhancement of Foldit, a Community Infrastructure Supporting Research on Knowledge Discovery Via Crowdsourcing in Computational Biology, Biosynthesis of (di)terpene scaffolds, G7697847498, Biocatalytic alkylation: fitting enzymes for selective C-C bond formations, G8278764384, G8581368535, CAREER: Engineering redox metabolism using unnatural cofactors, Multi-scaled Modeling of Electrostatic and Polarization Effects in Biomolecules
OpenAlex ID: W4293109393