CO 2 -elevated cell-free protein synthesis.

Gases are the vital nutrition of all organisms as the precursor of metabolism pathways. As a potential biological process, protein synthesis is inevitably regulated by gas transport and utilization. However, the effect of carbon dioxide (CO) present in many metabolic pathways on protein synthesis has not been studied well. In this work, carbon dioxide combined with oxygen was employed for cell-free protein synthesis (CFPS) in the tube-in-tube reactor with precise control of gas concentration. In this system, gases could directly affect the protein synthesis process without transmembrane transport. Varied concentrations of carbon dioxide (0-1%) and constant oxygen concentration (21%) were employed for CFPS to assess the effects. The cell-free reactions with 0.3% CO and 21% O showed the highest protein yields. The combined effect of CO and O also resulted in relatively high protein expression under high oxygen conditions (0.3% CO and 100% O). Moreover, metabolomics assays were performed to gain insight into metabolic changes, which showed that CO slightly improved energy metabolism and redox balance. In particular, the extra supplied CO activated the decarboxylating reactions and removed toxic metabolites to recover the protein synthesis activity. The exploration of CO on protein synthesis could provide guiding implications for basic studies and biomanufacturing.