Cytochromes

Overview

Figure 1: Extracellular electron transfer via cytochromes in E. coli with a minimal set of genes with a minimal set of genes from Shewanella oneidensins MR-1.

To enable transfer of electrons from the general metabolism to the outside of the cell the mtrCAB gene cluster from Shewanella oneidensis MR-1 was heterologously expressed in E. coli. The cluster encodes for three genes that form a electron shuttle pathway via different c-type cytochromes an a β -barrel membrane protein. These genes represent a minimal set of genes for a working pathway.
For correct heme insertion the cytochrome c maturation machinery is required. The corresponding genes are naturally expressed in E.coli under anaerobic cultivation, for aerobic expression they have to be expressed via plasmid. The mtrCAB cluster contains two illegal restriction sites, which where removed by a silent mutation via overlap-extension-PCR.
The resulting three fragments were combined and ligated with pSB1C3 via Gibson assembly. Subsequently this gene cluster was combined with three promotors and ribosome binding sites of varying strength form the parts registry. Furthermore it was attempted to clone the ccmAH cluster as well was unsuccesfull. This is, however a minor issue, since the microbial fuel cell will work under anareobic conditions. The proteins could not be functional characterized via SDS-PAGE and absorption spectrometry.

Theory

Cell membranes work as a natural insulator and prevent the flow from electrons out of the cell. To enable transfer of electrons from the general metabolism to the outside of the cell we had to alter the membrane of our organism E. coli without disturbing cell growth, stability and metabolism. Some species from the genera Shewanella and Geobacter have developed different mechanisms to allow extracellular electron transfer. In Shewanella oneidensis MR-1 this is achieved via different c-type cytochromes, which shuttle the electrons along a defined molecular route from the cytoplasma and the inner membrane to the outside of the cell during anaerobic respiration. This pathway is very well understood and characterized.

Previous work suggests that a working electron transfer chain can be achieved by a minimal set of three genes, tthe periplasmatic decaheme MtrA, the outer membrane β -barrel protein mtrB and the outer membrane cytochromes MtrC. MtrA interacts with at least one native redox protein, f.e. CymA and can therefore start the transfer of electrons.
Additionally another set of genes, the cytochrome c maturation genes (ccmABCDEFGH), is required for correct protein localization and heme insertion into MtrA and MtrC. Under anaerobic conditions these genes are naturally expressed in E. coli, whereas under aerobic conditions we had to co-express them. For aerobic growth the cells were transformed with both plasmids, containing the cytochrome-cluster, as well as the ccm-cluster . By this approach extracellular electron transfer should be possible in E.coli and allow the use of this genetically engineered strain in a microbial fuel cell.

Genetic Approach

Results

References