Scalable production of sequence-defined biopolymers containing multiple distinct non-canonical amino acids in recoded cell and cell-free systems

Engineered biological systems enable template-directed synthesis of biopolymers, such as proteins and peptides, with desirable material properties and functions. However, ribosome-mediated production is constrained to 20 canonical amino acids, with limited ability to encode unnatural monomers. Harnessing the translation apparatus to manufacture biopolymerbased products beyond these natural limits remains underexploited, and the efficiency and scalability of existing systems is constrained. This work will expand nature’s repertoire of ribosomal monomers to non-canonical amino acids (ncAAs) in a scalable production system to yield new classes of materials, chemicals, and enzymes with diverse genetically encoded chemistries. Our Phase I work demonstrated robust incorporation of two distinct ncAAs in a single biopolymer, in both cellbased and cell-free production systems. Additionally, we established a framework for rational optimization of biopolymer production titers. This framework will be leveraged in Phase II to achieve increased production levels of a prototype biopolymer containing two distinct ncAAs 10x relative to initial production titers. Furthermore, a novel genomically recoded organism with 3+ open codons will be harnessed to incorporate three distinct ncAAs in parallel. Both cell-based and cell-free production systems will be developed in Phase II, enabling versatile approaches to achieve manufacturing-relevant titers of designer biopolymers in Phase III.