Modifying Cytochrome c Maturation Can Increase the Bioelectronic Performance of Engineered Escherichia coli

Abstract

Genetic circuits that encode extracellular electron transfer (EET) pathways allow the intracellular state of Escherichia coli 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 c (cyts c) from Shewanella oneidensis MR-1, we hypothesized that lower expression levels of cyt c may explain this low EET flux and may be caused by the differences in the cyt c maturation (ccm) machinery between these two species. Here, we constructed random mutations within ccmH by error-prone PCR and screened for increased cyt c production. We identified two ccmH mutants, ccmH-132 and ccmH-195, that exhibited increased heterologous cyt c expression, but had different effects on EET. The ccmH-132 strain reduced WO 3 nanoparticles faster than the parental control, whereas the ccmH-195 strain reduced more slowly. The same trend is reflected in electrical current generation: ccmH-132, which has only a single mutation from WT, drastically increased current production by 77%. The percentage of different cyt c proteins in these two mutants suggests that the stoichiometry of the S. oneidensis cyts c is a key determinant of current production by Mtr-expressing E. coli. Thus, we conclude that modulating cyt c maturation effectively improves genetic circuits governing EET in engineered biological systems, enabling better bioelectronic control of E. coli.

Precise electronic control of redox reactions insideEscherichia coliusing a genetic module

“…To prepare E. coli for cathodic conditions, individual strains were first grown aerobically, incubated in potentiostatic-controlled bioreactors under anaerobic, anodic conditions (ΔV=+200 mV Ag/AgCl ) for at least one day. Under these conditions, the CymAMtr -E. coli strain produced a significant steady-state current, while Ccm -E. coli and Mtr -E. coli produced much lower currents ( Figure 2A ), reinforcing that CymA is important for current production 16–18 . As anaerobic conditions were maintained, the electrode bias was then switched to cathodic conditions (ΔV= −560 V Ag/AgCl ), fumarate was added, and current consumption was measured.…”

“…To couple an electrode to specific redox molecules in a bacteria of our choosing, we and others have introduced genes from the Mtr pathway from Shewanella oneidensis MR-1 into heterologous bacterial hosts 14–18 . Under anaerobic conditions, S. oneidensis can use the Mtr pathway to transfer electrons from catabolism to produce a current at an extracellular electrode ( Figure 1A ).…”

“…We have previously shown that oxidation of intracellular lactate can be coupled to current production in Escherichia coli that heterologously express cymAmtrCAB 16,18 . This led us to hypothesize that the Mtr pathway could couple oxidation of a cathode to reduction of intracellular biomolecules.…”

A hybrid cytcmaturation system enhances the bioelectrical performance of engineeredEscherichia coliby improving the rate-limiting step

“…Expressing these cyt c requires the cytochrome c maturation (Ccm) system, which covalently ligates heme groups to the apo-protein (Sanders et al, 2010; Verissimo et al, 2011). We and others have found that expressing components of the Mtr pathway and the Ccm system enables E. coli to perform EET (Jensen et al, 2016; Su et al, 2020; TerAvest et al, 2014). In the Ccm systems of Gram-negative bacteria, CcmH is essential to the heme ligation process (Sanders et al, 2010), which includes translocating heme groups, and formatting a thioether bond between the heme group and the apo cyt c. We recently found that mutations in the C-terminal domain of CcmH can significantly change the EET efficiency by changing the stoichiometry of the Mtr cyt c (Su et al, 2020).…”

“…We and others have found that expressing components of the Mtr pathway and the Ccm system enables E. coli to perform EET (Jensen et al, 2016; Su et al, 2020; TerAvest et al, 2014). In the Ccm systems of Gram-negative bacteria, CcmH is essential to the heme ligation process (Sanders et al, 2010), which includes translocating heme groups, and formatting a thioether bond between the heme group and the apo cyt c. We recently found that mutations in the C-terminal domain of CcmH can significantly change the EET efficiency by changing the stoichiometry of the Mtr cyt c (Su et al, 2020). This finding suggested that optimizing the heme ligation process by redesigning CcmH could remodel the stoichiometry of cyt c and enhance the bioelectrical performance of Mtr-expressing E. coli.…”