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

Abstract

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.

Development of a Pseudomonas putida cell-free protein synthesis platform for rapid screening of gene regulatory elements

“…Finally, we explored the impact of magnesium concentration of CFPS yields, which has been previously shown to be perhaps the most critical component of CFPS reactions ( 23 , 24 , 30 , 45 ). Magnesium is used to balance the charge present from nucleic acid phosphate groups and other anionic species.…”

“…In this study, we described the development and optimization of a P. putida- based CFPS system, and we were able to achieve sfGFP yields of ∼200 µg/ml in 4 h batch reactions. For comparison, although the current yield is lower than that of the E. coli -based CFPS system (>1000 µg/ml), which has been developed over the past 20 years ( 52 ), it is ten times higher than the recently developed S. cerevisiae -based CFPS system (<20 µg/ml) ( 45 ). The high yielding CFPS capacity, together with the heterologous protein expressing capability of P. putida , makes it a valuable addition to the current existing CFPS platforms.…”

An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts

“…Notably, we found that metabolic rewiring in these strains improves desired productivities in vitro. This reflects previous efforts to engineer strains for CFE to increase protein yields 27 , 57 , 58 and enable specialized applications 60 – 62 through gene knockouts and/or complementation, but the focus here was on reshaping metabolic flux for small molecule synthesis rather than translation. To our knowledge, this is the first report using multiplexed dCas9 modulation to enhance cell-free biosynthesis, using multiple gRNAs that can be implemented and adjusted more rapidly than knocking out enzymes in vivo 63 or selectively removing them in vitro 64 .…”

A PEGDA/DNA Hybrid Hydrogel for Cell-Free Protein Synthesis

“…In order to improve the life time of the template and the yield of protein for CFPS, many efforts have been taken from the perspective of inhibiting the activity of the nuclease, preparing an extract from genetically modified strains, improving energy regeneration and byproducts recycling, etc. (Caschera and Noireaux, 2014;Fujiwara and Doi, 2016;Schoborg et al, 2016). These efforts, indeed, improved the protein production of CFPS to a certain degree.…”