Combinatorial Assembly and Optimisation of Designer Cellulosomes - A Galactomannan Case Study

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.

Topological sorting and well-formed strings1

A topological sorting of a tree is a linear ordering of its elements that preserves and extends their partial ordering in the tree. Any such arrangement of tree elements has a characteristic property, involving collective weights of initial or final substrings. Such properties have been investigated in connection with random walks, combinatorial arrangements, and the well-formed strings of parenthesis-free notations. Topological sortings of directed acyclic graphs exhibit a similar structure.

Construction of BIBD'S from OA'S and Combinatorial Arrangements Analogous to OA'S

Summary The paper considers a generalisation of the result by Ramanujacharyulu (1966). Orthogonal arrays of type II defined by Rao (1961) are utilised for the purpose. It also contains a generalisation of a theorem by Shrikhande and Bose (1960) on the construction of BIBD's with the help of OA's of strength 2.