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Scalable Production of Engineered Designer Proteins Containing Multiple Distinct Non-canonical Amino Acids






OBJECTIVE: Develop cellular or cell-free synthetic processes from genomically recoded organisms to achieve robust, reliable and cost-effective production of engineered proteins containing multiple distinct non-canonical amino acids that are scalable to manufacturing-relevant levels.


DESCRIPTION: Biological systems are capable of synthesizing long sequence-defined protein biopolymers with extremely high efficiency and accuracy by employing templates to provide sequence information.  The precisely defined sequence of the amino acid building blocks directs the folding and assembly of these proteins into higher-order structures capable of a variety of advanced functions evolved to solve biological challenges such as catalysis, sensing and signal transduction. These higher-ordered natural protein structures also display diverse material properties spanning highly flexible and extensible to extremely tough and fracture resistant.  However, natural protein biopolymers are assembled using a limited library of only 20 amino acid monomer building blocks.  In contrast, synthetic polymers boast a vast array of monomer building blocks with functionality and properties not accessible to natural proteins.  Yet, chemical routes to the synthesis of long-chain polymers with control over monomer sequence have remained elusive. Co-opting the genetic code and the natural translation machinery to accept non-biological monomers is an attractive approach to access the diverse functionality afforded by non-biological chemical monomers while maintaining the precision polymer sequence control provided by biology [1,2]. This approach has the potential to develop novel hybrid biological-abiological polymers with functionalities not accessible either in natural biological systems or via traditional chemical synthesis.


To enable the translation machinery to incorporate monomer building blocks beyond the 20 canonical amino acids, researchers have been exploring expansion of the genetic code through reassignment of degenerate codons to direct the incorporation of non-canonical amino acids [3,4,5]. Using genomically recoded organisms in which one or more codons were reassigned to a non-canonical amino acid, researchers have demonstrated the incorporation of a variety of non-canonical amino acids at a single site within a protein.  Building upon these results, researchers have recently demonstrated the incorporation of multiple instances of a single non-canonical amino acid within a protein [6,7] as well as the incorporation of up to three distinct non-canonical amino acids into the same protein [8].  While these results demonstrate the promise of genomic recoding to access complex hybrid biological-abiological polymers with novel functionalities, the efficiency of incorporation of multiple non-canonical amino acids is currently very low, requiring highly sensitive techniques for detection of the targeted engineered protein product. 


The objective of this topic is to develop cellular or cell-free biotechnology processes from genomically recoded organisms that support efficient and accurate incorporation of multiple distinct non-canonical amino acids into engineered proteins.  Designed processes must consider scaling of protein production to manufacturing-relevant levels for protein-based materials (i.e., exceeding protein production levels that would be relevant for a protein-based therapeutic).


PHASE I: Build on recent advances to design methods for cellular or cell-free synthetic processes from genomically recoded organisms to efficiently and accurately incorporate two (2) distinct non-canonical amino acids in an engineered protein at lab-relevant scales.  Design a framework to be implemented in Phase II that will enable increased production levels of proteins containing multiple non-canonical amino acids by at least a factor of 10 relative to the current state-of-the-art.


PHASE II: Further develop approaches from Phase I to increase the number of multiple distinct non-canonical amino acids incorporated into engineered proteins to three (3) or more.  Implement the framework designed in Phase I and demonstrate increased production levels of proteins containing multiple instances of a single non-canonical amino acid and/or proteins containing two or more distinct non-canonical amino acids by at least a factor of 10 relative to the current state-of-the-art.


PHASE III DUAL USE APPLICATIONS: Phase 3 efforts will optimize and develop approaches to incorporate >3 distinct non-canonical amino acids and scale production of hybrid biological-abiological protein polymers to manufacturing-relevant levels for protein-based materials.  A variety of dual-use applications would benefit from an efficient production pipeline for engineered hybrid biological-abiological protein polymers, including synthetic enzymes for catalysis, scaffolds for molecular encoding and data storage, nanoelectronics, and self-healing materials.



  1. Kofman, C. et al. Engineering molecular translation systems. Cell Systems 12, 593-607 (2021);
  2. Shapiro, D.M. et al. Protein nanowires with tunable functionality and programmable self-assembly using sequence-controlled synthesis. Nat Commun 13, 829 (2022).
  3. Ostrov, N. et al. Design, synthesis, and testing toward a 57-codon genome. Science 353, 819-822 (2016).
  4. Sanders, J. et al. New opportunities for genetic code expansion in synthetic yeast. Curr Opin Biotechnol 75, 102691 (2022).
  5. Tang, H. et al. Recent Technologies for Genetic Code Expansion and their Implications on Synthetic Biology Applications. J Mol Biol 167382 (2021).
  6. Amiram, M. et al. Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. Nat Biotechnol 33, 1272–1279 (2015).
  7. Martin, R.W. et al. Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids. Nat Commun 9, 1203 (2018).
  8. Robertson, W., et al. Sense codon reassignment enables viral resistance and encoded polymer synthesis. Science 372, 1057-1062 (2021).


KEYWORDS: Protein engineering; non-canonical amino acids; genomically recoded organisms; genetic code expansion; sequence-defined polymers; biotechnology

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