ABSTRACT Cell‐free protein synthesis (CFPS) is a powerful and versatile platform that supports a wide range of applications, from fundamental studies of the genetic code to scalable and rapid protein production. The recently developed Pichia pastoris CFPS combines advantages of both prokaryotic and eukaryotic systems, including a rapid growth rate, inexpensive cultivation media, a well‐established genetic toolbox, and the capability to perform post‐translational modifications (PTMs). As such, it represents a promising alternative for both academic research and biopharmaceutical manufacturing. However, its broader application has been limited by relatively low protein yields and high reagent costs. In this study, building on a previously optimized reaction protocol, we further advanced the P. pastoris CFPS towards a more economical and efficient platform by reducing the cost of protein production. Through systematic screening of chemical additives and their combinations, we identified the most effective stabilizers and crowding agents to be incorporated in the reaction. Additionally, we applied a machine learning model to predict translation initiation rates and optimized the Kozak sequence for enhanced expression. We also evaluated lower‐cost glycolytic intermediates as alternative substrates for ATP regeneration to reduce the cost of goods. Compared with the initial baseline condition using the unoptimized CrP/CrK system, the optimized system, incorporating a modified Kozak sequence and the addition of PEG‐6000 and spermidine, resulted in a 10‐fold increase in protein yield, while reducing the cost per gram of protein by 89%. This work underscores the importance of protein‐stabilizing additives and the role of rationally designed DNA sequences with minimized mRNA structural complexity to enhance yield in CFPS. Our demonstration of glycolytic intermediates as a potential secondary energy system additionally provides the foundation for the development of a cost‐effective P. pastoris CFPS.
Wu et al. (Mon,) studied this question.