Identification of factors limiting the efficiency of transplanting extracellular electron transfer chains in Escherichia coli.

Abstract

Research in electro-microbiology provides unique opportunities to study and exploit microbial physiology. Several efforts have been made to transplant the extracellular electron transport chain from the native exoelectrogenic model organism Shewanella oneidensis into Escherichia coli. However, systematic comparisons between donor and recipient strain configurations are largely missing. Hence, the proposed minimal protein set, consisting of the c-type cytochromes cytoplasmic membrane protein A (CymA), small tetraheme cytochrome (STC), MtrA, and MtrC, as well as the β-barrel protein MtrB, was heterologously expressed in E. coli in different expansion stages. These stages were compared to corresponding S. oneidensis strains in terms of anthraquinone-2,6-disulfonate (AQDS) and ferric citrate reduction rates. This revealed that transplantation of heterologous extracellular electron transfer (EET) chains is associated with a tremendous decrease in electron transfer rates. As the acquired electron transfer rates were not competitive to S. oneidensis, it was hypothesized that protein localization and maturation might be affected by heterologous expression. Hence, the type II secretion system from S. oneidensis was also transplanted into an E. coli strain. The latter allowed the secretion of the terminal reductase MtrC onto the cell surface of E. coli for the first time. This was correlated with significantly increased but still insufficient extracellular electron transfer rates. Further experiments suggest that the correct folding of MtrB might be a further bottleneck.IMPORTANCEResearch on transplanting extracellular electron transfer (EET) chains into non-native exoelectrogens is vital for advancing bioenergy and bioremediation technologies. Enabling these organisms to transfer electrons to external surfaces like anodes can enhance microbial fuel cell efficiency and electricity generation from organic waste. This approach can broaden the range of substrates and products for biotechnological applications, offering innovative solutions for sustainable production. Our work shows that transplanting the EET chain of Shewanella oneidensis into Escherichia coli is more complex than previously suggested. The heterologous expression of only c-type cytochromes and the β-barrel protein MtrB is insufficient for competitive reduction rates. Predominantly, MtrC and MtrB require specific proteins for transport and folding, necessitating co-expression and maturation. We could identify the type II secretion system of S. oneidensis as crucial for MtrC secretion in E. coli. Thereby, this work highlights the substrate specificity of bacterial type II secretion systems, suggesting methods to optimize protein production and secretion in bioelectrochemical applications.