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.
Highlights
The non-natural needs of industrial applications often require new or improved enzymes
Reports published during the present study applied such selection using acetoin[11,13], but E. coli contains native acetoin reductase activity[11,15], and our present study found acetoin alone was sufficient to rescue the metabolic defect, so acetoin cannot be considered isolated from native E. coli metabolism
Synthetic defects in nicotinamide adenine dinucleotide (NAD)+ regeneration abolishing anaerobic growth can be rescued by metabolically isolated foreign enzyme-substrate pairs
Summary
The non-natural needs of industrial applications often require new or improved enzymes. A strong artificial selection pressure could be applied to libraries of enzyme variants for activity with substrates that are not produced within cells, linking restoration of growth by anaerobic fermentation to the presence of a variant possessing a desired new or improved activity with a non-natural substrate (Fig. 1b). This broader scope would be compatible with the development of biocatalysts active upon non-biogenic compounds for which no biosynthetic pathway exists in any organism, so cannot be produced using introduced heterologous pathways, which represent the great majority of chemotypes used as precursors and intermediates in synthetic chemistry. Reports published during the present study applied such selection using acetoin[11,13], but E. coli contains native acetoin reductase activity[11,15], and our present study found acetoin alone was sufficient to rescue the metabolic defect, so acetoin cannot be considered isolated from native E. coli metabolism
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