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

Abstract

Cellulosomes are multi-enzyme complexes produced by specialised micro-organisms. The spatial proximity of synergistically acting enzymes incorporated in these naturally occurring complexes supports the efficient hydrolysis of lignocellulosic biomass. Several functional designer cellulosomes, incorporating naturally non-cellulosomal cellulases, have been constructed and can be used for cellulose saccharification. However, in lignocellulosic biomass, cellulose is tightly intertwined with several hemicelluloses and lignin. One of the most abundant hemicelluloses interacting with cellulose microfibrils is xyloglucan, and degradation of these polymers is crucial for complete saccharification. Yet, designer cellulosome studies focusing on the incorporation of hemicellulases have been limited. Here, we report the conversion of the free Cellvibrio japonicus xyloglucan degradation system to the cellulosomal mode. Therefore, we constructed multiple docking enzyme variants of C. japonicus endoxyloglucanase, β-1,2-galactosidase, α-1,6 xylosidase and β-1,4-glucosidase, using the combinatorial VersaTile technique dedicated to the design and optimisation of modular proteins. We individually optimised the docking enzymes to degrade the xyloglucan backbone and side chains. Finally, we show that a purified designer xyloglucanosome comprising these docking enzymes was able to release xyloglucan oligosaccharides, galactose, xylose and glucose from tamarind xyloglucan. KEY POINTS: • Construction of xyloglucan-degrading designer cellulosome. • Conversion of free Cellvibrio japonicus enzymes to cellulosomal mode. • Type of linker inserted between dockerin and enzyme module affects docking enzyme activity.

Keywords: DNA assembly; Designer cellulosome; Hemicellulase; Multi-enzyme complex; VersaTile; Xyloglucan.