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

Abstract

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 nextgeneration molecules provide otherwise nonexistent opportunities to develop improved biologic therapies, bioremediation tools, and biodegradable plasticlike 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 β 2 -hydroxy acids possessing both (R) and (S) absolute configuration are substrates for pyrrolysyl-tRNA synthetase (PylRS) enzymes in vitro and that (S)-β 2 -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)-β 2 -hydroxy acid residues at internal positions. Metadynamics simulations provide a rationale for the observed preference for the (S)-β 2 -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 β 2 -hydroxy acid substrate and the first example of a protein hetero-oligomer containing multiple expanded-backbone monomers produced in cellulo.