Combinatorial assembly and optimisation of designer cellulosomes: a galactomannan case study

Julie Vanderstraeten, Maria João Maurício da Fonseca, Philippe De Groote, Dennis Grimon, Hans Gerstmans, Amaranta Kahn, Sarah Moraïs, Edward A. Bayer, Yves Briers

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Background: Designer cellulosomes are self-assembled chimeric enzyme complexes that can be used to improve lignocellulosic biomass degradation. They are composed of a synthetic multimodular backbone protein, termed the scaffoldin, and a range of different chimeric docking enzymes that degrade polysaccharides. Over the years, several functional designer cellulosomes have been constructed. Since many parameters influence the efficiency of these multi-enzyme complexes, there is a need to optimise designer cellulosome architecture by testing combinatorial arrangements of docking enzyme and scaffoldin variants. However, the modular cloning procedures are tedious and cumbersome. Results: VersaTile is a combinatorial DNA assembly method, allowing the rapid construction and thus comparison of a range of modular proteins. Here, we present the extension of the VersaTile platform to facilitate the construction of designer cellulosomes. We have constructed a tile repository, composed of dockerins, cohesins, linkers, tags and enzymatically active modules. The developed toolbox allows us to efficiently create and optimise designer cellulosomes at an unprecedented speed. As a proof of concept, a trivalent designer cellulosome able to degrade the specific hemicellulose substrate, galactomannan, was constructed and optimised. The main factors influencing cellulosome efficiency were found to be the selected dockerins and linkers and the docking enzyme ratio on the scaffoldin. The optimised designer cellulosome was able to hydrolyse the galactomannan polysaccharide and release mannose and galactose monomers. Conclusion: We have eliminated one of the main technical hurdles in the designer cellulosome field and anticipate the VersaTile platform to be a starting point in the development of more elaborate multi-enzyme complexes.

Original languageEnglish
Article number60
JournalBiotechnology for Biofuels and Bioproducts
Volume15
Issue number1
DOIs
StatePublished - 1 Dec 2022

Keywords

  • DNA assembly
  • Designer cellulosome
  • Galactomannan
  • Galactosidase
  • Hemicellulase
  • Mannanase
  • Mannosidase
  • Multi-enzyme complex
  • Scaffoldin
  • VersaTile

ASJC Scopus subject areas

  • Biotechnology
  • Energy (miscellaneous)
  • Renewable Energy, Sustainability and the Environment
  • Management, Monitoring, Policy and Law
  • Applied Microbiology and Biotechnology

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