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Synthetic NAD(P)(H) Cycle for ATP Regeneration
1Department of Chemical Engineering, Columbia University, New York, New York 10027, United States.
ACS Synthetic Biology
|June 27, 2023
Summary
This study presents a novel enzymatic cascade for regenerating adenosine triphosphate (ATP), the cell's energy currency. This method offers a sustainable solution for biotechnology applications like synthetic cells.
Area of Science:
- Biochemistry
- Synthetic Biology
- Enzymology
Background:
- Adenosine triphosphate (ATP) is essential for cellular functions.
- Efficient ATP regeneration is crucial for emerging biotechnologies, including synthetic cells.
- Current methods for ATP regeneration often require complex conditions or expensive components.
Purpose of the Study:
- To design and construct a membraneless enzymatic cascade for ATP regeneration.
- To utilize NAD(P)(H)-dependent oxidoreductases and substrate-specific kinases for efficient ATP synthesis.
- To demonstrate the application of this cascade in cell-free protein synthesis.
Main Methods:
- Assembled a membraneless enzymatic cascade using selected NAD(P)(H)-dependent oxidoreductases and kinases.
- Engineered enzyme specificity to prevent cross-reactions within the NAD(P)(H) cycle.
- Drove the cascade using irreversible fuel oxidation, initially formate, later methanol.
- Utilized NADH phosphorylation to NADPH and subsequent phosphate transfer to ADP via NAD+ kinase.
Main Results:
- Achieved high ATP regeneration rates (up to 0.74 mmol/L/h) sustained over hours.
- Demonstrated over 90% conversion of ADP to ATP using monophosphate.
- Successfully employed the regenerated ATP in cell-free protein synthesis reactions.
- Enhanced ATP production rates when powered by methanol oxidation.
Conclusions:
- Developed a simple, efficient in vitro ATP regeneration system using an enzymatic cascade.
- The system avoids the need for pH gradients or costly phosphate donors.
- This NAD(P)(H) cycle-based approach offers a versatile platform for biotechnological applications requiring sustained ATP supply.
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