translating-extracellular-electron-transfer-activities-with-organic

Translating Extracellular Electron Transfer Activities with Organic Electrochemical Transistors

Extracellular electron transfer (EET) is a process through which certain microorganisms can transfer electrons across their cell membranes to external ...

University of Texas at Austin

Stanford University

Metabolic engineering has facilitated the production of pharmaceuticals, fuels, and soft materials but is generally limited to optimizing well-defined metabolic pathways. We hypothesized that the reaction space available to metabolic engineering could be expanded by coupling extracellular electron transfer to the performance of an exogenous redox-active metal catalyst. Here we demonstrate that the electroactive bacterium  can control the activity of a copper catalyst in atom-transfer radical polymerization (ATRP) via extracellular electron transfer. Using , we achieved precise control over the molecular weight and polydispersity of a bioorthogonal polymer while similar organisms, such as , showed no significant activity. We found that catalyst performance was a strong function of bacterial metabolism and specific electron transport proteins, both of which offer potential biological targets for future applications. Overall, our results suggest that manipulating extracellular electron transport pathways may be a general strategy for incorporating organometallic catalysis into the repertoire of metabolically controlled transformations.

 as a living electrode for controlled radical polymerization.

Redox interactions between electroactive bacteria and inorganic materials underpin many emerging technologies, but commonly used materials (e.g., metal oxides) suffer from limited tunability and can be challenging to characterize. In contrast, metal-organic frameworks exhibit well-defined structures, large surface areas, and extensive chemical tunability, but their utility as microbial substrates has not been examined. Here, we report that metal-organic frameworks can support the growth of the metal-respiring bacterium Shewanella oneidensis, specifically through the reduction of Fe(III). In a practical application, we show that cultures containing S. oneidensis and reduced metal-organic frameworks can remediate lethal concentrations of Cr(VI) over multiple cycles, and that pollutant removal exceeds the performance of either component in isolation or bio-reduced iron oxides. Our results demonstrate that frameworks can serve as growth substrates and suggest that they may offer an alternative to metal oxides in applications seeking to combine the advantages of bacterial metabolism and synthetic materials.

Microbial reduction of metal-organic frameworks enables synergistic chromium removal.

Extracellular electron transfer (EET) pathways, such as those in the bacterium , interface cellular metabolism with a variety of redox-driven applications. However, designer control over EET flux in  has proven challenging because a functional understanding of its EET pathway proteins and their effect on engineering parametrizations (e.g., response curves, dynamic range) is generally lacking. To address this, we systematically altered transcription and translation of single genes encoding parts of the primary EET pathway of , CymA/MtrCAB, and examined how expression differences affected model-fitted parameters for Fe(III) reduction kinetics. Using a suite of plasmid-based inducible circuits maintained by appropriate  knockout strains, we pinpointed construct/strain pairings that expressed , , and  with maximal dynamic range of Fe(III) reduction rate. These optimized EET gene constructs were employed to create Buffer and NOT gate architectures that predictably turn on and turn off EET flux, respectively, in response to isopropyl β-D-1-thiogalactopyranoside (IPTG). Furthermore, we found that response functions generated by these logic gates (i.e., EET activity vs inducer concentration) were comparable to those generated by conventional synthetic biology circuits, where fluorescent reporters are the output. Our results provide insight on programming EET activity with transcriptional logic gates and suggest that previously developed transcriptional circuitry can be adapted to predictably control EET flux.

Tuning Extracellular Electron Transfer by  Using Transcriptional Logic Gates.

Enhancing materials with the qualities of living systems, including sensing, computation, and adaptation, is an important challenge in designing next-generation technologies. Living materials address this challenge by incorporating live cells as actuating components that control material function. For abiotic materials, this requires new methods that couple genetic and metabolic processes to material properties. Toward this goal, we demonstrate that extracellular electron transfer (EET) from  can be leveraged to control radical cross-linking of a methacrylate-functionalized hyaluronic acid hydrogel. Cross-linking rates and hydrogel mechanics, specifically storage modulus, were dependent on various chemical and biological factors, including  genotype. Bacteria remained viable and metabolically active in the networks for a least 1 week, while cell tracking revealed that EET genes also encode control over hydrogel microstructure. Moreover, construction of an inducible gene circuit allowed transcriptional control of storage modulus and cross-linking rate via the tailored expression of a key electron transfer protein, MtrC. Finally, we quantitatively modeled hydrogel stiffness as a function of steady-state  expression and generalized this result by demonstrating the strong relationship between relative gene expression and material properties. This general mechanism for radical cross-linking provides a foundation for programming the form and function of synthetic materials through genetic control over extracellular electron transfer.

Genetic Control of Radical Cross-linking in a Semisynthetic Hydrogel.

Spatial concentration gradients of antibiotics are prevalent in the natural environment. Yet, the microbial response in these heterogeneous systems remains poorly understood. We used a microfluidic reactor to create an artificial microscopic ecosystem that generates diffusive gradients of solutes across interconnected microenvironments. With this reactor, we showed that chemotaxis toward a soluble electron acceptor (nitrate) allowed Shewanella oneidensis MR-1 to inhabit and sustain metabolic activity in highly toxic regions of the antibiotic ciprofloxacin (>80× minimum inhibitory concentration, MIC). Acquired antibiotic resistance was not observed for cells extracted from the reactor, so we explored the role of transient adaptive resistance by probing multidrug resistance (MDR) efflux pumps, ancient elements that are important for bacterial physiology and virulence. Accordingly, we constructed an efflux pump deficient mutant (∆mexF) and used resistance-nodulation-division (RND) efflux pump inhibitors (EPIs). While batch results showed the importance of RND efflux pumps for microbial survival, microfluidic studies indicated that these pumps were not necessary for survival in antibiotic gradients. Our work contributes to an emerging body of knowledge deciphering the effects of antibiotic spatial heterogeneity on microorganisms and highlights differences of microbial response in these systems versus well-mixed batch conditions.

The role of chemotaxis and efflux pumps on nitrate reduction in the toxic regions of a ciprofloxacin concentration gradient.

Consumption of unsafe water is a major cause of morbidity and mortality in developing regions. Pasteurizing or boiling water to remove pathogens is energy-intensive and often impractical to off-grid communities. Therefore, low capital cost, rapid and energy-efficient water disinfection methods are urgently required to address global challenges of safe water access. Here, anti-bacterial hydrogels (ABHs) with catechol-enabled molecular-level hydrogen peroxide generators and quinone-anchored activated carbon particles are designed for effective water treatment. The bactericidal effect is attributed to the synergy of hydrogen peroxide and quinone groups to attack essential cell components and disturb bacterial metabolism. ABHs can be directly used as tablets to achieve >99.999% water disinfection efficiency within 60 min without energy input. No harmful byproducts are formed during the treatment process, after which the ABH tablets can be easily removed without residues. Taking advantage of their excellent photothermal and biofouling-resistant properties, ABHs can also be applied as solar evaporators to achieve stable water purification under sunlight (≤1 kW m ) after months of storage and operation in bacteria-containing river water. The ABH platform offers reduced energy and chemical demands for point-of-use water treatment technologies in remote areas and emergency rescue applications.

Molecular Engineering of Hydrogels for Rapid Water Disinfection and Sustainable Solar Vapor Generation.

Bacteria that perform extracellular electron transfer (EET) are central to redox-driven biotechnologies, including microbial fuel cells, bioremediation, and bioelectrosynthesis. However, engineerable EET strains have been restricted to well-characterized, Gram-negative model species. Light et al. identified a previously unknown but widely conserved EET pathway in the Gram-positive bacterium Listeria monocytogenes.

Tapping the potential of Gram-positive bacteria for bioelectrochemical applications.

Organic electrochemical transistors (OECTs) are ideal devices for translating biological signals into electrical readouts and have applications in bioelectronics, biosensing, and neuromorphic computing. Despite their potential, developing programmable and modular methods for living systems to interface with OECTs has proven challenging. Here we describe hybrid OECTs containing the model electroactive bacterium  that enable the transduction of biological computations to electrical responses. Specifically, we fabricated planar p-type OECTs and demonstrated that channel de-doping is driven by extracellular electron transfer (EET) from . Leveraging this mechanistic understanding and our ability to control EET flux via transcriptional regulation, we used plasmid-based Boolean logic gates to translate biological computation into current changes within the OECT. Finally, we demonstrated EET-driven changes to OECT synaptic plasticity. This work enables fundamental EET studies and OECT-based biosensing and biocomputing systems with genetically controllable and modular design elements.

A Hybrid Transistor with Transcriptionally Controlled Computation and Plasticity.

Organic electrochemical transistors (OECTs) are ideal devices for translating biological signals into electrical readouts and have applications in bioelectronics, biosensing, and neuromorphic computing. Despite their potential, developing programmable and modular methods for living systems to interface with OECTs has proven challenging. Here we describe hybrid OECTs containing the model electroactive bacterium Shewanella oneidensis that enable the transduction of biological computations to electrical responses. Specifically, we fabricated planar p-type OECTs and demonstrated that channel de-doping is driven by extracellular electron transfer (EET) from S. oneidensis. Leveraging this mechanistic understanding and our ability to control EET flux via transcriptional regulation, we used plasmid-based Boolean logic gates to translate biological computation into current changes within the OECT. Finally, we demonstrated EET-driven changes to OECT synaptic plasticity. This work enables fundamental EET studies and OECT-based biosensing and biocomputing systems with genetically controllable and modular design elements.

A hybrid transistor with transcriptionally controlled computation and plasticity.

Engineered living materials combine the advantages of biological and synthetic systems by leveraging genetic and metabolic programming to control material-wide properties. Here, we demonstrate that extracellular electron transfer (EET), a microbial respiration process, can serve as a tunable bridge between live cell metabolism and synthetic material properties. In this system, EET flux from Shewanella oneidensis to a copper catalyst controls hydrogel cross-linking via two distinct chemistries to form living synthetic polymer networks. We first demonstrate that synthetic biology-inspired design rules derived from fluorescence parameterization can be applied toward EET-based regulation of polymer network mechanics. We then program transcriptional Boolean logic gates to govern EET gene expression, which enables design of computational polymer networks that mechanically respond to combinations of molecular inputs. Finally, we control fibroblast morphology using EET as a bridge for programmed material properties. Our results demonstrate how rational genetic circuit design can emulate physiological behavior in engineered living materials.

Article	Year
as a living electrode for controlled radical polymerization. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29666254	2018
Microbial reduction of metal-organic frameworks enables synergistic chromium removal. Nature communications\| PubMed ID: 31740677	2019
Tuning Extracellular Electron Transfer by Using Transcriptional Logic Gates. ACS synthetic biology\| PubMed ID: 32786362	2020
Genetic Control of Radical Cross-linking in a Semisynthetic Hydrogel. ACS biomaterials science & engineering\| PubMed ID: 33313392	2020
The role of chemotaxis and efflux pumps on nitrate reduction in the toxic regions of a ciprofloxacin concentration gradient. The ISME journal\| PubMed ID: 33927341	2021
Molecular Engineering of Hydrogels for Rapid Water Disinfection and Sustainable Solar Vapor Generation. Advanced materials (Deerfield Beach, Fla.)\| PubMed ID: 34292641	2021
Tapping the potential of Gram-positive bacteria for bioelectrochemical applications. Trends in biotechnology\| PubMed ID: 36535817	2023
A Hybrid Transistor with Transcriptionally Controlled Computation and Plasticity. bioRxiv : the preprint server for biology\| PubMed ID: 37645977	2023
A hybrid transistor with transcriptionally controlled computation and plasticity. Nature communications\| PubMed ID: 38383505	2024
Transcriptional regulation of living materials via extracellular electron transfer. Nature chemical biology\| PubMed ID: 38783133	2024

Christopher M. Dundas

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