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ATP Energy Storage and Release01:31

ATP Energy Storage and Release

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ATP is a highly unstable molecule. Unless quickly used to perform work, ATP spontaneously dissociates into ADP and inorganic phosphate (Pi), and the free energy released during this process is lost as heat. The energy released by ATP hydrolysis is used to perform work inside the cell and depends on a strategy called energy coupling. Cells couple the exergonic reaction of ATP hydrolysis with endergonic reactions, allowing them to proceed.
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In cellular metabolism (the complete breakdown of glucose to extract energy),  glycolysis is the first step. Glycolysis takes place in the cytoplasm of both prokaryotic and eukaryotic cells. Glucose enters heterotrophic cells in two ways. One method is through secondary active transport, where the transport takes place against the glucose concentration gradient. The other mechanism uses a group of integral proteins called GLUT proteins, also known as glucose transporter proteins. These...
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Cell-Free Reaction System for ATP Regeneration from d-Fructose.

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Summary
This summary is machine-generated.

This study presents a cost-effective cell-free adenosine triphosphate (ATP) regeneration system using d-fructose. Optimized enzymes achieve high ATP yields, making industrial bioprocesses more feasible.

Keywords:
ATP cofactor regenerationacetyl phosphate synthesisin vitro biocatalysismolecular modelingphosphoketolasesemirational enzyme engineering

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Area of Science:

  • Biotechnology
  • Enzyme Engineering
  • Metabolic Engineering

Background:

  • Adenosine triphosphate (ATP)-dependent in vitro bioprocesses are crucial for industrial applications like cell-free protein synthesis.
  • The high cost of ATP is a major limitation for the widespread implementation of these bioprocesses.
  • Developing efficient and economical ATP regeneration systems is essential for advancing these technologies.

Purpose of the Study:

  • To develop and demonstrate a novel cell-free ATP regeneration system using low-cost substrates.
  • To engineer an improved phosphoketolase enzyme for enhanced ATP production efficiency.
  • To optimize the overall enzymatic cascade for maximizing ATP yield and productivity.

Main Methods:

  • Designed a cell-free system utilizing acetyl phosphate generated from d-fructose and inorganic phosphate.
  • Employed a semirational engineering approach to optimize Bifidobacterium adolescentis phosphoketolase (Bad.F6Pkt).
  • Integrated engineered Bad.F6Pkt, acetate kinase, glycerol kinase, and l-rhamnose isomerase into a complete ATP regeneration cascade.

Main Results:

  • Engineered Bad.F6Pkt variant H548N showed significantly increased activity for d-fructose, d-erythrulose, and glycolaldehyde.
  • Demonstrated ATP regeneration from d-fructose with a stoichiometry of 1 mol ATP per mol C6 ketose.
  • The complete system achieved an ATP yield of 2.53 mol ATP per mol d-fructose with a productivity of 7.2 mM/h.

Conclusions:

  • The developed cell-free ATP regeneration system effectively utilizes low-cost d-fructose.
  • Enzyme engineering of phosphoketolase is critical for improving ATP production efficiency.
  • This system offers a cost-effective solution for industrial ATP-dependent bioprocesses.