||The recalcitrance of lignocellulosic biomass to enzymic digestion remains a significant obstacle to the adoption of an environmentally and economically sustainable strategy for the synthesis of biomaterials. Traditional industrial pre-treatments are harsh, require significant investments of energy and money, and tend to produce degradation products which inhibit downstream processes. Carbohydrate Active enZymes (CAZymes) may reduce recalcitrance, through heterologous expression directly in the lignocellulosic biomass. CAZymes from extremely thermophilic organisms are not normally active at the mesophilic temperatures, allowing for accumulation in the biomass without negatively affecting the growth and development of the plant. Harvested biomass could then be heat-treated, activating the CAZymes and inducing hydrolysis of the biomass. Additionally, chimeric thermostable enzymes could be constructed from extremely thermophilic CAZyme domains, tailored to target specific biopolymers and perform directed modifications. However, while full-length CAZymes have been investigated, the extent of lignocellulose degrading capacity of extremely thermophilic CAZyme domains has not been assessed and the ability to produce and express chimeric CAZymes in planta has not been determined. In this thesis, the CAZyme domain content of extremely thermophilic organisms was surveyed and capacity for degradation of lignocellulose was assessed. A list of CAZyme domains from extremely thermophilic organisms was produced via HMMER analysis. There were differences in CAZyme composition between extremely thermophilic archaea and bacteria, which could be mainly attributed to differences in nutritional strategy as well as synthesis, composition and structure of the cell walls in the organisms. Many putative lignocellulose degrading and targeting domains were present in the dataset, identified mostly in bacteria, though some were found only in archaea. It was also seen that more CAZyme domain variants and CAZyme domain classes are likely to be identified as more genomes of extremely thermophilic organisms are sequenced.
Additionally, a chimeric CAZyme consisting of a thermostable GH11 domain and plant-derived CBM22 domains designated Xyl22L was designed, synthesised and heterologously expressed in Arabidopsis thaliana. The effect on growth and development of the plant as well as recalcitrance to enzymic digestion of the biomass was determined. Xyl22L did not retain catalytic xylanase activity but was able to accumulate in transgenic plant biomass, and expression of Xyl22L was strongly correlated with an increase in transgenic plant biomass. Fluorescent confocal microscopy showed that Xyl22L was associated with the secondary cell wall (SCW) in transgenic plants, indicating that the CBM22 domains retained function. Finally, transgenic plant lines showed increased recalcitrance to enzymic digestion, possibly through Xyl22L adhering to the SCW and preventing access of hydrolytic enzymes.
This work provides a list of extremely thermophilic CAZyme domains, providing insight into the survival and evolution of extremely thermophilic organisms as well as a toolbox of thermostable domains for the synthesis of custom chimeric enzymes. Additionally, this work provides an example of such an enzyme, and provides proof of concept that plant-based CBMs may be used to target enzymes to specific biopolymers or locations in plant biomass. Together, these findings could be applied to white biotechnological processes, allowing for cheaper and more energy efficient bioproduct synthesis, enabling a transition away from a petrochemical-based products.