Evolving LPMOs for Enhanced (Bio-)polymer Modifications

Our everyday life is heavily dependent on the use of plastics and synthetic polymers, but their resistance to natural breakdown leads to widespread pollution and creates significant environmental challenges. However, Nature might provide powerful tools to address these problems - lytic polysaccharide monooxygenases (LPMOs). These unique enzymes, produced by various organisms, are crucial for breaking down resistant natural materials like wood and chitin by selectively modifying strong carbon-hydrogen bonds. Their ability to act directly on solid, crystalline regions of polymers makes them particularly promising for industrial applications. However, applying LPMOs to new, non-natural materials has proven difficult. Currently engineered LPMO versions often become unstable or lose their activity, especially if they do not bind strongly enough to the target material. Existing engineering methods struggle with the enzyme`s complex interaction with its substrates and its tendency for self-inactivation. This project proposes a novel approach to overcome these limitations and expand LPMO`s capabilities. Using laboratory evolution and machine-learning applying "protein language models" plausible mutations will be identified that improve how LPMOs bind to and break down various substances. A key strategy involves re-shaping the enzyme`s binding site and fusing LPMOs with different binding domains. This approach aims to direct the enzyme`s powerful oxidative chemistry more effectively against the substrate, increasing activity and preventing rapid self-inactivation. Thousands of enzyme variants will be efficiently screened using a yeast cell surface display setup. This allows us to identify those variants with improved binding properties on diverse materials, including natural biopolymers (like cellulose and chitin), modified natural derivatives (such as chitosan), and even challenging synthetic polymers (like polyethylene and polystyrene). The most promising variants will be further evaluated for their enzymatic activity using newly developed electrochemical detection methods, which provide real-time insights into LPMO catalysis. Our ultimate goal is to create stable and highly efficient LPMOs with novel functionalities. This will provide a new platform for developing enzymes capable of modifying and degrading a broad range of natural and synthetic polymers. The results of this research are expected to contribute to further sustainable developments, fostering a greener future, a circular bioeconomy and to reduce our environmental footprint.