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Updated: Dec 30, 2025

Protein Engineering by Yeast Surface Display
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Constructing a yeast to express the largest cellulosome complex on the cell surface.

Marimuthu Anandharaj1,2,3, Yu-Ju Lin1, Rizwana Parveen Rani1

  • 1Biodiversity Research Center, Academia Sinica, 11529 Taipei, Taiwan.

Proceedings of the National Academy of Sciences of the United States of America
|January 19, 2020
PubMed
Summary

Scientists engineered a yeast to produce a powerful cellulosome, nature's enzyme complex for breaking down cellulose. This engineered cellulosome efficiently degrades plant material and produces higher ethanol yields than previous systems.

Keywords:
anchoring proteincellulose-binding modulecellulosomescaffoldin protein

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Area of Science:

  • Biotechnology
  • Synthetic Biology
  • Enzyme Engineering

Background:

  • Cellulosomes are highly efficient natural enzyme complexes for cellulose degradation.
  • Constructing functional cellulosomes in industrial yeasts is challenging due to gene size and complexity.

Purpose of the Study:

  • To engineer a probiotic yeast (*Kluyveromyces marxianus*) for cellulosome production.
  • To overcome challenges in synthesizing and expressing large cellulosome genes in a yeast host.

Main Methods:

  • Synthesized scaffoldin (CipA) and anchoring protein (OlpB) genes from *Clostridium thermocellum*.
  • Engineered *K. marxianus* to display dockerin-fused fungal cellulases on its surface.
  • Utilized confocal microscopy and fluorescence-activated cell sorting for cell-surface display confirmation.

Main Results:

  • Achieved efficient cell-surface display of the anchoring protein (OlpB-ScGPI) in 81% of yeast cells.
  • Confirmed cellulosome complex formation using gel electrophoresis and mass spectrometry.
  • Engineered cellulosome accommodated up to 63 enzymes, significantly larger than previous constructs.
  • Demonstrated enhanced degradation of cellulosic substrates and higher ethanol production (3.09 g/L from avicel, 8.61 g/L from phosphoric acid-swollen cellulose).

Conclusions:

  • Successfully constructed a highly efficient, chromosomally integrated cellulosome in *K. marxianus*.
  • Highlighted the importance of cohesin numbers and cellulose binding modules (CBM) for cellulosome function.
  • The engineered yeast presents a promising platform for advanced biofuel production and biomass degradation.