Redesigning Translation Systems for Bioproduction of New Catalysts, Therapeutics, and Materials

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Abstract

The standard genetic code utilizes 64 codons to encode the 20 canonical amino acids across domains of life, in a process involving the translation system, which consists of mRNA, ribosomes, tRNAs, aminoacyl tRNA synthetases (aaRSs), and other translation factors. New-to-nature genetic codes with reassigned open codons enable new chemistries, therapeutics, materials, and ecosystems engineering, but recoding an organism’s genome is complex and challenging to predict. I will describe efforts to redesign translation systems to enable modular on-demand genetic code reassignment and sequence-defined polymer synthesis. In order to rapidly build new genetic codes, we developed Automated Genetic tRNA Expansion (AGENTEX). AGENTEX allows multiplexed robotic prototyping of genetic codes in cell-free translation systems and without the need for genome synthesis or editing of host genomes. We investigated whether we could engineer components of the translation system: ribosomes, tRNAs, and aaRSs for mutual recognition and orthogonality to the native translation system. For this, we developed multiplexed and automated methods for reading of aminoacylation in libraries of synthetic tRNAs (tSCAN). We discovered that the tRNA 3′ end shows remarkable flexibility to mutation, allowing aminoacylation of most tRNAs with non-CCA 3’ ends by all Escherichia coliaaRSs. Building on our discovery, we developed custom cell lysates and performed translation of peptides using a compressed genetic code enabling 34 aaRSs for 34 codons. Using AGENTEX we evaluated two compressed genetic codes with codon reassignment and nonstandard amino acid incorporation. Our findings have implications for the design of radically new translation systems, synthesis of biopolymers with multiple instances of nonstandard monomers, and understanding possible past and future genetic codes.

Bio:

Felix Radford was awarded a PhD at Yale University, where his research focused on the study and evolution of ribosomes and development of new genome editing techniques. He is currently a postdoctoral research fellow in the laboratory of George Church at Harvard Medical School, where his work explores systems-level evolution of the Central Dogma and combines machine learning and robotics with protein engineering.

Felix’s work has led to the development of automated methods for profiling the aminoacylation of thousands of tRNAs simultaneously, enabling the discovery and engineering of novel orthogonal translation systems. He has also developed AGENTEX, a platform for rapidly prototyping new genetic codes and evolving biopolymers containing multiple non-natural monomers. This work has broad potential applications in the development of therapeutics, catalysts, and biomaterials with precisely tunable properties. Today, Felix will talk about his work on translation system engineering and genetic code expansion.