Abstract
The 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.
Highlights
The ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts
Prior work in SQ171 complementation using fully native heterologous rRNA operons has been extended to Salmonella typhimurium (96.8% 16S rRNA sequence identity to E. coli) and Proteus vulgaris rRNA (93.2%)[18]
We observed a substantial growth defect in SQ171 cells complemented by rRNA derived from A. baumannii despite the minor difference in sequence identity to E. coli rRNA as compared to P. aeruginosa
Summary
The 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. Extensive efforts towards engineering translation have yielded researcherdictated, specialized functions in vivo: parallel genetic circuits[4], augmented polypeptide diversity using non-canonical amino acids[5], expanded genetic codes incorporating quadruplet codons[6], and linked ribosomal subunits for improved cellular orthogonality[7,8,9]. These results establish a quantitative and extensible method for the engineering of heterologous ribosome activity in vivo, facilitating the development of diverse ribosomes for synthetic biology applications
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