skip to main content


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
NSF-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
More Like this
  1. null (Ed.)
    Abstract Background Noncanonical redox cofactors are emerging as important tools in cell-free biosynthesis to increase the economic viability, to enable exquisite control, and to expand the range of chemistries accessible. However, these noncanonical redox cofactors need to be biologically synthesized to achieve full integration with renewable biomanufacturing processes. Results In this work, we engineered Escherichia coli cells to biosynthesize the noncanonical cofactor nicotinamide mononucleotide (NMN + ), which has been efficiently used in cell-free biosynthesis. First, we developed a growth-based screening platform to identify effective NMN + biosynthetic pathways in E. coli . Second, we explored various pathway combinations and host gene disruption to achieve an intracellular level of ~ 1.5 mM NMN + , a 130-fold increase over the cell’s basal level, in the best strain, which features a previously uncharacterized nicotinamide phosphoribosyltransferase (NadV) from Ralstonia solanacearum. Last, we revealed mechanisms through which NMN + accumulation impacts E. coli cell fitness, which sheds light on future work aiming to improve the production of this noncanonical redox cofactor. Conclusion These results further the understanding of effective production and integration of NMN + into E. coli . This may enable the implementation of NMN + -directed biocatalysis without the need for exogenous cofactor supply. 
    more » « less
  2. Abstract

    Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN+) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. Here, we establish a growth selection platform to evolve enzymes to utilize NMN+-based reducing power. This is based on an orthogonal, NMN+-dependent glycolytic pathway inEscherichia coliwhich can be coupled to any reciprocal enzyme to recycle the ensuing reduced NMN+. With a throughput of >106variants per iteration, the growth selection discovers aLactobacillus pentosusNADH oxidase variant with ~10-fold increase in NMNH catalytic efficiency and enhanced activity for other NCBs. Molecular modeling and experimental validation suggest that instead of directly contacting NCBs, the mutations optimize the enzyme’s global conformational dynamics to resemble the WT with the native cofactor bound. Restoring the enzyme’s access to catalytically competent conformation states via deep navigation of protein sequence space with high-throughput evolution provides a universal route to engineer NCB-dependent enzymes.

     
    more » « less
  3. Abstract

    Many biocatalytic processes inside cells employ substrate channeling to control the diffusion of intermediates for improved efficiency of enzymatic cascade reactions. This inspirational mechanism offers a strategy for increasing efficiency of multistep biocatalysis, especially where the intermediates are expensive cofactors that require continuous regeneration. However, it is challenging to achieve substrate channeling artificially in vitro due to fast diffusion of small molecules. By mimicking some naturally occurring metabolons, nanoreactors are developed using P22 virus‐like particles (VLPs), which enhance the efficiency of nicotinamide adenine dinucleotide (NAD)‐dependent multistep biocatalysis by substrate channeling. In this design, NAD‐dependent enzyme partners are coencapsulated inside the VLPs, while the cofactor is covalently tethered to the capsid interior through swing arms. The crowded environment inside the VLPs induces colocalization of the enzymes and the immobilized NAD, which shuttles between the enzymes for in situ regeneration without diffusing into the bulk solution. The modularity of the nanoreactors allows to tune their composition and consequently their overall activity, and also remodel them for different reactions by altering enzyme partners. Given the plasticity and versatility, P22 VLPs possess great potential for developing functional materials capable of multistep biotransformations with advantageous properties, including enhanced efficiency and economical usage of enzyme cofactors.

     
    more » « less
  4. Abstract

    The efficient fixation of excess CO2from the atmosphere to yield value‐added chemicals remains crucial in response to the increasing levels of carbon emission. Coupling enzymatic reactions with electrochemical regeneration of cofactors is a promising technique for fixing CO2, while producing biomass which can be further transformed into biofuels. Herein, a bioelectrocatalytic system was established by depositing crystallites of a mesoporous metal–organic framework (MOF), termed NU‐1006, containing formate dehydrogenase, on a fluorine‐doped tin oxide glass electrode modified with Cp*Rh(2,2′‐bipyridyl‐5,5′‐dicarboxylic acid)Cl2complex. This system converts CO2into formic acid at a rate of 79±3.4 mm h−1with electrochemical regeneration of the nicotinamide adenine dinucleotide cofactor. The MOF–enzyme composite exhibited significantly higher catalyst stability when subjected to non‐native conditions compared to the free enzyme, doubling the formic acid yield.

     
    more » « less
  5. Abstract

    The efficient fixation of excess CO2from the atmosphere to yield value‐added chemicals remains crucial in response to the increasing levels of carbon emission. Coupling enzymatic reactions with electrochemical regeneration of cofactors is a promising technique for fixing CO2, while producing biomass which can be further transformed into biofuels. Herein, a bioelectrocatalytic system was established by depositing crystallites of a mesoporous metal–organic framework (MOF), termed NU‐1006, containing formate dehydrogenase, on a fluorine‐doped tin oxide glass electrode modified with Cp*Rh(2,2′‐bipyridyl‐5,5′‐dicarboxylic acid)Cl2complex. This system converts CO2into formic acid at a rate of 79±3.4 mm h−1with electrochemical regeneration of the nicotinamide adenine dinucleotide cofactor. The MOF–enzyme composite exhibited significantly higher catalyst stability when subjected to non‐native conditions compared to the free enzyme, doubling the formic acid yield.

     
    more » « less