Dynamic synthesis and transport of phenazine-1-carboxylic acid to boost extracellular electron transfer rate

Electron shuttle plays a decisive role in extracellular electron transfer (EET) of exoelectrogens. However, neither identifying the most efficient electron shuttle molecule nor programming its optimal synthesis level that boosts EET has been established. Here, the phenazine-1-carboxylic acid (PCA) biosynthesis pathway is first constructed to synthesize PCA at an optimal level for EET in Shewanella oneidensis MR-1. To facilitate PCA transport, the porin OprF is expressed to improve cell membrane permeability, the cytotoxicity of which, however, impaired cell growth. To mitigate cytotoxicity, PCA biosensor is designed to dynamically decouple PCA biosynthesis and transport, resulting in the maximum output power density reaching 2.85 ± 0.10 W m^-2, 33.75-fold higher than wild-type strain. Moreover, extensive analyses of cellular electrophysiology, metabolism, and behaviors reveal PCA shuttles electrons from cell to electrode, which is the dominant mechanism underlying PCA-boosted EET. We conclude dynamic synthesis and transport of PCA is an efficient strategy for enhancing EET.