Orthogonal translation enables heterologous ribosome engineering in E. coli

Author:

Kolber Natalie S.ORCID,Fattal Ranan,Bratulic SinisaORCID,Carver Gavriela D.ORCID,Badran Ahmed H.ORCID

Abstract

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.

Funder

National Aeronautics and Space Administration

Broad Institute

Publisher

Springer Science and Business Media LLC

Subject

General Physics and Astronomy,General Biochemistry, Genetics and Molecular Biology,General Chemistry

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