Phosphoserine as an Alternative Energy Source for E. coli Cell-Free Protein Synthesis with Increased Yield and Prolonged Activity
Year: 2026
Type: article
Abstract: High Resolution Image Download MS PowerPoint Slide 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.
Source: ACS Synthetic Biology
Institutions: Technion – Israel Institute of Technology, Lake Erie College of Osteopathic Medicine, Massachusetts Institute of Technology, Universidade do Porto, i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Instituto de Ciências Biomédicas Albel Salazar, Cooperativa de Ensino Superior Politécnico e Universitário, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
Funders: European Commission, Weizmann Institute of Science, Israel Science Foundation, Lorry I. Lokey Interdisciplinary Center for Life Sciences and Engineering, Technion-Israel Institute of Technology, Council for Higher Education, Technion-Israel Institute of Technology, Cooperativa de Ensino Superior Politécnico e Universitário, Universidade do Porto, Russell Berrie Nanotechnology Institute, Technion-Israel Institute of Technology, European Research Council, Fundação para a Ciência e a Tecnologia
OpenAlex ID: W7165143828