Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids

Abstract

Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein production for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled production of elastin-like-polypeptides with 30 nsAA residues at high yields (~50 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

“…evolving more efficient aaRSs in a genomically recoded E. coli strain, have greatly increased the suppression efficiency allowing the multi-site incorporation of a single ncAA at up to 30 positions in protein-polymers with high yields. [20] Multiple site-specific incorporation of different ncAAs, however, is still dependent on the number of o-pairs available and, especially, restricted to three stop codons. It is expected that this limitation will be overcome in the future by recoding sense codons.…”

Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes

“…High efficiencies (>10%) were achieved by using the phage λ Red Beta ssDNA annealing protein (SSAP) to facilitate ssODN annealing at the lagging strand during DNA replication (Costantino and Court, 2003). With the advent of multiplex automated genome engineering (MAGE), this approach was enhanced to generate multi-site genome modifications with bp precision at increased efficiencies (>30%) and used for pathway diversification (Wang et al, 2009), whole genomic recoding (Lajoie et al, 2013), and molecular evolution of proteins (Amiram et al, 2015). In S. cerevisiae homologous recombination (HR) of ssODNs results in low gene targeting efficiencies (~10 −4 –10 −3 %), which limits the scope of applications to single locus modifications (Kow et al, 2007; Storici and Resnick, 2003).…”

Portable, On-Demand Biomolecular Manufacturing

“…Abs have recently begun to revolutionize therapeutics by enabling targeted treatment of diseases, including cancer (Scott et al, 2012), immune disorders (Wang et al, 2015), viral (Ng et al, 2010), and bacterial infections (Kontermann, 2012), as well as targeted delivery of antibiotics (Lehar et al, 2015). Abs have been expressed using laboratory-based CF systems, however, their structural complexity, disulphide bonds, and post-translational modification requirements make their production challenging (Patel et al, 2011;Ryabova et al, 1997).…”

“…In recent years, in vitro biosynthesis from fresh or frozen lysates has developed remarkably, including the biomanufacture of difficult molecules that cause cell toxicity and the incorporation of non-canonical elements (Amiram et al, 2015;Dudley et al, 2015;Karim and Jewett, 2016;Sullivan et al, 2016;Welsh et al, 2012;Zawada et al, 2011). These advances have thus far been tied to laboratory settings where the necessary skills and equipment are found.…”