Engineered bacterial host for genetic encoding of physiologically stable protein nitration

Abstract

Across scales, many biological phenomena, such as protein folding or bioadhesion and cohesion, rely on synergistic effects of different amino acid side chains at multiple positions in the protein sequence. These are often fine-tuned by post-translational modifications that introduce additional chemical properties. Several PTMs can now be genetically encoded and precisely installed at single and multiple sites by genetic code expansion. Protein nitration is a PTM of particular interest because it has been associated with several diseases. However, even when these nitro groups are directly incorporated into proteins, they are often physiologically reduced during or shortly after protein production. We have solved this problem by using an engineered Escherichia coli host strain. Six genes that are associated with nitroreductase activity were removed from the genome in a simple and robust manner. The result is a bacterial expression host that can stably produce proteins and peptides containing nitro groups, especially when these are amenable to modification. To demonstrate the applicability of this strain, we used this host for several applications. One of these was the multisite incorporation of a photocaged 3,4-dihydroxyphenylalanine derivative into Elastin-Like Polypeptides. For this non-canonical amino acid and several other photocaged ncAAs, the nitro group is critical for photocleavability. Accordingly, our approach also enhances the production of biomolecules containing photocaged tyrosine in the form of ortho-nitrobenzyl-tyrosine. We envision our engineered host as an efficient tool for the production of custom designed proteins, peptides or biomaterials for various applications ranging from research in cell biology to large-scale production in biotechnology.

Orthogonal translation with 5‐cyanotryptophan as an infrared probe for local structural information, electrostatics, and hydrogen bonding

“…5‐nitro‐Trp may be of particular interest and the new 5CNW_RS can usefully complement the existing orthogonal pairs for nitro‐containing ncAAs. The site‐specific incorporation of these ncAAs displays a useful tool for the investigation of diseases related to reactive nitrogen species (RNS) (Koch et al, 2022). In summary, we succeeded to generate an orthogonal enzyme with a remarkable amino acid substrate tolerance, an aaRS feature known as substrate promiscuity or even “poly‐specificity.” (Guo et al, 2014).…”

“…The orthogonality in this context means that directed evolution of aminoacyl‐tRNA synthetases (aaRSs) should yield robust enzyme variants capable of selectively recognizing their cognate tRNA and ncAAs in the pool of endogenous tRNAs, aaRSs, metabolic intermediates (e.g., homoserine, homocysteine, citrulline, ornithine, etc.) and 20 canonical amino acids (Koch et al, 2022). In this study, we were able to generate an orthogonal 5CNW_RS variant that meets these criteria and has high activity towards 5CNW, which is also a substate for cellular uptake systems and metabolically well tolerated in the E. coli cytosol.…”

Genomically integrated orthogonal translation inEscherichia coli, a new synthetic auxotrophic chassis with altered genetic code, genetic firewall, and enhanced protein expression

“…Having eliminated the UAG-recognising termination factor (RF-1) along with five intracellular nitroreductases, the NK53 strain was equipped with a fivefold integrated OTS to decode the TAG amber codons for the synthetic amino acid m -oNB-Dopa. As a feature of NK53 strain, the deletion of the nitroreductase genes 20 in the engineered cell suppresses the reduction of the NO 2 group of m -oNB-Dopa analogues to ensure stability of oNB protective scaffold (prior to deprotecting). It is well known that the use of m -oNB-Dopa in mussel foot proteins to produce photoactive wet-bioadhesive materials as demonstrated elsewhere 31 .…”

“…RDB13711)) with no specific assignment to the UAG codon was previously modified by deletion of six nitroreductase genes to generate an attenuated nitroreductase activity strain (NK53) responsible for reduction of the nitro group on m-o NB-Dopa incorporated expressed protein. The genotype of new modified E. coli strain is as follow: E. coli B-95.ΔAΔfabR; ΔNfsA::FRT; Δ NfsB ::FRT; Δ AzoR ::FRT; Δ Ydja ::FRT; Δ NemA ::FRT; Δ RutE ::FRT, hereinafter is called NK53 strain 20 . A full list of E. coli strains used for genome editing experiments is provided in Supplementary Table S3.…”

“…We harnessed the Type I-F Tn7-like transposase from Vibrio cholera (Tn6677) as a highly efficient tool to integrate the OTS into recoded E. coli B-95.ΔA, specifically the B-95.ΔA NK53 strain 20 , which is a nitroreductase-deficient strain derived from the Escherichia coli B95ΔA strain (originally derived from E. coli BL21(DE3)) 21,22 . In this study, we used the integration of an orthogonal Mj TyRS (oNB-DopaRS)-tRNA CUA cassette (O-pair) as an example of OTS integration in E. coli for site-specific incorporation of m -oNB-Dopa ( meta -(ortho-(2-nitrobenzyl))-3,4-dihydroxyphenylalanine) 23 by in-frame amber stop codon readthrough.…”