A Phosphite-Based Sscreening Platform for Identification of Enzymes Favoring Nonnatural Cofactors

Background: Enzymes with dedicated cofactor preference are essential for advanced biocatalysis and biomanufacturing. However, directed evolution of an enzyme to switch its cofactor preference is often hindered by the lack of efficient and affordable method for screening as the cofactor per se or the substrate can be prohibitively expensive. Here, we developed a growth-based selection platform to identify nonnatural cofactor-dependent oxidoreductase mutants.ResultsThe growth-based selection platform was designed by coupling with nonnatural cofactor-dependent phosphite dehydrogenase (Pdh) mediated the conversion of non-metabolizable phosphite into phosphate in the culture media. Thus, Pdh variant that strongly favors nicotinamide cytosine dinucleotide (NCD), a NAD analogue, the feasibility of this strategy was successfully demonstrated using derived NCD-active malic enzyme as well as for the directed evolution of NCD synthetase in Escherichia coli .ConclusionsHere, we built a phosphite-based screening platform for identification of enzymes favoring nonnatural cofactor NCD. In the future, once Pdh variants favoring other biomimetic or nonnatural cofactors are available this selection platform may be readily redesigned to attain new enzyme variants with anticipated cofactor preference, providing opportunities to further expand the chemical space of redox cofactors in chemical biology and synthetic biology.