Creating a bilingual organism with Two mutually Orthogonal genetic CODES

Objetivo

Ribosomes have the potential to polymerize many types of building blocks beyond the L-alpha amino acids we observe in nature. However, the evolution of essential genes towards new or enhanced activities is often confounded by interactions with host machinery. A long-standing aim of the ribosome engineering field is the complete decoupling of ribosome function from the host, freeing a population of ribosomes within a live cell for researchers to modify, engineer, or evolve towards new polymerization chemistries. Previous attempts to decouple or “orthogonalize” ribosome sub-populations have come through the creation of orthogonal 16S ribosomal RNA – messenger RNA pairs. Unfortunately, this does not orthogonalize the ribosome large subunit where polymerization occurs. Here, we propose a path towards an organism with two mutually orthogonal genetic decoding systems (specialized ribosomes), creating two discrete ribosome–transfer RNA (tRNA) subpopulations, and freeing a population of ribosomes to evolve for new functions. We aim to decouple the large ribosomal subunit by engineering the universal ribosome–tRNA tail interaction. To achieve this, we will investigate the expression of tRNAs bearing non-CCA 3’ ends in live cells (WP1). We will test the aminoacylation of non-CCA-end tRNAs by natural and engineered aminoacyl-tRNA synthetases (WP2). Finally, we will develop a platform for directed evolution of the ribosome large subunit rRNA (WP3). Achieving complete ribosome–tRNA pool orthogonalization will generate new knowledge in basic ribosome biology and open the potential for DNA-encoded synthesis of biopolymers with new sidechain and backbone chemistries for drug discovery and biomanufacturing. The far-reaching implications go beyond biopolymer synthesis and microbial-based manufacturing to complement biocontainment strategies in the emerging field of genome writing.