Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform
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) + .
- Publication Date:
- Research Org.:
- Univ. of California, Irvine, CA (United States)
- Sponsoring Org.:
- USDOE Advanced Research Projects Agency - Energy (ARPA-E); National Science Foundation (NSF); National Institutes of Health (NIH); Alfred Sloan Research fellowship; National Institute of Environmental Health Sciences; European Research Council (ERC); European Union’s Horizon 2020
- OSTI Identifier:
- 1883631
- Alternate Identifier(s):
- OSTI ID: 1904184
- Grant/Contract Number:
- AR0001508; 1847705; DP2 GM137427; P42ES004699; R01 GM 076324-11; 1627539; 1805510; 1827246; GM130367; 949910
- Resource Type:
- Published Article
- Journal Name:
- Nature Communications
- Additional Journal Information:
- Journal Name: Nature Communications Journal Volume: 13 Journal Issue: 1; Journal ID: ISSN 2041-1723
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United Kingdom
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; applied microbiology; enzyme mechanisms; enzymes
Citation Formats
Zhang, Linyue, King, Edward, Black, William B., Heckmann, Christian M., Wolder, Allison, Cui, Youtian, Nicklen, Francis, Siegel, Justin B., Luo, Ray, Paul, Caroline E., and Li, Han. Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform. United Kingdom: N. p., 2022.
Web. doi:10.1038/s41467-022-32727-w.
Zhang, Linyue, King, Edward, Black, William B., Heckmann, Christian M., Wolder, Allison, Cui, Youtian, Nicklen, Francis, Siegel, Justin B., Luo, Ray, Paul, Caroline E., & Li, Han. Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform. United Kingdom. https://doi.org/10.1038/s41467-022-32727-w
Zhang, Linyue, King, Edward, Black, William B., Heckmann, Christian M., Wolder, Allison, Cui, Youtian, Nicklen, Francis, Siegel, Justin B., Luo, Ray, Paul, Caroline E., and Li, Han. Fri .
"Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform". United Kingdom. https://doi.org/10.1038/s41467-022-32727-w.
@article{osti_1883631,
title = {Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform},
author = {Zhang, Linyue and King, Edward and Black, William B. and Heckmann, Christian M. and Wolder, Allison and Cui, Youtian and Nicklen, Francis and Siegel, Justin B. and Luo, Ray and Paul, Caroline E. and Li, Han},
abstractNote = {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) + .},
doi = {10.1038/s41467-022-32727-w},
journal = {Nature Communications},
number = 1,
volume = 13,
place = {United Kingdom},
year = {Fri Aug 26 00:00:00 EDT 2022},
month = {Fri Aug 26 00:00:00 EDT 2022}
}
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