Substrate replenishment and byproduct removal improve yeast cell-free protein synthesis

Abstract

Cell-free protein synthesis (CFPS) platforms are now considered a powerful tool for synthesizing a variety of proteins at scales from pL to 100 L with accelerated process development pipelines. We previously reported the advancement of a novel yeast-based CFPS platform. Here, we studied factors that cause termination of yeast CFPS batch reactions. Specifically, we characterized the substrate and byproduct concentrations in batch, fed-batch, and semi-continuous reaction formats through high-performance liquid chromatography (HPLC) and chemical assays. We discovered that creatine phosphate, the secondary energy substrate, and nucleoside triphosphates were rapidly degraded during batch CFPS, causing a significant drop in the reaction's energy charge (E.C.) and eventual termination of protein synthesis. As a consequence of consuming creatine phosphate, inorganic phosphate accumulated as a toxic byproduct. Additionally, we measured amino acid concentrations and found that aspartic acid was rapidly consumed. By adopting a semi-continuous reaction format, where passive diffusion enables substrate replenishment and byproduct removal, we achieved over a 70% increase in active superfolder green fluorescent protein (sfGFP) as compared with the batch system. This study identifies targets for the future improvement of the batch yeast CFPS reaction. Moreover, it outlines a detailed, generalized method to characterize and improve other CFPS platforms.

Keywords: Cell-free biology; Cell-free protein synthesis; In vitro translation; Protein expression; Saccharomyces cerevisiae.