Anaerobic Conditioning of E. coli cell Lysate for Enhanced in Vitro protein Synthesis

Cell free protein expression (CFPS) from E. coli cell lysate is an established chemical biology technique especially suited for synthetic biology prototyping. Common efforts to improve synthesis capacity, such as strain engineering and lysate preparation process improvements, have overlooked the opportunity to increase productivity by reducing the dependence on limited, dissolved oxygen. Here we demonstrate conditioning E. coli cells for anaerobic respiration prior to standard lysate preparation which increases the initial protein expression rate by four-fold and titer by 50% as compared to a panel of aerobically cultured cell lysates (custom and commercial) when using sfGFP as a reporter protein in CFPS reactions run at atmospheric conditions. In this work we demonstrate that the advantage of using anaerobically conditioned lysate is even more pronounced when CFPS reactions are conducted in oxygen-depleted environments that mimic larger vessel CFPS experiments or oxygen-limited microdroplet reactors. For the first time, we demonstrate the possibility of increasing CFPS productivity through conditioning cells to use alternative terminal acceptors (TEA) such as nitrate. Further improvements in preconditioning cell lysate are possible through optimizing expression levels of anaerobic metabolism enzymes and screening other TEA candidates.