Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid

Abstract

Genetic code expansion technology allows for the use of noncanonical amino acids (ncAAs) to create semisynthetic organisms for both biochemical and biomedical applications. However, exogenous feeding of chemically synthesized ncAAs at high concentrations is required to compensate for the inefficient cellular uptake and incorporation of these components into proteins, especially in the case of eukaryotic cells and multicellular organisms. To generate organisms capable of autonomously biosynthesizing an ncAA and incorporating it into proteins, we have engineered a metabolic pathway for the synthesis of Omethyltyrosine (OMeY). Specifically, we endowed organisms with a marformycins biosynthetic pathway-derived methyltransferase that efficiently converts tyrosine to OMeY in the presence of the co-factor S-adenosylmethionine. The resulting cells can produce and site-specifically incorporate OMeY into proteins at much higher levels than cells exogenously fed OMeY. To understand the structural basis for the substrate selectivity of the transferase, we solved the X-ray crystal structures of the ligand-free and tyrosine-bound enzymes. Most importantly, we have extended this OMeY biosynthetic system to both mammalian cells and the zebrafish model to enhance the utility of genetic code expansion. The creation of autonomous eukaryotes using a 21st amino acid will make genetic code expansion technology more applicable to multicellular organisms, providing valuable vertebrate models for biological and biomedical research.

Generalized biomolecular modeling and design with RoseTTAFold All-Atom

“…Unlike previous methods, RFAA is able to jointly predict interactions between proteins and multiple nonprotein ligands in a single forward pass. Figure 2D shows three examples of recently solved structures with three or more components for which RFAA predictions had <2-Å ligand RMSD (when the proteins a dimeric tyrosine methyltransferase (PDB ID 7UX8; seq ID 28%; CASP15 target: T1124) with an S-adenosyl homocysteine and tyrosine interaction; and a DNA polymerase (PDB ID 7U7W; seq ID 100%) bound to DNA, a nucleotide, and a metal ion (31,66,67). The following color scheme is used in all panels: Predicted protein structure (aligned to native) is indicated in transparent teal, predicted ligand conformation in teal, and native ligand conformation in gray.…”

“…( D ) Three examples of successful predictions with multiple biomolecules. Shown from left to right are fatty acid decarboxylase (PDB ID 8D8P; seq ID 34%; from CAMEO) with a heme cofactor and a lipid substrate; a dimeric tyrosine methyltransferase (PDB ID 7UX8; seq ID 28%; CASP15 target: T1124) with an S -adenosyl homocysteine and tyrosine interaction; and a DNA polymerase (PDB ID 7U7W; seq ID 100%) bound to DNA, a nucleotide, and a metal ion ( 31 , 66 , 67 ). The following color scheme is used in all panels: Predicted protein structure (aligned to native) is indicated in transparent teal, predicted ligand conformation in teal, and native ligand conformation in gray.…”

Protein target highlights in CASP15: Analysis of models by structure providers

“…coli , mammalian HEK293T cells, and zebrafish through genetic code expansion and metabolic engineering that included production of MfnG. 27 This demonstrates that it is possible to generate cells and organisms that can incorporate ncAAs through exogenous biosynthesis of the ncAAs instead of high‐concentration feeding.…”

Recent developments in the cleavage, functionalization, and conjugation of proteins and peptides at tyrosine residues

“…This yield was nearly 4 times larger when compared with the 1.5 mg L −1 yield of the same protein produced by feeding the bacteria with 1 mM exogenous tyrosine- O -sulfate. Other successful examples of this technology include the site-specific incorporation of O -methyltyrosine, 115 and 3,4-dihydroxyphenylalanine ( l -DOPA). 116,117…”