Periodic Reporting for period 2 - DENZUAC (Designer enzymes featuring unnatural amino acids as catalytic residue)
Período documentado: 2022-03-01 hasta 2023-08-31
Biocatalysis is a key component of the transition towards a more sustainable and “greener” chemistry. Surprisingly, natural enzymes use only a relatively small section of “reaction space”, that is, only limited number of reaction classes. This in marked contrast to the vast reaction space available to the synthetic chemist. Therefore, it is highly desirable to have enzymes available for the catalysis of these abiological reactions. With the advent of robust expanded genetic code methodologies, it is now feasible to introduce a wide variety of unnatural amino acids into proteins. We envision that the time has come to use this breakthrough technology to create enzymes that contain abiological reactive groups as catalytic residue, for the catalysis of reactions that are not possible using canonical amino acids only. The global aim of this project is the creation and application of designer enzymes with genetically encoded unnatural amino acids as catalytic residue for novel and new-to-nature catalysis.
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.
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 the project to date, we have made progress towards all research objectives.
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.
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.
the artificial enzymes containing catalytically active non-canoncial amino acids created in this project allow to access a fully new area of "reaction space", i.e. biocatalytic reaction scope, that was not achievable with natural enzymes until now. These enzymes will be further developed and will make a major contribution towards the important goal of making organic synthesis more sustainable. Moreover, the first integration of artificial enzymes into biocatalytic cascades in vivo shows that the dream of a hybrid metabolism, where unnatural chemistry is integrated into biological synthesis to access new structural diversity is achievable. This will be further pursued in the second phase of the project, to move beyond "proof of principle studies" but focus on biocatalytic cascades of synthetic relevance.