Synthetic biology strategies for recombinant production of Lytic Polysaccharide Monooxygenases

Our modern society faces a severe global environmental crisis caused by our dependency on fossil-based energy. Therefore, finding alternative energy sources that can reduce or even displace the current fossil fuel dependency is of utter priority. Synthetic biology aims to tackle environmental issues by engineering life's building blocks to develop more efficient and sustainable sources of energy technologies. Thus,
strategic advances in synthetic biology are crucial to unlocking the full biological potential of organisms and the processes that govern them. A recent advance in biotechnology came from the discovery of Lytic Polysaccharide Monooxygenases (LPMOs), a new kind of enzyme with members in all kingdoms of life. Their roles on lignocellulose degradation place them as critical enzymes in the natural carbon cycle, and new emerging roles connect them directly with plant and human pathogenesis. Therefore, they are promising candidates for both the production of more sustainable energy alternatives and in connection with their roles in virulence and infection as targets for new drug discoveries. However, efficient production and characterization are challenging for these enzymes, hindering critical studies of their biological roles and, therefore, the full scope of their biotechnological potential.

The work presented in this thesis provides a universal cloning and protein production platform for LPMOs, facilitating the selection of the optimal enzymatic production. Furthermore, the performance of a DNA polymerase was optimized for advanced DNA engineering and diagnostics by applying a simple protein engineering approach. Moreover, a novel methyltransferase in the filamentous fungi Aspergillus nidulans
performing the N-terminal histidine methylation of fungal LPMOs was identified. Finally, the creation of a yeast strain that is able to perform N-terminal methylation on LPMOs is presented.