Orthogonal translation enables heterologous ribosome engineering in E. coli

Natalie S. Kolber, Ranan Fattal, Sinisa Bratulic, Gavriela D. Carver and Ahmed H. Badran ()
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Natalie S. Kolber: Broad Institute of MIT and Harvard
Ranan Fattal: Broad Institute of MIT and Harvard
Sinisa Bratulic: Broad Institute of MIT and Harvard
Gavriela D. Carver: Broad Institute of MIT and Harvard
Ahmed H. Badran: Broad Institute of MIT and Harvard

Nature Communications, 2021, vol. 12, issue 1, 1-12

Abstract: 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.

Date: 2021
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DOI: 10.1038/s41467-020-20759-z

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