Engineering natural and noncanonical nicotinamide cofactor-dependent enzymes: design principles and technology development

King, Edward J.; Maxel, Sarah; Li, Han

doi:10.1016/j.copbio.2020.08.005

Cited by 30 publications

(29 citation statements)

References 50 publications

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“…Another major factor that will require additional developments is cofactor costs. We will need to develop methods for making cofactors more cheaply or employ cheaper cofactor analogs 36 – 38 . CoA is particular problematic in this regard so lowering CoA costs would be important enabling technology for cell-free manufacturing and therefore should be a focus of research in cell-free systems 21 .…”

Section: Discussionmentioning

confidence: 99%

Expanding the use of ethanol as a feedstock for cell-free synthetic biochemistry by implementing acetyl-CoA and ATP generating pathways

Liu

Arbing

Bowie

2022

Sci Rep

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Ethanol is a widely available carbon compound that can be increasingly produced with a net negative carbon balance. Carbon-negative ethanol might therefore provide a feedstock for building a wider range of sustainable chemicals. Here we show how ethanol can be converted with a cell free system into acetyl-CoA, a central precursor for myriad biochemicals, and how we can use the energy stored in ethanol to generate ATP, another key molecule important for powering biochemical pathways. The ATP generator produces acetone as a value-added side product. Our ATP generator reached titers of 27 ± 6 mM ATP and 59 ± 15 mM acetone with maximum ATP synthesis rate of 2.8 ± 0.6 mM/h and acetone of 7.8 ± 0.8 mM/h. We illustrated how the ATP generating module can power cell-free biochemical pathways by converting mevalonate into isoprenol at a titer of 12.5 ± 0.8 mM and a maximum productivity of 1.0 ± 0.05 mM/h. These proof-of-principle demonstrations may ultimately find their way to the manufacture of diverse chemicals from ethanol and other simple carbon compounds.

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Section: Discussionmentioning

confidence: 99%

Expanding the use of ethanol as a feedstock for cell-free synthetic biochemistry by implementing acetyl-CoA and ATP generating pathways

Liu

Arbing

Bowie

2022

Sci Rep

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“…Directed evolution is an effective strategy to engineer enzymes with desired cofactor speci city. [11][12][13] However, rapid directed evolution of enzyme with desired properties is often hindered by the lack of e cient and affordable methods that can correctly identify rare positive hits from diverse mutant libraries, especially involving enormous mutant libraries. [14] Previous high-throughput screening strategies to identify dehydrogenase mutant activity on mNADs have been established depending on mass spectrometry and absorbance spectroscopic change during catalysis in multiple well plate.…”

Section: Introductionmentioning

confidence: 99%

“…[14] Previous high-throughput screening strategies to identify dehydrogenase mutant activity on mNADs have been established depending on mass spectrometry and absorbance spectroscopic change during catalysis in multiple well plate. [2,3,11,15] However, these approaches are labor-intensive and time-consuming resulting in a low throughput without the robotic systems. [16] By contrast, the growth complementation method, which couples the examined enzyme property with the tness of the host cell, is not dependent on intensive labor.…”

Section: Introductionmentioning

confidence: 99%

A Phosphite-Based Sscreening Platform for Identification of Enzymes Favoring Nonnatural Cofactors

Liu

Guo

et al. 2021

Preprint

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BackgroundEnzymes with dedicated cofactor preference are essential for advanced biocatalysis and biomanufacturing. However, directed evolution of an enzyme to switch its cofactor preference is often hindered by the lack of efficient and affordable method for screening as the cofactor per se or the substrate can be prohibitively expensive. Here, we developed a growth-based selection platform to identify nonnatural cofactor-dependent oxidoreductase mutants.ResultsThe growth-based selection platform was designed by coupling with nonnatural cofactor-dependent phosphite dehydrogenase (Pdh) mediated the conversion of non-metabolizable phosphite into phosphate in the culture media. Thus, Pdh variant that strongly favors nicotinamide cytosine dinucleotide (NCD), a NAD analogue, the feasibility of this strategy was successfully demonstrated using derived NCD-active malic enzyme as well as for the directed evolution of NCD synthetase in Escherichia coli.ConclusionsHere, we built a phosphite-based screening platform for identification of enzymes favoring nonnatural cofactor NCD. In the future, once Pdh variants favoring other biomimetic or nonnatural cofactors are available this selection platform may be readily redesigned to attain new enzyme variants with anticipated cofactor preference, providing opportunities to further expand the chemical space of redox cofactors in chemical biology and synthetic biology.

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“…There is increasing interest in replacing these expensive biological cofactors by synthetic, artificial cofactors, Figure 1a(ii). [1][2][3][4] Although these artificial cofactors can be produced cost-effectively, it is still undesirable to use them at stoichiometric levels due to poor atom economy imposed on the reaction, the cost of product clean-up and possible inhibition of enzymes at high cofactor concentrations. For these reasons, methods for recycling the artificial cofactors are required.…”

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confidence: 99%

A hydrogen-driven biocatalytic approach to recycling synthetic analogues of NAD(P)H

et al. 2022

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Engineering natural and noncanonical nicotinamide cofactor-dependent enzymes: design principles and technology development

Cited by 30 publications

References 50 publications

Expanding the use of ethanol as a feedstock for cell-free synthetic biochemistry by implementing acetyl-CoA and ATP generating pathways

Expanding the use of ethanol as a feedstock for cell-free synthetic biochemistry by implementing acetyl-CoA and ATP generating pathways

A Phosphite-Based Sscreening Platform for Identification of Enzymes Favoring Nonnatural Cofactors

A hydrogen-driven biocatalytic approach to recycling synthetic analogues of NAD(P)H

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