Efficient Genetic Code Expansion Without Host Genome Modifications

TL;DR: Metabolic engineering enables efficient production of chemicals and biofuels by rewiring cellular metabolism through hierarchical approaches, including part, pathway, network, genome, and cell level modifications, maximizing product titer, yield, and productivity.

TL;DR: Researchers develop a toolkit of 9 aminoacyl-tRNA synthetase/tRNA pairs to genetically encode 12 new histidine-like non-canonical amino acids, expanding the scope of histidine substitution for enzyme engineering and catalysis studies.

Abstract: Abstract Genetic code expansion with non-canonical amino acids (ncAAs) opens new opportunities for the function and design of proteins by broadening their chemical repertoire. Unfortunately, ncAA incorporation is limited both by a small collection of orthogonal aminoacyl-tRNA synthetases (aaRSs) and tRNAs and by low-throughput methods to discover them. Here, we report the discovery, characterization, and engineering of a UGA suppressing orthogonal translation system mined from metagenomic data. We developed an integrated computational and experimental pipeline to profile the orthogonality of >200 tRNAs, test >1,250 combinations of aaRS:tRNA pairs, and identify the AP1 TrpRS:tRNA Trp UCA as an orthogonal pair that natively encodes tryptophan at the UGA codon. We demonstrate that the AP1 TrpRS:tRNA Trp UCA is highly active in cell-free and cellular contexts. We then use Ochre , a genomically recoded Escherichia coli strain that lacks UAG and UGA codons, to engineer an AP1 TrpRS variant capable of 5-hydroxytryptophan incorporation at an open UGA codon. We anticipate that our strategy of integrating metagenomic bioprospecting with cell-free screening and cell-based engineering will accelerate the discovery and optimization of orthogonal translation systems for genetic code expansion.

TL;DR: A unique transfer RNA/aminoacyl-tRNA synthetase pair has been generated that expands the number of genetically encoded amino acids in Escherichia coli and should provide a general method for increasing the genetic repertoire of living cells to include a variety of amino acids with novel structural, chemical, and physical properties not found in the common 20 amino acids.

TL;DR: The ability to selectively replace amino acids in a protein with a wide variety of structural and electronic variants should provide a more detailed understanding of protein structure and function.

TL;DR: It is suggested that the slow "ramp" at the beginning of mRNAs serves as a late stage of translation initiation, forming an optimal and robust means to reduce ribosomal traffic jams, thus minimizing the cost of protein expression.

TL;DR: An orthogonal aminoacyl-tRNA synthetase/tRNA pair is evolved that makes possible the in vivo incorporation of p-benzoyl-l-phenylalanine into proteins in Escherichia coli in response to the amber codon, TAG.