Incorporation of multiple beta 2-backbones into a protein invivo using an orthogonal aminoacyl-tRNA synthetase

Synthesis of sequence-defined biomaterials whose monomer backbones diverge from canonical -amino acids represents the next frontier in protein and biomaterial evolution with the potential to yield better biological therapeutics, bioremediation tools, and biodegradable plastic-like materials. One monomer family of particular interest for biomaterials are {beta}-hydroxy acids. Many natural products contain isolated {beta}-esters, and polymeric {beta}-esters are found in polyhydroxyalkanoate (PHA) polyesters under development as bioplastics and drug encapsulation/delivery systems. Here we report that {beta}2-hydroxy acids possessing both (R) and (S) absolute configuration are excellent substrates for pyrrolysyl-tRNA synthetase (PylRS) enzymes in vitro, and that (S)-{beta}2-hydroxy acids are substrates in cellulo. Using the MaPylRS/MatRNAPyl pair, in conjunction with wild-type E. coli ribosomes and EF-Tu, we report the cellular synthesis of model proteins containing two (S)-{beta}2-hydroxy acid residues at internal positions. Metadynamics simulations provide a rationale for the observed enantioselective preference of the ribosome for the (S)-{beta}2-hydroxy acid backbone and mechanistic insights that inform future ribosomal engineering efforts. As far as we know, this finding represents the first example of an orthogonal synthetase that accepts a {beta}-backbone substrate and the first example of a protein hetero-oligomer containing multiple expanded-backbone monomers produced in cellulo.