title: Optimized plasmid systems for the incorporation of multiple different unnatural amino acids by evolved orthogonal ribosomes.
abstract: Incorporation of multiple different unnatural amino acids into the same polypeptide remains a significant challenge. Orthogonal ribosomes, which are evolvable as they direct the translation of a single dedicated orthogonal mRNA, can provide an avenue to produce such polypeptides routinely. Recent advances in engineering orthogonal ribosomes have created a prototype system to enable genetically encoded introduction of two different functional groups, albeit with limited efficiency. Here, we systematically investigated the limiting factors of this system by using assays to measure the levels and activities of individual components; we identified Methanosarcina barkeri PylRS as a limiting factor for protein yield. Balancing the expression levels of individual components significantly improved growth rate and protein yield. This optimization of the system is likely to increase the scope of evolved orthogonal ribosome-mediated incorporation of multiple different unnatural amino acids.
authors.affiliation: Free Floater (Junior) Research Group "Applied Synthetic Biology", Institute for Microbiology and Genetics, Georg-August University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany).
authors.forename: Christoph
authors.initials: C
authors.lastname: Lammers
authors.forename: Liljan E
authors.initials: LE
authors.lastname: Hahn
authors.forename: Heinz
authors.initials: H
authors.lastname: Neumann
mesh_terms: D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D017021:Methanosarcina barkeri / Q000201:enzymology* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D014176:Protein Biosynthesis; D015202:Protein Engineering; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D000604:Amino Acyl-tRNA Synthetases
keywords: amino acids; click chemistry; genetic code expansion; optimization; orthogonal ribosomes; unnatural amino acids