European Synthetic Cell Initiative (@syncelleu.bsky.social)

Publication Energy regeneration in CFPS systems is challenging. The article shows that phosphoserine is a promising energy source for E. coli lysate-based CFPS systems. It also validated the use of these optimised CFPS compositions within bilayer lipid vesicles edu.nl/3qjde #SyntheticCell #SynCell

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Phosphoserine as an Alternative Energy Source for E. coli Cell-Free Protein Synthesis with Increased Yield and Prolonged Activity Energy-supplying molecules are essential for biological processes, particularly for transcription and translation. Cell-free protein synthesis (CFPS) systems are powerful tools for in vitro protein production, offering flexibility for applications ranging from high-throughput protein screening to therapeutic protein production. However, energy regeneration in CFPS remains a key challenge, particularly for large-scale or resource-constrained settings. In this study, we introduce phosphoserine (PS) as a simple, cost-effective alternative secondary energy source, capable of partially or fully replacing 3-phosphoglycerate (PGA), the commonly used energy donor in E. coli lysate-based CFPS, whose availability is often limited. By supplementing CFPS reactions with PS, we demonstrate significant improvements in protein yield and cost-efficiency, achieving a 2-fold increase in protein production. Importantly, PS enhancement is maintained across lysate batches and protein targets. Furthermore, we offer affordable CFPS compositions that retain protein synthesis, making the system more accessible for resource-limited settings. Additionally, we show that higher PS concentrations, while reducing final protein yield, extend the reaction duration by more than 2-fold. Therefore, the incorporation of PS as an alternative energy donor enables a tunable modality for balancing protein yield, reaction longevity, and cost. Our data support a model in which PS enhances CFPS via the serine biosynthesis pathway by modulating flux between serine production and glycolysis to support ATP regeneration. Lastly, we validate the use of these optimized CFPS compositions within synthetic cells (SCs). This study establishes PS as a promising energy source for E. coli lysate-based CFPS systems, paving the way for enhanced and economical protein synthesis platforms tailored to diverse clinical, biotechnological, and industrial needs. https://edu.nl/3qjde