Implementation of a flavin biosynthesis operon improves extracellular electron transfer in bioengineered Escherichia coli

Bioelectrochemical systems (BES) are promising for energy, sensing, environmental, and synthesis applications. Escherichia coli were previously bioengineered for application in BES by introduction of extracellular electron transfer (EET) pathways. Inspired by the metal-reducing (Mtr) pathway of Shewanella oneidensis MR-1, several of its cytochromes were heterologously expressed in E. coli, leading to increased EET rates and successful application in BES. Besides direct electron transfer, S. oneidensis MR-1 is known to secrete flavins that act as redox mediators and are crucial for high EET rates. Here we co-express the Mtr pathway and a flavin biosynthesis pathway in E. coli, to enhance EET in engineered strains. The secretion of both flavin mononucleotide and riboflavin was increased up to 3-fold in engineered strains. Chronoamperometry revealed an up to ~3.4-fold increase in current over the wild type when co-expressing cytochromes and flavin biosynthesis genes, and a ~2.3-fold increase when expressing flavin biosynthesis genes on their own. Thus, the introduction of flavin biosynthesis genes yields in a distinct, yet complementary EET mechanism, and holds promise for application in BES.