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

University of California San Francisco

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.

Performing radical polymerizations under ambient conditions is a major challenge because molecular oxygen is an effective radical quencher. Here we show that the facultative electrogen Shewanella oneidensis can control metal-catalysed living radical polymerizations under apparent aerobic conditions by first consuming dissolved oxygen via aerobic respiration, and then directing extracellular electron flux to a metal catalyst. In both open and closed containers, S. oneidensis enabled living radical polymerizations without requiring the preremoval of oxygen. Polymerization activity was closely tied to S. oneidensis anaerobic metabolism through specific extracellular electron transfer proteins and was effective for a variety of monomers using low (parts per million) concentrations of metal catalysts. Finally, polymerizations survived repeated challenges of oxygen exposure and could be initiated using lyophilized or spent (recycled) cells. Overall, our results demonstrate how the unique ability of S. oneidensis to use both oxygen and metals as respiratory electron acceptors can be leveraged to address salient challenges in polymer synthesis.

Aerobic radical polymerization mediated by microbial metabolism.

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.

Extracellular electron transfer (EET) is an anaerobic respiration process that couples carbon oxidation to the reduction of metal species. In the presence of a suitable metal catalyst, EET allows for cellular metabolism to control a variety of synthetic transformations. Here, we report the use of EET from the electroactive bacterium  for metabolic and genetic control over Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC). CuAAC conversion under anaerobic and aerobic conditions was dependent on live, actively respiring  cells. The reaction progress and kinetics were manipulated by tailoring the central carbon metabolism. Similarly, EET-CuAAC activity was dependent on specific EET pathways that could be regulated via inducible expression of EET-relevant proteins: MtrC, MtrA, and CymA. EET-driven CuAAC exhibited modularity and robustness in the ligand and substrate scope. Furthermore, the living nature of this system could be exploited to perform multiple reaction cycles without regeneration, something inaccessible to traditional chemical reductants. Finally,  enabled bioorthogonal CuAAC membrane labeling on live mammalian cells without affecting cell viability, suggesting that  can act as a dynamically tunable biocatalyst in complex environments. In summary, our results demonstrate how EET can expand the reaction scope available to living systems by enabling cellular control of CuAAC.

Extracellular Electron Transfer Enables Cellular Control of Cu(I)-Catalyzed Alkyne-Azide Cycloaddition.

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
Aerobic radical polymerization mediated by microbial metabolism. Nature chemistry\| PubMed ID: 32424254	2020
Genetic Control of Radical Cross-linking in a Semisynthetic Hydrogel. ACS biomaterials science & engineering\| PubMed ID: 33313392	2020
Extracellular Electron Transfer Enables Cellular Control of Cu(I)-Catalyzed Alkyne-Azide Cycloaddition. ACS central science\| PubMed ID: 35233456	2022
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

Austin J. Graham

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