Improving Electron Transfer in Cytochrome C-Expressing Escherichia coli by Modulating Cytochrome C Maturation

Heterologously expressing Shewanella oneidensis MR-1 cytochromes c (cyt c) in Escherichia coli enables cellular states to be monitored and controlled through electron flow. However, the electron flux in E. coli is significantly less than Shewanella oneidensis MR-1, thus limiting the extent of our electronic control. We hypothesize that differences in the cytochrome c maturation (ccm) machinery between these two species may be responsible for the lower expression levels of cyt c and extracellular electron transfer (EET) efficiency. In this research, we constructed random mutations within ccm genes by prone error PCR and screened for increases in cyt c production in E. coli in 96-well plates. Two ccmH mutation strains were shown to exhibit increased cyt c expression. Since these two mutations occurred in the C-terminal domain of ccmH, which has a similar structure of ccmI in the Shewanella oneidensis MR-1, we also constructed a hybrid ccmH comprising the N-terminal domain of Escherichia coli and the C-terminal domain of Shewanella oneidensis MR-1 (ccmH^N:ccmI). Indeed, cells from this engineered ccmH^N:ccmI strain possess a redder color and higher expression of cyt c. We measured the electron flux out of these modified variants using three-electrode microbial electrochemical experiments and showed that the ccmH^N:ccmI strain produces ~40% more current per cell. Thus, modulating maturation of cyt c is not only possible, but can improve electron transfer in synthetic biological systems.