Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids

Abstract

Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein production for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled production of elastin-like-polypeptides with 30 nsAA residues at high yields (∼50 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.

Figure 1

Evaluation of multi-site nsAA incorporation and expression profiles on the activity of M. jannaschii derived pAcF orthogonal translation system (OTS). (a) Schematic illustration of reporter proteins for incorporation of 3, 10 and 30 nsAAs and equivalent control wild-type (WT) protein. (b) Incorporation of 3, 10 and 30 nsAAs (pAcF or pAzF) in a single protein by the plasmid-based M. jannaschii–derived pAcF OTS in the genomically recoded organism. (c) Production of superfolder GFP containing three UAG sites (GFP(3UAG)) by pAcFRS and tRNA_CUA expressed by plasmid or chromosomal integration. GFP(3UAG) is drastically reduced as a result of reduction in OTS transcript copy number, and partially rescued by supplementation of plasmids bearing either aaRS or tRNA_CUA. n = 3, error bars; mean ± s.d.

Figure 2

Evolution of chromosomally integrated aaRS variants. The genomically recoded organism (GRO) is engineered to contain a single chromosomal copy of the aaRS for diversification using MAGE, a negative-selection marker for removal of nonorthogonal translation systems (OTS) (capable of incorporation of natural amino acids), and a GFP marker for fluorescence-based identification and isolation of improved variants. Site-directed mutagenesis of chromosomally integrated translation components by MAGE generates a highly diversified population, which is subsequently subjected to tolC- and colicin E1–mediated negative selection in the absence of nsAAs. UAG suppression in GFP(3UAG) enables FACS of orthogonal aaRS libraries in the presence of the desired nsAA to identify improved variants. The selected aaRS variants are evaluated for multi-site nsAA incorporation, in vitro activity and protein purity.

Figure 3

Evolution and characterization of chromosomally integrated pAcFRS and pAzFRS variants with improved efficiencies. (a) Crystal structure of MjTyrRS in complex with tRNA_CUA and tyrosine. Insets highlight the amino acid binding pocket and the tRNA_CUA anticodon binding interface with a schematic representation of the libraries generated from pAcFRS (a mutant of the MjTyrRS) and aaRS variants isolated following each library diversification and selection steps. (b,c) pAcFRS (b) and pAzFRS (c) variants assayed by GFP(3UAG) fluorescence. n = 3; error bars, mean ± s.d. *P < 0.05 indicates comparison of each evolved aaRS with pAcFRS in the +pAcF or +pAzF condition. (d,e) Specificities of pAcFRS and pAzFRS variants for pAcF, pAzF (d), and 14 phenylalanine derivatives (e), as assayed by GFP(3UAG) fluorescence. Variant pAcFRS.2.t1 was evolved from variant pAcFRS.1.t1 for reduced pAzF incorporation (marked by black arrows). n = 3; error bars, mean ± s.d. *P < 0.05 indicates comparison of pAcFRS.2 with the progenitor pAcFRS.1.

Figure 4

Evaluation of multi-site nsAA incorporation by evolved aaRS variants expressed on multi-copy plasmids. Production of GFP(3UAG) (a), ELP(10UAG)-GFP (b) and ELP(30UAG)-GFP (c) by progenitor and evolved orthogonal translation systems expressed on multi-copy plasmids in the genomically recoded organism compared with WT (no UAG) proteins. Production of ELP(30UAG)-GFP by progenitor and evolved orthogonal translation systems expressed on multi-copy plasmids in the genomically recoded organism in the presence of variable pAcF (d) or pAzF (e) concentrations (n = 3, error bars, s.d. *P < 0.05 indicates comparison of evolved aaRS with pAcFRS in the +pAcF or +pAzF condition). (f) Efficiency and specificity of progenitor and evolved pAcFRS and pAzFRS variants for 14 phenylalanine derivatives, as assayed by ELP(30UAG)-GFP fluorescence. Data shown are the average of two independent experiments each with n = 3.

Figure 5

Quantitative MS evaluation of the purity of multi-site nsAA incorporation by evolved aaRS variants expressed on multi-copy plasmids. (a,b) Relative intensities of reporter peptides originating from ELP(10UAG) (a) and ELP(30UAG) (b) reporters containing pAcF, produced by progenitor and evolved orthogonal translation systems expressed on multi-copy plasmids in the genomically recoded organism. n = 4; error bars represent confidence interval calculated at the 95% confidence level. (c) Partial top-down mass spectrum of recombinant ELP(10UAG), after removal of the GFP UAG by trypsin digestion; the isotopically resolved 14+ charge state [M+14H]¹⁴⁺ is shown. Mass values are for the most abundant species, incorporating 10 pAcF residues (isotopic distribution in red: theoretical: 13,245.68 Da, experimental: 13,245.62 Da ± 4.5 p.p.m.). To the left of the main peak, species incorporating 9 pAcF and 1 Tyr residue (theoretical: 13,219.66 Da, experimental: 13,219.57 Da ± 6.8 p.p.m.) or 9 pAcF and 1 Phe residues (theoretical: 13,203.67 Da, experimental: 13,203.67 Da ± 7.6 p.p.m.) are colored black and blue, respectively. Species marked with *, ** and *** are M+O (+16 Da), M+2O (+32 Da) and M+3O (+48 Da), respectively, typical artifacts of analysis in electrospray MS. (d) Partial top-down mass spectrum of recombinant ELP(30UAG), after removal of the GFP UAG by trypsin digestion; the isotopically resolved 46+ charge state [M+46H]⁴⁶⁺ is shown. Mass experimental vs. mass theoretical (1.8 p.p.m. error) values are for the most abundant species, incorporating 30 pAcF residues (isotopic distribution in red: theoretical: 38,198.30 Da, experimental: 38,198.37 Da ± 1.8 p.p.m.), and species incorporating 29 pAcF and 1 Tyr residues (theoretical: 38,172.28 Da, experimental: 38,172.35 Da ± 1.8 p.p.m.) or 29 pAcF and 1 Phe (theoretical: 38,156.29 Da, experimental: 38,156.30 Da ± 0.3 p.p.m.) residues are colored black and blue, respectively.