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Related Concept Videos

Other Glycolytic Pathways01:24

Other Glycolytic Pathways

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The pentose phosphate pathway (PPP) operates in parallel with glycolysis, facilitating the metabolism of both pentoses and glucose. This pathway consists of two distinct phases: the oxidative and non-oxidative phases. While it does not directly generate ATP, the intermediates formed during the process can integrate into glycolysis, contributing to cellular energy metabolism when required.Oxidative Phase: NADPH ProductionThe oxidative phase of the pentose phosphate pathway is primarily...
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Integrated Protein Engineering and Expression Balancing Enable Efficient d-Allulose Production.

Xue Cai1,2,3, Cheng-Hao Hu1,2, A-Yang Wu1,2

  • 1The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, P. R. China.

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|September 28, 2025
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Summary

Engineered a thermostable enzyme, d-allulose 3-epimerase (DAE), to improve allulose production. This enhanced biocatalysis enables efficient conversion of agricultural waste into valuable sugars, advancing green biomanufacturing.

Keywords:
cascade biocatalysiscoexpression systemcorncob hydrolysated-alluloseenzyme engineeringthermostability

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Area of Science:

  • Biotechnology and Biocatalysis
  • Enzyme Engineering
  • Green Chemistry

Background:

  • Allulose is a rare sugar with significant food and health applications.
  • Current biocatalytic production of allulose using d-allulose 3-epimerase (DAE) faces challenges in enzyme stability and cascade efficiency, limiting industrial scalability.
  • Low-cost substrates like agricultural residues are desirable for sustainable allulose production.

Purpose of the Study:

  • To engineer a thermostable DAE for enhanced allulose synthesis.
  • To develop an efficient one-pot cascade reaction for allulose production using low-cost substrates.
  • To improve the overall yield and sustainability of allulose biomanufacturing.

Main Methods:

  • Semirational design was used to engineer a thermostable DAE from *Clostridium cellulolyticum*.
  • A triple mutant DAE (F155Y/D281G/C289R) was created, exhibiting increased melting temperature and optimal temperature.
  • A modular coexpression system was employed for balanced expression of DAE and thermophilic glucose isomerase in a one-pot cascade.

Main Results:

  • The engineered DAE showed a 12.0 °C increase in melting temperature (Tm to 73.2 °C) and an optimal temperature shift to 60 °C.
  • The conversion rate of the engineered DAE improved from 27.5% to 33.3%.
  • The one-pot cascade achieved high d-allulose titers from glucose (18.5%), F42 fructose syrup (17.4%), and corncob hydrolysate (7.5%), outperforming existing benchmarks.

Conclusions:

  • The engineered thermostable DAE and optimized cascade system significantly enhance the efficiency and scalability of allulose production.
  • This approach enables the conversion of agricultural residues into high-value sugars, offering a cost-effective and sustainable alternative to traditional methods.
  • The study advances green biomanufacturing by improving process continuity and reducing costs associated with allulose production.