Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids

Abstract

Synthesis of proteins with noncanonical amino acids (ncAAs) enables the creation of protein-based biomaterials with diverse new chemical properties that may be attractive for material science. Current methods for large-scale production of ncAA-containing proteins, frequently carried out in Escherichia coli, involve the use of orthogonal aminoacyl-tRNA synthetases (o-aaRSs) and tRNAs (o-tRNAs). Although o-tRNAs are designed to be orthogonal to endogenous aaRSs, their orthogonality to the components of the E. coli metabolism remains largely unexplored. We systematically investigated how the E. coli tRNA modification machinery affects the efficiency and orthogonality of o-tRNASep used for production of proteins with the ncAA O-phosphoserine (Sep). The incorporation of Sep into a green fluorescent protein (GFP) in 42 E. coli strains carrying deletions of single tRNA modification genes identified several genes that affect the o-tRNA activity. Deletion of cysteine desulfurase (iscS) increased the yield of Sep-containing GFP more than eightfold, while overexpression of dimethylallyltransferase MiaA and pseudouridine synthase TruB improved the specificity of Sep incorporation. These results highlight the importance of tRNA modifications for the biosynthesis of proteins containing ncAAs, and provide a novel framework for optimization of o-tRNAs.

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

“…For instance, the NAAs with bulky or extremely charged chemical group are unable to effectively cross cellular membrane, thus, the successful approach to evolve orthogonal pairs and site-specifically incorporate such a NAAs would be either propeptide strategy utilizing NAAs in dipeptide form [114] or modification of the host organism transport system, such as engineering of periplasmic binding protein, for optimized uptake of the desired NAA into the cell [115]. Very recently the significance of heterologous cellular background, particularly the enzymes modifying the orthogonal tRNA, for NAA-containing protein synthesis was demonstrated [116]. Another important issue to consider is the permissivity of the mutation site, i.e., sequence flexibility representing an ability to tolerate a large number of mutations at a selected locus without significant loss in protein activity [117].…”

Gene miaA for post‐transcriptional modification of tRNAXXA is important for morphological and metabolic differentiation in Streptomyces

“…It is unclear why ∆ miaA produces more antimycins and novel compounds as compared to parental SAM2 strain. At present we are not aware of any reports, where deficiency in modification of natural tRNA would lead to improved expression of a certain trait (however, precedents are known for heterologous tRNA that mediate incorporation of nonstandard amino acids in E. coli ; Crnković et al , ). As in the related species S. albus S4, antimycin and candicidin gene clusters are grouped together in J1074 genome (see Fig.…”

Versatility of Synthetic tRNAs in Genetic Code Expansion

“…A genetic approach to addressing this issue involves monitoring ncAA incorporation and reporter protein yields across E. coli or yeast strain collections containing deletions and/or the overexpression cassettes of metabolic genes. Recently it was shown that the yield and specificity of O- phosphoserine incorporation is significantly improved by the deletion of cysteine desulfurase and the overexpression of E. coli dimethylallyltransferase (MiaA) and pseudouridine synthase (TruB) [ 65 ]. Furthermore, a yeast study involving the removal of modifications by single gene deletions from U34, U35, A37, U47 and C48 in the anticodon stem-loop impairs nonsense suppression, with the strongest effect observed for U34 and A37.…”