Engineering synthetic metabolism for enhanced cell-free protein synthesis in the PURE system

Abstract

This thesis presents advancements in the Protein synthesis Using Recombinant Elements (PURE) system, a minimal, cell-free protein synthesis platform composed entirely of purified components. PURE is widely used in synthetic biology due to its defined nature, which allows for precise control over biochemical processes. However, one of the limitations of the PURE system is its relatively low protein yield and short reaction lifetime compared to lysate-based systems, which may result from inefficient ATP recycling mechanisms or the accumulation of inhibitory by-products such as inorganic phosphate.

To address this limitation, this work integrates an ATP regeneration system into PURE, utilizing a synthetic pathway that combines the activities of pyruvate oxidase, acetate kinase, and catalase. The integrated pathway regenerates ATP by converting pyruvate, phosphate, and oxygen into acetyl phosphate, which in turn rephosphorylates ADP to ATP. Remarkably, the addition of a high initial phosphate concentration (∼10 mM), which is necessary for the reaction, does not negatively impact the protein synthesis activity of the PURE system. This finding opens new possibilities for enhancing energy efficiency in cell-free systems.

Furthermore, the ATP regeneration pathway can function both independently and in combination with the existing creatine phosphate/creatine kinase (CP/CK) system, leading to significant improvements in protein production yields. The CP/CK system utilizes creatine phosphate as a substrate to regenerate ATP through the action of the creatine kinase enzyme. In particular, the combined system is capable of synthesising up to 233 µg/ml of mCherry, representing a 78% increase in yield compared to using the creatine system alone. The robustness of this approach is demonstrated through reproducible results across multiple batches of homemade PURE, and importantly, the system’s general applicability is shown through successful implementation in the commercial PURE system, PURExpress®.

Additionally, preliminary experiments show that alternative ATP regeneration pathways, such as those based on glycolysis, could also be integrated into the PURE system to further expand its capabilities. Overall, this thesis provides a systematic approach to enhancing the efficiency of the PURE system by introducing rational, modular ATP regeneration strategies. The results lay the groundwork for broader applications of PURE in areas such as cell-free synthetic biology, metabolic engineering, and the construction of synthetic cells, where controlled and sustained energy supply is crucial for functional, long-term protein synthesis.