Understanding the underlying electron transport phenomena of biocatalytic processes is key to engineering effective and scalable bio-electrochemical systems and devices. This electron transfer occurs largely through small-molecule flavin mediators, however these mediators perform one-electron transfers in abiotic systems but concerted two-electron transfer in biological systems, rendering abiotic systems less efficient. To boost efficiency, the principles guiding flavin electron transfer must be elucidated, necessitating a tunable system. Ionic liquids (ILs) are a class of organic molten salts with remarkable electrochemical stability and diverse chemical compositions that make them attractive materials to study bio-electrochemical systems. Herein, we apply model IL 1-ethyl-3-methylimidazolium ([Emim][BF4]) as a biosimilar electrolyte to study the electron transfer behavior of these flavin mediators. We observe a concerted two-electron transfer between flavin mononucleotide (FMN) at an unmodified glassy carbon electrode surface in contrast to the one-electron transfer observed in standard inorganic electrolytes. This improved electron transfer efficiency demonstrates the potential of ILs to study the mechanistic features of biological electron transfer with the aim of improving electrochemical technologies based on these bio-electrochemical processes.

Electroactive microbes that can release or take up electrons are essential components of nearly every ecological niche and are powerful tools for the development of alternative energy technologies. Small-molecule mediators are critical for this electron transfer but remain difficult to study and engineer because they perform concerted two-electron transfer in native systems but only individual, one-electron transfers in electrochemical studies. Here, we report that electrode modification with ion- and electron-conductive polymers yields biosimilar, concerted two-electron transfer from Shewanella oneidensis via flavin mediators. S. oneidensis biofilms on these polymers show significantly improved per-microbe current generation and morphologies that more closely resemble native systems, setting a new paradigm for the study and optimization of these electron transfer processes. The unprecedented concerted electron transfer was found to be due to altered mediator electron transfer thermodynamics, enabling biologically relevant studies of electroactive biofilms in the lab for the first time. These important findings pave the way for a complete understanding of the ecological role of electroactive microbes and their broad application in sustainable technologies.

Correction for ‘Imidazolium-based ionic liquids support biosimilar flavin electron transfer’ by Grace I. Anderson et al., Mater. Adv., 2024, https://doi.org/10.1039/d4ma00558a.

Organic waste streams contain a significant amount of latent energy that is difficult to harvest. Microbial electrochemical systems (MESs), which extract electrons through microbial metabolism, are a promising technology to recover this energy. Despite advances in these technologies, microbial electrocatalysis in MESs remains limited by inefficient electron transfer between living cells and electrodes. Here, we demonstrate significant improvement in this interfacial electron transfer through the incorporation of electron- and ion-conductive polymers. We have designed these polymers with a conductive backbone and side chains that mimic ligands of catalytic centers in redox-active enzymes. Specifically, methylimidazolium groups mimic protonated histidines prevalent in enzyme active sites. We show that these polymers substantially enhance mediated electron transfer and overall current generation by Shewanella oneidensis, an electroactive microbe often used in MESs. Our work highlights the importance of materials engineering applied to the biotic-abiotic interface to improve charge transfer in bioelectronic systems.

Bio-electrochemical systems (BESs) are promising for renewable energy generation but remain hindered by inefficient electron transfer at electrode surfaces. As the toolbox of bio-anode materials increases, rigorous electrochemical characterization of emerging materials is needed. Here, we holistically characterize the electrochemical interaction of flavin mononucleotide (FMN), an electron shuttle in biological systems and a cofactor for oxidoreductase enzymes, with the bio-inspired mixed conducting polymer poly{3-[6′-(N-methylimidazolium)hexyl]thiophene} (P3HT-Im+). The behavior of this polymer is compared to the equivalent polymer without the histidine-like imidazolium. We find improved conductivity and charge storage in imidazolium-containing polymers beyond what is explained by differences in the electroactive area. The P3HT-Im+ further shows internal charge storage but with negligible faradaic contribution, indicating that charge storage capacity may translate to improved biocatalysis non-intuitive ways. Finally, one-electron transfer is observed between FMN and glassy carbon, while a bio-similar two-electron transfer is observed for the P3HT-Im+. To our knowledge, this is the first example of a concerted two-electron transfer between FMN and an electrode interface, which we attribute to the bio-inspired, histidine-like imidazolium functional groups in the polymer. These studies demonstrate the importance of bio-relevant materials characterization when such materials are deployed in BESs.

Microbial electrocatalysis is an emerging technology which enables electricity generation directly from organic feedstocks, many of which are otherwise difficult to harness for renewable energy. While microbes possess unique advantages for these applications, their use in practical settings is currently limited by inefficient electron transfer from cells to electrodes. To address this bottleneck, we have developed improved electrode materials that combine principles of conductive polymer design with lessons from enzymatic electron transfer reactions. Our polymer electrodes exhibit superior nanoscale electroactive area and enable concerted two-electron transfer from the electron carrier flavin mononucleotide (FMN) to an abiotic surface, a high efficiency mechanism which was previously restricted to biological contexts. Electroactive microbes exhibited greatly improved current production on polymer electrodes in quantitative agreement with in vitro electrochemical properties. Our findings establish bio-inspired functionalization as a useful paradigm to bridge the gap between microbial metabolism and abiotic electrochemistry.

Recent attempts to create synthetic Escherichia coli methylotrophs identified that de novo biosynthesis of amino acids, in the presence of methanol, presents significant challenges in achieving autonomous methylotrophic growth. Previously engineered methanol-dependent strains required co-utilization of stoichiometric amounts of co-substrates and methanol. As such, these strains could not be evolved to grow on methanol alone. In this work, we have explored an alternative approach to enable biosynthesis of all amino acids from methanol-derived carbon in minimal media without stoichiometric coupling. First, we identified that biosynthesis of threonine was limiting the growth of our methylotrophic E. coli. To address this, we performed adaptive laboratory evolution to generate a strain that grew efficiently in minimal medium with methanol and threonine. Methanol assimilation and growth of the evolved strain were analyzed, and, interestingly, we found that the evolved strain synthesized all amino acids, including threonine, from methanol-derived carbon. The evolved strain was then further engineered through overexpression of an optimized threonine biosynthetic pathway. We show that the resulting methylotrophic E. coli strain has a methanol-dependent growth phenotype with homoserine as co-substrate. In contrast to previous methanol-dependent strains, co-utilization of homoserine is not stoichiometrically linked to methanol assimilation. As such, future engineering of this strain and successive adaptive evolution could enable autonomous growth on methanol as the sole carbon source. • Adaptive evolution of E. coli enables biosynthesis of all amino acids from methanol. • Overexpression of threonine biosynthesis pathway improves methanol assimilation. • Methanol-dependent growth is seen in minimal media with homoserine as co-substrate.

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methylotrophs have proved successful, autonomous methylotrophy, that is, the ability to utilize methane or methanol as sole carbon and energy substrates, has not yet been realized. Here, we address an important limitation of autonomous methylotrophy in E. coli : the inability of the organism to synthesize several amino acids when grown on methanol. We targeted global and local amino acid regulatory networks. Those include removal of amino acid allosteric feedback inhibition ( argA H15Y , ilvA L447F , hisG E271K , leuA G462D , proB D107N , thrA S345F , trpE S40F ), knockouts of transcriptional repressors ( ihfA, metJ ); and overexpression of amino acid biosynthetic operons ( hisGDCBHAFI, leuABCD, thrABC, trpEDCBA ) and transcriptional regulators ( crp, purR ). Compared to the parent methylotrophic E. coli strain that was unable to synthesize these amino acids from methanol carbon, these strategies resulted in improved biosynthesis of limiting proteinogenic amino acids (histidine, leucine, lysine, methionine, phenylalanine, threonine, tyrosine) from methanol carbon. In several cases, improved amino acid biosynthesis from methanol carbon led to improvements in methylotrophic growth in methanol minimal medium supplemented with a small amount of yeast extract. This study addresses a key limitation currently preventing autonomous methylotrophy in E. coli and possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via global and local regulatory modifications.

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methylotrophs have proved successful, autonomous methylotrophy, i.e. the ability to utilize methane or methanol as sole carbon and energy substrates, has not yet been realized. Here, we address an important limitation of autonomous methylotrophy in E. coli: the inability of the organism to synthesize several amino acids when grown on methanol. By activating the stringent/stress response via ppGpp overproduction, or DksA and RpoS overexpression, we demonstrate improved biosynthesis of proteinogenic amino acids via endogenous upregulation of amino acid synthesis pathway genes. Thus, we were able to achieve biosynthesis of several limiting amino acids from methanol-derived carbon, in contrast to the control methylotrophic E. coli strain. This study addresses a key limitation currently preventing autonomous methylotrophy in E. coli and possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via stringent/stress response activation.

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methanol auxotrophs have proved successful, these studies focused on scarce and expensive co-substrates. Here, we engineered E. coli for methanol-dependent growth on glucose, an abundant and inexpensive co-substrate, via deletion of glucose 6-phosphate isomerase (pgi), phosphogluconate dehydratase (edd), and ribose 5-phosphate isomerases (rpiAB). Since the parental strain did not exhibit methanol-dependent growth on glucose in minimal medium, we first achieved methanol-dependent growth via amino acid supplementation and used this medium to evolve the strain for methanol-dependent growth in glucose minimal medium. The evolved strain exhibited a maximum growth rate of 0.15 h−1 in glucose minimal medium with methanol, which is comparable to that of other synthetic methanol auxotrophs. Whole genome sequencing and 13C-metabolic flux analysis revealed the causative mutations in the evolved strain. A mutation in the phosphotransferase system enzyme I gene (ptsI) resulted in a reduced glucose uptake rate to maintain a one-to-one molar ratio of substrate utilization. Deletion of the e14 prophage DNA region resulted in two non-synonymous mutations in the isocitrate dehydrogenase (icd) gene, which reduced TCA cycle carbon flux to maintain the internal redox state. In high cell density glucose fed-batch fermentation, methanol-dependent acetone production resulted in 22% average carbon labeling of acetone from 13C-methanol, which far surpasses that of the previous best (2.4%) found with methylotrophic E. coli Δpgi. This study addresses the need to identify appropriate co-substrates for engineering synthetic methanol auxotrophs and provides a basis for the next steps toward industrial one-carbon bioprocessing.