Flavin Biosynthesis Enhances Extracellular Electron Transfer in Bioengineered Escherichia coli

Microbial electronics are promising for energy, sensing, environmental, and synthesis applications. E. coli are engineered with extracellular electron transfer (EET) pathways from a microbe that naturally produces current to enable bioelectronics based on E. coli. Abstract Advancements in bioengineering have unlocked new microbial electrochemical applications in energy, sensing, remediation, and synthesis. Key to realizing these technologies is the engineering of conduits in metabolically versatile microbes like Escherichia coli to enable efficient charge exchange with the electrode. Inspired by mechanisms found in natural exogelectrogens, previous studies have largely focused on introducing conduits based on the metal‐reducing (Mtr) pathway in Shewanella oneidensis MR‐1. This study explores the concomitant expression of flavin secretion pathways for mediated charge transfer to complement the direct charge transfer from the bioengineered Mtr pathway. The engineered strains show a 3‐fold increase in the total secretion of flavin mononucleotide (FMN) and riboflavin compared to a state‐of‐the‐art Mtr‐expressing strain lacking flavin overexpression. The concomitant flavin secretion further contributes up to a ≈3.4‐ and ≈1.5‐fold increase in current compared to unmodified cells and the previous Mtr‐expressing cells, respectively, with the greatest currents achieved for the strain favoring riboflavin secretion over FMN secretion. The introduction of flavin biosynthesis genes to Mtr‐expressing strains thus reveals a distinct, yet complementary, EET mechanism for robust and multi‐modal microbial applications. Microbial electronics are promising for energy, sensing, environmental, and synthesis applications. E. coli are engineered with extracellular electron transfer (EET) pathways from a microbe that naturally produces current to enable bioelectronics based on E. coli. Abstract Advancements in bioengineering have unlocked new microbial electrochemical applications in energy, sensing, remediation, and synthesis. Key to realizing these technologies is the engineering of conduits in metabolically versatile microbes like Escherichia coli to enable efficient charge exchange with the electrode. Inspired by mechanisms found in natural exogelectrogens, previous studies have largely focused on introducing conduits based on the metal-reducing (Mtr) pathway in Shewanella oneidensis MR-1. This study explores the concomitant expression of flavin secretion pathways for mediated charge transfer to complement the direct charge transfer from the bioengineered Mtr pathway. The engineered strains show a 3-fold increase in the total secretion of flavin mononucleotide (FMN) and riboflavin compared to a state-of-the-art Mtr-expressing strain lacking flavin overexpression. The concomitant flavin secretion further contributes up to a ≈3.4- and ≈1.5-fold increase in current compared to unmodified cells and the previous Mtr-expressing cells, respectively, with the greatest currents achieved for the strain favoring riboflavin secretion over FMN secretion. The introduction of flavin biosynthesis genes to Mtr-expressing strains thus reveals a distinct, yet complementary, EET mechanism for robust and multi-modal microbial applications. Advanced Science, Volume 13, Issue 2, 9 January 2026.