Evolution of translation initiation sequences using in vitro yeast ribosome display

We report a novel in vitro yeast ribosome display method based on cell‐free protein synthesis (CFPS) using linear DNA templates. We demonstrate that our platform can enrich a target gene from a model library by 100‐fold per round of selection. We demonstrate the utility of our approach by evolving cap‐independent translation initiation (CITI) sequences, which result in a 13‐fold increase in CFPS yields after four rounds of selection, and a threefold further increase by placing the beneficial short sequences in tandem. We also show that 12 of the selected CITI sequences permit precise control of gene expression in vitro over a range of up to 80‐fold by enhancing translation (and not as cryptic promoters). These 12 sequences are then shown to tune protein expression in vivo, though likely due to a different mechanism. Looking forward, yeast ribosome display holds promise for evolving libraries of proteins and DNA regulatory parts for protein engineering and synthetic biology. Biotechnol. Bioeng. 2016;113: 1777–1786. © 2016 Wiley Periodicals, Inc.