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A Programmable Bidirectional Dynamic Switch Overcomes Reversible Isomerization Reaction for Efficient d-Allulose

Wei Zhang1, Feng Yang1, Shiqiang Yue1

  • 1Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education; Tianjin Key Laboratory of Industrial Microbiology; College of Biotechnology, National Engineering Laboratory for Industrial Enzymes, Tianjin University of Science and Technology, Tianjin 300457, P. R. China.

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Summary
This summary is machine-generated.

Researchers engineered a novel redox-driven pathway for d-allulose biosynthesis in single cells. This method overcomes thermodynamic limitations, achieving high yields of the rare sugar d-allulose using programmable protein switches.

Keywords:
D-allulosedynamic regulationmultienzyme cascade catalysispost-translational regulationprotease-based programmable toolbox

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Area of Science:

  • Biotechnology
  • Metabolic Engineering
  • Synthetic Biology

Background:

  • d-Allulose biosynthesis via D-fructose isomerization is thermodynamically limited, leading to low yields.
  • Enzyme incompatibility in cascade reactions hinders efficient d-allulose production.
  • Developing robust biosynthetic pathways requires precise control over enzyme activity and levels.

Purpose of the Study:

  • To redesign a redox-driven cascade pathway for enhanced d-allulose biosynthesis in single-cell systems.
  • To utilize post-translational protein-level reprogramming tools to overcome enzyme incompatibility.
  • To establish a controllable system for rewiring reverse carbon flows.

Main Methods:

  • Engineered a protease-based programmable OFF/ON-switch toolbox for protein-level reprogramming.
  • Optimized the OFF/ON-switch by adjusting expression levels of mf-Lon, degron, and repressors.
  • Controlled enzyme expression using a thermosensitive regulator for precise switching within 2 hours.
  • Integrated specific enzymes (KEase, RDH, FDH, ADH, NOX) under the control of the OFF/ON switches.

Main Results:

  • Achieved a high d-allulose titer of 190.7 g/L.
  • Reached a conversion rate of 95.4%, significantly overcoming thermodynamic equilibrium limitations.
  • Demonstrated simultaneous switching of protein abundance to desired states using the optimized OFF/ON modules.
  • Engineered strain T3 successfully synthesized d-allulose via the constructed redox-driven pathway.

Conclusions:

  • The developed redox-driven cascade pathway and programmable protein switch toolbox enable efficient d-allulose biosynthesis.
  • This approach provides a versatile strategy for controlling enzyme levels and rewiring metabolic pathways.
  • The study showcases the potential of post-translational reprogramming for overcoming biosynthetic challenges in microbial systems.