The goal of the LightCas project proposal was to develop a (light-)regulated enzyme cascade avoiding side-reactivity of the enzymes. As an ultimate goal, self-control of this cascade was envisaged.
Details and outcome with respect to the planned work packages:
Work Package “Enzyme entrapment in stimuli-switchable microgels”: Here, a reversible and selective on/off switching of enzymes involved in multi-step cascade reactions using photo switchable microgels was envisaged. These hydrogels swell and shrink with stimuli shifts. As the mesh size of the material changes in the process, it was expected that the activity of the enzyme is influenced by less flexible movement with smaller mesh size. In collaborations with experts on polymeric materials, we tested several materials for enzyme immobilisation but with unsatisfactory results. Besides light and temperature, also salts and even the substrates stimulated the swelling degree of the immobilisates. Anyhow, we understood the underlying principle of enzyme entrapment in these material (Kappauf K. et al. 2021. ChemBioChem 12 (4): 1190). Furthermore, a highly suitable online NMR-detection device was bought, conducting on-line analytics even more sensitive selective and reliable than expected (Claaßen C. et al. 2020. ChemCatChem. 12(4): 1190-119). This on-line NMR-detection formed the basis for self-regulation in biocatalytic reactions.
Work package “Light-induced enzyme deactivation using genetically encoded photosensitizers”: This WP on the regulation of enzyme cascades with genetically encoded photosensitisers was very successful. We could (i) set-up a toolbox of different photosensitizers being appropriate for enzyme inactivation, (ii) therewith regulated enzyme activity as desired and (iii) additionally show, that for some enzymes even no additional photosensitizer needs to be attached, as they already inactivate by simple light exposure e.g. due to naturally included photosensitizers, e.g. the cofactor PLP. Latter is of importance for a broad range of researchers from our discipline (Gerlach T. et al. 2021. ChemCatChem 13: 2398-2406; Gerlach T. et al. 2022. Frontiers Catal. 2:835919).
Work package “light-regulated enzyme reactor”: All puzzle pieces of the LightCas project work were bundled in a final light regulated enzyme cascade in a 50 mL reactor. We combined online-analytics with the NMR with automated feeding strategies and light switches for a self-controlled regulation of multi-step biotransformation. This is very new, exciting ground, as no one succeeded in self-controlling enzyme activities in one-pot cascades so far. The 1 mL process could be successfully scaled into a 50 mL reactor. Software and hardware regulated blue-light exposure and enzyme feeding depending on threshold given by automatically analysed substrate/intermediate/product concentrations. It was finally demonstrated, that with the automatically self-regulated light-controlled cascade by-product formation could be avoided.
Final outcome: As hoped for, the overall enzyme cascade for the synthesis of metaraminol and tetrahydroisoquinolines in a regulated manner avoiding side-product formation could be set-up. Due to online analytics and suitable soft- and hardware, this process was even performed in a self-controlled way. The outcome is even more fruitful and ground-breaking than expected, as automation seemed not to be possible within the frame of the ERC-STG-757320.
The feedback from the scientific community on the results and ideas of the LightCas project was amazing. Our review on stimulus-responsive enzyme regulation was selected as a 'very important article' by the journal Advanced Synthesis and Catalysis. The principle PI Dörte Rother was invited to present at renowned national and international conferences in the field of biocatalysis, e.g. at BioCat2022 (about 350 participants) and Biotrans2023, (about 700 participants). Further, due to the LightCas results, Dörte Rother is invited to give a talk at the Gordon Conference in Biocatalysis in summer 2024. Two prestigious prizes were awarded: Biotrans Junior Prize 2019 (international prize of the biocatalytic community) and DECHEMA Prize 2018 (prize of the German Society for Chemical Engineering and Biotechnology).
