Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

Linyue Zhang, Edward King, William B. Black, Christian M. Heckmann, Allison Wolder, Youtian Cui, Francis Nicklen, Justin B. Siegel, Ray Luo, Caroline E. Paul and Han Li ()
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Linyue Zhang: University of California Irvine
Edward King: University of California Irvine
William B. Black: University of California Irvine
Christian M. Heckmann: Delft University of Technology
Allison Wolder: Delft University of Technology
Youtian Cui: University of California, Davis, One Shields Avenue
Francis Nicklen: University of California Irvine
Justin B. Siegel: University of California, Davis, One Shields Avenue
Ray Luo: University of California Irvine
Caroline E. Paul: Delft University of Technology
Han Li: University of California Irvine

Nature Communications, 2022, vol. 13, issue 1, 1-12

Abstract: 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)+.

Date: 2022
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DOI: 10.1038/s41467-022-32727-w

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