(470c) Biomimetic Platform to Explore Conductive Outer Membrane Proteins from Acidophilic Biomining Bacteria

Many microorganisms can exchange electrons with surrounding environments, providing the fundamental biotechnology support for the increasingly demands of green energy and sustainable environment, such as biofuel cells, bioleaching, and bioelectrocatalysis. Direct extracellular electron transfer (EET) is the key process to link these electroactive bacteria (EAB) with microenvironment. Direct EET relies on outer membrane proteins (OMPs) which couple internal metabolism with external electron donor/receptor. Outer membrane proteins (OMPs) usually serve as conductive electron wires through the insulating bacterial cell-membrane, also serve as channels for controlling ions and small molecule transport with microenvironment. However, the molecular details of the functional components in conductive membrane proteins are not fully characterized, especially due to most of the electroactive bacteria are non-model hosts.

Bio-electrochemical systems offer the opportunities to integrate biological and electrochemical components together. Bacterial outer membrane vesicles (OMVs) fused supported lipid bilayers (SLB) systems are ideal biomimetic platforms to study outer membrane proteins while keep them in the cell-membrane environment. Thus, we hypothesis to use OMV-SLB system, combining with protein engineering, to investigate and engineer EET protein, to understand the mechanism.

In this work, Cyc2 protein, a c-type cytochrome outer membrane protein from Acidithiobacillus ferrooxidans was selected as model protein. Acidithiobacillus ferrooxidans is a widely studied biomining and bioleaching bacterium and Cyc2 protein has been reported as the first electron carrier during bioleaching process that mediates reversible electron transfer between intercellular metabolism and extracellular environment.

The Cyc2 protein was first recombinantly overexpressed in hypervesiculating Escherichia coli. OMVs containing Cyc2 were then deposited on an electrode surface via SLB fusion formation. Significant conductivity difference was detected between Cyc2 enriched and native bacterial membranes. Several site-directed mutations were made on core components in the Cyc2 protein electron-wire to explore the molecular determinants of electron conduction. Furthermore, this biomimetic platform provides novel perspective to study biotic/abiotic interfaces, as well as membrane protein interactions with inorganic microenvironments.