Modulation of Intracellular O₂ Concentration in Escherichia coli Strains Using Oxygen Consuming Devices

The use of cell factories for the production of bulk and value-added compounds is nowadays an advantageous alternative to the traditional petrochemical methods. Nevertheless, the efficiency and productivity of several of these processes can improve with the implementation of micro-oxic or anoxic conditions. In the industrial setting, laccases are appealing catalysts that can oxidize a wide range of substrates and reduce O₂ to H₂O. In this work, several laccase-based devices were designed and constructed to modulate the intracellular oxygen concentration in bacterial chassis. These oxygen consuming devices (OCDs) included Escherichia coli's native laccase (CueO) and three variants of this protein obtained by directed evolution. The OCDs were initially characterized in vitro using E. coli DH5α protein extracts and subsequently using extracts obtained from other E. coli strains and in vivo. Upon induction of the OCDs, no major effect on growth was observed in four of the strains tested, and analysis of the cell extract protein profiles revealed increased levels of laccase. Moreover, oxygen consumption associated with the OCDs occurred under all of the conditions tested, but the performance of the devices was shown to be strain-dependent, highlighting the importance of the genetic background even in closely related strains. One of the laccase variants showed 13- and 5-fold increases in oxidase activity and O₂ consumption rate, respectively. Furthermore, it was also possible to demonstrate O₂ consumption in vivo using l-DOPA as the substrate, which represents a proof of concept that these OCDs generate an intracellular oxygen sink, thereby manipulating the redox status of the cells. In addition, the modularity and orthogonality principles used for the development of these devices allow easy reassembly and fine-tuning, foreseeing their introduction into other chassis/systems.