Modifying Cytochrome <i>c</i> Maturation Can Increase the Bioelectronic Performance of Engineered <i>Escherichia coli</i>

Abstract

Genetic circuits that encode extracellular electron transfer (EET) pathways allow the intracellular state of <i>Escherichia coli</i> to be electronically monitored and controlled. However, relatively low electron flux flows through these pathways, limiting the degree of control by these circuits. Since the EET pathway is composed of multiple multiheme cytochromes <i>c</i> (cyts <i>c</i>) from <i>Shewanella oneidensis</i> MR-1, we hypothesized that lower expression levels of cyt <i>c</i> may explain this low EET flux and may be caused by the differences in the cyt <i>c</i> maturation (<i>ccm</i>) machinery between these two species. Here, we constructed random mutations within <i>ccmH</i> by error-prone PCR and screened for increased cyt <i>c</i> production. We identified two <i>ccmH</i> mutants, <i>ccmH</i>-132 and <i>ccmH</i>-195, that exhibited increased heterologous cyt <i>c</i> expression, but had different effects on EET. The <i>ccmH</i>-132 strain reduced WO₃ nanoparticles faster than the parental control, whereas the <i>ccmH</i>-195 strain reduced more slowly. The same trend is reflected in electrical current generation: <i>ccmH</i>-132, which has only a single mutation from WT, drastically increased current production by 77%. The percentage of different cyt <i>c</i> proteins in these two mutants suggests that the stoichiometry of the <i>S. oneidensis</i> cyts <i>c</i> is a key determinant of current production by Mtr-expressing <i>E. coli</i>. Thus, we conclude that modulating cyt <i>c</i> maturation effectively improves genetic circuits governing EET in engineered biological systems, enabling better bioelectronic control of <i>E. coli</i>.