Biotechnology is considered as one of the key-enabling technologies to create a sustainable, bio-based economy. Besides well-established applications of enzymes in food and laundry industry, enzyme catalysis is an especially powerful tool for the industrial production of fine chemicals (cosmetics, pharmaceuticals). Due to their selectivity, the formation of unwanted side products can be avoided. Furthermore, mild reaction conditions can be employed (room temperature and aqueous reaction media) accompanying with preventing of toxic reagents, and solvents as well as the use of heavy metals as catalysts. This makes enzymes very attractive for environmentally benign syntheses routes.
Although nature has demonstrated its great potential to evolve diverse enzymes to construct complex pathways, a limited set of enzymatic reactivities have been developed during evolution. Enzymes are composed out of 20 amino acids, which allow a restricted set of catalytic mechanisms. With the help of (a limited number of) small organic molecules – cofactors – nature expanded this range significantly. However, chemists often desire reactions that are not known in nature. The creation of non-natural enzymes with an expanded reaction scope is therefore an important step to increase the impact of biotechnology. An expanded collection of available enzymes will facilitate a greater contribution of biocatalysis in industrial synthesis, having an important economical and ecological potential. One example of desired but not yet available enzymatic reactions are transformations driven by light that can be catalyzed by small organic dyes. Light is a cheap and renewable energy source, which can fuel reactions that otherwise would need reagents providing high energy. Furthermore, light-driven reactions often enable a complementary range of reactivities, which cannot be obtained via classical chemical approaches. However, it is not possible to render photo-organo-catalyzed reactions stereoselective in most cases, which is a prerequisite for applying these reaction for the production of pharmaceutical ingredients or intermediate building blocks that are required at high optical purity.
Therefore, this research program aims to expand the chemical repertoire of naturally evolved enzymes for selective photocatalyzed conversions of small organic molecules. We want to combine the natural ability of enzymes to create selectivity to a catalyzed reaction by incorporating organic photocatalysts as non-natural cofactors. These hybrid proteins will then catalyse light-driven reactions, which are not known in nature. The protein scaffold is required to induce the desired selectivity to the reaction which is not observed if the photo catalyst is used alone. To this end, an important goal is to combine laboratory evolution and protein design methods to optimize these newly designed hybrid proteins. This requires work on the following subgoals: (i) to identify and establish model reaction, (ii) to employ and develop molecular techniques to integrate the small organic photocatalysts into the protein, (iii) to create and optimize the production of hybrid proteins in E. coli cells, (iv) to develop instrumental and high throughput analytics that facilitate the screening, (v) to create variants and identify ones with improved properties, and (vi) to optimize a selected reaction for preparative scale.
