Versatile selective evolutionary pressure using synthetic defect in universal metabolism

• Published in: Nature communications 2021-11, Vol.12 (1), p.6859-15, Article 6859
• Main Authors: Sellés Vidal, Lara, Murray, James W., Heap, John T.
• Format: Article
• Language: English
• Subjects: 140/131; 38/23; 38/70; 38/77; 42/44; 42/47; 631/45/603; 631/553/1886; 631/553/338/469; 631/553/338/552; 631/61/318; 82/80; 82/83; Adenine; Alcohol dehydrogenase; Anaerobic conditions; Cell growth; Cofactors; Design; Directed Molecular Evolution - methods; E coli; Enzymes; Escherichia coli - genetics; Escherichia coli - metabolism; Humanities and Social Sciences; Imines - metabolism; Inactivation; Industrial applications; Metabolic Engineering - methods; Metabolic networks; Metabolic Networks and Pathways; Metabolic pathways; Metabolism; Mimicry; multidisciplinary; Mutation; NAD - chemistry; NAD - metabolism; NADP; NADP - chemistry; NADP - metabolism; NADPH-diaphorase; Nicotinamide; Nicotinamide adenine dinucleotide; Nitroreductase; Oxidoreductases - chemistry; Oxidoreductases - genetics; Oxidoreductases - metabolism; Reductases; Regeneration; Science; Science (multidisciplinary); Selectivity; Substitutes; Substrates; Synthetic Biology
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-27266-9

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. Rational design of enzymes with new or improved properties is rarely straightforward, and artificial selection pressure approaches that link an improvement in the target to cell growth are an alternative. Here, the authors show that diverse enzymes sharing the ubiquitous cofactor NAD(P) + can substitute for defective NAD + regeneration, representing a very broadly-applicable artificial selection.