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Citrate as Cost-Efficient NADPH Regenerating Agent.

Reinhard Oeggl1,2, Timo Neumann1, Jochem Gätgens1

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|January 12, 2019
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Summary
This summary is machine-generated.

Efficiently regenerate nicotinamide adenine dinucleotide phosphate (NADPH) using citrate and native TCA cycle enzymes in whole cells, cell extracts, or lyophilized cells. This simple method enhances cofactor regeneration for enzymatic applications.

Keywords:
NADPH regenerationcitrate oxidationcofactor regenerationnicotinamide cofactoroxidoreductase screeningreduction equivalent regeneration

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Area of Science:

  • Biotechnology
  • Enzyme Catalysis
  • Metabolic Engineering

Background:

  • Efficient cofactor regeneration is crucial for NAD(P)H-dependent enzymes.
  • Current methods often involve substrate supplementation or additional enzymes.
  • Whole-cell nicotinamide adenine dinucleotide phosphate (NADPH) regeneration is a simpler alternative.

Purpose of the Study:

  • To present an easy-to-apply whole-cell cofactor regeneration approach using citrate.
  • To demonstrate the utility of this method in screening applications.
  • To investigate the mechanism of NADPH regeneration by TCA cycle enzymes.

Main Methods:

  • Utilizing citrate or citrate/phosphate buffer for NADPH regeneration via native TCA cycle enzymes.
  • Applying the method with viable-culturable cells, lyophilized cells, and crude cell extracts.
  • Investigating the regeneration mechanism using transient isotopic labeling with [1,5-13C]citrate.

Main Results:

  • Citrate addition effectively regenerates NADPH using native enzymes in various cell preparations.
  • The method was successfully applied to the synthesis of 1-phenylethanol from acetophenone.
  • Isotopic labeling revealed optimization potential by modifying the glyoxylate shunt and glutamate dehydrogenase pathways.

Conclusions:

  • Citrate-based NADPH regeneration is a versatile and simple method for enzymatic applications.
  • This approach is applicable across different cell states, including lyophilized cells and extracts.
  • Genetic modification of specific metabolic pathways can further enhance cofactor regeneration efficiency.