Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform
Linyue Zhang (University of California)
Edward King (University of California)
William B. Black (University of California)
Christian M. Heckmann (TU Delft - BT/Biocatalysis)
Allison Wolder (TU Delft - BT/Biocatalysis)
Youtian Cui (University of California)
Francis Nicklen (University of California)
Justin B. Siegel (University of California)
Caroline E. Paul (TU Delft - BT/Biocatalysis)
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Abstract
Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN+). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN+ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN+ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA+), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)+.
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