Incorporation of Multiple β2-Hydroxy Acids into a Protein In Vivo Using an Orthogonal Aminoacyl-tRNA Synthetase

The programmed synthesis of sequence-defined biomaterials whose monomer backbones diverge from those of canonical α-amino acids represents the next frontier in protein and biomaterial evolution. Such next-generation molecules provide otherwise nonexistent opportunities to develop improved biologic therapies, bioremediation tools, and biodegradable plastic-like materials. One monomer family of particular interest for biomaterials includes β-hydroxy acids. Many natural products contain isolated β-hydroxy acid monomers, and polymers of β-hydroxy acids (β-esters) are found in polyhydroxyalkanoate (PHA) polyesters under development as bioplastics and drug encapsulation/delivery systems. Here we report that β²-hydroxy acids possessing both (R) and (S) absolute configuration are substrates for pyrrolysyl-tRNA synthetase (PylRS) enzymes in vitro and that (S)-β²-hydroxy acids are substrates in cellulo. Using the orthogonal MaPylRS/MatRNA^Pyl synthetase/tRNA pair, in conjunction with wild-type E. coli ribosomes and EF-Tu, we report the cellular synthesis of model proteins containing two (S)-β²-hydroxy acid residues at internal positions. Metadynamics simulations provide a rationale for the observed preference for the (S)-β²-hydroxy acid and provide mechanistic insights that inform future engineering efforts. As far as we know, this finding represents the first example of an orthogonal synthetase that acylates tRNA with a β²-hydroxy acid substrate and the first example of a protein hetero-oligomer containing multiple expanded-backbone monomers produced in cellulo.

The aminoacyl-tRNA synthetase PylRS from M. alvus acylates tRNA^Pyl with β²-hydroxy acid substrates and supports their incorporation at multiple sites into a protein in vivo.