Synthetic enzymatic pathway for ATP regeneration

This technology is a synthetic enzymatic cascade that can regenerate ATP from ADP using polyphosphate as a phosphate donor for in vitro ATP regeneration.

Unmet Need: Flexible and affordable energy carrier regeneration for metabolic reactions

Energy carrier supply is critical for many emerging technologies, such as cell-free protein synthesis or artificial cell construction. Current technologies attempting to regenerate ATP in cell-free protein synthesis require a constant supply of expensive phosphate donors. Alternatively, complex biomimetic organelles such as ATP synthase-embedded membranes are necessary for ATP regeneration to power artificial cells. There are currently no flexible and affordable ATP regeneration methods for artificial cell development and protein synthesis.

The Technology: Low-cost ATP regeneration for synthetic protein production and pseudo-cell development

This technology is a synthetic enzymatic cascade that affordably regenerates ATP from ADP using polyphosphates, a cheaper alternative than the currently used phosphate donors. The reactions are driven by fuel oxidation using common and low-cost fuel resources such as methanol or glucose. This cascade exploits common biological cofactors such as NADPH and recombinant enzymes, which are advantageous for producing ATP for artificial cells. The technology generates cascades via expression of NADH-dependent dehydrogenases, polyphosphate NAD+ kinases, NADPH oxidases, and ATP-NAD+ kinases. The synthetic pathway does not rely on a membrane for proton gradient and thus could achieve cell-free and membrane-free protein production.

This technology has been validated with E. coli expressing the enzymatic cascade.

Applications:

• Solution for ATP regeneration from ADP
• Powering mechanism for artificial cells
• Synthetic pseudo-cell development for actuation and mobility
• Cell-free and membrane-free protein production
• Research platform for production of complex proteins
• Continuous ATP production for processes such as muscle contraction, nerve impulse transmission, protein synthesis, ion transport, and DNA replication

Advantages:

• High-throughput platform for protein production
• Cost-effective ATP regeneration with polyphosphates
• Versatile ATP regeneration through common fuel oxidation
• Does not require a membrane
• Continuous source ATP product
• Utilizes various fuel sources

Lead Inventor:

Scott Banta, Ph.D.

Patent Information:

Patent Pending(US20240318218)

Related Publications:

• Willet E, Banta S. “Synthetic NAD(P)(H) cycle for ATP generation.” ACS Synth Biol. 2023 Jul 21; 12(7): 2118-2126.

• Willett E, Jiang V, Koder RL, Banta S. “NAD+ kinase enzymes are reversible, and NAD+ product inhibition is responsible for the observed irreversibility of the human enzyme.” Biochemistry. 2022 Sep 6; 61(17): 1862-1873.

Tech Ventures Reference:

• IR CU20363

• Licensing Contact: Dovina Qu