Optimization of Quinone-Based, Extracellular Electron Transfer Mediators for Bioelectronic Applications

Abstract

To survive in anaerobic conditions, some bacteria have evolved to respire onto non-oxygen terminal electron acceptors (TEAs). A subset of these organisms, also known as metal reducing (MR) bacteria, can produce a usable electric current. Production of electric current makes MR bacteria of interest for the development of bioelectronic devices like biosensors and microbial fuel cells. The overall process of these bacteria passing electrons to a TEA is referred to as extracellular electron transfer (EET). One mechanism of EET is mediated by diffusible, redox-active small molecules, or electron shutting compounds (ESCs). Quinones, a class of cyclic, conjugated, di-keto compounds often serve as ESCs. Lactiplantibacillus plantarum, a commensal lactic acid bacterium, can utilize quinoline-mediated EET through a type II NADH dehydrogenase protein, Ndh2. In nature, quinone-based shuttling compounds are often decorated with different substituents (e.g. hydroxyls, halogens, amines) that alter their chemical properties. Recent studies have shown that quinone-based mediators of different chemical properties facilitate EET at different rates. In this project, a library of 40 unique quinone-based mediators was assembled from commercial, natural, and semi-synthetic sources. The library was screened in a high-throughput iron(III) oxide nanoparticle reduction assay, and top performers were evaluated in a bioelectronic system (BES). It was found through screening that Ndh2-dependent EET in L. plantarum activity strongly correlates to a mediators' lipophilicity [LogD (pH 7.4)] and predicted free energy of binding ∆Gcomp. A mediator promoting very stable current in a BES for 5 days, 3-Amine-menadione, was discovered. Additionally, a library of 1,4-naphthoquinones, substituted with various biogenic amines was assembled. This library was designed around probing at interactions in the L. plantarum Ndh2 active site that promotes EET. Screening for Ndh2-mediated EET in L. plantarum in the nanoparticle reduction assay showed that 1,4-naphthoquinones substituted with amines able to promote pi-stacking interactions with Tyr-403 were top performers. The compound with the highest EET rate was the only primary amine-substituted mediator, 3-Amine-menadione. Ndh2-dependent EET is strongly correlated to LogD (pH 7.4). A predictive EET metric, or composite score, was created based on in vivo data, molecular modeling, and ADME properties of all mediators. Lastly, highly active EET amine-substituted 1,4-naphthoquionones are proposed to be covalently bound to polymer networks. The redox-active polymers are to be embedded on an electrode for the purposes of enhancing EET in BES. Mediators with two functional groups, epoxides and free thiols, will be synthesized for the addition of chitosan and methacrylate/methyl acrylate polymers, respectively. The redox-active polymers will be screened for Ndh2-mediated EET L. plantarum in nanoparticle reduction assays and BES. Highly active quinone-based mediators will be used to make bioelectronic communication faster and more efficient.