Yeast knockout library allows for efficient testing of genomic mutations for cell-free protein synthesis

Cell-free protein synthesis (CFPS) systems from crude lysates have benefitted from modifications to their enzyme composition. For example, functionally deleting enzymes in the source strain that are deleterious to CFPS can improve protein synthesis yields. However, making such modifications can take substantial time. As a proof-of-concept to accelerate prototyping capabilities, we assessed the feasibility of using the yeast knockout collection to identify negative effectors in a Saccharomyces cerevisiae CFPS platform. We analyzed extracts made from six deletion strains that targeted the single deletion of potentially negative effectors (e.g., nucleases). We found a statistically significant increase in luciferase yields upon loss of function of GCN3, PEP4, PPT1, NGL3, and XRN1 with a maximum increase of over 6-fold as compared to the wild type. Our work has implications for yeast CFPS and for rapidly prototyping strains to enable cell-free synthetic biology applications.