Microbial compositions and methods of use thereof

Abstract

This invention is directed to microbial compositions and methods of use thereof.

Description

MICROBIAL COMPOSITIONS AND METHODS OF USE THEREOF

[0001] For countries that permit incorporation by reference, all patents, patent applications and publications cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted being prior art.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records but otherwise reserves any and all copyright rights.CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This international application claims the benefit of priority to U.S. Provisional Application No. 63 / 569,659, filed on March 25, 2024, entitled “MICROBIAL COMPOSITIONS AND METHODS OF USE THEREOF” and to U.S. Provisional Application No. 63 / 652, 127, filed on May 27, 2024, entitled “MICROBIAL COMPOSITIONS AND METHODS OF USE THEREOF”FIELD OF THE INVENTION

[0004] This invention is directed to microbial compositions and methods of use thereof.BACKGROUND OF THE INVENTION

[0005] Most bioproduction processes grow microbes under anaerobic conditions to enhance product yield and eliminate the need for expensive oxygenation. However, producing these chemicals under anaerobic conditions often comes with the constraint of maintaining a redox balance with the starting materials.SUMMARY OF THE INVENTION

[0006] Aspects of the invention are drawn towards a method of stimulating extracellular electron transfer (EET) by a microorganism, the method comprising: providing the microorganism with an electron donor; exogenously supplementing the microorganism with a small molecule redox mediator; and subjecting the microorganism to an electron sink, therebystimulating EET in the microorganism. In embodiments, the redox mediator is supplemented at a concentration of less than 1 pM, about 1 pM, about 2.5 pM, about 5 pM, about 7.5 pM, about 10 pM, about 12.5 pM, about 15 pM, about 20 pM, about 25 pM, or greater than about 25 pM. In embodiments, the redox mediator is a naphthoquinone. In embodiments, the naphthoquinone is selected from the group consisting of 2-hydroxy-l,4-naphthoquinone (HNQ), 2-methyl-l,4-naphthoquinone (MNQ), or l,4-dihydroxy-2-naphthoic acid (DHNA). In embodiments, the microorganism is a bacteria. In embodiments, the bacteria is E. coli. In embodiments, the bacteria is an exoelectrogen or is not an exoelectrogen. In embodiments, the electron sink is an anode. In embodiments, the electron donor comprises glucose, glycerol, pyruvate, or any combination thereof. In embodiments, the method overcomes direct EET feedstock selectivity of direct EET. In embodiments, the method reduces the required electrode surface area compared to direct EET, utilizes the whole reactor volume, does not require the formation of biofilm on the electrode, or any combination thereof. In embodiments, the microorganism is genetically engineered to increase EET output. In embodiments, the genetically engineered bacteria partially, substantially, or completely deletes, silences, inactivates, or down-regulates a gene or fragment thereof that encodes for an anaerobic respiratory pathway, a fermentative pathway, or a combination thereof. In embodiments, the genetically engineered bacteria is AnoxicNull EnapFDAGHBC EnarGHI EnarZYW EdmsABC EynfEFGH EtorCAD EtorYZ EfrdABCD EnirBDC EnrfABCDEFG EiiorVW Ehmp \ccp . FermNull (EmgsA EadhE EldhA or any combination thereof.

[0007] Aspects of the disclosure are drawn towards a genetically modified microorganism, wherein a gene or fragment thereof that encodes for an anaerobic respiratory pathway, a fermentative pathway, or a combination thereof is partially, substantially, or completely deleted, silenced, inactivated, or down-regulated in the genetically modified microorganism. In embodiments, the microorganism is genetically engineered to increase extracellular electron transfer (EET). In embodiments, the genetically modified microorganism is AnoxicNull EnapFDAGHBC EnarGHI EnarZYW EdmsABC EynfEFGH EtorCAD EtorYZ EfrdABCD EnirBDC EnrfABCDEFG EnorVW Ehmp \ccp). FermNull (EmgsA EadhE EldhA or any combination thereof. In embodiments, the microorganism is a bacteria. In embodiments, the bacteria is E. coli. In embodiments, the bacteria is an exoelectrogen or is not an exoelectrogen.

[0008] Other objects and advantages of this invention will become readily apparent from the ensuing description.BRIEF DESCRIPTION OF THE FIGURES

[0009] FIG. 1 shows non-limiting exemplary schematics and data indicating HNQ-mediated EET is coupled with catabolism. (Panel A) A schematic illustrating that HNQ serves as a redox shuttle between cells and anode. Elucidating this EET mechanism is challenging because several cellular redox pools, such as NADH, NADPH, quinones, glutathione, and thioredoxin, can reduce redox shuttles either enzymatically or non-enzymatically. (Panel B) The GlyNull strain was generated by deleting the glycerol consumption pathways in the BioD strain. (Panel C) Chronoamperometry of BioD, GlyNull, and GldA+strains under resting conditions, showing that only strains capable of consuming the available carbon source can perform EET. The strains in this experiment were supplemented with 20 mM glycerol in both pre-culture and biomass-generating cultures. (Panel D) Only the BioD and GldA+strains are capable of consuming glycerol, while GlyNull, lacking the glycerol oxidation pathways, does not. The data in (Panel C) and (Panel D) represent mean ± standard deviation obtained in duplicate BES experiments. The P-value is calculated using Welch’s t-test. (Panel E) Differential gene expression analysis (DEG) of BioD in DMSO alone and with HNQ under open circuit conditions, showing that [the presence of HNQ upregulates a relatively small amount of gene expression]. This data indicates that HNQ reduction is coupled with the induction of key electron transfer genes (trxC) and the quinone reductase, azoR. Activation of SoxS indicates activation of oxide-reducing proteins. (Panel F) A Treemap of the explained variance of each DEG in the transcriptome of HNQ-treated cells, showing that differentially expressed genes are enriched with redox-associated genes. The evidence points to NQ degradation comprising the largest gene expression changes, followed by redox stress, NADH, and NADPH gene sets. (Panel G) Differential iModulon activity (DiMA) analysis of HNQ-treated cells, showing that redox stress and metal stress gene sets are differentially regulated, along with the nrf operon. UC-1 is a complex iModulon containing prophage and redox genes. EvgA is associated with an acid and drug resistance two-component sensor. The data in (Panel E), (Panel F), and (Panel G) represent mean ± standard deviation obtained in a triplicate no-EET BES setup, where the potentiostat was not connected to the BES. The BESs did not have anode, and cells were injected at the final ODeoo of 0.25. The cells were treated with HNQ for three hours under resting conditions and harvested for RNA extraction.

[0010] FIG. 2 shows non-limiting, exemplary data indicating NADH is critical for HNQ- mediated EET. (Panel A) A schematic illustrating electron flow from the carbon catabolism into the anaerobic respiratory and fermentative pathways. Diverse cellular electron carriers like the NADH pool, the NADPH pool, and the quinone pool orchestrate this electron flow into different electron sinks. (Panel B) (top) The E. coli respiratory oxidoreductases that reduce theknown terminal electron acceptors are deleted in the AnoxicNull strain, (bottom) Chronoamperometry of the AnoxicNull and BioD strains under resting conditions, showing that the peak and steady-state current density in AnoxicNull is higher than that in BioD. This data suggests that enzymes responsible for nitrate, nitrite, nitric oxide, DMSO, TMAO, fumarate, and peroxide reduction do not distinctly reduce HNQ. The data represent mean ± standard deviation obtained in triplicate and duplicate BES experiments of BioD and AnoxicNull, respectively. (Panel C) (top) The E. coli NADH dehydrogenases that transfer electrons from NADH to quinones are deleted in the NdhNull strain, (bottom) Under resting conditions, NdhNull has a higher peak current density than the BioD strain, suggesting that the NADH dehydrogenases do not reduce HNQ. (Panel D) (top) The E. coli fermentative enzymes that transfer electrons from NADH to pyruvate, acetyl-CoA, and MGO are deleted in the FermNull strain, (bottom) Under resting conditions, FermNull maintains higher peak and steady current density levels than the AnoxicNull strain. This data indicates that higher availability of NADH enhances the HNQ-mediated EET. The strains in this experiment were supplemented with 20 mM glucose in the biomass-generating culture and tested with 2 mM glucose as the electron donor in the BES. 20 mM pyruvate and 20 mM glycerol were added to the biomass-generating culture of the strains investigated in (Panel B) and (Panel C). 2 mM of pyruvate as the electron donor was added in the BES to stimulate EET. The data in (Panel C) and (Panel D) represent mean ± standard deviation obtained in triplicate BES experiments.

[0011] FIG. 3 shows non-limiting, exemplary data indicating NADPH, glutathione, and thioredoxins are not critical for HNQ-mediated EET, the NADPH pool does not stimulate the HNQ-mediated EET pathway directly, and eliminating glutathione biosynthesis has a pleiotropic effect on HNQ-mediated EET. (Panel A) (Left) A schematic of cellular redox reactions powered by NADPH in E. coli. NADPH generated from carbon catabolism is indispensable for cell growth and global redox homeostasis. (Right) Eliminating NADPH- generating enzymes Zwf, led, PntAB, and MaeB renders the E. coli-NADPHnull strain into a gluconate (GNT) auxotroph. (Abbreviations: G6P, glucose 6-phosphate; 6PG, 6- phosphogluconate; Ru5P, ribulose 5-phosphate; ICT, D-threo-isocitrate; 2KG, a-ketoglutarate, MAL, malate; PYR, pyruvate; Zwf, Glucose-6-phosphate dehydrogenase; Gnd, 6- phosphogluconate dehydrogenase; led, Isocitrate dehydrogenase; MaeB, Malate dehydrogenase; PntAB, Pyridine nucleotide transhydrogenase, PPP, Pentose Phosphate Pathway; TCA, Tricarboxylic Acid cycle; GNG, Gluconeogenesis; TH, Transhydrogenase; GNT, Gluconate). (Panel B) A schematic to show that the NADPHnull strain is incapable of both NADPH production and growth when either pyruvate (PYR) or glucose (GLC) is thecarbon source. However, cells can generate NADPH and grow biomass with gluconate (GNT) as the carbon source. (Panel C) OD600 of Anoxi cNull and NADPHnull strains after 16 hours of aerobic growth, showing that NADPHnull does not grow on 2x-YT media, whereas NADPHnull strain accumulated cell biomass in 20 mM gluconate-supplemented 2x-YT media. This data confirms that NADPHnull is a gluconate auxotroph. The strains in the experiment were pre-grown in LB supplemented with 20 mM of gluconate and 1 mM MgSO4. The data represent mean ± standard deviation obtained in four biological replicates. The P-value is calculated using Welch’s t-test. (Panel D) Chronoamperometry of NADPHnull and AnoxicNull strains under resting conditions, showing NADPHnull reached saturation current levels earlier than AnoxicNull, although with a smaller value of peak current density. The strains in this experiment were supplemented with 20 mM gluconate and 20 mM pyruvate in the biomass-generating culture. The data represent mean ± standard deviation obtained in triplicate BES experiments. (Panel E) (Left) An illustration of the glutathione biosynthetic pathway in E. coli. Deletion of gshA abolishes GSH biosynthesis. (Right) GSH is known to reduce HNQ both enzymatically and non-enzymatically. (Panel F) Under resting conditions, GSHnull generates a lower current density than the AnoxicNull strain. The attenuated current level is attributed to a trickle-down effect of the gshA deletion. The data represent mean ± standard deviation obtained in triplicate BES experiments. (Panel G) Chronoamperometry investigation of abiotic electron transfer from GSH to HNQ, showing that 40 pM GSH is oxidized at the anode poised at 200 mV vs. Ag / AgCl with and without 20 pM HNQ. This data suggests that GSH does not influence HNQ-mediated EET via abiotic electron transfer. The data represent mean ± standard deviation obtained in triplicate BES experiments. The strains in (Panel F) and (Panel G) were supplemented with 20 mM pyruvate and 20 mM glycerol in the biomass-generating culture. The EET levels in (Panel F) were tested in BES with 2 mM of pyruvate as the electron donor.

[0012] FIG. 4 shows non-limiting, exemplary data indicating that nitroreductase enables HNQ-mediated EET under resting condition. (Panel A) A schematic showing that the QRedNull strain lacks the known and the putative quinone reductases in E. coli. (Panel B) Chronoamperometry of QRedNull and AnoxicNull strains under resting conditions. QRedNull produces insignificant current density levels compared to AnoxicNull, suggesting that quinone reductases are the key enzymes facilitating HNQ-mediated EET. The strains in this experiment were supplemented with 20 mM glycerol and 20 mM pyruvate in the biomass-generating culture. The cells were first provided with 2 mM pyruvate as the electron donor in the BES. Once the pyruvate-powered current levels dropped for the AnoxicNull strain, 1 mM glucosewas injected into the BES. The data represent mean ± standard deviation obtained in duplicate BES experiments. (Panel C) Chronoamperometry of the NfsB+ and QRedNull-Ll strains under resting conditions, demonstrating that plasmid complementation of NfsB restores the HNQ- mediated EET in the QRedNull-Ll strain. The HNQ-mediated EET restoration is seen with 2 M each of glycerol, pyruvate, and glucose as carbon sources. The data represent mean ± standard deviation obtained in triplicate BES experiments. (Panel D) The QRedNull strain can grow on native fermentative pathways and does not perform HNQ-mediated EET. (Panel E) The EsinkNull strain cannot grow on native fermentative pathways and does not perform HNQ- mediated EET. (Panel F) The EsinkNull strain generates insignificant current density levels compared to the AnoxicNull and the QRedNull strains. The loss of EET in EsinkNull is due to blocked electron flow into both the HNQ-mediated EET and the native fermentative pathways. The data represent mean ± standard deviation obtained in triplicate BES experiments. (Panel G) OD600 of the AnoxicNull, the QRedNull, and the EsinkNull strains under growth conditions, showing that the EsinkNull strain cannot grow in anaerobic conditions. The data represent mean ± standard deviation obtained in triplicate BES experiments. The P-value is calculated using Welch’s t-test. (Panel H) The Charge (Panel Q) to OD600 ratio of the QRedNull and the EsinkNull strain, showing equivalent charge transfer onto anode per unit cell of both these strains. The data represent mean ± standard deviation obtained in triplicate BES experiments. The P-value is calculated using Welch’s t-test. (Panel I) Working model of HNQ-mediated EET in E. coli.

[0013] FIG. 5 shows non-limiting, exemplary indicating transcriptomics analysis reveals HNQ-induced response under growing conditions. (Panel A) The strains with varied growth and EET capabilities are tested on their ability to reduce amaranth (redox dye) using HNQ- mediated EET under anaerobic conditions. The cell cultures in this experiment were supplemented with 20 mM glucose and 5 g / L of NZ-amine as the electron donor and 12.5 pM amaranth as the terminal electron acceptor. The amaranth assay was subjected to constant shaking in an anaerobic chamber for 1.5 hours. (Panel B) Biplot for principal component analysis of the activity matrix A computed with Independent Component (iModulon) Analysis for AnoxicNull, QRedNull, and ESinkNull with and without HNQ supplementation. (Panel C) Different gene expression plots for AnoxicNull, QRedNull, and ESinkNull, with highly perturbed genes within the LPS iModulon highlighted. Gene expression was centered on an aerobic glucose reference condition. (Panel D) Different gene expression plots for AnoxicNull, QRedNull, and ESinkNull, with highly perturbed genes within the gcvB iModulon highlighted. Gene expression was centered on an aerobic glucose reference condition. (Panel E) Heatmapof redox stress iModulon activities across strains and conditions. (Panel F) Boxplot of expression of a naphthoquinone degradation iModulon across an E. coli expression database (PRECISE1K) and the data generated within this project (Electric coli) (Panel G) Expression of genes within the naphthoquinone degradation iModulon.

[0014] FIG. 6 shows non-limiting, exemplary data indicating HNQ-mediated EET enables E. coli to respire and grow on anode. (Panel A) Flux balance analysis of E. coli strains under anaerobic growth on glucose shows the recovery of anaerobic growth via naphthoquinone and NfsB. Fluxes in mmol / gDW / hr units, growth rate (GR) in 1 / hr units. The listed fluxes represent >95% of the turnover of NAD / NADH. Reaction abbreviations - GAPD: Glyceraldehyde 3- phosphate Dehydrogenase. THD: Transhydrogenase. ACALD: Acetaldehyde Dehydrogenase. ALCD: Alcohol Dehydrogenase. MDH: Malate Dehydrogenase. PDH: Pyruvate Dehydrogenase. HNQ: Naphthoquinone Reductase (Panel B) A schematic showing that the FermNull strain cannot grow on fermentative pathways; however, the QReda are intact to enable HNQ-mediated EET. (Panel C) Chronoamperometry of the FNO and the EsinkNull strains under growth conditions, showing that the FNO strain carries over the EET phenotype gained in the extended BES run of the FermNull strain. This indicates that this gain of the EET phenotype is genetically / metabolically encoded. The data represent mean ± standard deviation obtained in triplicate BES experiments. (Panel D) OD600 and (Panel E) Glucose oxidized by FNO at the end of the BES run were significantly higher than EsinkNull. These results indicate that HNQ-mediated EET drives catabolism and biomass growth by respiring on the anode. The data represent mean ± standard deviation obtained in triplicate BES experiments. The P-value is calculated using Welch’s t-test. (Panel F) [Genome alignment] (Panel G) Biplot for principal component analysis of the activity matrix A computed with Independent Component (iModulon) Analysis for FermNull and adapted-FermNull strains. (Panel H) Volcano plot for gene expression of adapted FermNull FN1 vs. FermNull.

[0015] FIG. 7 shows non-limiting, exemplary data related to Figure 1. (Panel A) Chronoamperometry of BioD with and without HNQ under resting conditions, showing that HNQ stimulates EET upon the addition of glycerol as the electron donor. (B) Glycerol consumption and the metabolite profile of the spent media, showing that cells under both conditions are metabolically active. The data in (Panel A) and (Panel B) represent mean ± standard deviation obtained in triplicate BES experiments. The P-value is calculated using Welch’s t-test. (Panel C) Cyclic voltammogram (CV) of BioD with HNQ and DMSO, showing that HNQ serves as a redox shuttle between metabolically active cells and the anode. The CV waveforms are plotted from three independent BES experiments. (Panel D) E. coli consumesglycerol via three routes: GlpD and GlpABC are glycerol 3-phosphate dehydrogenases that relay electrons to the quinone pool, and GldA is a glycerol dehydrogenase that relays electrons to the NADH pool. GlpD is the preferred route under aerobic conditions, whereas GlpABC is the preferred route of anaerobic glycerol oxidation. GldA expression, on the other hand, is independent of aerobicity. (Panel E) Genotypic representation of GlyNull and GldA+ strains, showing that GlyNull is devoid of all the three routes of glycerol consumption whereas GldA+ has intact gldA, under a synthetic constitutive promoter, as the sole route of glycerol consumption. (Panel F) A schematic showing the catabolism of glycerol and pyruvate as the electron donors in the PoxNull strain. Since glycerol is more reduced than pyruvate, it can release 4 extra electrons. So, a higher EET output is expected on the complete oxidation of glycerol compared to pyruvate. (Panel G) PoxNull generates similar EET outputs with either glycerol or pyruvate as electron donors under resting conditions. The strains in this experiment were supplemented with 20 mM glycerol and 20 mM pyruvate in both pre-culture and biomassgenerating cultures. (Panel H) Carbon source consumption and the corresponding metabolite profile of the spent media, showing that PoxNull consumed all of the pyruvate (~ 2mM), whereas it consumed only about 25% of the total glycerol provided. The data in (Panel G) and (Panel H) represent mean ± standard deviation obtained in triplicate BES experiments. (Panel I) Activity levels of stress-related imodulons, showing that only OxyR activity difference is statistically significant between the samples, and no other stresses are reflected in the transcriptome. P values are obtained using Welch’s t-test. (Panel J) Higher expression levels of key NADH and other electron carrier-generating enzymes are observed with HNQ-enabled EET. aceF and sdhC subunits of pyruvate and succinate dehydrogenases are expressed higher, which release NADH and UQH2 in addition to the acnB subunit of aconitate hydratase, which catalyzes the formation of isocitrate, which is further catalyzed to release NADH.

[0016] FIG. 8 shows a non-limiting, exemplary data related to Figure 2. (Panel A) OD600 of BioD and Anoxi cNull strains after 30 hours of anaerobic growth on various respiratory electron acceptors in M9P-2xYT media, showing that AnoxicNull consistently achieves lower growth levels than BioD in the presence of TEAs but has similar growth levels as BioD with no electron acceptor conditions. This data confirms that nitrate, TMAO, DMSO, and fumarate respiratory pathways are successfully eliminated in the AnoxicNull strain. (Panel B) The total charge deposited by BioD and AnoxicNull on the anode with 2 mM pyruvate as the electron donor, showing that AnoxicNull yields higher electron transfer onto the anode than BioD. The P-value is calculated using Welch’s t-test. (Panel C) A schematic showing the menaquinone pool that can reduce HNQ is deleted in the MqNull strain (Panel D) OD600 of BioD andMqNull strains after 30 hours of anaerobic growth on DMSO, fumarate, and no TEA conditions in M9P-2xYT media. The lack of a menaquinone pool prevents DMSO and fumarate from being used in anaerobic respiration as TEA, confirming that menaquinone biosynthesis is successfully knocked out in the MqNull strain. (Panel E) Chronoamperometry of the BioD and MqNull strains under resting conditions, showing that the peak current density of MqNull is higher than that in BioD. This data suggests that the menaquinone pool is not involved in reducing HNQ under EET conditions. The data represent mean ± standard deviation obtained in duplicate BES experiments. (Panel F) Colony PCR confirmation of the ndh gene and the nuo operon knockout in NdhNull. The nuo operon size in BioD is very large to amplify and distinctly show on the agarose gel; thus, the nuo locus amplification is shown only in the strain with nuo operon knockout. (Panel G) OD600 of BioD and NdhNull after 10 hours of aerobic growth in M9P-LB media, showing that lack of NADH dehydrogenases abates biomass growth. This growth assay phenotypically validates the genotype of NdhNull. (Panel H) The total charge deposited by BioD and NdhNull on the anode with 2 mM pyruvate as the electron donor, showing that NdhNull facilitates higher electron transfer onto the anode than BioD. The P-values are calculated using Welch’s t-test. (Panel I) Anaerobic growth of Anoxi cNull and FermNull in M9P-LB media, showing that the lack of fermentation pathways reduces biomass growth of the FermNull strain. This anaerobic growth assay phenotypically confirms the deletion of fermentative pathways in the FermNull strain. The data represent mean ± standard deviation obtained in three biological replicates. The P-value is calculated using Welch’s t-test. (Panel J) The total charge deposited by AnoxicNull and FermNull on the anode with 2 mM glucose as the electron donor, showing that FermNull yields higher electron transfer onto the anode than AnoxicNull. (Panel K) The metabolite profile of the spent BES media, showing that there is no difference between the amount of formate and acetate generated by the AnoxicNull and FermNull strains. The P-values are calculated using Welch’s t-test.

[0017] FIG. 9 shows a non-limiting, exemplary data related to Figure 3. (Panel A) The total charge deposited by AnoxicNull and NADPHnull on the anode with 2 mM pyruvate as the electron donor, showing that both strains deposit similar amounts of electrons on the anode. The P-value is calculated using Welch’s t-test. (Panel B) Transhydrogenases that can reduce HNQ are deleted in the Thnull strain (Panel C) Chronoamperometry of AnoxicNull and THnull strains under resting conditions, showing no visible differences in the EET performance. This result indicates that the transhydrogenases do not reduce HNQ. (Panel D) Colony PCR confirmation of the gshA gene knockout in GSHnull (Panel E) The total charge deposited by AnoxicNull and GSHnull on the anode with 2 mM pyruvate as the electron donor, showingthat GSHnull yields lower electron transfer onto the anode compared to Anoxi cNull. The P- value is calculated using Welch’s t-test. (Panel F) Reduced glutathione can abiotically react with oxidized naphthoquinones (NQ) to form an NQ-SG adduct and a reduced version of the NQ. (Panel G) The NADPH pool maintains the reduced form of the thioredoxin (TRX), which can transfer electrons to HNQ. (Panel H) Colony PCR confirmation of the trxA, trxB, and trxC gene knockouts in TRXnull (Panel I) Chronoamperometry of AnoxicNull and TRXnull strains under resting conditions, showing no visible differences in the EET performance. This result indicates that the thioredoxin pool does not reduce HNQ in the conditions tested. The data represent mean ± standard deviation obtained in triplicate BES experiments.

[0018] FIG. 10 shows a non-limiting, exemplary data related to figure 4. (Panel A) Chronoamperometry of the AnoxicNull, the AzoNull, and the NfsNull strains under resting conditions. The EET levels in NfsNull are substantially diminished compared to the AnoxicNull and AzoNull strains. This data indicates that quinone reductases NfsA and NfsB reduce HNQ. The data represent mean ± standard deviation obtained in duplicate BES experiments. (Panel B) A schematic showing the plasmid expression design of NfsB in the QRedNull-Ll strain. (Panel C) The total charge deposited by QRedNull-Ll and NfsB+ on the anode with different electron donors, showing that complementing NfsB on a plasmid in the QRedNull-Ll strain enables higher charge deposition on the anode. The P-values are calculated using Welch’s t-test. (Panel D) The total charge deposited by BioD, AnoxicNull, QRedNull, and EsinkNull on the anode under growing conditions, demonstrating that the amount of charge deposited on the anode is directly proportional to the strains’ ability to grow using fermentative pathways. (Panel E) pH measured at the end of the BES run, suggesting that EsinkNull did not significantly oxidize electron donors and perform EET. (Panel F) Glucose consumed by the end of the BES run suggesting that EsinkNull is limited in oxidizing glucose due to a lack of available catabolic electron sink. The (Panel G) pyruvate, (Panel H) lactate, and (Panel I) acetate profiles of the spent BES media of BioD, AnoxicNull, QRedNull, and EsinkNull, suggesting that all strains are metabolically active. (Panel J) A schematic depicting that all three formate dehydrogenases are deleted in the FDHnull strain. (Panel K) Chronoamperometry of the BioD and FDHnull strains under resting conditions, showing that BioD maintains a higher steady current density level than the FDHnull strain. This data indicates the flow of electrons in the formate catabolism contributes to HNQ-mediated EET. (Panel L) The metabolite profile of the spent BES media, showing that there are different levels of formate retention in the BioD and the FDHnull strains. This data indicates that BioD performs additional EET at the expense of formate. The data in (Panel K) and (Panel L)represent mean ± standard deviation obtained in duplicate BES experiments. The P-values are calculated using Welch’s t-test.

[0019] FIG. 11 shows a non-limiting, exemplary data related to Figure 5. (Panel A) The percentage reduction of amaranth by AnoxicNull, QRedNull, and EsinkNull in the presence of DMSO and HNQ, showing that, using HNQ, the AnoxicNull strain significantly reduces amaranth, whereas QRedNull and EsinkNull barely perform measurable EET in 1.5 hours. (Panel B) Glucose consumed in the amaranth assay of AnoxicNull, QRedNull, and EsinkNull at the end of 1.5 hours, showing that both AnoxicNull and QRedNull consume glucose, whereas EsinkNull does not. This data indicates the presence of fermentative pathways in AnoxicNull and QRedNull allows anaerobic growth. The P-values in (Panel A) and (Panel B) are calculated using Welch’s t-test. (Panel C) Lactate measured in the amaranth assay of AnoxicNull, QRedNull, and EsinkNull at the end of 1.5 hours, showing that QRedNull produces a higher lactate than AnoxicNull and EsinkNull. This result validates the genetic background of the strains and matches with the lactate production trend previously seen in the BES condition. The P-values are calculated using one-way ANOVA. The data in (Panel A) to (Panel C) represents mean ± standard deviation obtained in triplicate experiments. (Panel D) Volcano plot for gene expression of AnoxicNull. (Panel E) Volcano plot for gene expression of QRedNull. (Panel F) Volcano plot for gene expression of EsinkNull. (Panel G) Scatter plot of differential iModulon activity for AnoxicNull with and without HNQ supplementation. (Panel H) Scatter plot of differential iModulon activity for QRedNull with and without HNQ supplementation. (Panel I) Scatter plot of differential iModulon activity for EsinkNull with and without HNQ supplementation. (Panel J) Expression of the ATP-generating genes ackA and pgk. pvalues were calculated using the Welch’s t-test with a significance threshold of 0.05. (Panel K) The activity of the LPS and gcvB iModulons across the PRECISElk database of E. coli gene expression. Samples from this project and other projects with high activity of these iModulons are highlighted. (Panel L) Different gene expression plots for AnoxicNull, QRedNull, and ESinkNull, with highly perturbed genes within the Uncharacterized- 1 (UC-1) iModulon highlighted. Gene expression was centered on an aerobic glucose reference condition.

[0020] FIG. 12 shows non-limiting, exemplary data related to Figure 6. (Panel A) Flux balance analysis of the FermNull+nfsB strain under anaerobic growth on glucose shows the ATP production pathways supporting elevated growth. Fluxes in mmol / gDW / hr units, growth rate (GR) in 1 / hr units. The listed fluxes represent >95% of the turnover of ATP / ADP. Reaction abbreviations - PGK: Phosphoglycerate Kinase. ACK: Acetate Kinase. PYK: Pyruvate Kinase.PFK: Phosphofructokinase. ATPS: ATP Synthase. GAM: Growth-associated Maintenance. NGAM: Non-growth-associated Maintenance (Panel B) A stacked bar chart that displays the mass fraction of the proteome allocated to ETC, Glycolysis, Fermentation sectors, as well as NfsB. This is done for a wild-type strain undergoing nitrate respiration, fermentation only, and the FermNull strain with and without the addition of NfsB. (Panel C) A bar chart that shows the percent decrease in the mass fraction of the proteome allocated to five stress-related iModulons upon the addition of a QRed (NfsB) to FermNull. (Panel D) Chronoamperometry of the FermNull and the EsinkNull strains under growth conditions, showing that FermNull transforms into generating a high EET output after several hours in the BES. This indicates that the FermNull strain is undergoing changes that increase the flux through the HNQ- mediated EET pathway. The data represent mean ± standard deviation obtained in triplicate BES experiments. (Panel E) The total charge deposited on the anode by FermNull and EsinkNull over time, showing that FermNull consistently deposits a higher charge value over time than EsinkNull. The data represent mean ± standard deviation obtained in triplicate BES experiments. (Panel F) OD600 of the FermNull and the EsinkNull strains post BES run under growth conditions, showing FermNull grew to higher biomass than EsinkNull. This indicates that HNQ-mediated EET can enable growth under anaerobic conditions. The data represent mean ± standard deviation obtained in triplicate BES experiments. The P-value is calculated using Welch’s t-test. (Panel G) pH measured at the end of the BES run, suggesting that as compared to EsinkNull, adapted-FermNull has conducted higher catabolism and EET. (Panel H) Acetate produced by the end of the BES run, showing that EsinkNull has a higher carbon- flux going into acetate biosynthesis than the adapted-FermNul strain. The data represented in (Panel G) and (Panel H) are the means ± standard deviations obtained in triplicate BES experiments. The P-values are calculated using Welch’s t-test. (Panel I) The FNO strain can utilize QReds and HNQ to respire on amaranth. (Panel J) The percentage reduction of amaranth by FNO, showing that in the presence of HNQ, FNO can significantly reduce amaranth in 1.5 hours. This result, along with the fig 11 panel A, corroborates with the EET phenotypes seen in the BES set with FNO and EsinkNull. (Panel K) Glucose consumed in the amaranth assay of FNO at the end of 1.5 hours, showing that FNO significantly oxidizes glucose only in the presence of HNQ. The P-values in (Panel J) and (Panel K) are calculated using Welch’s t-test. (Panel L) Volcano plot for gene expression of FNO with and without HNQ supplementation. (Panel M) Scatter plot of differential iModulon activity for FNO with and without HNQ supplementation. (Panel N) Expression of the ATP-generating genes ackA and pgk. (Panel O)Shows non-limiting, exemplary data described herein. (Panel P) Shows non-limiting, exemplary data described herein.

[0021] FIG. 13 shows non-limiting, exemplary methods described herein. (Panel A) Chronoamperometry of AnoxicNull at pH 6.5 and 7 under resting conditions, showing that the HNQ stimulates higher EET levels at pH 6.5 (Panel B) Chronoamperometry of AnoxicNull with glycerol as the electron donor under resting conditions, showing that the HNQ stimulates EET whereas DMSO does not (Panel C) Glycerol consumed by AnoxicNull in (Panel B), showing that HNQ-mediated EET drives oxidation of glycerol. Although, AnoxicNull consumed only about 25% of the provided glycerol (2 mM). The AnoxicNull in (Panel B) and (Panel C) is precultured in M9P-LB, followed by biomass-generating culture in M9P-2xYT with 20 mM glycerol supplementation. The data in (Panel A), (Panel B), and (Panel C) represent mean ± standard deviation obtained in triplicate BES experiments. The P-value in (Panel C) is calculated using Welch’ s t-test. (Panel D) Chronoamperometry of AnoxicNull with increasing HNQ concentrations, showing that 20 pM HNQ stimulates a significant EET output. In this experiment, the cells were precultured in glycerol, and 20 mM glycerol was supplemented in the BES media. The data represents mean ± standard deviation obtained in triplicate BES experiments. (Panel E) Cell viability assay of AnoxicNull with increasing concentrations of lawsone, showing that 20 pM HNQ does not negatively impact the viability of cells. The experiment was initiated with 1 mL of 0.5 ODeoo cells in M9P (+2 mM glycerol) and incubated with different concentrations of HNQ for about 40 hours before taking the reported ODeoo measurements. (Panel F) ODeoo of AnoxicNull in the presence of DMSO and 20 pM HNQ after 20 hours of anaerobic growth in M9P complete-growth medium with 20 mM glucose, showing that 20 pM of HNQ has no negative effect on the fermentative growth. The data represent mean ± standard deviation obtained in eight biological replicates. The P- value is calculated using Welch’s t-test. (Panel G) A schematic showing the structures of rose- red colored amaranth and its reduced colorless products. The degree of EET is assessed by measuring the loss of absorbance of amaranth at 518 nm. (Panel H) The percentage reduction of amaranth by AnoxicNull, QRedNull, and EsinkNull in growth media, showing that the AnoxicNull strain reduces amaranth to the highest level, QRedNull at an intermediate level, and EsinkNull at the lowest level. This data corroborates with the EET capabilities of the strains previously elucidated from BES experiments. The data represent mean ± standard deviation obtained in three biological replicates. The P-values are calculated using one-way ANOVA. The metabolite profile of the spent BES media, showing that EsinkNull shows a similar amount of (Panel I) pyruvate consumption yet different levels of (Panel J) formate and (Panel K) acetategenerated, compared to AnoxicNull and QRedNull strains. This data validates the genetic backgrounds of AnoxicNull, QRedNull, and EsinkNull. The data represent mean ± standard deviation obtained in triplicate experiments. The P-values are calculated using one-way ANOVA.

[0022] FIG. 14 shows non-limiting, exemplary methods described herein. (Panel A) A schematic showing the two-step gene deletion in BioD using the GKX sei ection / countersel ection cassette. (Panel B) A schematic showing the BES setup consisting of two chambers and three electrodes (anode, cathode, and reference).

[0023] FIG. 15 provides nonlimiting, exemplary data to support that HNQ-mediated anode respiration as the sole electron sink can drive growth in E. coli. (A) Chronoamperometry of OmpC*, FN1, and FermNull in complete media, showing that OmpC* has a similar EET phenotype to FN1. This data suggests that the adaptation in OmpC leads to the gain in EET output in FN1. Chronoamperometry of OmpC*, OmpCnull, and FermNull under non-growth conditions with 2 mM glucose as the electron donor, showing that OmpCnull has similar (B) peak current density and (C) charge deposited on the anode to OmpCnull. The data in (C), (D), and (E) represent mean ± standard deviation obtained in duplicate BES experiments. The P- values in (C) are calculated using one-way ANOVA, with a significance threshold of 0.05. The dotted lines indicate the time of peak current for each condition.

[0024] FIG. 16 shows non-limiting, exemplary data related to Figure 105. (A) A schematic showing the electron fluxes from catabolism to the potential electron sinks in the AnodeSole strain, where the anode is the sole respiratory electron sink. (B) OD600 of AnoxicNull and AnodeSole after 24 hours of anaerobic cultures on complete media consisting of 20 mM of carbon source, 5 g / L NZ- Amine, and 1% yeast extract, showing that AnodeSole does not grow in biomass under anaerobic conditions. (C) Chronoamperometry of the OmpC* and the AnodeSole strains under growth conditions, showing similar EET output phenotypes. AnodeSole shows similar (D) charge and (E) biomass growth to OmpC*, indicating that eliminating hydrogen as a potential electron sink does not significantly impact the HNQ- mediated EET output. The data in (C), (D), and (E) represent mean ± standard deviation obtained in duplicate BES experiments. The P-values are calculated using Welch’s t-test, with a significance threshold of 0.05.

[0025] FIG. 17 provides a non-limiting, exemplary schematic for interfacing metabolism with electrochemistry.

[0026] FIG. 18 provides a non-limiting, exemplary schematic and data showing that a redox mediator stimulates EET in the presence of a carbon source.

[0027] FIG. 19 provides a non-limiting, exemplary schematic and data showing that NADH is the primary electron donor for EET.

[0028] FIG. 20 shows a non-limiting, exemplary schematic and data showing that anaerobic carbon flux and the EET pathway are intricately linked.

[0029] FIG. 21 shows non-limiting, exemplary data and a schematic the robust means for enabling unbalanced fermentation under one embodiment of the present disclosure.

[0030] FIG. 22 shows a schematic providing non-limiting, exemplary applications of the present disclosure.

[0031] FIG. 23 provides a non-limiting exemplary schematic showing aerobic respiration in E. coli. In embodiments, electron flow in metabolic and respiratory pathways enable generation of certain cellular currencies such as ATP, PMF, and cofactor turnover.

[0032] FIG. 24 shows non-limiting, exemplary location of electron sinks in E. coli. As can be seen terminal electron acceptors can be present in either cytoplasm or periplasm.

[0033] FIG. 25 provides non-limiting, exemplary data showing that quinone stimulates extracelluar electon transfer inDETAILED DESCRIPTION OF THE INVENTION

[0034] Abbreviations and Definitions

[0035] Detailed descriptions of one or more preferred embodiments are provided herein. It is to be understood, however, that the present invention can be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0036] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0037] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.

[0038] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0039] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0040] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

[0041] Described herein are cytoplasmic enzymes that enable or promote EET. In embodiments, the cytoplasmic enzymes enable or promote EET by reducing naphthoquinones. For example, nitroreductase NfsB can reduce HNQ. However, other cytoplasmic enzymes can reduce other types of naphthoquinones promote or enable EET. In embodiments, the naphthoquinone comprises any one or more of menadione (MNQ), l,4-dihydroxy-2-naphthoic acid (DEINA), and HNQ. Embodiments include enzymes and mediators that are homologous to any of the various cytoplasmic enzymes and mediators (such as napthoquinone) disclosed herein.

[0042] Aspects of the disclosure are drawn towards a method of stimulating EET in a microorganism comprising: providing the microorganism with an electron donor; exogenously supplementing the microorganism with a small molecule redox mediator; and subjecting the microorganism to an electron sink, thereby stimulating EET in the microorganism.

[0043] As used herein, the term “supplemented” can refer to the addition or supply of a composition.

[0044] As used herein, the term “redox mediator” can refer to a molecule or composition that can be reversibly reduced and oxidized. For example, the redox mediator can act as an electron shuttle. The redox mediator can comprise a quinone compound. For example, the redox mediator is a naphthoquinone. In certain embodiments, the naphthoquinones comprise exogenously added naphthoquinones.

[0045] For example, the naphthoquinone is selected from the group consisting of 2-hydroxy- 1,4-naphthoquinone (HNQ), 2-methyl-l,4-naphthoquinone (MNQ), or l,4-dihydroxy-2-naphthoic acid (DHNA). In embodiments, the naphthoquinone is biosynthesized within the microbe. In one embodiment, HNQ is biosynthesized within E. Coli. Further, in one embodiment, naphthoquinone-mediated EET promotes catabolism and growth in E. coli. This can allow for bioelectrocatalysis and bioproduction in the absence of oxygen. The naphthoquinone-mediated EET pathway disclosed herein can be employed in any microbe that is capable of electron transmission.

[0046] In embodiments, the redox mediator is supplemented at a concentration of up to about 500 pM. The redox mediator can be supplemented at a concentration of less than about 1 pM. In certain embodiments, the redox mediator is supplemented at a concentration of between about 1 pM and 100 pM. The redox mediator can be supplemented at a concentration of between about 5 pM and 50 pM. The redox mediator can be supplemented at a concentration of between about 10 pM and 30 pM. In embodiments, the redox mediator is supplemented at a concentration of less than 1 pM, about 1 pM, about 2.5 pM, about 5 pM, about 7.5 pM, about 10 pM, about 12.5 pM, about 15 pM, about 20 pM, about 25 pM, or greater than about 25 pM. The redox mediator can be supplemented at a concentration of about 20 pM.

[0047] In embodiments, the microorganism is a bacteria. For example, the bacteria is E. coli.In certain embodiments, the bacteria is an exoelectrogen. In alternative embodiments, the bacteria not an exaelectron. In embodiments, a non-exoelectrogen bacteria refers to a bacteria that is not capable of directly transferring electrons to an external electron sink. In embodiments, non-exoelectrogen bacteria do not naturally possess specialized EET pathways to respire on extracellular electron sinks. In certain embodiments, a non-exoelectrogen bacteria relies on indirect EET to transfer one or more electrons to an electron sink.

[0048] As used herein, the term “electron sink” can refer to a molecule or ion that can accept a bond or lone pair of electrons. In embodiments, the electron sink is an anode. In embodiments, the electron sink can be compounds such as metal hydroxides. The electron sink can be insoluble Fe(III), tellurite, selenite, and azo dyes, or any combination thereof.

[0049] In embodiments, the electron donor is any substance that is capable of donating one or more electrons during a microbial metabolic process. Electron donors can comprise organic compounds that are capable of being metabolized by microbes in a manner that, during such metabolism, an electron is released therefrom. In embodiments, the electon donor an organic substance. The electron donor can comprise a sugar, a carbohydrate, or a combination thereof. In embodiments, the electron donor comprises glucose, fructose, sucrose, glycerol, pyruvate, or any combination thereof. The electron donor can comprise a short-chain fatty acid. In certain embodiments, the electron donor comprises acetate, lactate, formate, and any other organicacid that serves as an electron donor in microbial systems. The electron donor can comprise hydrogen gas (H2). In certain embodiments, the electron donors comprise a humic substance. In certain embodiments, the electron donor comprise sulfate, such for applications of a sulfatereducing processes in wastewater treatment systems.

[0050] In embodiments, the method overcomes direct EET feedstock selectivity of direct EET. For example, there are limited feedstock (electron donors) options for certain microbes (such as exoelectrogens including but not limited to Shewanella and Geobacter species) that perform direct EET. This can be attributed not to the direct EET pathway but to the catabolism abilities of these exoelectrogens. Thus, researchers have heterologously expressed the direct EET pathways from these exoelectrogens in microbes that are not exoelectrogens but can utilize diverse feedstocks. However, these prior engineering efforts have not come to fruition with high efficiency. However, the naphthoquinone-mediated EET pathway method described herein directly interfaces with the NADH pool of the microbes. Thus, as long as the microbes have the ability to utilize the feedstock of choice, the microbes can perform EET using exogenously added naphthoquinone and naphthoquinone reductase.

[0051] In embodiments, the method reduces the required electrode surface area compared to direct EET, utilizes the whole reactor volume, does not require the formation of biofilm on the electrode, or any combination thereof.

[0052] As used herein, the term “genetically engineered” can refer to a process that uses laboratory or synthetic technologies to alter the genetic makeup of an organism. For example, the genetic makeup can be the DNA. In embodiments, the microorganism is genetically engineered to increase EET output. For example, the genetically engineered bacteria partially, substantially, or completely deletes, silences, inactivates, or down-regulates a gene or fragment thereof that encodes for an anaerobic respiratory pathway, a fermentative pathway, or a combination thereof. The genetically engineered bacteria can be a fermentative-deficient bacterial strain. For example, the genetically engineered bacteria is AnoxicNull napFDAGHBC AnarGHI EnarZYW AdmsABC AynfEFGH AtorCAD AtorYZ f rd A BCD AnirBDC AnrfABCDEFG AnorVW Ehmp \ccp), FermNull (AmgsA AadhE AldhA or any combination thereof.

[0053] Aspects of the disclosure are drawn towards a genetically modified microorganism, wherein the genetically modified microorganism partially, substantially, or completely deletes, silences, inactivates, or down-regulates a gene or fragment thereof that encodes for an anaerobic respiratory pathway, a fermentative pathway, or a combination thereof. In embodiments, the microorganism is genetically engineered to increase extracellular electrontransfer (EET). In embodiments, the genetically modified organism is a fermentative-deficient strain. The genetically modified organism can be any one or more of the strains listed in Table 1. The genetically modified organism can have any of the various characteristics or properties recited in Tables 2 or 3. The genetically modified organism can be AnoxicNull (AnapFDAGHBC AnarGHI AnarZYW EdmsABC EynfEFGH \lorCAD \lorYZ EfrdABC EnirBDC EnrfABCDEFG AnorVW \hmp Eccp QRedNull (EazoR \nfsA \nfsB \qorA \qorB \wrbA \yieF \kefFC \kefGB \nemA \aegA \mdaB-ygiN RbtuE \yqhl) \med- \cys.JIH-ygc operons \ydi operon Efix operon). EsinkNull (EpoxB \ldhA \fdhF EadhE \hyc-hyp operons \fdhE-fdo-fdhI)) FermNull (EmgsA EadhE \ldhA). or a combination thereof. In embodiments, the genetically modified microorganism is AnoxicNull (EnapFDAGHBC EnarGHI EnarZYW EdmsABC EynfiEFGH EtorCAD EtorYZ EfirdABCD EnirBDC EnrfABCDEFG EnorVW Ehmp \ccp). FermNull (EmgsA EadhE EldhA). or any combination thereof.

[0054] In certain embodiments, the present disclosure relates to methods of leveraging EET pathways in A. coli. Without being bound by theory, the present disclosure provides methods for evaluating and harnessing the mechanisms of the mediated EET pathway in a microorganism.

[0055] In embodiments, the EET pathway disclosed herein is orthogonal to cell-native respiratory and fermentative pathways.

[0056] In certain embodiments, the EET output varies with the concentration and type of electron donor, the number of cells, the concentration of exogenously added redox mediator, the identity of the redox mediator, or any combination thereof.

[0057] In certain embodiments, the starting cell OD600 is of 0.1 and the concentration of HNQ is about 20 pM.

[0058] In embodiments, the identity electron donor depends on the type of experiment or the exogenous conditions. Such effects of experimental conditions are further discussed in the methods below (e.g., see “Rationale - Choices of nitrogen sources under HNQ-mediated EET conditions” and “Rationale - Choices of carbon sources (electron donors) under HNQ-mediated EET conditions”).

[0059] Embodiments of the present disclosure relate to a respiratory pathway, which connects a living microbe with a non-living anode. This EET pathway can independently drive catabolism and growth. In various embodiments, the disclosure provided herein can be applied to any system or application that relates to or otherwise involves anaerobic metabolism and bacterial growth. In embodiments the disclosure relates to an EET pathway that exhibits a highcurrent output, high signal / noise ratio, is genetically encodable, can drive respiration, or any combination thereof.

[0060] In certain aspects, the present disclosure relates to methods of tracking of electron flow through metabolism.

[0061] Further aspects of the present disclosure relate to methods of stimulating EET in a microorganism by reducing competition with the microorganism’s natural fermentation pathways. In certain embodiments, such methods include partially, substantially, or completely deleting, silencing, inactivating, or down-regulating competing NADH sinks within fermentative pathways.

[0062] Without being bound by theory, non-limiting, exemplary applications of the present disclosure include medical applications, environmental applications, and biotechnological applications. Exemplary medical applications include: influencing, investigating, or exploiting the electricity of pathogens (such as to develop treatments directed to eliminating or reducing such the pathogenic activity); developing drug targets and infection mitigation strategies, direct metabolic monitoring, and screening for therapeutics. Non-limiting, exemplary environmental applications include: biogeochemical cycling, switching between electroactive and nonelectroactive metabolism, EET under stress conditions, and electro activity in extreme environments. In addition, exemplary biotechnological applications can include, but are not limited to: electro-fermentation and food sensory profiles, off-grid power and biosensing in remote regions (e.g., cold regions), and oxygen scavenging and substrate breakdown in BES. Without wishing to be bound by theory, additional or alternate applications of the present disclosure include space exploration. Such space exploration applications can include growing microbes in space using electronics, extracting electrical energy from organic waste, and biosensing of analytes (e.g., sugar) with electrical readout.

[0063] In embodiments, any of the various genetically modified organisms disclosed herein stimulate extracelluar electron transfer under resting conditions. The electron transfer can be coupled to catabolism in the genetically modified organism.

[0064] In one embodiment, the redox mediator draws and electron from NADH.

[0065] In embodiments the electron flow to the redox mediator does not pass through or otherwise any one or more of: anaerobic respiration, NADPH, GSH, and TRX.

[0066] In certain embodiments, cytoplasmic reductases contribute to redox-mediated EET. Redox-mediated electron transfer can be coupled to cellular growth of the microorganism. One embodiment of the present disclosure is characterized by anode respiration in E. coli. In suchembodiments, the electron sink comprises an anode, and such anode respiration contributes to growth of the E. coli.EXAMPLES

[0067] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.EXAMPLE 1

[0068] Overcoming redox limitations in microbial bioproduction using electronic control

[0069] The bioproduction of chemicals and fuels is experiencing a surge, driven by its potential for sustainability and facilitating a circular economy. Most bioproduction processes grow microbes under anaerobic conditions to enhance product yield and eliminate the need for expensive oxygenation. However, producing these chemicals under anaerobic conditions often comes with the constraint of maintaining a redox balance with the starting materials. Competition from the cell's natural fermentation pathways for the same starting material makes controlling the desired bioproduction pathway even harder. We have engineered E. coli with a unique metabolic twist to overcome these challenges. Instead of relying on its natural anaerobic metabolism, we've built in a completely synthetic alternative. This system uses a small molecule called HNQ and an electrode to maintain the crucial redox balance, allowing E. coli to grow and produce valuable chemicals without oxygen. This platform strain, combined with our electrochemistry methods, holds immense potential. We envision its use in sustainable chemical production, improved food fermentation processes, and bioremediation efforts. This innovative approach can revolutionize bioproduction and pave the way for a greener future.

[0070] E. coli, a facultative anaerobe, is traditionally not classified as an exoelectrogen. Exoelectrogen is a microbe that uses extracellular terminal electron acceptors for respiration and growth. However, E. coli secretes redox-active molecules (RAMs) and uses extracellular electron acceptors like anode for respiration. In certain, non-limiting examples, 2 -hydroxy- 1,4- naphthoquinone (HNQ) serves as the RAM, which enables E. coli to perform extracellular electron transfer (EET). The mechanism of the RAM-mediated EET remains largely unknown. We used an in-house developed library of redox mutant strains to elucidate the mechanism of HNQ-mediated EET. We discovered that (1) Increasing the availability of the NADH pool canpromote the EET levels, and (2) Quinone reductase NfsB is an exemplary cytoplasmic oxidoreductase enzyme that can mediate anodic respiration via HNQ. In such embodiments, NfsB uses NADH as the electron donor. With HNQ-mediated EET as the only respiration route, cells can generate anaerobic biomass. In summary under these embodiments, HNQ- mediated anode-assisted respiration is like anaerobic respiration on inorganic substrates like nitrate and serves the same function in generating ATP, like fermentation. However, a benefit of employing this EET pathway is that the pathway permits the electronical control of the redox homeostasis and growth condition of microbes used for bioproduction in real-time. From a microbial bioelectronics’ perspective, the HNQ-mediated EET pathway generates the highest current output reported in E. coli. Intriguingly, we achieved these results without heterologous expression of any pathway. E. coli expresses all the enzymatic components required to respire on anode.

[0071] Microbial electrochemistry relies heavily on RAMs. But how these RAMs do their job - shuttling electrons from microbes to electrodes - remains a mystery in many cases. Our research takes a step towards cracking this code. We've built an “instruction manual” and a library of redox mutant strains for understanding RAM-mediated electron transfer in any microbe. The present disclosure provides toolbox that combines genetic engineering and electrochemical experiments, shedding light on these hidden processes. In certain embodiments, the present disclosure can be adapted to explore new ways microbes use RAMs for anaerobic respiration - breathing without oxygen. This instruction manual will open the door to newer versions of this technology that can change the current purview of bioproduction and bioremediation.

[0072] The present disclosure can be applied to all the applications associated with extracellular electron transfer and microbial bioelectronics. The innovations will revolutionize the specificity and efficiency of EET pathways that are highly desirable for at least the following applications -

[0073] -Microbial fuel cell technology to treat organic waste (aquacycl.com / services / carbon- removal / )

[0074] -Bioremediation of toxic metal wastes.

[0075] -Real-time biosensing for healthcare and environmental pollutants

[0076] -Biosynthesis and bioconversion for sustainable production of value-added chemicals

[0077] -El ectrofermentati on

[0078] The current ideas of bioproduction using EET often hinge on the species of Geobacter and Shewaneta's direct EET pathways. While these microbes are equipped for direct passageof electrons to electrodes, their limitations hamper large-scale, economical bioproduction under oxygen-free conditions. Firstly, their anode respiration comes at the cost of being picky eaters, restricting their feedstock options. Cheap, readily available feedstocks are crucial for cost-effective bioproduction. Secondly, scaling up these systems is a hurdle. They often require vast electrode surfaces, have a slow growth rate, and use significant cellular resources to maintain their cytochrome-c pathways. They operate under long timescales with precise bioreactor conditions (e.g., biofilm formation). In embodiments of the present disclosure, HNQ-mediated EET can operate on its own or be imparted as an ancillary electronic pathway to existing bioproduction-relevant microbes.

[0079] Herein we describe a strategy for extracellular electron transfer (EET) in bioproduction, circumventing the inherent limitations of direct EET observed in species like Geobacter and Shewanella. The HNQ-mediated EET pathway is orthogonal to cell metabolism and can seamlessly integrate in engineered bioproduction strains. This approach overcomes the feedstock selectivity and scalability challenges associated with direct EET. The simplicity of HNQ-mediated EET reduces the requirement of vast electrode surface area and cellular energy requirements compared to conventional systems. The current approaches mostly use microbes as a black box for microbial electrochemistry applications. However, the mechanism of the HNQ-mediated EET pathway enables further optimization and targeted strain engineering for enhanced bioproduction efficiency and stability. These discoveries fundamentally reshape the bioproduction landscape by unveiling an improved EET route and methodological framework, unlocking a spectrum of previously unattainable applications and efficiencies.

[0080] While a desired EET response is possible with externally added HNQ, a sustainable long-term solution can be achieved using HNQ biosynthesis.. E. coli represents one suitable organism for the biosynthesis HNQ in such systems. Intriguingly, the structure of HNQ is quite similar to DHNA (l,4-dihydroxy-2-naphthoic acid), a precursor to the menaquinone biosynthetic pathway in E. coli. Without being bound by theory, HNQ can be derived from DHNA. HNQ is also a plant secondary metabolite. HNQ is the main component of a widely used hair and skin dye called Henna, sourced from the plant Law sonia inermis. Using the tools from the field of natural products research to unravel the HNQ-biosynthetic pathway is an achievable target.

[0081] Without wishing to be bound by theory, we can indicate the bioproduction capabilities of HNQ-mediated EET in E. coli and industry relevant bioproduction chassis. E.g., acetoin electrofermentation. An additional application of the present disclosure further includeswastewater treatment to sustainably control biological oxygen demand (BOD) and dissolved effluent organic matter (dEfOM) levels.

[0082] Non-Limiting Advantages Over Existing Technology

[0083] Current chemical production, heavily reliant on natural gas, faces a triple threat: dwindling resources, escalating costs, and a burgeoning carbon footprint. Bioproduction offers a sustainable alternative, but feedstock conversion and process control limitations often hinder its scalability. Here, microbial electrochemistry acts as a bridge between microbes and their environment. This innovative approach unlocks a circular economy of waste as feedstock, enabling efficient conversion of diverse materials into value-added chemicals. Imagine transforming agricultural waste into bioplastics or industrial effluents into valuable biofuels - all in a closed-loop system that minimizes waste and dependence on fossil fuels. Microbial electrochemistry empowers bioproduction to compete with petrochemical giants and surpass them in sustainability and economic viability.

[0084] Without wishing to be bound by theory, we can produce:

[0085] Synthetic bacteria with applications in bioproduction and bioremediation

[0086] Bioproduction of chemicals, food ingredients, and pharmaceuticals

[0087] References Cited Herein

[0088] Moscoviz, R., Toledo- Alarcon, J., Trably, E., and Bernet, N. (2016). ElectroFermentation: How To Drive Fermentation Using Electrochemical Systems. Trends in Biotechnology 34, 856-865. 10.1016 / j.tibtech.2016.04.009.

[0089] Yamada, S., Takamatsu, Y., Ikeda, S., Kouzuma, A., and Watanabe, K. (2022). Towards Application of Electro-Fermentation for the Production of Value-Added Chemicals From Biomass Feedstocks. Frontiers in Chemistry 9.

[0090] Gong, Z., Yu, H., Zhang, J., Li, F., and Song, H. (2020). Microbial electro-fermentation for synthesis of chemicals and biofuels driven by bi-directional extracellular electron transfer. Synthetic and Systems Biotechnology 5, 304-313. 10.1016 / j.synbio.2020.08.004.EXAMPLE 2

[0091] Electronic respiration as a new energy metabolism in bacteria

[0092] Abstract

[0093] Certain microbes utilize redox shuttles to exchange electrons with their environment, a process known as mediated extracellular electron transfer (EET). Mediated EET maintains redox homeostasis and supports anaerobic survival across diverse microbial communities. While this phenomenon has been harnessed in microbial electrochemical technologies, afundamental question remains: how do these redox shuttles undergo reduction within the cells, and what are the associated bioenergetic implications? The lack of understanding in this area limits our ability to fully comprehend the physiological roles of mediated EET in diverse microbes and develop efficient microbial electrochemical technologies. To address this gap, we devised a methodology integrating genome editing and electrochemistry to probe the cellular electron fluxes, thereby elucidating the mechanism of mediated EET in bacteria. Using this methodology, we elucidate a native EET mechanism in Escherichia coli, a discovery previously impeded by the absence of suitable methods. We discovered that 2-hydroxy-l,4- naphthoquinone (HNQ) drives energy metabolism in E. coli by enabling it to respire on an extracellular electrode via cytoplasmic nitroreductase. Without being bound by theory, indicate EET metabolism offers an energetically favorable route to supporting anaerobic growth. In embodiments, transcriptomics can be used to characterize the elevated carbon / nitrogen metabolism and adaptation in response to HNQ-EET. This work shows that heterotrophs can grow independently of classical electron transport chains and fermentative pathways, revealing a new facet of anaerobic microbial physiology.

[0094] Introduction

[0095] Electron flow drives energy metabolism, sustaining life at its core1. All energy metabolisms transfer electrons to an electron sink through diverse respiratory and fermentative pathways. Escherichia coli, a microbe suitable for studying energy metabolism2, reduces intracellular electron sinks such as oxygen and nitrate. Exoelectrogens, conversely, are specialized microbes that reduce extracellular electron sinks, such as metal (hydro)oxides, through a process called extracellular electron transfer (EET)3,4. EET is pivotal in diverse environments, contributing to Earth’s biogeochemical cycles5 7and microbial community dynamics in mammalian guts8,9. Moreover, EET pathways can connect electronics and living cells, enabling the electrical modulation of energy metabolism, gene expression, and overall cellular physiology10 l3.

[0096] E. coli is conventionally regarded as a non-exoelectrogen and, because of its importance as a biotechnology workhorse, has been the focus of extensive engineering efforts to introduce EET14 l 9. However, the synthetically introduced EET registered only a modest electrical output, and unlike in model exoelectrogens, EET is uncoupled with growth20 22 8. Intriguingly, E. coli can evolve to perform EET when interfaced with an extracellular electrode23 32. The reported EET levels from evolved E. coli are robust and comparable to pure cultures of model exoelectrogens33. The evolved EET is mediated by 2-hydroxy-l,4-naphthoquinone (HNQ), which without wishing to be bound by theory can be biosynthesized and secreted by E. coli.These observations are of great interest as there can represent latent exoelectrogenic mode of energy metabolism for anaerobic sustenance. Moreover, the ability of E. coli to perform HNQ- mediated EET coupled with growth can pave the way for constructing robust biosensors and synthetic metabolisms for carbon capture. Despite these promises, many questions remain around the mechanism and bioenergetic implications of HNQ-mediated EET.34,35.

[0097] Described herein, we addressed this gap by integrating high-fidelity genome editing, systems biology, and microbial electrochemistry to elucidate mechanisms of mediated EET in E. coli. With these tools, we dissected electron flow in E. coli metabolism, genetically altering the electron flux network and gauging the impact of the modified network on EET levels (Figure 1 A). Using this approach, we exhaustively characterized the molecular mechanism of HNQ-mediated EET and its associated energy metabolism in E. coli. We found that cytoplasmic HNQ reductases selectively facilitate HNQ-mediated EET, efficiently driving catabolism by enabling NAD+regeneration. Additionally, HNQ-mediated EET allows E. coli to respire on an anode and support unexpectedly robust anaerobic growth. Without being bound by theory, EET-mediated anaerobic metabolism offers bioenergetic advantages over canonical fermentative pathways.

[0098] Transcriptomic response to HNQ revealed perturbation of redox pathways and a shift to support growth through anaplerotic catabolism of peptides. In summary, and without being bound by theory, the discovered naphthoquinone-mediated EET pathway reveals a new facet of anaerobic microbial physiology.

[0099] Importantly, this study lays the foundation for a minimal and generalizable method of engineering EET in microbes to improve planetary and human health.

[0100] Non-Limiting, Exemplary Results

[0101] The HNQ-mediated EET pathway depends on the oxidation of a carbon source.

[0102] Without wishing to be bound by theory, exogenous HNQ can stimulate EET in E. coli21, 6. To determine if HNQ can stimulate EET in E. coli, we tested a (parent MG1655 derived) BioDesignER37strain’s (BioD) ability to generate current in a bioelectrochemical system (BES) using glycerol as an electron donor and HNQ as a redox shuttle. Under resting conditions, adding glycerol to the cells with HNQ resulted in immediate current generation (Fig 7 panel A). In the presence of HNQ, BioD consumed 1.33 ± 0.03 mM glycerol and showed a peak current level of 101.8 ± 4.7 mA / m2. In contrast, without HNQ, BioD consumed 1.51 ± 0.04 mM glycerol and registered a peak current level of only 0.68 ± 0.02 mA / m2(Fig 7 panels A and B)38. To validate the shuttling of HNQ, we performed a cyclic voltammogram analysis on BioD with HNQ and DMSO. The cyclic voltammetry of BioD showed that HNQ behaveslike a redox shuttle and has a midpoint potential of -323 mV (Fig 7 panel C). Consistent with prior studies, HNQ as a redox shuttle enables E. coli to perform EET using an anode as an extracellular electron sink.

[0103] To validate that the E. coli strains depend on oxidizing electron donors to generate EET using HNQ (Fig 1 panel B), we deleted the three glycerol consumption routes (Fig 7 panel D)39to create a GlyNull strain (Fig 7 panel E). We then compared the ability of BioD and GlyNull to generate EET and oxidize glycerol in a BES under resting conditions. BioD consumed 1.30 ± 0.04 mM glycerol and showed steady-state current levels of 74.8 ± 2.1 mA / m2. In contrast, GlyNull consumed ~ one-tenth as much glycerol (0.141 ± 0.033 mM) and recorded only 2.3 ± 0.72 mA / m2(Figure 1 panel C and D). These results strongly suggest that GlyNull does not perform EET due to a lack of glycerol consumption. To confirm this loss of function was not due to polar effects, we reintroduced the / t / 4 gene at its native genomic locus under a constitutive synthetic promoter (Fig. 7 panel E, Methods). This GldA± strain exhibited restored glycerol consumption and EET levels, consuming 0.85 ± 0.07 mM glycerol and generating a steady-state current density of 54 ± 2 mA / m2(Figure 1 panels B and C). This data indicates that oxidation of a carbon source is crucial for HNQ-mediated EET.

[0104] Glycerol oxidation simultaneously initiates electron fluxes from the cytoplasm (NADH) and the inner membrane (reduced quinone). Thus, to simplify the investigation process of the HNQ-mediated EET mechanism, we chose pyruvate as an electron donor rather than glycerol for further investigation, as it can initiate electron fluxes only from the cytoplasm. To validate the choice of pyruvate as the electron donor, we deleted the ubiquinonedependent40poxB gene to generate the PoxNull strain (Fig 7 panel F) since PoxB oxidizes pyruvate and reduces the quinone pool. We then investigated PoxNull ’s ability to generate EET with complete consumption of the provided pyruvate and glycerol as the control electron donor. The PoxNull strain consumed nearly all the provided pyruvate while consuming only 25% of the glycerol (Fig 7 panel H). Although glycerol is a more reduced electron donor than pyruvate and is expected to stimulate higher EET, the PoxNull strain generated similar EET levels with glycerol or pyruvate as electron donors (Fig 7 panel G). This result indicates that pyruvate is a more suitable choice of electron donor for investigating the EET mechanism.

[0105] E. coli upregulates diverse redox pathways in response to HNQ

[0106] With data supporting that HNQ-dependent EET is linked to catabolism, we next sought to identify the biomolecule that catabolism reduces and serves as an electron donor to HNQ. Such identification is often recognized as a formidable challenge because catabolic pathways are intricately linked to many redox-active biomolecules. To address this challenge,we first profiled the transcriptome of HNQ-treated cells under no EET (BES) condition to infer which cellular redox pools HNQ engages. The HNQ-treated cells show only 2.1% differentially expressed genes (DEG), yet perturbed genes involve several redox biomolecules (Figure 1 panel E). Upregulated gene transcripts include pathways for naphthoquinone degradation, naphthoquinone sensing transcription factors, and enzymes that oxidize NAD(P)H (Figure 1 panel F). Regulon-level transcriptional analysis suggested simulation of the oxidative stress regulator OxyR41,42(Figure 1 panel F, Fig 7 panel I). Within central metabolism, HNQ upregulates the expression of enzyme subunits involved in pyruvate dehydrogenase and oxidative TCA cycle (Fig 7 panel J). The transcriptomics results encouraged us to investigate all the major cellular redox pools under EET conditions to elucidate the mechanism of HNQ- mediated EET.

[0107] NADH enables HNQ-mediated EET

[0108] As the redox perturbations induced by HNQ were broad, we set out to experimentally probe each of the major cellular redox pools (NADH, NADPH, quinones, glutathione, and thioredoxin) that can interact with the HNQ-mediated EET pathway. Since these redox pools are interconnected, perturbing any of these redox pools can alter the EET levels. However, we aimed to pinpoint the exact redox biomolecule that reduces HNQ through extensive deletions of redox-related genes. Thus, we established the following criteria to assess the outcome of perturbing the redox pool levels: (1) Gain in EET levels suggests that the deleted gene(s) compete with HNQ for electron fluxes, (2) Less than 50% loss in EET levels suggests that the deleted gene(s) do not directly reduce HNQ and is instead a pleiotropic effect, (3) More than 50% loss in EET levels indicates that the deleted gene(s) directly reduce HNQ. The fold change in the total charge deposited on the anode by the mutant and parent strains was utilized to assess the extent of EET gain or loss.

[0109] Our first line of inquiry explored the possibility of the inner membrane respiratory reductases reducing HNQ. The anaerobic respiratory reductases can reduce naphthoquinones in vitro43,44. To understand if these reductases can reduce HNQ in vivo, we generated the AnoxicNull strain lacking native anaerobic respiratory pathways (Figure 2 panel B, top). To confirm the phenotype, we show that AnoxicNull does not benefit anaerobic growth while respiring on the electron acceptors (Fig 8 panel A). Next, we evaluated AnoxicNull’ s ability to produce current with HNQ compared to BioD under resting conditions. AnoxicNull showed a gain in current density peak with 90.6 ± 3.8 mA / m2, whereas BioD generated a current density peak of 67.7 ± 5.4 mA / m2(Figure 2 panel B, bottom). AnoxicNull deposited a 1.7-fold higher charge on the anode than BioD (Fig 8 panel B). These data support thatrespiratory reductases do not reduce HNQ and instead suggest that the respiratory reductases compete with HNQ for electrons.

[0110] Naphthoquinones can be reduced by non-respiratory oxidoreductases that partner with menaquinone (Fig 8 panel C) and by the menaquinone pool in a non-enzymatic fashion45. To understand if the menaquinone pool significantly assists in reducing HNQ, we generated the MqNull strain by deleting the menaquinone biosynthetic pathway (Fig 8 panel D). We then tested MqNull’ s ability to produce current using HNQ alongside BioD under resting conditions. MqNull strain generated a higher peak current density of 91.3 ± 5.5 mA / m2than the BioD strain (74.6 ± 0.9 mA / m2), although it showed only a minor difference in the overall charge deposited on the anode. This data indicates that the menaquinone pool can be weakly compete for electron fluxes with the HNQ-mediated EET pathway.

[0111] We next sought to investigate if NADH dehydrogenases directly reduce HNQ. NADH dehydrogenase is a critical reductase enabling mediated EET in other exoelectrogens35,46’47. Moreover, naphthoquinones like HNQ are structurally similar to menaquinone and are known to be reduced by NADH dehydrogenases48 51. To elucidate if NADH dehydrogenases enable HNQ-mediated EET in E. coh. we generated the NdhNull strain that does not encode the two NADH dehydrogenases NDH-I and NDH-II in E. coli (Figure 2 panel C top, Fig 8 panels F-G). We then tested the HNQ-mediated EET in NdhNull and BioD under resting conditions. The NdhNull strain generated a 1.3-fold higher charge deposition on the anode than the BioD strain (Fig 8 panel H). The NdhNull showed a higher current density peak (127 ± 7.810 mA / m2) than BioD (81.4 ± 1.824 mA / m2) (Figure 2 panel C, bottom). These data indicate that NADH dehydrogenases NDH-I and NDH-II do not reduce HNQ. Importantly, AnoxicNull, MqNull, and NdhNull, designed to have limited electron fluxes in the inner membrane, show higher EET output, strongly suggesting that the electron donor(s) to HNQ are present in the cell cytoplasm.

[0112] To further show NADH’s involvement, without wishing to be bound by theory, deleting competing NADH sinks within fermentative pathways can boost the electron flux through the HNQ-mediated EET pathway52,53. We deleted the enzymes catalyzing the formation of lactate, ethanol, and 1,2-propanediol in AnoxicNull to generate the FermNull strain (Figure 2 panel D, top, and Fig 8 panel I). To test the EET performances of FermNull and AnoxicNull, we chose glucose over pyruvate as the electron donor since it yields more NADH. Under resting conditions, FermNull generated a 1.4-fold higher charge deposition on the anode than AnoxicNull (Fig 8 panel J). FermNull showed a higher current density peak (102.56 ± 3.38 mA / m2) than AnoxicNull (72.6 ± 0.6 mA / m2) (Figure 2 panel D). Interestingly,both these strains showed similar formate and acetate profiles at the end of the BES run (Fig 8 panel K). Together with prior data (Figure 2 panel C), these results strongly suggest that NADH supplies electrons to the HNQ-mediated EET.

[0113] NADPH, glutathione, and thioredoxin are not the key players in HNQ-mediated EET

[0114] Having established that NADH is important for reducing HNQ, we next examined the relationship of NADPH with HNQ-mediated EET. While NADPH is mandatory for cell growth (Figure 3 panel A), cells also use its reductive power to detoxify electrophiles like naphthoquinones54 56. To probe if NADPH enables HNQ-mediated EET, we engineered AnoxicNull to generate the NADPHnull strain that can only generate NADPH when gluconate is present (Figure 3 panel B). To show the gluconate auxotrophy, we show that NADPHNull achieved aerobic growth with gluconate supplementation but not in the 2xYT media (Figure 3 panel C). We then probed the EET performance of NADPHnull and AnoxicNull with pyruvate as the electron donor. NADPHnull with 52.8 ± 5.8 mA / m2showed a lower peak current density than AnoxicNull (72.4 ± 0.602 mA / m2) (Figure 3 panel D). However, the difference in the charge deposited on the anodes by NADPHnull (5.963 ± 0.638 coulombs) and AnoxicNull (6.143 ± 0.153 coulombs) was not significant (Fig 9 panel A). This data indicates that NADPH does not play a dominant role in reducing HNQ. We also investigated whether transhydrogenases can reduce HNQ via their promiscuous activity57. We deleted both the transhydrogenases in the AnoxicNull strain to generate a THnull strain (Fig 9 panel B) and saw no notable loss of EET, indicating that SthA does not reduce HNQ (Fig 9 panel C).

[0115] The glutathione58 63and the protein thiols64 67are known to reduce naphthoquinones enzymatically and form adducts68. We also found that HNQ stimulates the upregulation of protein thiols like aphC and trxC (Figure 1 panel F). Thus, without wishing to be bound by theory that glutathione and protein thiols like thioredoxins can reduce HNQ and enable EET (Figure 3 panel E). To elucidate the role of GSH in HNQ-mediated EET, we generated the GSHnull strain by deleting the first gene gshA of the GSH biosynthetic pathway (Fig 9 panel D). We then compared the EET performance of the AnoxicNull and the GSHnull strains under resting conditions. The GSHnull strain deposited a 0.8-fold lower charge on the anode than the AnoxicNull strain (Fig 9 panel E). GSHnull showed a smaller current density peak (68.4 ± 0.7 mA / m2) than AnoxicNull (99 ± 4.8 mA / m2) (Figure 3 panel F).With our defined rubrics, this data suggests that the GSH has a pleiotropic effect on HNQ-mediated EET. However, there is also a possibility that GSH abiotically reduces HNQ. To test if GSH reduces HNQ abiotically (Fig 9 panel F), we mixed GSH and HNQ in a BES and monitored the current.In this abiotic chronoamperometry experiment, adding 40 pM GSH to HNQ and DMSO gave a peak current density of 4.43 ± 1.08 mA / m2and 7.4 ± 1.5 mA / m2respectively (Figure 3 panel G). This data indicates that GSH is directly oxidized by the anode poised at 200 mV, regardless of the presence of HNQ. Although this result does not efficiently probe the abiotic reduction of HNQ by GSH, we conclude that the partial loss of EET in GSHnull is due to a pleiotropic effect.

[0116] Further, to elucidate the role of TRX in HNQ-mediated EET, we generated the TRXnull strain by deleting trxA, trxB, and trxC genes in the AnoxicNull strain (Fig 9 panels G and H). We then compared the EET performance of the TRXnull and the AnoxinNull strains under resting conditions. We saw no loss of EET in the TRXnull strain, indicating that the thioredoxin pool does not reduce HNQ (Fig 9 panel I).

[0117] Cytoplasmic quinone reductases facilitate HNQ-mediated EET under resting conditions

[0118] The experimental observations so far suggest that NADH assists in reducing HNQ, and other cellular electron carriers like quinones, NADPH, glutathione, and thioredoxin do not directly reduce HNQ. Without wishing to be bound by theory, NADH-utilizing quinone reductases can catalyze HNQ-mediated EET. E. coli hosts several such quinone reductases (QReds)69 84, and these QReds are essential players in xenobiotic metabolism85. To understand the role of quinone reductases in reducing HNQ, we generated the QRedNull strain by deleting several characterized and putative quinone reductases in the AnoxicNull strain (Figure 4 panel A). We then compared the EET performance of QRedNull and AnoxicNull with different electron donors under resting conditions. QRedNull showed a current density peak of 13.2 ± 1.9 mA / m2, about 7.5-fold lower than AnoxicNull’ s 98.3 ± 1.7 mA / m2, using 2 mM pyruvate as the electron donor. With an additional 1 mM glucose, QRedNull continued to maintain the current density level of 13.8 ± 0.3 mA / m2, whereas AnoxicNull generated 97.6 ± 2.9 mA / m2, indicating the substantial loss of EET extends to other electron donors (Figure 4 panel B). This result indicates that QReds reduce HNQ.

[0119] Knowing that QReds are central to HNQ-mediated EET, we investigated which specific QReds reduce HNQ. The DEG analysis of HNQ-treated cells previously showed upregulated azoR expression levels (Fig 1 panels E and F). Thus, we first deleted azoR in AnoxicNull to generate the AzoNull strain. AzoNull showed only a minor loss of EET compared to AnoxicNull (Fig 10 panel A)86. Next, we deleted the nitroreductases nfsA and nfsB in the AzoNull strain87, leading to a significant loss of EET compared to AnoxicNull (Fig 10 panel A). Literature reports have established that NfsA activity is NADPH-dependent88,whereas NfsB is compatible with both NADH and NADPH as electron donors89. Thus, based on the conditions under which we performed the experiments, we concluded that NfsB is likely the key QRed facilitating HNQ-mediated EET.

[0120] Next, to confirm the role of nf B. we reintroduced the nfsB gene under the Para. AM promoter on a low copy plasmid (Fig 10 panel B). The NfsB+strain generated peak current densities of 77.1 ± 3.0 mA / m2with 2 mM glycerol, 96.0 ± 2.4 mA / m2with 2 mM pyruvate, and 204.3 ± 5.5 mA / m2with 2 mM glucose, restoring the EET levels in the QRedNull-Ll strain (Figure 4 panel C). The NfsB± strain deposited 1.8, 2.7, and 6.6 fold higher charge than QRedNull-Ll with glycerol, pyruvate, and glucose as the electron donors, respectively (Fig 10 panel C). This data illustrates that the nitroreductase NfsB facilitates the HNQ-mediated EET by oxidizing NADH. In summary, HNQ-mediated EET occurs through cytoplasmic HNQ-reductases under resting conditions. Moreover, HNQ-mediated EET restores NAD+as the sole electron sink, the hallmark of a respiratory pathway (Figure 2 panel D).

[0121] Additional routes of HNQ-mediated EET exist under fermentative growth conditions

[0122] Having established the HNQ-mediated EET mechanism under resting conditions, we aimed to elucidate the behavior of E. coli HNQ-mediated EET during growth conditions. We postulated that, under anaerobic growth conditions, additional NAD+regenerating HNQ-reductases might be expressed. To identify any additional HNQ-reductases, we took the strategy to first eliminate fermentative pathways that serve as confounding factors by facilitating NAD+regeneration during anaerobic growth. Consequently, we generated the EsinkNull strain by deleting the fermentative pathways in QRedNull (Figure 4 panel E). We then tested the EET output of the AnoxicNull, the QRedNull, and the EsinkNull strains under growth conditions (More details in Methods). The AnoxicNull, the QRedNull, and the EsinkNull strains showed current density peaks of 682 ± 9.1 mA / m2, 400.6 ± 9.3 mA / m2, and 22 ± 1.4 mA / m2respectively (Figure 4 panel F). This data strongly indicates that EsinkNull no longer contains a pathway for HNQ-mediated EET. AnoxicNull and QRedNull oxidized all the provided glucose to achieve anaerobic growth and perform EET (Figure 4 panel G and Fig 10 panels F to I). Even though EsinkNull is metabolically active (Fig 10 panel I), it oxidized -10- fold less glucose than the other strains and did not show biomass growth (Figure 4 panel G and Fig 10 panel F). QRedNull, on the other hand, showed 0.6-fold and 14.1-fold charge deposition on the anode compared to AnoxicNull and EsinkNull, respectively (Fig 10 panel D). These observations suggest that the fermentative pathways enabling growth in QRedNull also enablecells to perform HNQ-mediated EET. Intriguingly, the difference in the values of charge deposited on the anode per unit cell of the QRedNull and the EsinkNull strains was insignificant (Figure 4 panel H). This analysis indicates that a baseline HNQ-mediated EET occurs under growth conditions independent of QReds.

[0123] To address the source of the EET output of QRedNull under growth conditions, without wishing to be bound by theory, the enzymes involved in formate and hydrogen metabolism can reduce HNQ. Hence, we deleted the three formate dehydrogenase operons in BioD to generate the FDHnull strain and tested its EET performance alongside BioD under resting conditions. BioD and FDHnull showed similar peak current density levels with pyruvate as the electron donor. However, BioD maintained a 6-fold higher steady-state current density than FDHnull (Fig 10 panels J and K).

[0124] Additionally, metabolite measurements of spent media showed that BioD had ~0.6-fold less formate than FDHnull (Fig 10 panel L). These results suggest that some EET output comes from the formate / hydrogen metabolism, which proportionally increases during the BES run with cell growth, as we see in QRedNull. Based on all the experimental evidence, we propose that selective electron transfer from the NADH pool to HNQ via quinone reductases facilitates HNQ-mediated anode respiration (Figure 4 panels H and I). At the same time, additional route(s) of EET is coupled with fermentative growth (Figure 4 panel I).

[0125] EET in growing cells is characterized by redox and stress expression perturbations

[0126] We next sought to determine the gene expression changes associated with EET in both growing and non-growing strains. Without wishing to be bound by theory, the gene expression changes between the constructed strains can further indicate the interconnectivity of anaerobic electron sink, redox stress, and biomass growth. The gene expression changes under EET can indicate whether the cells are experiencing significant stress, providing a lens into the physiological response to the EET -based metabolic state. Unfortunately, harvesting cells from BES is logistically challenging since cells localize in the anode during the chronoamperometry run. Thus, to efficiently collect cells to perform transcriptomics, we designed a representative colorimetric assay performed in the anaerobic chamber that investigates HNQ-mediated EET using amaranth, a redox dye, as an electron sink (Rationale and details in Methods).

[0127] Using the amaranth assay, we profiled the gene expression of AnoxicNull (which has both fermentative and nfsB-based routes to reduce HNQ), QRedNull (which has only fermentation), and EsinkNull (which has neither) in the presence of HNQ under growthconditions (Figure 5 panel A, Fig 11 panels A, B, C). A clear separation between gene expression across the strains was observed, with EsinkNull being farther separated from both AnoxicNull and QRedNull, consistent with the fact that EsinkNull is unable to grow (Figure 5 panel B). Transcriptional differences across strains are driven by a down-regulation of growth pathways (ppGpp, translation, thiamine, pyrimidine, arginine regulons) and up-regulation of stress (rpoS, soxS regulon) and energy starvation (Crp-1) pathways in the non-growing EsinkNull strain, consistent with a well-characterized growth / stress transcriptional tradeoff in E. coll. termed the ‘fear / greed’ tradeoff (Figure 5 panel B)90.

[0128] The introduction of HNQ had a pronounced and similar effect on AnoxicNull and QRedNull. In these strains, HNQ supplementation reduced rpoS activity and perturbed two primary transcription modules (Figure 5 panel B). The first perturbation was a downregulation of the hya operon, which is associated with redox response and microaerobic growth conditions42. The second perturbation was a pronounced activation of a set of poorly characterized transcriptional modules containing lipopolysaccharide (LPS) genes (Figure 5 panel C), gcvB-associated genes (Figure 5 panel D), membrane-associated genes, prophage genes, and a large number of y-genes (Fig 11 panels G, H, L). While the functional association to HNQ is unclear, these LPS and gcvB-associated transcriptional modules show a high degree of correlation across an E. coli gene expression data compendium and are activated under redox stress-related conditions42(Fig 11 panel K). Notably, the gcvB regulon was downregulated on pyruvate under non-growing conditions with HNQ, and LPS was not significantly changed, indicating that this activation is specific to growing conditions in the presence of HNQ (Figure 1 panel G).

[0129] While AnoxicNull and QRedNull generally exhibited similar responses to HNQ, there were certain additional strain-specific responses to HNQ. In the AnoxicNull strain, the NAD biosynthesis gene nadB was downregulated in response to HNQ, suggesting an interaction between HNQ and the NADH pool (Fig 11 panel D). In the QRedNull strain, there was an HNQ-induced activation of a ubiquinone knockout-associated regulon that contains the deleted nfsB (Figure 5 panel E) (Fig 11 panel E). In the ESinkNull strain, a set of genes associated with HNQ degradation is up-regulated potentially to mitigate HNQ-mediated stresses when EET cannot be carried out (Figure 5 panels F, G). Additional activation of SoxS was observed that can indicate additional redox stress when cells are not able to perform EET (Fig 11 panels F, I), and the acetate kinase-encoding gene ackA is upregulated (Fig 11 panel J), consistent with measured acetate production under this condition.

[0130] Model simulations indicate that HNQ-mediated EET facilitates anaerobic growth.

[0131] We then put forward the question of whether there is a rational basis for the use of HNQ reduction as a part of anaerobic growth. This question is well suited to be explored with genome-scale constraint-based metabolic modeling, which computes optimal metabolic states under defined conditions91. Flux balance analysis using the genome-scale metabolic model for E. coli, iML151592, revealed distinct metabolic adaptations to genetic modifications across strains. Under anaerobic conditions with a glucose uptake rate of 10 mmol / gDW / hr, BioD and Anoxi cNull demonstrated a wild-type-like growth rate of 0.15 hr’1, utilizing mixed acetate fermentation pathways. In stark contrast, with most NAD-regenerating enzymes removed and the transhydrogenase (pntAB) activity capped to WT levels, the FermNull and EsinkNull strains exhibited a severely reduced growth rate of 0.01 hr’1, utilizing the Entner- Doudoroff pathway for reduced NADH production and Malate Dehydrogenase for NAD regeneration. Strikingly, integrating a quinone reduction enzyme (QRed) into the FermNull strain restored growth to 0.19 hr’1, outperforming the WT strain (Figure 6 panel A). Simulations indicate the growth advantage of HNQ-mediated anaerobic metabolism is largely due to the ability of the cell to secrete acetate rather than lactate, which is normally the preferred route to regenerate NAD, thereby enabling greater ATP production through acetate kinase (Fig 12 panel A). Additionally, the inclusion of proteome allocation considerations through simulations with metabolism and macromolecular expression (ME) models suggested a reduction in both stress- related proteome and total proteome requirements in FermNull+nfsB compared to WT (Fig 12 panels B, C)93,94. This analysis indicates that HNQ-mediated EET can not just rescue anaerobic growth in fermentation-impaired strains but can even offer a more efficient pathway both in terms of stoichiometric and proteomic efficiency, assuming the HNQ can be efficiently recycled.

[0132] E. coli adapts to BES conditions to grow on the anode as the sole respiratory electron sink

[0133] Based on modeling results, we postulated that HNQ-mediated EET can fuel an anaerobic energy metabolism without fermentation-driven NAD+regeneration and restore growth in a fermentation -impaired E. coli strain. To elucidate if HNQ-mediated EET can conserve energy to enable growth in E. coli, we probed the FermNull strain that does not grow anaerobically but performs HNQ-mediated EET with QReds (Figure 6 panel B). We tested the EET output of the FermNull and the EsinkNull strains under growth conditions. Surprisingly, the EET levels in FermNull significantly increased only after 39 hours (Fig 12 panel D).FermNull showed a delayed peak current density of 1670 ± 124.9 mA / m2, whereas EsinkNull showed a consistent current density level lower than ~23 mA / m2(Fig 12 panel D). The net charge deposited by FermNull by the end of the BES run was 55 folds higher than EsinkNull (Fig 12 panel E). Thus, we labeled this strain as the adapted-FermNull strain. The FermNull showed a 6.6-fold higher ODeoo than EsinkNull (Fig 12 panel F). This data indicates that HNQ- mediated EET facilitates biomass growth.

[0134] We passaged and saved a glycerol stock of the adapted-FermNull (FNO) strain from the BES run. We further probed the FNO strain for EET and found that cells performed high levels of EET as soon as they were introduced in the BES. FNO showed peak current density levels of 2493.3 ± 111.5 mA / m2, whereas EsinkNull showed a maximum current density of 24.2 ± 1.5 mA / m2(Figure 6 panel C). The FNO strain showed about 4-fold higher growth higher than EsinkNull (Figure 6 panel D). This high EET performance, coupled with growth, showed a drop in the pH of the BES spent medium (Fig 12 panel G), consumed 4.3- fold higher glucose (Figure 6 panel E), and generated only 0.12-fold acetate as compared to the EsinkNull strain (Fig 12 panel H). We further passaged and saved a glycerol stock of FNO at the end of the BES run and labeled it as FN1. This is the first-ever demonstration of redox shuttle-mediated anaerobic growth on the anode as the sole electron sink.

[0135] We expression profiled FermNull, FNO, and FN1 to determine transcriptional changes associated with adaptation underlying improved growth under no EET condition. Two of the previously determined growth-associated fear transcription modules (rpoS, Crp) were higher in the unadapted strains, consistent with previous results where these transcription modules were higher expressed on the non-growing ESinkNull strain (Figure 6 panel G). The hya operon that we previously associated with HNQ supplementation was also found to be higher in the adapted strains, and an FHL-associated transcription module that was deleted in other strains was activated. In terms of new transcriptional modules, there were three modules most perturbed by the adaptation, specifically ArcA, Ubiquinone-KO, and Fur, indicating a possible derepression of the TCA cycle to generate more NADH. Notably, the HNQ-associated transcriptional module (LPS+gcvB) identified earlier was not perturbed by the growth adaptation. Looking at individual genes, tnaAB, amtB, sdhA, and pckA are up-regulated in the adapted strain, while pflB is down-regulated (Figure 6 panel H). These perturbations to amino acid and anaplerotic metabolism genes indicate a metabolic reprogramming towards adapting to utilizing the provided peptide amino acids to sustain growth. Interestingly, adaptation did not result in the increased acetate production predicted by the model -computed optimal metabolic state (Fig 12 panel K); instead, adapted-FermNull showed higher substrate-level phosphorylation activity in glycolysis (Fig 12 panel N). Finally, we observed a perturbation of a NadR-associated NAD biosynthesis component, consisting of nadAB and pnuC, broadly across strains generated in this project (Fig 12 panel O). The FermNull strain exhibited strong activation of this regulon on glucose growth, consistent with free NAD levels being altered from the deletion of many NAD-dependent enzymes, and this perturbation was attenuated following adaptation (Fig 12 panel P). These results suggest that E. coli can adapt to support EET-driven growth; however, the adaptations observed in this experiment appeared to support growth through peptide incorporation and stress down-regulation rather than to induce the model-predicted metabolic shift towards greater acetate secretion or to enhance EET. Thus, it is possible that further optimization of the strain can be done by continued laboratory evolution.

[0136] Next, we proposed that the acquired mutation in ompC enhanced HNQ accessibility in FermNull cells. To test this, we introduced the D126V mutation in ompC within the FermNull background, generating the OmpC* strain (see Table 1). Interestingly, OmpC* exhibited an EET phenotype comparable to FN1 under BES growth conditions (Figure 15 A). To further evaluate the role of OmpC in HNQ transport, we deleted ompC in FermNull, creating the OmpCnull strain. Notably, under non-growth BES conditions, OmpCnull displayed a current density peak similar to FermNull (-100.75 ± 16 mA / m2), while OmpC* demonstrated a significantly enhanced EET output (-399.5 ± 19 mA / m2; Figure 15B). Moreover, OmpC* deposited -2.5-fold more charge on the anode than FermNull and OmpCnull (Figure 115C). This data indicates that OmpC is not the primary HNQ transport route in FermNull, but the D126V mutation in OmpC substantially improves the EET phenotype.

[0137] Finally, we investigated whether E. coli could utilize an anode as the sole electron sink for biomass growth. We deleted the co-localized hyc-hyp operons in the OmpC* background, thereby abolishing hydrogenase biosynthesis and generating the AnodeSole strain that is unable to utilize reduced carbon or hydrogen as electron sinks for biomass growth (Figure 16A-B). Intriguingly, under BES growth conditions with mannitol (see Rationale II in STAR methods) as the electron donor, AnodeSole exhibited EET output and biomass growth comparable to OmpC* (Figure 16C-E). This experiment is the first to confirm that A. coli can respire and grow using an anode as its sole respiratory electron sink.

[0138] Discussion

[0139] Herein we describe a new energy metabolism in E. coli in which extracellular shuttling of HNQ enables respiration and growth on an anode. Using a reductionist approach, we found that cytoplasmic quinone reductase, NfsB, is the key player in enabling HNQ-mediated EETunder resting conditions. NfsB oxidizes NADH generated by catabolism and reduces HNQ. Model simulations indicate that an HNQ-mediated metabolism can support anaerobic growth in A. coli.

[0140] This new energy metabolism even offers advantages over canonical mixed acid fermentation due to improved bioenergetics. We further experimentally validated that HNQ- mediated EET can use an anode as an extracellular electron sink to drive glucose consumption and growth under anaerobic conditions. In this experiment, cells were found to adapt to the BES conditions and generate A / m2levels of EET output, two orders of magnitude higher than previously reported in E. coli. Genome sequencing and transcriptome analysis suggested these adaptations were enabled by perturbed amino acid metabolism and activation of anaplerosis- driven TCA cycle. In summary, our findings suggest that E. coli is equipped with a latent mediated EET pathway that facilitates energy conservation.

[0141] A blueprint for elucidating mechanisms and bioenergetic implications of mediated EETs

[0142] Prior efforts to understand the intracellular interaction between redox shuttles and cell metabolism have relied on transposon insertion mutant libraries and in vitro biochemical methods34,35. These approaches have proven time-consuming and often overlooked crucial physiological aspects, such as redox shuttle transport across the membrane to perform EET, thus limiting the utility of the study. We have adopted another approach to address these limitations. The electron flux on the anode through the EET pathway can be influenced by the electron flux directed to cell growth, a competing electron sink95. Thus, we first investigated whole-cell EET performance in BES in the absence of a nitrogen source, wherein cells do not divide. Under this condition, we realized that the electron flux through the EET pathway is intricately linked with the carbon catabolism and the redox state of the cellular electron carriers: NADH, NADPH, quinones, glutathione, and thioredoxins. Thus, we systematically eliminated gatekeeping enzymes that influence the electron fluxes through these electron carriers and pinpointed the key electron carrier responsible for reducing the redox shuttle. We then targeted the electron carrier-dependent oxidoreductases to elucidate the mechanism of the mediated EET pathway. Next, we realized that functional fermentative pathways can complicate the assessment of EET’ s influence on the cell bioenergetics. Therefore, to evaluate the implications of EET on cellular redox homeostasis and energy conservation under growth conditions, we investigated EET in fermentative-deficient strains. We also augmented the above experimental strategies with the transcriptomic characterizations of cellular objectives under EET conditions. We used both an unsupervised learning-based approach to identify perturbedtranscriptional regulatory modules as well as differential gene expression to pinpoint specific genes responsive to mediated EET metabolism. Our approach offers a universal methodological blueprint for exploring the mechanisms of mediated EET pathways in target microbes.

[0143] A minimal module coupling cell metabolism and anode respiration for growth

[0144] Through the workflow we established, we elucidated a minimal EET pathway exhibiting robust electrical output and made progress toward a comprehensive understanding of its energy metabolism. Previous engineering efforts assumed that E. coli can seamlessly integrate heterologously expressed EET pathways with its metabolism18,22. However, these engineered EET pathways did not yield the degree of anode respiration required to drive catabolism, highlighting the necessity for design rules to better integrate with cell metabolism. Through HNQ-mediated EET, we establish systems-level design rules for electrically wiring cell metabolism with an extracellular electrode. The HNQ-mediated EET pathway’s simplicity appears portable and generalizable when interfaced with metabolically active living systems. A portable single-enzyme EET pathway promises efficient bioelectrocatalysis, overcoming the limitations of current synthetic EET pathways for carbon remediation. Additionally, it lays the foundation for designing simplified electrosynthesis pathways96and building synthetic minimal cells97.

[0145] The frameworks of HNQ-mediated anode respiration and energy conservation are unprecedented

[0146] The HNQ-mediated pathway introduces a new topology of anode-assisted energy metabolism. While Shewanella species uses the direct EET pathways that interface with the inner membrane and phenazines in Pseudomonas aeruginosa interface with enzymes in both the cytoplasm and the inner membrane, the HNQ-mediated EET pathway directly interfaces with the cytoplasmic NADH pool. This topology has critical implications for the associated energy metabolism. Firstly, since HNQ does not oxidize the quinone pool, catabolic reactions using inner membrane dehydrogenases that reduce the quinone pool will eventually cease. Secondly, since HNQ-mediated EET does not utilize inner membrane reductases, it cannot benefit from PMF-generating NADH dehydrogenase for cell growth. Nonetheless, when provided with glucose and peptides, HNQ-mediated EET drives catabolism and generates ATP to achieve biomass growth. ATP generated via substrate-level phosphorylation is inherent to microbial catabolism under anaerobic conditions. Without wishing to be bound by theory, the movement of reduced HNQ across the inner membrane generates PMF.

[0147] The framework of energy metabolism driven by HNQ-mediated EET is distinct from classical anaerobic respiratory pathways and mixed acid fermentation. As supported by model simulations, any route to externally oxidize NADH frees the cell from the forced use of fermentation pathways to regenerate NAD, enabling greater metabolic flexibility in anaerobic environments. Quinone reductases in association with soluble naphthoquinones can provide an economical mode of heterotrophy under nutrient-limited anaerobic conditions98. Theoretically, as long as oxidized HNQ can be sufficiently regenerated, there are several motivating factors for A. coli to engage in this metabolic activity, even when fermentation alone is possible. First, our constraint-based modeling suggests that EET is stoichiometrically and proteometrically more favorable than mixed acid fermentation. Second, there is less risk of growth-inhibiting fermentative metabolite accumulation. Third, the reliance on HNQ oxidation by external factors can create the basis for the community exchange of valuable metabolic resources, as predicted in other community models, leading to even more favorable metabolic scenari •os 112 ’ 113.

[0148] Physiologically relevant HNQ-mediated EET and biosynthesis of HNQ

[0149] Our findings raise the important question of which potential terminal electron acceptors naphthoquinones can reduce to benefit the anaerobic survival of microbes. Interestingly, reduced HNQ can abiotically transfer electrons to insoluble Fe(III)99,100, tellurite, selenite101,102, and azo dyes103,104. Without wishing to be bound by theory, E. coli can use HNQ to respire on these diverse electron sinks for redox homeostasis, allowing energy conservation in the gut and environmental niches. Similar anaerobic sustenance mechanisms have been described in Shewanella species, which use ACNQ to reduce extracellular humic acids105-107; in lactic acid bacteria, which use ACNQ and DHNA to reduce extracellular Fe(III)46,108; and in P. aeruginosa, which uses diverse phenazines to reduce extracellular Fe(III). Additionally, E. coli might utilize the endogenous quinones as a respiratory electron sink, as previously shown with melanin in Shewanella algae BrY109,110.

[0150] HNQ biosynthesis intuitively seems to originate from the DHNA biosynthetic pathway, which is a precursor for menaquinone biosynthesis. Although several reports show that E. coli evolves to secrete HNQ for EET, we have yet to understand the physiological conditions necessary for native HNQ biosynthesis in E. coli. From a broader perspective, the biosynthesis of HNQ in E. coli can give more critical insights into the physiological relevance of this new energy metabolism. However, the quantitative analysis of redox shuttle biosynthesis shows that it poses only a minor metabolic cost111. Considering an alternative perspective, E. coli doesn’t need to synthesize HNQ for physiologically relevant electrogenicenergy metabolism. As long as E. coli can obtain HNQ exogenously or from its ecological niche, it can still perform EET. Some exoelectrogens, like Lactiplantibacillus plantarum, utilize this communal resource strategy to perform EET without synthesizing the naphthoquinones they employ112,113. Although HNQ levels in ecological niches are unknown, it is a well-characterized secondary metabolite produced by various plants, facilitating interactions between plants and microbes114,115. In summary, E. coli can use the HNQ-mediated energy metabolism in ecological niches with the communal presence of exogenous HNQ.

[0151] Table 1: Non-Limiting Example of Certain Genetically Modified Strains

[0152] Table 2: Exemplary, Non-Limitig Characteristics of Certain Genetically ModifiedStrains

[0153] Table 3: Current Output of Certain Genetically Modified Strains at Resting

[0154] Experimental model and subject details

[0155] Escherichia coli BioDesignER

[0156] To comprehensively probe the mechanism of the HNQ-mediated EET in E. coli, we leveraged the BioDesignER (BioD) strain to make fast-paced genome editing37. BioD encodes a refactored A- Red recombination machinery in its genome, enabling highly efficient recombineering with dsDNA and ssDNA. The k-Red machinery is encoded under an anhydrotetracycline (aTc) inducible TetR-Pteto2 transcriptional control.

[0157] Cell culturing conditions

[0158] E. coli strains subjected to genome editing were cultured in LB. The E. coli strains investigated for EET performances were cultured, washed, and tested in the M9P buffered conditions. The M9P buffer (pH 6.5) comprises 42 mM Na2HPO4.7H2O, 24 mM KH2PO4, and 9 mM NaCl. A pH of 6.5 was chosen since E. coli showed higher HNQ-mediated EET performance in the BES (Figure 16 panel A). The cell cultures were grown with aerobic shaking at 250 rpm at either 37°C or 30°C. All the cell culture media were sterilized using a vacuum filtration system.

[0159] Preparation of cell biomass to test for HNQ-mediated EET

[0160] The cell biomass used to investigate EET performance was cultured in two steps. In step one, the pre-cultures inoculated from glycerol stocks were grown for 10 hours at 37°C in M9P-LB media containing 10 g / L tryptone, 5 g / L yeast extract, and 1 mM MgSCh. In step two, these pre-cultures were then diluted by a factor of 100 and transferred to M9P-2xYT media containing 16 g / L tryptone and 10 g / L yeast extract, where they were grown for 12 hours at 30°C to generate the cell biomass. These cell biomass-generating cultures were supplemented with 1 mM MgSO4, lx trace metal mix (T1001) purchased from Teknova, 20 mM glycerol, and additional carbon sources catered to the experimental design. The additional carbon source enabled cells to adapt to the carbon source (electron donor) tested under the EET conditions. The cell biomass was prepared by serial washing in a chilled M9P buffer for two rounds (centrifuged at 4000 rpm at 4°C for 15 minutes, and the supernatant was discarded). Each round of washing volume equals twice the cell culture volume. The washed cells were added to a final ODeoo of either 0.1 or 0.25 to test their EET performances under anaerobic conditions.

[0161] Rationale - Choices of nitrogen sources under HNQ-mediated EET conditions

[0162] To simplify elucidating the mechanism of HNQ-mediated EET, the E. coli strains were first probed for EET performance under resting conditions. The resting conditions media comprised only the M9P buffer and 2 mM carbon source. This approach was carefully designed to avoid electron flux originating from multiple carbon / nitrogen assimilation pathways and streamline the efforts in probing redox pools elicited by the carbon source. However, once the predominant mechanism of EET under resting conditions was unveiled (Figure 4A-C), acomplete-growth medium was used to investigate the additional routes of HNQ-mediated EET. The growth media buffered with M9P (pH 6.5) contained 5 g / L NZ-Amine, 1 mM MgSCh, 0.002% v / v Antifoam 204 (Sigma, A8311), and 20 mM glucose.

[0163] Rationale - Choices of carbon sources (electron donors) under HNQ-mediated EET conditions

[0164] Glycerol was the preferred choice of electron donor due to its high degree of reduction116, which encourages cells to use EET pathways. Our preliminary experiments found that when cells are pre-cultured in M9P-LB with 20 mM glycerol, they used both GlpD and GldA routes of glycerol oxidation under EET testing conditions (Fig 7 panel D, Figure 1 panel C). However, when cells are precultured in M9P-LB without glycerol, our data indicates that the cells use only the GlpD route of glycerol oxidation under EET testing conditions (Figure 13 panel B). Under these conditions, cells can oxidize only a fraction of the provided glycerol, indicating that HNQ-mediated EET does not oxidize the reduced ubiquinone pool (Figure 13 panel C). To benchmark the ideal conditions for investigating the EET mechanism using chronoamperometry (CA) under resting state, we prioritized two conditions: (1) Cells can completely oxidize the limited amount of 2 mM electron donor in about a day, where we expect a bell curve shaped current density vs. time, and (2) The electron flux can originate from the cytoplasm, preferably via NADH pool, since the NADH pool is connected to the rest of the redox biomolecules in the cell. This strategy was expected to reduce the complexity of probing the mechanism of HNQ-mediated EET. While glucose did not fulfill the former condition, pyruvate as the electron donor satisfied both conditions. Thus, unless otherwise mentioned, all the experiments to investigate the mechanism of HNQ-mediated EET under resting conditions were done with pyruvate. However, glucose was used as the electron / carbon source in the experiments with actively growing cells.

[0165] Rationale - Concentration of HNQ used to stimulate EET in A. coli

[0166] Throughout this work, 20 pM HNQ was used to stimulate EET in E. coli. This concentration was chosen since it gave maximum EET output and did not affect the cell viability under resting / growth conditions (Figure 13 panels D-F). HNQ (Sigma H46805) was dissolved in 100% DMSO. A primary stock of 20 mM HNQ was prepared freshly for every experiment. Thus, with 20 pM HNQ in the EET probing experiments, the unavoidable 14.08 mM of DMSO was introduced in the HNQ-mediated EET experiments. The additional DMSO provides a potent respiratory electron acceptor under anaerobic conditions and can compete with HNQ-mediated EET. Moreover, DMSO’s presence can alter the cellular carbon and electron fluxes, influencing our experimental approach to understanding the mechanism ofHNQ-mediated energy metabolism. However, the most thoroughly investigated strains in this work: Anoxi cNull, FermNull, QRedNull, and EsinkNull have all the anaerobic respiratory pathways deleted, including DMSO.

[0167] Rationale - Transcriptomics analysis of cells harvested from amaranth assay over BES

[0168] Two aspects can influence the transcriptomics of cells performing EET: (1) The EET pathway and (2) the Redox potential of the electron sink. In the BES system, the extraction of cells performing EET is challenging for the following reasons: (1) In all our experiments, we have injected cells in the BES to a final ODeoo of 0.1, which is about 5-10 fold lesser than the conventional practice. The small amount of injected cells is initially planktonic, and as the CA experiment proceeds, cells localize in the anode made up of graphite felt (mesh). The time required to isolate an anode from a BES and harvest cells can be sufficient to expose cells to aerobic conditions. Additionally, it can introduce variance amongst replicates. (2) If we were to isolate EET-performing planktonic cells from the BES early, it can not accurately represent the underlying biological question. Since planktonic cells performing mediated EET in a 110 mL BES can exhibit heterogeneity in terms of rate of EET. Thus, to interrogate transcriptomics- associated questions, we designed a proxy EET assay. This assay was conducted in the anaerobic chamber, where instead of an anode, we used a water-soluble and color-changing redox dye called amaranth as the electron sink (Figure 13 panel G). However, the redox potential of amaranth is not the same as the anode poised at 200 mV vs. Ag / AgCl. Thus, amaranth will influence the transcriptome of the investigated strains differently. The amaranth assay is also done in an anaerobic chamber, where cells are exposed to a different gas mixture (96% N2, 2% CO2, 2% H2) compared to BES (100% N2). Considering these factors, while the results of the regulon-level transcriptomics analysis from the amaranth assay cannot perfectly align with those from the BES, they remain representative of the BES experimental conditions.

[0169] Methods Details

[0170] Gene deletion in BioD

[0171] A two-step selection-counterselection approach was utilized to perform clean gene deletions in the BioDesignER (BioD) strain. In the first recombineering step, a GKX selection cassette replaces nearly all of the CDS of the gene to be deleted. In the second step, the GKX cassette is excised by an ssDNA recombination event to achieve the targeted gene deletion (Figure 14 panel A). All the strains generated in this work thrive under aerobic conditions.

[0172] All the PCR amplifications of linear dsDNA were done with either the 2x Phanta Max master mix (Dye Plus, Vazyme, P525) or the 2x Phanta Flash Master Mix (Dye Plus, Vazyme,P510). The gel extractions of dsDNA were performed using the FastPure Gel DNA Extraction Kit (Vazyme).

[0173] dsDNA and ssDNA for homologous recombination

[0174] The selection cassette (dsDNA) GKX cassette comprises a constitutive expression of an sfGfp-KanR fusion protein alongside a cumate inducible cell lysis gene (phiX174). The GKX cassette is integrated into the genome of E. coli strain RE88037. The GKX selection donors were synthesized by PCR amplifying the GKX cassette from RE880’s genome with primers containing overhangs of 50 bp homology targeting the gene of interest37. The suitable PCR amplified selection donors were gel extracted for high purity. On the other hand, the countersei ection donor templates of 100 bp ssDNS oligonucleotides containing the tandemly stitched 50 bp homologies with the targeted gene of interest were obtained from Sigma Aldrich. The ssDNA oligonucleotides were designed to have 5’ phosphorothioate base modifications. In the cases of poor ssDNA recombineering efficiency, counterselections were successfully repeated with dsDNA donors, with at least 500 bp homologies obtained from Twist Biosciences.

[0175] Competent cell preparation and recombineering

[0176] BioD and its derivative strains were grown in LB Miller broth overnight. The saturated cultures were diluted 100-fold in LB Miller broth supplemented with aTc (100 ng / pL) to induce the A-Red recombination machinery. Further, the cells were harvested at 0.4-0.6 ODeoo and prepared for electroporation by washing the cells once with the same culture volume of chilled water and then twice with chilled 10% glycerol. The cells were finally resuspended in 1000'1fold of the initial culture volume with 10% glycerol. The electroporation-guided transformation was done with 50 pL of cells and 400 ng of selection dsDNA donor. The transformations were recovered for three hours and plated on kanamycin LB Miller agar plates. The transformed colonies were confirmed with sfGFP fluorescence and colony PCR. The confirmed colonies were passaged for the next step of clean deletion with kanamycin supplementation in the growth media. The counterselection was performed as described above with either ssDNA or dsDNA, and the transformation recovery was selected on 100 pM cumate. Successful counterselections were confirmed with the loss of sfGFP fluorescence.

[0177] Confirmation of gene deletion

[0178] The knockout strains were confirmed with colony PCRs and functional assays. The key strains were genome sequenced and confirmed. The phenotypic confirmation of respiratory and fermentation mutants was performed with anaerobic growth assays. The cell cultures ondifferent anaerobic electron sinks (Nitrate, DMSO, TMAO, and fumarate) were performed in 96-deep well plates in an anaerobic chamber (Whitley A45 anaerobic workstation). The ODeoo measurements were done using a microplate reader in the anaerobic chamber (Byonoy, Absorbance 96).

[0179] DNA-seq and mutation analysis pipeline

[0180] Breseq was run two ways to predict and verify possible genetic mutations in the adapted FermNull strains compared to the FermNull strain117. In the first way, both DNAseq reads from FermNull and adapted FermNull strains were compared to the E. coli MG1655 reference genome. Mutation predictions specific to adapted FermNull were then considered as genetic adaptations. In the second method a genome was assembled for the FermNull strain based on its DNAseq reads using the spades assembler and prokka to get an annotated genbank file118,119. Reads from the adapted FermNull strain were then compared to the assembled FermNull genome. Both methods showed consistent predicted mutations in the adapted FermNull strains.

[0181] Synthetic control on the expression of native genes

[0182] In the GldA+ strain, the 5’ region of the genomic gldA CDS was replaced with synthetic transcriptional genetic parts for constitutive expression. A strong RBS was designed using the RBS calculator tool on the De Novo platform120. A strong and constitutive PSHO39 promoter was chosen from the non-repetitive synthetic promoter library121. A double terminator DT42 was also included upstream of the promoter PSHO39 (Fig 7 panel E)122. The promoter swap was implemented in the BioD derivative strain using the above-described protocol.

[0183] The synthetic expression of HNQ-reductase nfsB was encoded under an arabinose inducible promoter (Para AM)123(Sup Fig 4B). The transcriptional activator (AraC) of Para.AM is constitutively expressed in the stains derived from BioD. The nfsB expressing cassette was ordered from Twist Biosciences and integrated into a low-copy part vector using a Golden Gate assembly. The part vector comprises a chloramphenicol -resistant gene and pSClOl origin.

[0184] Testing HNQ-mediated EET using bioelectrochemical systems (BES)

[0185] A BES reactor comprises two chambers and a three-electrode configuration (Methods Figure 2B). The two chambers, cathodic and anodic, are filled with 110 mL of M9P buffer / media and separated by a cation exchange membrane (CMI-7000, Membranes International). The cathodic chamber consists of a counter electrode with a 0.5 mm radius of titanium wire (Alfa Aesar). The anodic chamber consists of the working electrode made of titanium wire, which is extended onto a graphite felt with a geometric surface area of 16 cm2(Alfa Aesar), serving as an anode compatible with E. coli cells. The anodic chamber is maintained in anaerobic conditions by continuous sparging of N2 gas and is also held under constant stirring conditions of 220 rpm by magnetic stir bars (IKA RO 10 Magnetic Stirrers). The anodic chamber is also interfaced with an Ag / AgCl reference electrode (CHI 11, CH Instruments)) saturated with 3 M KC1. Six such water-jacketed BES reactors are connected in series to a temperature-controlled water bath (Adams & Chittenden Scientific Glass; ECO E 4S, Lauda Brinkmann). The temperature of the BES reactors was maintained at 30°C throughout the experiment. Each BES reactor was connected to a potentiostat (Bio-Logic Science Instruments, TN, USA, model - VSP300) to perform electrochemical measurements.

[0186] All the electrochemical measurements were done with the anode poised at 200 mV vs. Ag / AgCl at the potential resolution of 100 pV. In the cyclic chronoamperometry (CA) measurements, the current levels were recorded every 36 seconds. In the cyclic voltammetry (CV) measurements, the scan rate of 1 mV / s was used over the potential window of -500 mv to 0 mV. The E. coli strains under investigation were added roughly 12-14 hours of nitrogen gas spurging in the BESs. This timeframe ensures the complete elimination of oxygen from the BES setup and stabilizes current baseline levels. The EET enabling components, consisting of cells, HNQ, and the electron donor, were added in this order. This sequence was chosen to facilitate the oxidation of reducing equivalents accumulated from the prior aerobic growth. This approach allowed us to evaluate the EET output in response to the electron donor oxidized in the anaerobic BES conditions.

[0187] The EET output from CA experiments is interpreted in three ways: (1) Peak current density (mA / m2; Ampere, meter) - The highest current value corresponding to a state where the highest fraction of the provided HNQ is reduced (2) Stead state current density (mA / m2) - The current value at the end of the CA run (3) Charge (Q; Coulombs) - The total area under CA plot (current vs. time) corresponding to the total charge deposited on the anode by the end of the CA run. All these values are generated in the software (Bio-Logic, EC -lab) that operates the potentiostat.

[0188] As the CA experiments progress, the cells localize in the graphite felt. Thus, to measure the final ODeoo, the BES is disconnected from the potentiostat and the temperature- controlled water bath system, followed by vigorous shaking to dislodge the cells. The cell ODeoo was then measured on a spectrophotometer (Agilent, Cary Series UV-Vis).

[0189] Testing HNQ-mediated EET using a colorimetric assay

[0190] The colorimetric assay utilized amaranth, an azo dye (Sigma Al 016), as the electron sink. This colorimetric assay was initially validated with an assay media containing 125 pMamaranth in the M9P complete-growth medium with 20 mM pyruvate. In this assay, cells were inoculated at ODeoo of 0.25 and incubated in a shaker (IKA, VXR basic Vibrax) in the anaerobic chamber for 12 hours. However, the cells exposed to amaranth at 125 pM for a long time were found to have RNA degradation, especially in the E. coli strains (FermNull and EsinkNull) with impaired anaerobic growth. Thus, the assay was redesigned to use 12.5 pM amaranth dye with shaking under anaerobic conditions for 2 hours. This redesigned assay was used to collect cells for transcriptomics analysis. The degree of amaranth reduction was measured by probing absorbance at 518 nm (Abssis) of the spent assay media using a spectrophotometer (Agilent, Cary Series UV-Vis). The reported percentage amaranth reduction was calculated as follows -

[0191] Amaranth reduction (%) = [(No cell control)Abs5i8 - (E. coli strain) Abs5 is] / (No cell control)Abs5i8

[0192] Note: To validate if the amaranth reduction assay85,87mimics the EET phenotype shown by the strains in BES experiments, we probed the AnoxicNull, QRedNull, and EsinkNull strains (Figure 5 panel A). The amaranth reduction was measured by quantifying the decreased absorbance of the spent media at 518 nm. The AnoxicNull, QRedNull, and EsinkNull show amaranth reduction of 59.7%, 30.7%, and 15.9%, respectively (Figure 13 panel H). This data indicates EsinkNull has the lowest HNQ reduction ability and is consistent with the EET assessments in BES. In this assay, all the tested strains consumed similar pyruvate levels (Figure 13 panel I); however, EsinkNull retained ~30-fold higher formate concentrations (Figure 13 panel J). The retention of formate is attributed to the formate dehydrogenase deletions in EsinkNull. EsinkNull also generated ~1.7-fold higher acetate levels (Figure 1 panel K), suggesting that AnoxicNull and QRedNull have relatively lower carbon flux through ATP- generating acetyl-CoA / acetate node. These data indicate that the amaranth can serve as a proxy for the EET tests in BES, and the cells can be effortlessly harvested for transcriptomics analysis.

[0193] Measuring metabolite concentrations using HPLC

[0194] Quantifications of glycerol, glucose, and other organic acids were done using HPLC (Shimadzu, Kyoto, Japan). An isocratic mobile phase of 30 mM H2SO4 was operated on the Aminex Organic Acid Analysis column (Bio-Rad, HPX-87H 300 x 7.8 mm) with a flow rate of 0.3 L min ' . The Aminex column was held at 60°C, and the compounds of interest were separated with discernible peaks. Glycerol and glucose were measured using a refractive indexdetector (Shimadzu, RID-20A, 120V), whereas the organic acids were measured using a UV detector (Shimadzu, SPD-M20A). No gaseous products were measured in this study.

[0195] Modeling of HNQ-mediated EET

[0196] We performed Flux Balance Analysis using the latest genome-scale metabolic model of A. coli, iML1515124. We generated three strains for analysis: 1) wild type, which is identical to the base iML1515 model; 2) EsinkNull, which has the complete set of redox reaction knockouts described in this manuscript (see Strain list); and 3) EsinkNull + nfsB, which has the EsinkNull reaction set knocked out but the addition of the reactions for naphthoquinone reduction by NADH (NADH + Oxidized HNQ NAD + Reduced HNQ), and regeneration by the anode (Reduced HNQ Oxidized HNQ). FBA was performed in a standard manner91using the COBRA Toolbox125in Matlab with the Gurobi solver version 9.1.1. We utilized growth as the primary objective for simulations, with glucose uptake constrained to 10 mmol / gDW / hr, oxygen constrained to 0 mmol / gDW / hr (anaerobic), with a secondary quadratic objective to minimize the Euclidean norm of the flux state while constrained to the maximal growth rate. Additionally, the NAD(P) transhydrogenase flux was capped to predicted wildtype levels of 2 mmol / gDW / hr in all simulations to prevent the use of futile redox cycles to oxidize NADH freely. Genes were knocked out respective of logical relationships in reaction gene-to-protein relationships (GPRs), such that a reaction flux was constrained to 0 mmol / gDW / hr if and only if all of its possible catalyzing protein complexes have at least one essential gene knocked out. Simulations were performed in Matlab using the COBRA toolbox125.

[0197] We then analyzed the proteome allocation using the iJL1678b Metabolism and Macromolecular Expression (ME) model94. We generated three strains for analysis: 1) wild type, identical to the base iJL1678b model; 2) FermNull, as described above; and 3) FermNull + nfsB, as described above. We estimated the NfsB kefrvalue to be 57.4 in the model using the transcriptome-constrained wild-type flux state and experimental absolute proteomics from this study126. All strains were grown anaerobically, with growth being the primary objective for simulations. The Wild Type strain was simulated under two conditions: 1) Fermentation, which was grown anaerobically with glucose uptake constrained to 10 mmol / gDW / hr, and 2) Nitrate Respiration, which was grown similarly but additionally with nitrate uptake constrained to 30 mmol / gDW / hr. The FermNull and FermNull + nfsB strains were constrained to the same growth rates calculated from the iML1515 simulations. Proteome allocation was analyzed using the simulation results for ETC, Glycolysis, and Fermentation proteins. Proteome allocation related to stress iModulons (SoxS, OxyR, ppGpp, CpxR, and RpoS) were analyzedfor the FermNull strains by calculating the proteome allocated to the genes enriched in each iModulons. Simulations were performed using the COBRAme Python software package94.

[0198] RNA-seq and transcriptomics analysis pipeline

[0199] 6 ml of cell culture (ODeoo > 0.25) was immediately added to two volumes of RNA protection solution (RNAprotect Bacteria Reagent, Qiagen 76506), vortexed for 5 s, incubated at room temperature for 5 min, and immediately centrifuged for 15 min at 4000 rpm. The supernatant was decanted, and the cell pellet was stored at the -80 °C. Cell pellets were thawed, and total RNA was prepared using a PuroMAG™ Total RNA Purification Kit (Luna Nanotech Inc., catalog number NKM051-96), following the vendor’s manual, including a 15-minute DNase treatment at room temperature. RNA was quantified using a Nanodrop, and quality was assessed by running on an Agilent TapeStation. The rRNA was removed using the RiboRid procedure with oligonucleotide probes specific to E. coli ribosomal RNA. Following the manufacturer’s protocol, a KAPA RNA HyperPrep kit (catalog number 08098107702) was used to create sequencing libraries with an average insert length of around D300 bp. Libraries were run on an AVITI instrument (Element Biosciences).

[0200] RNAseq abundance computation, quality checks, processing and Independent component analysis (ICA) for calculating iModulons were done using the modulome workflow127. Differential gene expression was done using Deseq enabled with the pydeseq library in python. An adjusted pvalue of 0.05 and log2FoldChange of 1.5 was taken as the threshold for determining significant genes. For figure 5, due to a new ICA capturing strain level variance as signals, we inferred the iModulon activities using the existing E. coli structure by deleting all the genetic knock outs across the strains. This resulted in capturing within strain level differences across HNQ and DMSO better while preserving variance across strains. For figure 6, we ran ICA on all data up to figure 5. We then used this newly obtained iModulon structure to infer activities for data in figure 6. This was done to see what the newly obtained iModulons with figure 5 data can tell us about unseen / figure 6 data and how iModulon activities changed across the datasets.

[0201] Note: In some experiments, we compared the transcriptome of the strains under no- EET conditions. We can investigate the transcriptome makeup of the cells as they were introduced into the BES under EET conditions. Chronoamperometry experiments indicate that the EET machinery reducing HNQ in the respective strains (BioD, FermNull, and adapted FermNulls) was already present at the point of introducing these strains into the BES.

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[0330] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and proceduresdescribed herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

CLAIMSWhat is Claimed is:

1. A method of stimulating extracellular electron transfer (EET) in a microorganism, the method comprising: providing the microorganism with an electron donor; exogenously supplementing the microorganism with a small molecule redox mediator; and subjecting the microorganism to an electron sink, thereby stimulating EET in the microorganism.

2. The method of claim 1, wherein redox mediator is supplemented at a concentration of less than 1 pM, about 1 pM, about 2.5 pM, about 5 pM, about 7.5 pM, about 10 pM, about 12.5 pM, about 15 pM, about 20 pM, about 25 pM, or greater than about 25 pM.

3. The method of claim 1, wherein the redox mediator is a naphthoquinone.

4. The method of claim 3, wherein the naphthoquinone is selected from the group consisting of 2-hydroxy-l,4-naphthoquinone (HNQ), 2-methyl-l,4-naphthoquinone (MNQ), or l,4-dihydroxy-2-naphthoic acid (DHNA).

5. The method of claim 1, wherein the microorganism is a bacteria.

6. The method of claim 5, wherein the bacteria is E. coli.

7. The method of claim 5, wherein the bacteria is an exoelectrogen or is not an exoelectrogen.

8. The method of claim 1, wherein the electron sink is an anode.

9. The method of claim 1, wherein the electron donor comprises glucose, glycerol, pyruvate, or any combination thereof.

10. The method of claim 1, wherein the method overcomes direct EET feedstock selectivity of direct EET.

11. The method of claim 1, wherein the method reduces the required electrode surface area compared to direct EET, utilizes the whole reactor volume, does not require the formation of biofilm on the electrode, or any combination thereof.

12. The method of claim 1, wherein the microorganism is genetically engineered to increase EET output.

13. The method of claim 12, wherein the genetically engineered bacteria partially, substantially, or completely deletes, silences, inactivates, or down-regulates a gene or fragment thereof that encodes for an anaerobic respiratory pathway, a fermentative pathway, or a combination thereof.

14. The method of claim 13, wherein the genetically engineered bacteria is AnoxicNull (jYnapFDAGHBC EnarGHI AnarZYW EJmsA C EynfEFGH EorCAI) EtorYZ EfrdABCD EnirBDC EnrfABCDEFG EnorVW Ehmp !Accp FermNull (EngsA adhE EldhA or any combination thereof.

15. A genetically modified microorganism, wherein the genetically modified microorganism partially, substantially, or completely deletes, silences, inactivates, or down-regulates a gene or fragment thereof that encodes for an anaerobic respiratory pathway, a fermentative pathway, or a combination thereof.

16. The genetically modified microorganism of claim 15, wherein the microorganism is genetically engineered to increase extracellular electron transfer (EET).

17. The genetically modified microorganism of claim 14, wherein the genetically modified microorganism is AnoxicNull (EiapFI)AGHBC EnarGHI EnarZYW EdmsABC EynfEFGH EorCAD EtorYZ EfrdABCD EnirBDC EnrfABCDEFG EnorVW Ehmp \ccp . FermNull (Engs A EidhE EldhA or any combination thereof.

18. The genetically modified microorganism of claim 14, wherein the microorganism is a bacteria.

19. The genetically modified microorganism 18, wherein the bacteria is E. coli.

20. The genetically modified microorganism of claim 18, wherein the bacteria is an exoelectrogen or is not an exoelectrogen.

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