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  • Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform
Title: Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform
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 inEscherichia colito 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)+.  more » « less
Award ID(s):
1847705
PAR ID:
10370089
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ;
Publisher / Repository:
Nature Publishing Group
Date Published:
Journal Name:
Nature Communications
Volume:
13
Issue:
1
ISSN:
2041-1723
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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