Engineering of a synthetic electron conduit in living cells

Abstract

Engineering efficient, directional electronic communication between living and nonliving systems has the potential to combine the unique characteristics of both materials for advanced biotechnological applications. However, the cell membrane is designed by nature to be an insulator, restricting the flow of charged species; therefore, introducing a biocompatible pathway for transferring electrons across the membrane without disrupting the cell is a significant challenge. Here we describe a genetic strategy to move intracellular electrons to an inorganic extracellular acceptor along a molecularly defined route. To do so, we reconstitute a portion of the extracellular electron transfer chain of Shewanella oneidensis MR-1 into the model microbe Escherichia coli. This engineered E. coli can reduce metal ions and solid metal oxides ∼8× and ∼4× faster than its parental strain. We also find that metal oxide reduction is more efficient when the extracellular electron acceptor has nanoscale dimensions. This work demonstrates that a genetic cassette can create a conduit for electronic communication from living cells to inorganic materials, and it highlights the importance of matching the size scale of the protein donors to inorganic acceptors.

Fig. 1.

(A) Schematic of proposed extracellular electron transfer pathway in Shewanella oneidensis MR-1 where ES denotes the extracellular space, P denotes the periplasm, and C denotes the cytoplasm. The silver and black spheres represent extracellular iron oxide. (B) Schematic of plasmids used to create the ccm, mtrA, and mtrCAB strains in E. coli. (C) Schematic of the engineered mtrA and mtrCAB strains for soluble and extracellular metal reduction.

Fig. 2.

Expression of full-length redox-active MtrA and MtrC in E. coli. Heme-stained SDS-PAGE gels of (A) periplasmic fractions and (B) membrane fractions of the WT, ccm, mtrA, and mtrCAB strains. (C) Anti-MtrB immunoblot of membrane fractions of the WT, ccm, mtrA, and mtrCAB strains. (D) Absorption spectra of the periplasmic fraction of the mtrA strain under oxidizing and reducing conditions. (E) Absorption spectra of the membrane fraction of the mtrCAB strain under oxidizing and reducing conditions.

Fig. 3.

Reduction of 10 mM Fe(III) citrate to Fe(II) as a function of time for the WT, ccm, mtrA, and mtrCAB E. coli strains. Error bars represent the standard deviation between triplicates from separate starting cultures.

Fig. 4.

Direct link of MtrA redox state to Fe(III) citrate reduction. (A) Absorption spectra showing the α-band of MtrA in high-density, anaerobic cell suspensions of the mtrA strain before and after the addition of Fe(III) citrate. MtrA begins reduced (black line, strong α-band absorption), but is oxidized upon the addition of 50 μM Fe(III) citrate (red line). Over time, the α-band absorbance recovers (colored dotted lines). (B) ΔA_552 nm and Fe(II) concentration immediately before and after Fe(III) citrate addition as measured by the ferrozine assay.

Fig. 5.

NapC is not the sole electron donor to MtrA. Reduction of 10 mM Fe(III) citrate to Fe(II) by WT, ccm, ΔnapC ccm, mtrA, and ΔnapC mtrA strains.

Fig. 6.

MtrCAB reduces solid α-Fe₂O₃. (A) Brightfield optical image of bulk α-Fe₂O₃, d ∼ 5 μm. (B) The concentration of bulk α-Fe₂O₃ reduced by WT, cmm, mtrA, mtrCAB strains normalized by colony forming units after 24 d. (C) Transmission electron microscopy of crystalline α-Fe₂O₃ nanoparticles, d = 13 nm. (B) The concentration of α-Fe₂O₃ nanoparticles reduced by WT, ccm, mtrA, mtrCAB strains normalized by colony forming units after 24 d.