Enhancing the Performance of Microbial Fuel Cells via Metabolic Engineering of Escherichia coli for Phenazine Production

One of the central roles in the design, development,and applicationadvances in mediator-based microbial electrochemical systems, suchas microbial fuel cells (MFCs), is the establishment of efficientand successful communication between conductive electrode surfacesand microorganisms via modes of extracellular electron transfer (EET).Most microbial-based systems require the use of artificial electroactivemediators in order to facilitate and/or enhance electron transfer.Our previous work established an exogenous phenazine-based libraryas a mediator system to enable EET from the model microorganism Escherichia coli as a promising biotechnological host. However,the addition of exogenous mediators to a microbial electrochemicalsystem has certain limiting downsides, specifically with regard tomediator toxicity to cells and increased operational expenses. Herein,we demonstrate the metabolic and genetic engineering of E. coli to self-generate phenazine metabolites endogenouslyby introducing the phenazine biosynthetic pathway from P.aeruginosa into E. coli. This biosynthetic pathway contains a phenazine cluster of sevengenes, namely, phzABCDEFG (phzA-G), responsible for the synthetic conversion of phenazine-1-carboxylicacid (PCA) from chorismic acid, and two additional phenazine accessorygenes phzM and phzS to catalyzethe transformation of PCA to pyocyanin (PYO). We present the characterizationof the engineered E. coli cellsthat were collected via electrochemical measurements, RNA sequencing,and microscopy imaging. Finally, the engineered E. coli cells were used for the design of a microbialfuel cell with enhanced performances, demonstrating a maximum powerdensity increase from 127 & PLUSMN; 5 mW m(-2) with nonengineered E. coli cells to 806 & PLUSMN; 7 mW m(-2) with genetically engineered, phenazine-producing E. coli. Our results indicate that theintroduction of a heterologous electron shuttle into E. coli is not only an efficient, but also a promisingstrategy toward establishing efficacious electron mediation in livingbioelectrochemical systems and enhancing the overall MFC performancerelated to the MFC current generation and power output. Metabolic engineering of E. coli to produce phenazine electron mediator carriers for enhancing thepower outputs of microbial fuel cells.