Shewanella oneidensis as a living electrode for controlled radical polymerization

Abstract

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 Shewanella oneidensis can control the activity of a copper catalyst in atom-transfer radical polymerization (ATRP) via extracellular electron transfer. Using S. oneidensis, we achieved precise control over the molecular weight and polydispersity of a bioorthogonal polymer while similar organisms, such as Escherichia coli, 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.

Keywords: extracellular electron transport; metabolic engineering; polymerization.

Fig. 1.

S. oneidensis enabled ATRP and initial polymerization kinetics. (A) Electron equivalents generated from S. oneidensis MR-1 reduce a metal catalyst from an inactive state (M^OX) to an active state (M^RED). The active catalyst reacts with a halogenated initiator or polymer chain to produce a radical (gray arrow) that can polymerize olefins. The radical can also react with the now-deactivated catalyst (M^OX) to form a dormant chain (black arrow, Right). (B) ATRP initiator (HEBIB) and macromonomer (OEOMA₅₀₀) used in this study. (C) Monomer conversion after 24 h under various conditions with (white) and without (purple) trace metal mix added to bacterial media. (D) Kinetics of monomer conversion in MR-1 or E. coli culture using Cu(II)-EDTA as catalyst (E) Extracellular Cu(II) reduction monitored with the Cu(I) specific fluorescent dye CF4. Data show mean ± SD of three independent experiments. **P < 0.01.

Fig. 2.

Kinetics and properties of polymers formed with MR-1. (A) First-order kinetics for conversion of monomer over time using Cu(II)-TPMA with MR-1. (B) Molecular weight and polydispersity of poly(OEOMA₅₀₀) as a function of monomer conversion. (C) Repeated addition of OEOMA₅₀₀ monomer showing first-order kinetics and living nature of polymerization. Data show mean ± SD of three independent experiments.

Fig. 3.

Polymerization activity is controlled by electroactive metabolism. (A) Simplified carbon metabolism of S. oneidensis. (B) Polymerization kinetics for MR-1 supplied with different carbon sources using Cu(II)-TPMA as catalyst. Data show mean ± SD of three independent experiments. Statistical analysis for B is presented in SI Appendix, Fig. S24.

Fig. 4.

Electron transfer proteins impact polymerization kinetics. (A) Key proteins involved in extracellular electron transport in MR-1. (B) Effect of gene knockouts on polymerization activity using Cu(II)-TPMA. (C) Rescue of normal polymerization activity via complementation with a plasmid encoding MtrC, using Cu(II)-TPMA as a catalyst. Data show mean ± SD of three independent experiments. Statistical analysis for B and C is presented in SI Appendix, Figs. S25 and S26, respectively.