Evolving Bacterial Fitness with an Expanded Genetic Code

Drew S Tack,Austin C Cole,Raghav Shroff,Andrew D Ellington,Barrett R Morrow

doi:10.1038/s41598-018-21549-w

Abstract

Since the fixation of the genetic code, evolution has largely been confined to 20 proteinogenic amino acids. The development of orthogonal translation systems that allow for the codon-specific incorporation of noncanonical amino acids may provide a means to expand the code, but these translation systems cannot be simply superimposed on cells that have spent billions of years optimizing their genomes with the canonical code. We have therefore carried out directed evolution experiments with an orthogonal translation system that inserts 3-nitro-L-tyrosine across from amber codons, creating a 21 amino acid genetic code in which the amber stop codon ambiguously encodes either 3-nitro-L-tyrosine or stop. The 21 amino acid code is enforced through the inclusion of an addicted, essential gene, a beta-lactamase dependent upon 3-nitro-L-tyrosine incorporation. After 2000 generations of directed evolution, the fitness deficit of the original strain was largely repaired through mutations that limited the toxicity of the noncanonical. While the evolved lineages had not resolved the ambiguous coding of the amber codon, the improvements in fitness allowed new amber codons to populate protein coding sequences.

Highlights

Since the fixation of the genetic code evolution has been confined to the 20 canonical amino acids, with some incursions by selenocysteine and pyrrolysine
We assembled an Orthogonal translation systems (OTSs) for the incorporation of 3nY comprised of a Methanocaldococcus jannaschii tyrosyl-aminoacyl-tRNA synthetase (aaRS) variant that had previously been engineered to be specific for 3-iodo-L-tyrosine[10] but was compatible with 3nY16, and the corresponding M. jannaschii tyrosyl-tRNA in which the anticodon was complementary to the UAG amber stop codon
Two different approaches have been used to generate organisms with altered or expanded amino acid genetic codes; a bottom-up approach where components of the organism were engineered to function with a noncanonical amino acids (ncAAs), or a top-down approach, where an organism was allowed to evolve in the presence of a ncAA47

Summary

Introduction

Since the fixation of the genetic code evolution has been confined to the 20 canonical amino acids, with some incursions by selenocysteine and pyrrolysine. Orthogonal translation systems (OTSs) comprising aminoacyl-tRNA synthetase (aaRS)/suppressor tRNA pairs have been developed that do not significantly interact with the host translational machinery or interfere with already occupied portions of the genetic code[8,9,10] These OTSs allow the incorporation of noncanonical amino acids (ncAAs) by suppressing the amber stop codon (UAG). We utilize an engineered β-lactamase (bla) that is structurally dependent on OTS incorporation of the ncAA 3-nitro-L-tyrosine (3nY)[9] This ‘addicted’ bla has allowed us to overcome fitness deficits and carry out long term evolution experiments with an ambiguous amber codon without loss of the underlying OTS. We demonstrate that our system stably incorporates an ncAA for 2,000 generations of evolution, and for the first time identify the entire complement of genomic mutations that lead to improved fitness in the presence of an enforced 21 amino acid code

Methods

Results

Conclusion