Energizing Yeast Cell-Free Protein Synthesis with Glucose Metabolism

Energizing yeast cell free protein synthesis with glucose metabolism

Jessica C. Stark, Mark Anderson, C. Eric Hodgman, Michael C. Jewett Abstract Cell-free protein synthesis (CFPS) is a powerful technology with a growing number of applications, including high-throughput synthesis of protein libraries and characterization of genetic components. There currently exist high-yielding Escherichia coli CFPS platforms that avoid the use of expensive high-energy phosphate compounds. However, to date, eukaryotic CFPS systems generally utilize such compounds to regenerate the adenosine triphosphate (ATP) necessary to drive protein synthesis. Expensive reagent costs have prevented the widespread use and practical implementation of eukaryotic CFPS technology. In this study, we report the development of the first ever, to our knowledge, eukaryotic CFPS system powered by natural energy metabolism of non-phosphorylated energy substrates. To achieve this, we screened six different glycolytic intermediates for their ability to regenerate ATP and fuel protein synthesis in a Saccharomyces cerevisiae crude extract CFPS platform. We observed the synthesis of 1.05 ± 0.12 µg mL^-1 luciferase when using 16mM glucose as a secondary energy substrate and demonstrate that glycolysis is active by quantifying the production of ethanol during the reaction. With the addition of potassium phosphate, our yields using glucose increased approximately 3.5-fold to 3.64 ± 0.35 µg mL^-1. Although synthesis yields on a gram per liter basis remain lower than the CrP/CrK system previously developed, the cost-to-yield ratio is comparable. These promising developments provide the foundation for further development of a cost-effective eukaryotic CFPS system. Further, this work provides the first evidence that glycolytic metabolism is active in eukaryotic crude extract CFPS platforms. This demonstration may lead the way to development of cost-effective eukaryotic CFPS platforms from multiple host organisms for high throughput protein expression, synthetic biology, proteomics, and structural genomics applications.