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.
