초록
<P>Bacteria undergoing anaerobic fermentation must maintain redox balance. <I>In vivo</I> metabolic evolution schemes based on this principle have been limited to targeting NADH-dependent reactions. Here, we developed a facile, specific, and high-throughput growth-based selection platform for NADPH-consuming reactions <I>in vivo</I>, based on an engineered NADPH-producing glycolytic pathway in <I>Escherichia coli</I>. We used the selection system in the directed evolution of a NADH-dependent <SMALL>D</SMALL>-lactate dehydrogenase from <I>Lactobacillus delbrueckii</I> toward utilization of NADPH. Through one round of selection, we obtained multiple enzyme variants with superior NADPH-dependent activities and protein expression levels; these mutants may serve as important tools in biomanufacturing <SMALL>D</SMALL>-lactate as a renewable polymer building block. Importantly, sequence analysis and computational protein modeling revealed that diverging evolutionary paths during the selection resulted in two distinct cofactor binding modes, which suggests that the high throughput of our selection system allowed deep searching of protein sequence space to discover diverse candidates <I>en masse</I>.</P><BR>[FIG OMISSION]</BR>