Periodic Reporting for period 2 - DENZUAC (Designer enzymes featuring unnatural amino acids as catalytic residue)
Periodo di rendicontazione: 2022-03-01 al 2023-08-31
The following research objectives are key to achieving the overall aim:
1. Achieving incorporation of unnatural amino acids containing organocatalytic side chains in proteins.
2. Creation of a library of novel designer enzymes containing unnatural amino acids as catalytic residue.
3. Application of these designer enzymes in catalysis of important reactions that have no equivalent in nature.
4. Directed evolution of designer enzymes featuring unnatural amino acids as catalytic residue.
5. Application of designer enzymes containing UAAs as catalytic residue in biocatalytic cascades.
This highly ambitious project combines frontier chemical and biochemical research and will deliver completely new classes of enzymes that can access new and previously unexplored parts of biocatalytic “reaction space”. In this way, this project will contribute to achieving the important societal goal of achieving greener and more sustainable approaches to chemical synthesis.
In addition to the incorporation of p-aminophenylalanine, which we have reported before, we have now achieved the incorporation a wide variety of novel catalytically active non-canonical amino acids into protein scaffolds such as LmrR and QacR. These proteins are well established in our group and there is a lot of experience in their handling and a lot of mutants are available, which makes it easy to generate a library of variants for testing. But to further streamline the discovery process, we have made a novel screening platform that allows us to quickly screen a variety of orthogonal translations systems for incorporation of novel non-canonical amino acids. Also a number of new protein scaffolds for artificial enzyme design have been identified and are currently explored.
The catalytic potential of the novel artificial enzymes created has been explored, giving rise to efficient and (enantio)selective biocatalysis of reactions that have no equivalent in nature. Initial hits, i.e. artificial enzymes that show a basic level of activity, have been improved with directed evolution to become proficient enzymes. A notable example is the artificial enzyme catalyzed tandem conjugate addition/enantioselective protonation reaction: a unique reactions where the approach of a proton to a reactive intermediate is completely controlled, in water. A variety of other reactions have also been explored and will soon be reported.
Finally, one such an evolved artificial enzyme containing p-aminophenylalanine was found to be fast enough to be able to catalyze the hydrazone formation reaction in whole cells. This made it possible to create, for the first time, small biocatalytic cascades in vivo, in which the artificial enzyme catalyzed reaction was coupled with natural enzyme catalyzed reactions that made the substrates for the reaction (see figure, Angewandte Chemie, International Edition 2023, 62, e202214191). This represents one of the first steps towards a hybrid metabolism.