Designer enzymes featuring unnatural amino acids as catalytic residue

Información del proyecto

DENZUAC

Identificador del acuerdo de subvención: 885396

DOI

10.3030/885396

Proyecto cerrado

Fecha de la firma de la CE 21 Abril 2020

Fecha de inicio 1 Septiembre 2020

Fecha de finalización 31 Agosto 2025

Financiado con arreglo a

• EXCELLENT SCIENCE - European Research Council (ERC)

Coste total

€ 2 499 996,00

Aportación de la UE

€ 2 499 996,00

Coordinado por

RIJKSUNIVERSITEIT GRONINGEN
Netherlands

Periodic Reporting for period 4 - DENZUAC (Designer enzymes featuring unnatural amino acids as catalytic residue)

Período documentado: 2025-03-01 hasta 2025-08-31

Resumen del contexto y de los objetivos generales del proyecto

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.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

In this project, we developed the concept of designer enzymes featuring non-natural amino acids with catalytically active side chains, for the catalysis of chemical reactions that have no equivalent in nature.
A key element of our approach involves using expanded genetic code methods, especially stop codon suppression, to introduce these non-canonical amino acids by genetically encoding them with a stop codon in the gene of interest. Using this technology, we’ve built a library of designer enzymes, based on a set of protein scaffolds, with the ncAA at various positions. ncAAs employed during this project featured various unnatural catalytically active groups, ranging from organocatalytic groups such as anilines, thiophenols and aryl boronic acids, to ncAA-bound metal complexes based on copper and gold. Using these, a variety of biocatalytic reactions that have no equivalent in nature were developed. After the first screening, the initial “hits” were further developed using the powerful approach of directed evolutions, resulting in enzymes that gave high enantioselectivity in reactions, of which many could be run aa preparative scale.
Some of these enzymes were even applied in living cells, and integrated into biocatalytic cascades in vivo. This represents a first major step towards a hybrid metabolism, which integrates unnatural chemistry in biological synthesis.
All project objectives were achieved and the results have been published in leading scientific journals, with several publications underway.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

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 have been 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.

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