Abstract
Establishing an efficient and successful extracellular electron transfer (EET) between microorganisms and electrode surfaces plays a critical role in the design, development, and application of mediated microbial electrochemical technologies, such as microbial fuel cells (MFCs). Most microbial-based systems require the use of artificial, redox-active mediator systems in order to facilitate and/or increase electron transfer. Our previous work established an exogenous phenazine-based library as a mediator system to enable electron transfer from the model microorganism Escherichia coli. However, the addition of exogenous mediators to a microbial electrochemical system has certain limiting downsides, specifically with regard to mediator toxicity to cells and increased operational expenses. Herein, we demonstrate the metabolic and genetic engineering of E. coli to self-generate phenazine metabolites endogenously by introducing the phenazine biosynthetic pathway from Pseudomonas aeruginosa into E. coli. This pathway consists of a seven-gene phenazine biosynthetic cluster phzA-G responsible for the synthesis of phenazine-1-carboxylic acid (PCA) and two phenazine accessory genes phzM and phzS to produce pyocyanin (PYO). We present the characterization of the engineered E. coli cells via electrochemical measurements, RNA sequencing, and microscopy imaging. Finally, the engineered E. coli cells were used for the design of a microbial fuel cell with enhanced performances, demonstrating a maximum power density increase from 305 mW/m2 with non-engineered E. coli cells to 1555 mW/m2 with the genetically engineered, phenazine-producing E. coli. Our results indicate that introducing a heterologous electron shuttle into E. coli can be an efficient approach to establishing efficacious mediation in living bioelectrochemical systems and improving the performance of MFCs.