(792g) Characterizing and Alleviating Substrate and By-Product Limitations for Improved Yeast Cell-Free Protein Synthesis

Cell-free protein synthesis (CFPS) has become a powerful technology for high-throughput, rapid production of proteins. Towards the development of a cost-effective, high-yielding eukaryotic CFPS platform, we recently reported a system derived from Saccharomyces cerevisiae. Here, we study factors causing the early cessation of protein synthesis in a yeast CFPS batch reaction. Specifically, high-performance liquid chromatography and chemical assays were used to characterize the substrate and by-product concentrations during the CFPS reaction over time. We discovered that creatine phosphate, the secondary energy substrate, as well as nucleoside triphosphates were rapidly degraded during the cell-free reaction. This led to a significant drop in the energy charge of the reaction (from 1 to 0.4 in 3 hr), and as a consequence, the cessation of protein synthesis. Of the 20 amino acids, aspartic acid, asparagine, alanine, and proline were consumed.  Additionally, inorganic phosphate accumulated, which is a toxic by-product. Semi-continuous exchange reactions, where passive diffusion enables substrates to be replenished and by-products to be removed, led to over a 100% increase in active superfolder green fluorescent protein (sfGFP) expressed as compared to the batch system.  This yielded 17.9 ± 3.7 µg sfGFP mL^-1 in a 10 hr semi-continuous reaction. In addition to increasing sfGFP production, this characterization identified promising targets for future improvements to the batch reaction format.