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

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.

Conversion of the free Cellvibrio japonicus xyloglucan degradation system to the cellulosomal mode

“…To aid the interpretation of the results, a theoretical degradation map is visualised in the bottom right corner ◂ other polysaccharides present in the intricate lignocellulosic matrix. Before, we have constructed a designer cellulosome active on the major softwood hemicellulose, galactomannan (GM) (Vanderstraeten et al 2022). Three different enzyme modules were incorporated into this complex.…”

“…In our previous work, multiple parameters, linked to both docking enzyme and scaffoldin architecture, influencing the designer cellulosome efficiency were analysed (Vanderstraeten et al 2022). However, the expression and purification of the large number of proteins was an immense effort, especially since several tile combinations led to unstable proteins.…”

“…Each primer (Integrated DNA Technologies, Leuven, Belgium) contained the following necessary parts (listed starting from the coding sequence to outwards): a position tag, a BsaI recognition site and another type IIs (SapI, BpiI, BseRI, BsmBI, BtgZI or BfuAI) restriction and recognition site. Primers used to amplify cohesin, dockerin, CBM and linker tiles were listed previously (Vanderstraeten et al 2022). Primers used to amplify the tiles encoding XG-degrading enzymes are listed in Supplemental Table S1.…”

“…In our previous work (Vanderstraeten et al 2022), we demonstrated the substantial benefit of VersaTile at the DNA level to make multiple docking enzyme variants. However, expression of the high number of proteins remained a tedious task.…”

“…We have developed a threeway assembly system to construct docking enzymes and a five-way assembly system to construct scaffoldins. Recently, we applied the technique to illustrate the multiparametric optimisation of a cellulosome complex specifically designed for galactomannan degradation (Vanderstraeten et al 2022). There, we observed that the major determinants of docking enzyme efficiency are the selected dockerin and the type of linker inserted between the enzymatically active module and dockerin.…”