Scalable Biomanufacturing of Sequence-Defined Aramid Copolymers

OUSD (R&E) CRITICAL TECHNOLOGY AREA(S): Advanced Materials; Biotechnology OBJECTIVE: Develop a reliable, cost-effective cellular or cell-free process from genomically recoded organisms or other viable approaches supporting ribosomal incorporation of non-canonical amino acids to synthesize aramid copolymers with controlled monomer sequence. DESCRIPTION: High performance materials such as Kevlar or Twaron are aromatic polyamide (aramid) type polymers used in aerospace and military applications such as body-armor, composites and hull reinforcement, in addition to many other consumer applications. The structure of the polymer is based on alternating repeat units of two monomers that become highly oriented along the fiber axis upon processing because of significant hydrogen bonding and aromatic stacking between individual polymer chains. The strong intermolecular interactions give the materials a very high tensile strength to weight ratio. Despite the highly regular structure, only 10% of the theoretical possible strength is achieved due to challenges in getting many hydrogen bonds to align. More recently, a new aramid polymer, Kevlar EXOTM was developed which introduces a third monomer into the structure leading to increased alignment between chains and higher strength. The third monomer is randomly incorporated into the chain through a polycondensation reaction, potentially limiting the amount of alignment possible. An aramid polymer where the sequence of three or more monomers is precisely controlled could conceivably achieve even higher chain alignment and greater fiber tenacity. However, current synthetic approaches to preparing aramid polymers are not amenable to controlling the sequence of individual monomers. Biological protein translation machinery (ribosomes, tRNAs, aminoacyl-tRNA synthetases, and other translation factors) is capable of producing protein polymers with precisely defined sequences of individual amino acid monomers linked via amide bonds according to mRNA templates which encode the protein sequences. The sequence-defined protein polymers can fold and assemble into secondary and tertiary hierarchical structures to perform a variety of biological functions including catalysis, communication, transport, and structural reinforcement. While naturally-occurring protein polymers are assembled using a library of 20 amino acid monomers, recent advances in synthetic biology have enabled the engineering of the translation machinery to accept non-canonical amino acids not existing in natural biological systems. The objective of this topic is to develop cellular or cell-free biotechnology processes utilizing genomically recoded organisms or other viable approaches that support efficient and accurate ribosomal incorporation of non-canonical amino acids to synthesize sequence-defined aramid copolymers. Designed processes must consider the monomer composition and sequence required to realize enhanced material properties relative to the current state-of-the-art, as well as scaling of ribosomal-based synthesis of aramid copolymers to manufacturing-relevant levels. PHASE I: Develop cellular or cell-free synthetic processes utilizing genomically recoded organisms or other viable approaches that support efficient and accurate ribosomal incorporation of non-canonical amino acids to synthesize aramid copolymers comprised of p-phenylene terephthalamide with an AABB alternating structure and degree of polymerization of 50. Using computational modeling, identify additional candidate monomers that, when added to the aramid copolymer in a sequence defined manner to form a terpolymer or higher, improve polymer chain alignment for enhanced strength. Design a framework to be implemented in Phase II that will increase ribosomal production levels of non-biological polymers by at least a factor of 10 relative to the current state-of-the-art. PHASE II: Further develop biosynthetic approaches from Phase I to incorporate promising additional monomers with control over sequence along the p-phenylene terephthalamide copolymer structure and assess material properties of the sequence-defined aramid heteropolymers relative to current state-of-the-art aramid copolymers. Demonstrate ribosomal synthesis of terpolymers with degrees of polymerization of at least 100 and polydispersity < 1.2. Implement the framework designed in Phase I and demonstrate increased ribosomal production levels of non-biological polymers by at least a factor of 100 relative to the current state-of-the-art. PHASE III DUAL USE APPLICATIONS: Phase III efforts will optimize approaches for ribosomal synthesis of sequence-defined aramid copolymers with at least 3 distinct monomers with degrees of polymerization of at least 150 and scale production to manufacturing-relevant levels. A variety of dual-use applications would benefit from an efficient production pipeline for biomanufactured non-biological sequence-defined aramid copolymers, including personal armor, composites for automotive and aerospace and other applications requiring high tenacity fibers. REFERENCES: 1. Martin, R.W., Des Soye, B.J., Kwon, YC. et al. Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids. Nat Commun 9, 1203 (2018). https://doi.org/10.1038/s41467-018-03469-5 2. Lee, J., Schwarz, K.J., Kim, D.S. et al. Ribosome-mediated polymerization of long chain carbon and cyclic amino acids into peptides in vitro. Nat Commun 11, 4304 (2020). https://doi.org/10.1038/s41467-020-18001-x 3. Lee, J., Coronado, J.N., Cho, N. et al. Ribosome-mediated biosynthesis of pyridazinone oligomers in vitro. Nat Commun 13, 6322 (2022). https://doi.org/10.1038/s41467-022-33701-2 KEYWORDS: Sequence-defined polymers; non-canonical amino acids; biotechnology; fibers; crystallinity; aramid