Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Abstract

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

Aminoacyl-tRNA synthetases

“…changing field of aaRS synthetic biology is well-served by a steady stream of comprehensive reviews for the interested reader covering a broad field of topics; from general overviews (Melnikov and Söll 2019;Smolskaya and Andreev 2019), genetic code expansion Mukai et al 2017b;Arranz-Gibert et al 2018), adding amino acids with new chemistries (Wang et al 2009;Liu and Schultz 2010;Sakamoto et al 2014) or engineering of tRNA-aaRSs pairs (Mukai et al 2017b;Baumann et al 2018).…”

An Escherichia coli Chassis for Production of Electrically Conductive Protein Nanowires

“…The much broader range of strategies for introducing genes and controlling their expression in E. coli 22, 23 is expected to enable the design and expression of e-PNs with unique properties and functionalities that could not readily be fabricated with G. sulfurreducens . Most notably, E. coli is an excellent platform for modifying proteins with unnatural amino acids that can confer diverse new functionalities to proteins 24, 25 . The development of new e-PNs in the E. coli chassis for enhanced sensor, electronics, and energy-harvesting applications 1-8 is underway.…”

“…E. coli is a common platform for the commercial scale production of organic commodities 22, 23 . The substantial E. coli genetic toolbox, including the possibility of introducing unnatural amino acids 24, 25 , could provide broad options for designing e-PNs with different properties and functions. Non-pathogenic strains of E. coli typically do not express type IV pili.…”

Tackling Achilles’ Heel in Synthetic Biology: Pairing Intracellular Synthesis of Noncanonical Amino Acids with Genetic‐Code Expansion to Foster Biotechnological Applications

“…Therefore, the incorporation of ncAAs into proteins in vivo is a much more relevant tool for technological applications, and thus the focus of this report. Although the successful in vivo incorporation of ncAAs has been reported for various host systems, including yeast, plants, and mammalian cells, the bacterium Escherichia coli is still the most universally used platform for ncAA incorporation and heterologous protein expression given the numerous available molecular biology kits, protocols, plasmid catalogue, and well‐known industrial applications for this organism …”