Directed-evolution of translation system for efficient unnatural amino acids incorporation and generalizable synthetic auxotroph construction

Site-specific incorporation of unnatural amino acids (UAAs) with similar incorporation efficiency to that of natural amino acids (NAAs) and low background activity is extremely valuable for efficient synthesis of proteins with diverse new chemical functions and design of various synthetic auxotrophs. However, such efficient translation systems remain largely unknown in the literature. Here, we describe engineered chimeric phenylalanine systems that dramatically increase the yield of proteins bearing UAAs, through systematic engineering of the aminoacyl-tRNA synthetase and its respective cognate tRNA. These engineered synthetase/tRNA pairs allow single-site and multi-site incorporation of UAAs with efficiencies similar to those of NAAs and high fidelity. In addition, using the evolved chimeric phenylalanine system, we construct a series of E. coli strains whose growth is strictly dependent on exogenously supplied of UAAs. We further show that synthetic auxotrophic cells can grow robustly in living mice when UAAs are supplemented.

The Escherichia coli serS gene promoter region overlaps with the rarA gene

Deletion of the entire gene encoding the RarA protein of Escherichia coli results in a growth defect and additional deficiencies that were initially ascribed to a lack of RarA function. Further work revealed that most of the effects reflected the presence of sequences in the rarA gene that affect expression of the downstream gene, serS. The serS gene encodes the seryl aminoacyl-tRNA synthetase. Decreases in the expression of serS can trigger the stringent response. The sequences that affect serS expression are located in the last 15 nucleotides of the rarA gene.

SUMOylation of Arginyl tRNA Synthetase Modulates the Drosophila Innate Immune Response

SUMO conjugation of a substrate protein can modify its activity, localization, interaction or function. A large number of SUMO targets in cells have been identified by Proteomics, but biological roles for SUMO conjugation for most targets remains elusive. The multi-aminoacyl tRNA synthetase complex (MARS) is a sensor and regulator of immune signaling. The proteins of this 1.2 MDa complex are targets of SUMO conjugation, in response to infection. Arginyl tRNA Synthetase (RRS), a member of the sub-complex II of MARS, is one such SUMO conjugation target. The sites for SUMO conjugation are Lys 147 and 383. Replacement of these residues by Arg (RRSK147R,K383R), creates a SUMO conjugation resistant variant (RRSSCR). Transgenic Drosophila lines for RRSWT and RRSSCR were generated by expressing these variants in a RRS loss of function (lof) animal, using the UAS-Gal4 system. The RRS-lof line was itself generated using CRISPR/Cas9 genome editing. Expression of both RRSWT and RRSSCR rescue the RRS-lof lethality. Adult animals expressing RRSWT and RRSSCR are compared and contrasted for their response to bacterial infection by gram positive M. luteus and gram negative Ecc15. We find that RRSSCR, when compared to RRSWT, shows modulation of the transcriptional response, as measured by quantitative 3′ mRNA sequencing. Our study uncovers a possible non-canonical role for SUMOylation of RRS, a member of the MARS complex, in host-defense.