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Abstract Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g. library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing of combined ribosomal assembly and translation activity, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design and selection may serve as a powerful tool for rRNA engineering. 

Cell-free synthetic biology is a maturing field that aims to assemble biomolecular reactions outside cells for compelling applications in drug discovery, metabolic engineering, biomanufacturing, diagnostics, and education. Cell-free systems have several key features. They circumvent mechanisms that have evolved to facilitate species survival, bypass limitations on molecular transport across the cell wall, enable high-yielding and rapid synthesis of proteins without creating recombinant cells, and provide high tolerance towards toxic substrates or products. Here, we analyze ~750 published patents and ~2000 peer-reviewed manuscripts in the field of cell-free systems. Three hallmarks emerged. First, we found that both patent filings and manuscript publications per year are significantly increasing (five-fold and 1.5-fold over the last decade, respectively). Second, we observed that the innovation landscape has changed. Patent applications were dominated by Japan in the early 2000s before shifting to China and the USA in recent years. Finally, we discovered an increasing prevalence of biotechnology companies using cell-free systems. Our analysis has broad implications on the future development of cell-free synthetic biology for commercial and industrial applications.