Recombinant cellulase production in plants

Enzyme-catalyzed lignocellulose conversion is a promising pathway to increase the feasibility of second generation biofuel production. The development of viable processes for enzymatic lignocellulose utilization must address different issues to become economically feasible. One important issue is the production of appropriate and cost-effective enzymes to deconstruct the complex and heterogeneous structure of the lignocellulose. Plant based expression of cell wall degrading enzymes is a promising technique, but it currently faces a number of challenges, such as enzyme expression levels and auto-hydrolysis of the plant cell walls. This thesis deals with establishing and evaluating cellulase expression strategies in tobacco plants, including differential protein targeting, inducible protein expression and the implementation of enzymes with different biochemical properties, such as thermophile. Different approaches of in planta produced cellulases are evaluated by the analysis and their properties as well as by detailed phenotypical analyses, such as biochemical characterization of plant cell walls. Constitutive high level expression of a mesophilic cellulase led to detrimental changes in the plant phenotype, including growth and development. The retention of the enzyme inside the endoplasmic reticulum could not completely avoid this impact on the plant development. The application of an alcohol inducible promoter for cellulase production led to enzyme expression rate without any significant phenotypical effects on the plant. Hyperthermophilic cellulases are inactive under normal plant growth conditions, allowing their expression in plants without negative effects on growth and development. Subsequent heat-inducible activation enables lignocellulose degradation. The expression rate of these hyperthermophilic enzymes is at lower levels than that of mesophilic enzymes. However, this enzyme was shown to survive harsh pretreatment conditions. Therefore it is a promising candidate for an advanced enzymatic conversion of lignocellulosic biomass. This study established advanced basic knowledge for plant based production of cellulolytic enzymes and subsequently the developed and evaluated new production strategies for cellulases would provide an advanced enzymatic conversion processes of plant biomass