Online monitoring of the mitochondrial respiration activity and protein formation in the Almost Living Cell-free Expression (ALiCE) system

Cell-free protein synthesis (CFPS) is one approach to address the increasing demand for complex recombinant proteins in various applications, especially in the pharmaceutical sector. CFPS offers a variety of advantages like the ability to express cytotoxic proteins, no need for transformations or screening of strains and, thus, reduced production times. Often industrially relevant proteins require post-translational modifications (PTM). While disulfide bonds can be obtained with prokaryotic systems, some eukaryotic CFPS systems are able to perform glycosylation to a limited extent. However, scaling the production of a eukaryotic CFPS system and protein production has been a main challenge to enable the manufacturing of complex proteins with these CFPSs. One plant-based system that overcomes these limitations is the Almost Living Cell-free Expression (ALiCE) system, which is based on tobacco BY-2 cells and can produce protein titers of up to 3 µg/µL in batch mode. This study focuses on gaining a deeper understanding of oxygen demand, protein formation and the role of mitochondria in this CFPS system. Online monitoring was established in a combined µRAMOS-BioLector device to investigate the correlation of oxygen transfer, eYFP production and NADH levels during the ALiCE reaction. By varying the maximum oxygen transfer capacity, it was revealed that oxygen availability is tightly coupled to protein formation and that the eYFP production rate decreases with decreasing oxygen availability. Moreover, the mitochondrial inhibitors salicylhydroxamic acid (SHAM) and potassium cyanide (KCN) were added to ALiCE reactions to examine the influence and importance of mitochondrial alternative oxidase and cytochrome c oxidase on the ALiCE reaction. Inhibition of alternative oxidase and cytochrome c oxidase demonstrated that oxygen is consumed in the respiratory chain of intact mitochondria within the ALiCE system. In addition, the NADH balance and eYFP formation are highly dependent on oxygen availability. For the first time, a plant-based cell-free expression system was characterized concerning oxygen demand and the influence of oxygen availability on the kinetics of protein production. The new findings enable the design of ALiCE experiments in mL-scale with optimal oxygen supply for protein formation in the future and provide first insights into the energy metabolism of this plant-based CFPS system.