Engineering posttranslational proofreading to discriminate nonstandard amino acids

Abstract

Incorporation of nonstandard amino acids (nsAAs) leads to chemical diversification of proteins, which is an important tool for the investigation and engineering of biological processes. However, the aminoacyl-tRNA synthetases crucial for this process are polyspecific in regard to nsAAs and standard amino acids. Here, we develop a quality control system called "posttranslational proofreading" to more accurately and rapidly evaluate nsAA incorporation. We achieve this proofreading by hijacking a natural pathway of protein degradation known as the N-end rule, which regulates the lifespan of a protein based on its amino-terminal residue. We find that proteins containing certain desired N-terminal nsAAs have much longer half-lives compared with those proteins containing undesired amino acids. We use the posttranslational proofreading system to further evolve a Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) variant and a tRNA^Tyr species for improved specificity of the nsAA biphenylalanine in vitro and in vivo. Our newly evolved biphenylalanine incorporation machinery enhances the biocontainment and growth of genetically engineered Escherichia coli strains that depend on biphenylalanine incorporation. Finally, we show that our posttranslational proofreading system can be designed for incorporation of other nsAAs by rational engineering of the ClpS protein, which mediates the N-end rule. Taken together, our posttranslational proofreading system for in vivo protein sequence verification presents an alternative paradigm for molecular recognition of amino acids and is a major advance in our ability to accurately expand the genetic code.

Keywords: N-end rule; genetic code expansion; nonstandard amino acids; protein degradation; synthetic biology.

Fig. 1.

Posttranslational proofreading proof of concept. (A) Scheme for proofreading consisting of N-end exposure and recognition steps applied to synthetic substrates. Ub is cleaved by ubiquitin cleavase UBP1 to expose the target site as N-terminal. ClpS is the native N-recognin in E. coli and ClpAP forms an AAA⁺ protease complex for degradation by the N-end rule pathway. (B) nsAAs used in this study (full chemical names in SI Appendix). (C) Incorporation assay showing fluorescence resulting from GFP expression normalized by optical density (FL/OD) in the absence/presence of BipA and expression of various OTS or N-end rule components. “Over” indicates overexpression of natively expressed components. Error bars represent SD, n = 3. (D) Heatmap of FL/OD signals obtained from an nsAA panel arranged roughly in descending size from left to right without proofreading occurring in Top row and with proofreading occurring in Bottom row. Left reflects activity of the Bipyridylalanine OTS and Right reflects activity of the p-acetyl-phenylalanine OTS. Heatmap values here and elsewhere are average of n = 3.

Fig. 2.

Proofreading tunability achieved through rational ClpS engineering. (A) Cartoon generated from crystal structure of E. coli ClpS binding N-end Phe peptide (PDB ID code: 3O2B) showing four hydrophobic ClpS residues subjected to single-point mutations that sampled F/L/I/V. (B) Heatmap of FL/OD signals obtained using a ClpS⁻ host expressing UBP1, the p-acetyl-phenylalanine OTS, and variants of ClpS in the presence of different nsAAs. (C) Cartoon generated from crystal structure of C. crescentus ClpS binding N-end Trp peptide (PDB ID code: 3GQ1). (D) FL/OD heatmap resulting from expression of UBP1, the 5-hydroxytryptophan OTS, and ClpS variants in the presence/absence of 5-hydroxytryptophan. Scale as in B. (E) FL/OD heatmap resulting from expression of UBP1/ClpS in strains with Ub-X-GFP reporter genes expressing standard AAs in place of X.

Fig. 3.

Selective BipA OTS evolution using proofreading. (A) FACS evolution scheme with error-prone PCR aminoacyl-tRNA synthetase libraries transformed into hosts with posttranslational proofreading (“PTP”, using ClpS^V65I) genomically integrated, followed by three sorting rounds. (B) Evaluation of enriched evolved BipARS variants in clean backgrounds on a panel of nsAAs ([BipA] = 100 μM, [rest] = 1 mM, which are their standard concentrations). The parental variant is noted as “P.” (C) In vitro amino acid substrate specificity profile of BipA OTS variants. Error bars = SD, n = 3.

Fig. 4.

Effect of evolved BipA OTS on biocontainment strain escape and fitness. (A) Escape frequencies over time for adk.d6 strains transformed with constructs indicated in legend below plots. Green and yellow circles compare escape frequencies of parent and evolved variants and are most relevant for this study. Navy circles represent previously published data (from ref. 3). Gray circles for adk.d6 represent our repeat of previously published data. KA, kanamycin+arabinose; SCA, SDS+chloramphenicol+arabinose. Error bars in A–C represent SEM, n = 3. (B) Escape frequencies over time for tyrS.d8 strains. Lines represent assay detection limit in cases where no colonies were observed. (C) Escape frequencies over time for adk.d6/tyrS.d8 strains. (D) Doubling time for biocontained strains with parental (P) or variant 10 OTS. Error bars = SD, n = 3.