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Versatile selective evolutionary pressure using synthetic defect in universal metabolism

DOI: 10.1038/s41467-021-27266-9 DOI Help

Authors: Lara Sell├ęs Vidal (Imperial College London) , James W. Murray (Imperial College London) , John T. Heap (Imperial College London; The University of Nottingham)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature Communications , VOL 12

State: Published (Approved)
Published: November 2021
Diamond Proposal Number(s): 12579

Open Access Open Access

Abstract: 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 Keywords: Biocatalysis; Metabolic engineering; Protein design; Protein engineering; Synthetic biology

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography

Added On: 30/11/2021 10:05


Discipline Tags:

Biotechnology Biochemistry Catalysis Chemistry Structural biology Engineering & Technology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)