CO₂-elevated cell-free protein synthesis.

ABSTRACT: 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 (CO₂) 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 in vitro 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% CO₂ and 21% O₂ showed the highest protein yields. The combined effect of CO₂ and O₂ also resulted in relatively high protein expression under high oxygen conditions (0.3% CO₂ and 100% O₂). Moreover, metabolomics assays were performed to gain insight into metabolic changes, which showed that CO₂ slightly improved energy metabolism and redox balance. In particular, the extra supplied CO₂ activated the decarboxylating reactions and removed toxic metabolites to recover the protein synthesis activity. The exploration of CO₂ on protein synthesis could provide guiding implications for basic studies and biomanufacturing.

SUBMITTER: Lin X 

PROVIDER: S-EPMC9136254 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature