In Vivo Assembly of a Genetically Encoded Artificial Metalloenzyme for Hydrogen Production

The genetic encoding of artificial enzymes represents a substantial advantage relative to traditional molecular catalyst optimization, as laboratory-based directed evolution coupled with high-throughput screening methods can provide rapid development and functional characterization of enzyme librari...

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Bibliographic Details
Published in:ACS synthetic biology 2021-08, Vol.10 (8), p.2116-2120
Main Authors: Naughton, Kassandra J, Treviño, Regina E, Moore, Peter J, Wertz, Ashlee E, Dickson, J. Alex, Shafaat, Hannah S
Format: Article
Language:English
Subjects:
Escherichia coli - enzymology
Escherichia coli - genetics
Hydrogen - metabolism
Hydrogenase - genetics
Hydrogenase - metabolism
Protein Engineering
Rubredoxins - genetics
Rubredoxins - metabolism
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Summary:The genetic encoding of artificial enzymes represents a substantial advantage relative to traditional molecular catalyst optimization, as laboratory-based directed evolution coupled with high-throughput screening methods can provide rapid development and functional characterization of enzyme libraries. However, these techniques have been of limited utility in the field of artificial metalloenzymes due to the need for in vitro cofactor metalation. Here, we report the development of methodology for in vivo production of nickel-substituted rubredoxin, an artificial metalloenzyme that is a structural, functional, and mechanistic mimic of the [NiFe] hydrogenases. Direct voltammetry on cell lysate establishes precedent for the development of an electrochemical screen. This technique will be broadly applicable to the in vivo generation of artificial metalloenzymes that require a non-native metal cofactor, offering a route for rapid enzyme optimization and setting the stage for integration of artificial metalloenzymes into biochemical pathways within diverse hosts.
ISSN:2161-5063
2161-5063
DOI:10.1021/acssynbio.1c00177