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

Crnković, Ana; Vargas-Rodriguez, Oscar; Merkuryev, Anna; Söll, Dieter

doi:10.3390/bioengineering5010011

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

Journal Article · Fri Feb 02 00:00:00 EST 2018 · Bioengineering

DOI:https://doi.org/10.3390/bioengineering5010011· OSTI ID:2484059

; ; Merkuryev, Anna;

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.

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Research Organization:: Yale Univ., New Haven, CT (United States)

Sponsoring Organization:: USDOE; USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

Grant/Contract Number:: FG02-98ER20311

OSTI ID:: 2484059

Alternate ID(s):: OSTI ID: 1629897

Journal Information:: Bioengineering, Journal Name: Bioengineering Journal Issue: 1 Vol. 5; ISSN 2306-5354; ISSN BIOENG

Publisher:: MDPI AGCopyright Statement

Country of Publication:: Switzerland

Language:: English

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