Abstract
Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties. Such proteins serve in sophisticated biochemical and biophysical studies both in vitro and in vivo, they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes. In the Methanosarcinaceae the incorporation of the 22nd genetically encoded amino acid, pyrrolysine (Pyl), is facilitated by pyrrolysyl-tRNA synthetase (PylRS) and the cognate UAG-recognizing tRNAPyl. This unique aaRS•tRNA pair functions as an orthogonal translation system (OTS) in most model organisms. The facile directed evolution of the large PylRS active site to accommodate many ncAAs, and the enzyme's anticodon-blind specific recognition of the cognate tRNAPyl make this system highly amenable for GCE purposes. The remarkable polyspecificity of PylRS has been exploited to incorporate >100 different ncAAs into proteins. Here we review the Pyl-OT system and selected GCE applications to examine the properties of an effective OTS.
Highlights
New macromolecular functions often come from the expansion of chemical diversity
As neither the E. coli LeuRS or SerRS use the tRNA anticodon as a recognition element, these enzymes do not cross-react with tRNAPylCAG or tRNAPylACU, resulting in orthogonal tRNAPyl variants that are able to decode sense codons
The Pyl-orthogonal translation system (OTS) is a powerful tool for genetic code expansion
Summary
New macromolecular functions often come from the expansion of chemical diversity. In the field of synthetic biology, the increase in chemical complexity originates from a functional demand. A genomic code expansion approach to introduce ncAAs in vivo involves site-specific cotranslational incorporation through stop codon suppression (SCS, Fig 1). PylRS specific tRNA-binding domain 1 makes the majority of the interactions with the tertiary core of tRNAPyl. The PylRStRNAPyl structure confirms earlier biochemical data that bases at position
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