Orthogonal translation enables heterologous ribosome engineering in E. coli

• DOI: 10.1038/s41467-020-20759-z
• Publication Date: 2021-01-26T11:04:04Z
• Authors: Natalie S. Kolber; Ranan Fattal; Sinisa Bratulic; Gavriela D. Carver; Ahmed H. Badran
• Keywords: Ribosomal Proteins; 0303 health sciences; Science; Q; Article; RNA, Bacterial; RNA, Ribosomal, 23S; 03 medical and health sciences; Bacterial Proteins; Protein Biosynthesis; RNA, Ribosomal, 16S; Escherichia coli; Synthetic Biology; rRNA Operon; Ribosomes; Phylogeny

AbstractThe ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli. Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli-derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells.