Directed Evolution of the Methanosarcina barkeri Pyrrolysyl tRNA/aminoacyl tRNA Synthetase Pair for Rapid Evaluation of Sense Codon Reassignment Potential.

David G Schwark,John D Fisk,Margaret A Schmitt

doi:10.3390/ijms22020895

David G Schwark, John D Fisk + Show 1 more

Open Access

https://doi.org/10.3390/ijms22020895

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Abstract

Genetic code expansion has largely focused on the reassignment of amber stop codons to insert single copies of non-canonical amino acids (ncAAs) into proteins. Increasing effort has been directed at employing the set of aminoacyl tRNA synthetase (aaRS) variants previously evolved for amber suppression to incorporate multiple copies of ncAAs in response to sense codons in Escherichia coli. Predicting which sense codons are most amenable to reassignment and which orthogonal translation machinery is best suited to each codon is challenging. This manuscript describes the directed evolution of a new, highly efficient variant of the Methanosarcina barkeri pyrrolysyl orthogonal tRNA/aaRS pair that activates and incorporates tyrosine. The evolved M. barkeri tRNA/aaRS pair reprograms the amber stop codon with 98.1 ± 3.6% efficiency in E. coli DH10B, rivaling the efficiency of the wild-type tyrosine-incorporating Methanocaldococcus jannaschii orthogonal pair. The new orthogonal pair is deployed for the rapid evaluation of sense codon reassignment potential using our previously developed fluorescence-based screen. Measurements of sense codon reassignment efficiencies with the evolved M. barkeri machinery are compared with related measurements employing the M. jannaschii orthogonal pair system. Importantly, we observe different patterns of sense codon reassignment efficiency for the M. jannaschii tyrosyl and M. barkeri pyrrolysyl systems, suggesting that particular codons will be better suited to reassignment by different orthogonal pairs. A broad evaluation of sense codon reassignment efficiencies to tyrosine with the M. barkeri system will highlight the most promising positions at which the M. barkeri orthogonal pair may infiltrate the E. coli genetic code.

Highlights

Genetic code expansion, biosynthetic incorporation of non-canonical amino acids to expand the chemical properties of proteins, is a rapidly developing area of synthetic biology research [1,2]
30 E. coli codons and found that every sense codon evaluated could be partially reassigned by providing an orthogonal tRNA with an anticodon capable of Watson–Crick base pairing to the targeted codon [17,31,32,33]
We used a combination of site-directed and random mutagenesis to evolve a variant of the orthogonal M. barkeri pyrrolysyl aminoacyl tRNA synthetase capable of charging its cognate tRNA with tyrosine with an efficiency rivaling that of natural aaRSs

Summary

Introduction

Biosynthetic incorporation of non-canonical amino acids (ncAAs) to expand the chemical properties of proteins, is a rapidly developing area of synthetic biology research [1,2]. Engineering systems in which evolved variants of the M. jannaschii and M. barkeri orthogonal tRNA/aaRS pairs each target different codons will facilitate biosynthesis of proteins simultaneously containing multiple copies of multiple ncAAs. predicting which sense codons are most amenable to reassignment and which orthogonal translation machinery is best suited to each codon is challenging. We describe the directed evolution of a new variant of the orthogonal M. barkeri pyrrolysyl translation machinery for the rapid evaluation of sense codon reassignment using our previously reported fluorescence-based screen. Comparing the efficiency of sense codon reassignments by the two orthogonal systems should begin to reveal the contribution of orthogonal tRNA aminoacylation efficiency to sense codon reassignment

Results and Discussion

A Z domain with a UAC codon at codon at 5position

ESI-MS

Evaluation of

Materials and Methods

Conclusions