A novel recombination approach for cellulase engineering

Cellulose is the most abundant biopolymer on the planet and the hydrolyzation of cellulose to glucose is a rich source for industrially relevant biofuels and chemicals. Especially the development of second generation biofuels represent a promising attempt to reduce carbon dioxide emissions and increase the supply of liquid fuels. Nature has developed an array of different cellulases for the conversion of this polymer to glucose. From an industrial point of view, more thermostable cellulases for higher operating temperature would be desirable. Firstly, thermostable enzymes tend to inactivate more slowly and, as a result, produce more total product. Secondly, higher reaction temperatures raise the specific activity of enzymes (Arrhenius effect). Over the last decades major developments regarding the methodology and in understanding recombinant protein engineering have evolved. In parallel, the development of software aided approaches complemented novel protein recombination efforts. One elegant approach combining multiple sequence alignments, protein structure and software guidance for recombinant protein library design was developed in the Arnold Laboratory, referred to as SCHEMA. This facilitates the prediction of novel chimeric proteins with a very high probability of being functional. Based on this expertise, a novel recombinatorial protein engineering approach for the generation of improved cellulases will be investigated within the Arnold laboratory. The establishment of the novel non- contiguous fragment skipping approach enables the recombination of less closely related enzymes. This leads to the generation of more variable enzyme libraries. The new level of complexity obtained by this approach and the subsequent identification of stabilizing fragments will not only contribute to a more sophisticated understanding of protein engineering itself, but will also contribute to the development of refined computational approximations.

Recent years have seen a growing interest in developing more sustainable methods for chemical processes taking advantage of biological catalysts (enzymes). However, in many cases it is necessary to improve distinct enzyme characteristics such as stability, catalytic activity or specificity. Protein engineering offers several different approaches to improve such characteristics. In order to improve the understanding of thermal inactivation of cellulases as well as to investigate the catalytic activity of P450 enzymes for non-natural reactions we chose a rational design approach based on protein sequence alignments and available protein structures. For the engineering of terpene synthases however, we pursued two different approaches: We used SCHEMA recombination to explore if this method could be leveraged to generate chimeras with novel characteristics. In parallel we developed a novel high-throughput assay facilitating a directed evolution approach. Using this assay we screened several mutant libraries in order to isolate enzymes exhibiting the desired characteristics.