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Enzyme colocalisation on synthetic protein scaffolds : a VersaTile approach

(2022)
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(UGent) and (UGent)
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Abstract
In nature, enzymes are often colocalised to promote substrate channeling and to enhance synergism. Synthetic protein scaffolds are state-of-the-art multimodular proteins that can be employed to mimic this natural phenomenon and bring multiple enzymes in close proximity in a laboratory environment. A synthetic protein scaffold is composed of a range of cohesins, which are separated by linkers. Enzymes can be recruited to the scaffold by fusing them to dockerins that have a strong affinity for cohesins. Many different parameters have an influence on the efficiency of these multi-enzyme complexes. On the one hand, the selection of cohesin, dockerin, enzyme and linker domains has a major impact. On the other hand, protein engineers are also able to control the spatial organisation and enzyme stoichiometry within the scaffold. The optimisation of these multi-modular protein complexes is therefore a complex and often empirical process. Due to the high modularity of the interacting proteins and the extensive DNA work to prepare all components, this is a particularly tedious job. In this work, we describe the extension of the VersaTile technique, a rapid DNA assembly method for modular proteins, to allow the efficient construction of synthetic protein scaffolds and their corresponding docking enzymes. As a proof of concept, we illustrate the multiparametric optimisation of three distinct multi-enzyme complexes. Two of the constructed complexes are catabolic, allowing the complete degradation of hemicelluloses galactomannan and xyloglucan, respectively. The final complex is anabolic, incorporating three pathway enzymes to convert a simple glucose monomer to fructose-1,6-biphosphate. With VersaTile, we have removed a major hurdle in the field. However, the in vitro production and optimisation process remains labour intensive. Future research should therefore focus on the parallel production and characterisation of synthetic protein scaffolds. Tackling this last hurdle will allow us to fully exploit both the combinatorial power offered by VersaTile and the opulence provided by nature.

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MLA
Vanderstraeten, Julie. Enzyme Colocalisation on Synthetic Protein Scaffolds : A VersaTile Approach. Ghent University. Faculty of Bioscience Engineering, 2022.
APA
Vanderstraeten, J. (2022). Enzyme colocalisation on synthetic protein scaffolds : a VersaTile approach. Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium.
Chicago author-date
Vanderstraeten, Julie. 2022. “Enzyme Colocalisation on Synthetic Protein Scaffolds : A VersaTile Approach.” Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
Chicago author-date (all authors)
Vanderstraeten, Julie. 2022. “Enzyme Colocalisation on Synthetic Protein Scaffolds : A VersaTile Approach.” Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
Vancouver
1.
Vanderstraeten J. Enzyme colocalisation on synthetic protein scaffolds : a VersaTile approach. [Ghent, Belgium]: Ghent University. Faculty of Bioscience Engineering; 2022.
IEEE
[1]
J. Vanderstraeten, “Enzyme colocalisation on synthetic protein scaffolds : a VersaTile approach,” Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium, 2022.
@phdthesis{8734067,
  abstract     = {{In nature, enzymes are often colocalised to promote substrate channeling and to enhance synergism.
Synthetic protein scaffolds are state-of-the-art multimodular proteins that can be employed to mimic
this natural phenomenon and bring multiple enzymes in close proximity in a laboratory environment.
A synthetic protein scaffold is composed of a range of cohesins, which are separated by linkers.
Enzymes can be recruited to the scaffold by fusing them to dockerins that have a strong affinity for
cohesins. Many different parameters have an influence on the efficiency of these multi-enzyme
complexes. On the one hand, the selection of cohesin, dockerin, enzyme and linker domains has a
major impact. On the other hand, protein engineers are also able to control the spatial organisation
and enzyme stoichiometry within the scaffold. The optimisation of these multi-modular protein
complexes is therefore a complex and often empirical process. Due to the high modularity of the
interacting proteins and the extensive DNA work to prepare all components, this is a particularly
tedious job.
In this work, we describe the extension of the VersaTile technique, a rapid DNA assembly method for
modular proteins, to allow the efficient construction of synthetic protein scaffolds and their
corresponding docking enzymes. As a proof of concept, we illustrate the multiparametric optimisation
of three distinct multi-enzyme complexes. Two of the constructed complexes are catabolic, allowing
the complete degradation of hemicelluloses galactomannan and xyloglucan, respectively. The final
complex is anabolic, incorporating three pathway enzymes to convert a simple glucose monomer to
fructose-1,6-biphosphate. With VersaTile, we have removed a major hurdle in the field. However, the
in vitro production and optimisation process remains labour intensive. Future research should
therefore focus on the parallel production and characterisation of synthetic protein scaffolds. Tackling
this last hurdle will allow us to fully exploit both the combinatorial power offered by VersaTile and the
opulence provided by nature.}},
  author       = {{Vanderstraeten, Julie}},
  isbn         = {{9789463574730}},
  language     = {{eng}},
  pages        = {{XII, 285}},
  publisher    = {{Ghent University. Faculty of Bioscience Engineering}},
  school       = {{Ghent University}},
  title        = {{Enzyme colocalisation on synthetic protein scaffolds : a VersaTile approach}},
  year         = {{2022}},
}