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Versatile selective evolutionary pressure using synthetic defect in universal metabolism

Sellés Vidal, Lara; Murray, James W.; Heap, John T.

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Lara Sellés Vidal

James W. Murray

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Associate Professor


The non-natural needs of industrial applications often require new or improved enzymes. The structures and properties of enzymes are difficult to predict or design de novo. Instead, semi-rational approaches mimicking evolution entail diversification of parent enzymes followed by evaluation of isolated variants. Artificial selection pressures coupling desired enzyme properties to cell growth could overcome this key bottleneck, but are usually narrow in scope. Here we show diverse enzymes using the ubiquitous cofactors nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) can substitute for defective NAD regeneration, representing a very broadly-applicable artificial selection. Inactivation of Escherichia coli genes required for anaerobic NAD regeneration causes a conditional growth defect. Cells are rescued by foreign enzymes connected to the metabolic network only via NAD or NADP, but only when their substrates are supplied. Using this principle, alcohol dehydrogenase, imine reductase and nitroreductase variants with desired selectivity modifications, and a high-performing isopropanol metabolic pathway, are isolated from libraries of millions of variants in single-round experiments with typical limited information to guide design.

Journal Article Type Article
Acceptance Date Nov 4, 2021
Online Publication Date Nov 25, 2021
Publication Date Dec 1, 2021
Deposit Date Nov 12, 2021
Publicly Available Date Nov 25, 2021
Journal Nature Communications
Electronic ISSN 2041-1723
Peer Reviewed Peer Reviewed
Volume 12
Issue 1
Article Number 6859
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