CC BY 4.0Schloßhauer, JeffreyJeffreySchloßhauerDondapati, Srujan KumarSrujan KumarDondapatiKubick, StefanStefanKubickZemella, AnneAnneZemella2025-11-062025-11-062024https://publica.fraunhofer.de/handle/publica/498806https://doi.org/10.24406/publica-609210.3390/bioengineering1101009210.24406/publica-60922-s2.0-85183141442Cell-free systems are particularly attractive for screening applications and the production of difficult-to-express proteins. However, the production of cell lysates is difficult to implement on a larger scale due to large time requirements, cultivation costs, and the supplementation of cell-free reactions with energy regeneration systems. Consequently, the methylotrophic yeast Pichia pastoris, which is widely used in recombinant protein production, was utilized in the present study to realize cell-free synthesis in a cost-effective manner. Sensitive disruption conditions were evaluated, and appropriate signal sequences for translocation into ER vesicles were identified. An alternative energy regeneration system based on fructose-1,6-bisphosphate was developed and a ~2-fold increase in protein production was observed. Using a statistical experiment design, the optimal composition of the cell-free reaction milieu was determined. Moreover, functional ion channels could be produced, and a G-protein-coupled receptor was site-specifically modified using the novel cell-free system. Finally, the established P. pastoris cell-free protein production system can economically produce complex proteins for biotechnological applications in a short time.entruecell-free protein synthesisdesign of experimentsenergy regenerationorthogonal systemPichia pastorisprotein productionsignal sequenceyeastjournal article