Guiding electron transfer at the microbe-electrode interface: the role of MtrCAB

Project Information

MTRoute

Grant agreement ID: 101130784

DOI

10.3030/101130784

Project closed

EC signature date 8 May 2023

Start date 1 June 2023

End date 31 May 2025

Funded under

• Widening participation and spreading excellence

Total cost

No data

EU contribution

€ 172 618,56

Coordinated by

UNIVERSIDADE NOVA DE LISBOA
Portugal

Periodic Reporting for period 1 - MTRoute (Guiding electron transfer at the microbe-electrode interface: the role of MtrCAB)

Reporting period: 2023-06-01 to 2025-05-31

Summary of the context and overall objectives of the project

The project MTRoute investigated the molecular mechanisms of extracellular electron transfer (EET) mediated by the porin–cytochrome complex MtrCAB from different Shewanella species, as well as from more phylogenetically distant organisms such as Aeromonas hydrophila and Vibrio natriegens. This complex is composed of the decaheme cytochromes MtrA and MtrC, and the porin protein MtrB where MtrA is embedded (Figure). These complexes span the outer membrane of specialized microorganisms known as electroactive microbes and enable electrons to move from the cell interior to external acceptors such as minerals or electrodes. Understanding how these proteins facilitate electron transport is crucial for advancing bioelectrochemical systems (BES), a promising technology that harnesses electroactive microbes to transfer electrons produced by the oxidation of organic matter in wastewater to electrodes, thereby generating electricity. This particular BES system is generally called a Microbial Fuel Cell (MFC). However, the performance of BES is currently limited by incomplete knowledge of the structural and mechanistic factors that control and enhance microbial electron transfer at the cell–electrode interface.
The overall objective of MTRoute was to elucidate the structural and functional diversity of MtrCAB complexes and identify the molecular mechanisms that control electron transfer at the bacterial cell surface. Through an integrated approach combining molecular biology, structural biophysics (NMR spectroscopy, X-ray crystallography and analytical ultracentrifugation (AUC)), (bio)electrochemical analyses, and computational modelling, the project was crucial to:
1. Characterize the functional diversity of MtrCAB complexes from different electroactive bacteria;
2. Identify the molecular factors that define electron transfer rates;
3. Engineer the model electroactive microbe Shewanella oneidensis with enhanced electron transfer capabilities for improved BES performance.
MTRoute aims to generate new insights into the mechanistic basis of EET and to identify structural features that can be harnessed to rationally design high-performance electroactive microbes. These advances have the potential to accelerate the development of sustainable energy technologies to generate renewable electricity from wastewater. By enabling more efficient BES, the project directly supports the objectives of the European Green Deal and REPowerEU, which aim to reduce dependence on fossil fuels, expand renewable energy production, and drive the transition to a climate-neutral European economy.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The project successfully investigated the functional diversity of MtrCAB complexes from different electroactive bacteria. The engineered strains harboring variant MtrCAB complexes were evaluated for current production in bioelectrochemical systems (BES). Comparative BES experiments revealed that heterologous MtrCAB complex have the capacity of producing notably higher current when compared with native ones, particularly during the early stages of the experimental run. To further explore the molecular factors underlying extracellular electron transfer (EET), the corresponding MtrC subunits were overproduced and purified for detailed structural and functional studies. These included one-dimensional proton NMR spectroscopy to investigate specific interactions between MtrC and key electron shuttles such as FMN, PMS, AQDS, and riboflavin, as well as Saturation Transfer Difference (STD) NMR to map ligand-binding epitopes.
Crystallization and co-crystallization trials were performed to obtain three-dimensional structures of MtrC in both free and ligand-bound states. Well-formed crystals were obtained under several conditions, and X-ray diffraction analysis was performed to determine their high-resolution structures. Analytical ultracentrifugation (AUC) experiments further provided insights into the oligomeric state, overall shape, and potential complex formation of MtrC, complementing the structural data.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The MTRoute project delivers new mechanistic and structural insights into extracellular electron transfer (EET), advancing the state of the art in microbial bioelectrochemistry. By systematically comparing heterologous MtrCAB complexes in engineered Shewanella strains, the project identified a variant that produces higher current output in bioelectrochemical systems (BES), particularly during the early stages of operation, compared to the model organism Shewanella oneidensis MR-1. This outcome strengthens the mechanistic basis for selecting and engineering electron transfer pathways, contributing to ongoing efforts to improve the efficiency of microbial electrochemical technologies.
At the molecular level, the project provided structural evidence of how MtrC interacts with soluble electron shuttles such as FMN, PMS, AQDS, and riboflavin. One-dimensional proton NMR and Saturation Transfer Difference (STD) NMR revealed distinct binding epitopes for each ligand, highlighting diverse interaction modes. Complementary crystallization trials produced well-formed crystals of MtrC in both free and ligand-bound states, paving the way for high-resolution X-ray structures. Analytical ultracentrifugation (AUC) further clarified the potential complex formation of MtrC, providing an in-depth structural basis for understanding and manipulating EET.
Building on these advances, the project will continue to explore the structural and molecular factors underlying EET and translate these insights into the design of next-generation electroactive microbes, supporting future innovations in sustainable energy technologies.

Schematic representation of the MtrCAB complex across the outer membrane (modelled using PDB: 6R2Q)