Periodic Reporting for period 4 - DrEAM (Directed Evolution of Artificial Metalloenzymes for In Vivo Applications) Periodo di rendicontazione: 2021-04-01 al 2022-09-30 Sintesi del contesto e degli obiettivi generali del progetto Artificial metalloenzymes (ArMs) result from combining an organometallic catalyst in a protein scaffold. The resulting hybrid catalyst display advantageous features from both homogeneous catalyst (e.g. broad reaction repertoire) and enzymes (e.g. evolvable catalytic performance via genetic mutations). Within the DrEAM ERC project, we set out to assemble, evolve and exploit ArMs in a cellular environment, thus enabling the use of bacterial cells as molecular factories (or as "test-tubes") for the synthesis of high-added value chemicals.Today, chemical transformations are typically carried out in pure organic solvents and in a stepwise fashion. In stark contrast biochemical transformations proceed in a complex cellular environment and to not require the isolation of the chemical intermediates along the multistep reaction sequence. The latter strategy presents multiple advantages, both from an economic and environmental perspective: “using a cell as test-tube or a molecular factory”. During the DrEAM ERC project, we demonstrated ArMs can indeed be assembled, and evolved in a cellular environment. In addition, they can be combined with natural enzyme leading to efficient enzyme cascades, whereby a substrate is converted via multiple (artificial) enzymes into a product. These findings demonstrate that cells can indeed be used as test-tube whereby both natural enzymes and ArMs work in concert in a complex environment.To achieve these ambitious goals, two protein scaffolds were evaluated: human carbonic anhydrase II and streptavidin. The following ArMs-catalyzed new-to-nature reactions were implemented and optimized in a cellular environment: transfer hydrogenation, allylic substitution, cyclopropanation, olefin metathesis and hydroamination.In summary, the field of ArMs has enormously progressed, thanks to the DrEAM ERC grant. Gratifyingly, our work is widely acclaimed as has inspired many groups to follow our footsteps. Currently, more than fourty groups worldwide are active in the field. Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti Since initiation of the DrEAM ERC, we have unambiguously demonstrated that ArMs can be assembled and evolved within a cellular environment. These findings significantly broaden the scope of application of ArMs. Importantly, ArMs can catalyse new-to-nature reactions in vivo, thus endowing cells with new tools to synthesize high-added value products. ArMs are perceived by the biosynthetic community as an elegant means to broaden the reaction repertoire that is accessible to natural enzymes. Accordingly, this work is very well perceived, rewarded, and highlighted in various media. Biocatalysis-, bioinorganic- and synthetic biology- conferences typically devote an entire day to ArMs-related research. Some of the key players in the field include: Frances Arnold, John Hartwig, Todd Hyster, Vincent Pecoraro, Gerard Roelfes, Anthony Green, Katsunori Tanaka, etc. The ArMs community regularly publishes in highly-regarded journals. Thus far 21 publications acknowledge the support of the DrEAM-ERC. Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora) The ultimate aim of the research was to assemble and evolve an artificial metalloenzyme in an E. coli chassis to complement natural enzymes in vivo. With this goal in mind, we have demonstrated that both the periplasm and the outer membrane of E. coli are suitable to localize and evolve an ArM while maintaining the critical phenotype-genotype linkage. Thus far, we have achieved over one hundred turnovers in vivo for an ArM's-catalyzed reaction in vivo. To the best of our knowledge, this result far exceeds anything reported to date (typically single digit turnovers). To further highlight the potential in synthetic biology, ArMs were combined with a broad palette of natural enzymes including: hydroxylases, reductases, desaturases, amine oxidases, dehydrogenases etc. Accordingly, this DrEAM ERC has significantly contributed to expand the repertoire of reaction (cascades) that can be carried out in a cellular environment. We have unambiguously demonstrated that organometallic reactions catalyzed by ArMs can be optimized by directed evolution schemes. Artificial metalloenzymes result from combining a small molecule catalyst with a protein
