Non-photosynthetic biological CO2 reduction - Biochemistry (ACS

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Non-photosynthetic biological CO reduction Jake N Gonzales, Morgan M Matson, and Shota Atsumi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00937 • Publication Date (Web): 05 Nov 2018 Downloaded from http://pubs.acs.org on November 6, 2018

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Biochemistry

Non-photosynthetic biological CO2 reduction Jake N. Gonzales1, Morgan M. Matson2, and Shota Atsumi1,2,* 1Plant

Biology Graduate Group, University of California, Davis, One Shields Ave, Davis, CA,

95616, USA 2Department

of Chemistry, University of California, Davis, One Shields Ave, Davis, CA, 95616,

USA

*To whom correspondence may be addressed: Email: [email protected].

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Abstract Alarming changes in environmental conditions have prompted significant research into producing renewable commodities from alternative sources to fossil fuels. One such alternative is CO2, a determinate greenhouse gas with historically high atmospheric levels. If sequestered, CO2 could be used as a highly renewable feedstock for industrially relevant products and fuels. The vast majority of atmospheric CO2 fixation is accomplished by photosynthetic organisms, which have unfortunately proven difficult to utilize as chassis for industrial production. Nonphotosynthetic CO2 fixing microorganisms and pathways have recently attracted scientific and commercial interest. This perspective will review promising alternate CO2 fixation strategies and their potential to supply microbially produced fuels and commodity chemicals, such as higher alcohols. Acetogenic fermentation and microbial electrosynthesis are the primary focuses of this review.


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Introduction In response to alarmingly high atmospheric CO2 levels1-2, there has been an increased focus on investigating and engineering efficient means of converting atmospheric CO2 into industrially relevant fuels and commodities. As increasing population further strains the global food supply3, it has also become critical to secure carbon feedstocks that do not require crop biomass or arable land. Traditional microbial production strategies rely entirely on these feedstocks, such as sugars or secondary metabolites, for their carbon and energy. CO2 fixing microorganisms have the potential to produce valuable chemicals from atmospheric CO2 without the need for biomass-derived feedstocks. The vast majority of global CO2 fixation occurs in autotrophic photosynthetic organisms through the Calvin-Benson-Bassham cycle (CBB)4. Photosynthetic organisms, such as cyanobacteria and algae, are promising chassis organisms for chemical production from atmospheric CO2 but are not without their own challenges5-7. The cornerstone of CO2 fixation, the enzyme ribulose-1,5 bisphosphate carboxylase/oxygenase (RuBisCO), has a notoriously low catalysis rate. RuBisCO does not differentiate between O2 and CO2, requiring energetically wasteful photorespiration processes to avoid accumulation of toxic intermediates8. Attempts to engineer RuBisCO to increase its conversion rate have been met with little success, creating a hurdle for photosynthetic organisms as production platforms9-11. Thus, there is a need for finding alternative, nonphotosynthetic organisms that can utilize CO2 as a carbon building block. As reviewed below, organisms utilizing alternate CO2 fixation pathways may be suitable candidates for sustainable production of fuels and chemicals.


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Chemolithotrophs In contrast to photosynthetic organisms, chemolithotrophs derive the energy to synthesize ATP from oxidation of inorganic compounds12 instead of solar energy. Chemolithotrophs generate reducing power in the form of NAD(P)H by reversing the electron transport chain to accept electrons from high redox potential donors. Chemolithotrophs can therefore avoid production problems photosynthetic organisms frequently face, such as photorespiration and cell shading. They are not without their own production set-backs, such as requiring multiple substrates and complex physiochemical cellular environments. Acetogens, a class of chemolithotrophs, have been of commercial interest for many years due to their unique carbon fixation strategy, the Wood-Ljungdahl pathway (WLP)13. Under anaerobic conditions the WLP converts H2, CO2 and/or CO into acetyl-CoA, generating ATP and acetate for further carbon anabolism13-14 (Fig. 1). The WLP is split into two branches, the methyl branch and the carbonyl branch which merge at acetyl-CoA. The methyl branch of the WLP, sometimes referred to as the eastern branch, first reduces CO2 to formate via formate dehydrogenase. Formate is then converted to a methyl group through a series of formyl/methyl- tetrahydrofolate and cobalamin reactions. The carbonyl branch, the western branch, is driven by the carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) enzyme complex. CODH/ACS merges the two branches of the WLP by converting CO2 to CO and catalyzing the formation of acetyl-CoA in conjunction with the CoA bound methyl group generated in the methyl branch. Acetyl-CoA is then converted into acetyl phosphate by phosphate acetyl transferase which is in turn converted to acetate by acetate kinase.


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Figure 1. The autotrophic Wood-Ljungdahl pathway (WLP) CO2 is reduced to a THF bound methyl group by the methyl (eastern) branch. The carbonyl (western) branch reduces another CO2 to an enzyme bound CO group. The methyl and CO group are combined by the CODH/ACS complex to produce acetyl-CoA, which is then processed into acetate. Abbreviations are as follows, [H]: reducing equivalent, THF: tetrahydrofolate, CODH/ACS: carbon monoxide dehydrogenase/acetyl-CoA synthase, CoFeSP: corrinoid-iron-sulfur protein

Although acetate is the main product of the WLP, carbon fixed through the WLP can be used to synthesize other organic molecules such as 2,3-butanediol, butyrate, ethanol, or nbutanol14. Stoichiometric modeling indicates the WLP is likely the most efficient carbon fixation


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method due to its efficient use of reducing power to generate ATP and the low energy cost of generating acetyl-CoA15. As obligate anaerobes, acetogens can recapture CO2 generated from the decarboxylation step of glycolysis, enabling a closed loop which utilizes both the carbon substrates provided and the byproducts produced. It can also flexibly incorporate a variety of carbon sources into acetogenic fermentation, such as waste gas from steel mills16, syngas (a mix of CO, CO2 and H2)17, and CO2 generated from heterotrophic fermentation18. To establish a robust production platform based on acetogenic carbon fixation, the unique challenges of the WLP must be overcome. Theoretical carbon conversion yields are capped at 67% due the loss of one CO2 for every molecule of acetyl-CoA synthesized in glycolysis. Although the WLP can recapture CO2 from glycolysis, as mentioned above, it is difficult to generate the reducing power to do so because of the limited availability of NAD(P)H. Without improving re-assimilation, the WLP cannot achieve industrially viable/attractive yields. A secondary issue to contend with is the WLP is strictly limited to anaerobic conditions due to its dependence on ferredoxins to perform reduction chemistry and the severe oxygen sensitivity of acetyl-CoA synthase15. This limits the variety of products that can be synthesized in a one-pot bioreactor system, reducing the range of potential commercial applications. One method that alleviates the need for reducing power is mixotrophic fermentation. Mixotrophy is defined as concurrent utilization of organic substrates, such as sugars, and inorganic substrates, like CO2 and H2. This method provides additional electron donors and carbon sources, thereby increasing the reducing power available. Greater CO2 re-assimilation is therefore possible, allowing for an increase in carbon fixation efficiency, thus an increase in production yields and decrease in feedstock costs. Allowing for greater CO2 re-assimilation is 6 ACS Paragon Plus Environment

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critical for bringing acetogens to industrial scale production. While mixotrophy addresses the need for greater reducing power, it is not without complications. When introducing multiple substrates, such as carbohydrates and gas mixtures, to acetogens there is the possibility the organism will prefer one, usually the sugar, resulting in the underutilization of the other. This is called carbon catabolite repression (CCR) and has been attributed in multiple mixotrophic fermentation studies to have detrimental effects on production18-20. Described below are studies that attempt to find solutions to these challenges. To increase the reducing power available for acetone production, a syngas/fructose mixotrophic fermentation strategy in Clostridium Ijungdahlii was designed to utilize glycolysis generated CO221. Acetone is a prime target metabolite for acetogen production because it requires no reductive chemistry downstream of acetyl-CoA and therefore promotes greater CO2 re-assimilation. ATP is often the limiting factor for the growth of anaerobic bacteria such as acetogens. Mixotrophy provides glycolysis with a sugar substrate, enabling significantly higher ATP generation to further promote growth and production15, 17-18. To investigate the potential deleterious effect of CCR to mixotrophic fermentation, carbon labeling experiments were conducted. 12C labeled fructose and 13C labeled syngas were fed into C. ljungdahlii and Clostridium autoethanogenum. 13C labeled syngas substrates were significantly incorporated into final products, 73-80% in C. Ljungdahlii and 51-58% in C. autoethanogenum. No changes were found in WLP associated transcription and protein expression when compared to an autotrophic control. These results suggest that CCR is not occurring in this production scheme. CCR is likely dependent on variables such as fermentation conditions, host organism, and the target metabolite. After implementing production with cell recycling and high-density cultures, 7 ACS Paragon Plus Environment

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the final acetone titer was 10.8 g l-1 at a rate of 2.32 g l-1 h-1 in C. ljungdahlii. After augmenting syngas/fructose mixotrophic fermentation a 138% heterotrophic maximal yield of acetone was achieved, displaying the potential of mixotrophic fermentation. As mentioned above the WLP can only operate in anaerobic conditions, limiting the variety of products that can be synthesized and the available strategies to generate ATP. Atmospheric CO2 can be fixed by the WLP into a feedstock which can be fed to an aerobic host for further processing into high value commodities. To this end, a two-stage lipid biosynthesis process was designed in which acetate is produced from syngas anaerobically in the acetogen, Moorella acetecia, and fed to the oleaginous yeast, Yarrowia lipolytica, to synthesize C16-C18 triglycerides22. Triglycerides are a valuable industrial feedstock because they can be processed into biodiesel via transesterification23. First, each individual process was optimized then the two were coupled into a continuous syngas-to-lipid production scheme. After experimenting with CO/CO2 and H2/CO2 gas substrates on M. acetecia cultures growth was four times greater in the CO culture, but both had similar final acetate titers of over 30 g l-1. This suggests CO/CO2 gas is ideal for promoting growth, but H2/CO2 has a much higher specific productivity. Acetate production in M. aceticia was split into two stages, a growth promoting CO/CO2 phase and a high specific cell productivity of acetate H2/CO2 phase. This step-wise gas exchange strategy and fresh media cycling at the point of gas exchange produced a final acetate titer more than 30 g l-1 at a production rate of 0.9 g l- h-1. Dilute acetic acid (3% v v-1) was fed in Y. lipolytica stirred tanks and reached titers of 46 g l-1 with a productivity rate of 0.27 g l-1 h-1 and a lipid content of 59%. Finally, both processes were integrated to produce a titer of 18 g L-1 at a rate of 0.19 g l-1 h-1 with a lipid content of 38%. While the integrated process was significantly less productive 8 ACS Paragon Plus Environment

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Biochemistry

than the standalone reactions, it was shown that a two-step gas-to-lipids production scheme is possible. The utility of a multi-organism production strategy is further reinforced by a recent study of wild-type Clostridium ljungdahlii and Clostridium kluyveri co-culture producing long chain alcohols from syngas24. C. ljungdahlii fixes syngas efficiently into acetate/ethanol and C. kluyveri carries out chain elongation via the 𝛽-oxidation pathway, producing a variety of higher alcohols. A syngas mixture was pumped into a single stage 1 L bioreactor inoculated with C. ljungdahlii. C. kluyveri inoculation took place at the 670 hour mark of the 2200 hour study. A variety of hydrocarbons were produced during this time, acetate being the main product. Acetate was synthesized at a rate of 12.7 g L-1 d-1. The other products, n-butyrate, n-caproate, ethanol, n-butanol, n-hexanol, 2,3 butanediol, and n-octanol, were produced at rates between 0.7 mg L-1 d-1 and 2.8 g L-1 d-1 . Productivity and co-culture health appeared to be highly dependent on pH. Unfortunately, optimal pH for chain elongation and ethanol production are different, making any optimization process laborious. Product specificity is also a significant challenge in co-culture as chain elongation and biological reduction are constantly competing with each other, resulting in an uncontrollable variation in products. Despite these issues, this study strongly suggests that long term acetogenic co-culture can sustain hydrocarbon synthesis. These wild-type strains and production processes were not engineered nor optimized, leaving considerable room for improvements. For example, if a chain elongation strain was engineered to operate at pH 5-5.5, productivity would be significantly higher. While both of the above studies make a strong case for two step or co-culture production, there are inherent limitations that must be overcome to make these production 9 ACS Paragon Plus Environment

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strategies feasible. In the case of two-step processing, combining both production systems led to a significantly lower titer of lipids than the individual systems. In addition, a complex bioreactor transfer system was required to constantly feed substrate to the second culture. In the case of co-culturing, optimizing conditions that meet the requirements for both organisms is exceptionally difficult. For example, the optimal pH discrepancy between C. ljungdahlii growth and C. Kluyveri chain elongation. The production of one specific higher alcohol is also very difficult to achieve in a co-culture system because C. ljungdahlii and C. kluyveri are competing to reduce and elongate the same substrate. Despite these challenges, acetogenic fermentation remains an attractive production platform due to the efficiency of the WLP and the sheer variety of possible products co-culturing offers. Acetogenic fermentation can also take place at ambient temperatures and pressures, which significantly simplifies scale up.

Electrofuels Electrocatalysis can be used in conjunction with a wide range of novel chemistries to reduce CO2 into useful one carbon molecules. Forming C-C bonds electrocatalytically remains a significant challenge25-26. Small carbon molecules such as acetic acid have been synthesized electrocatalytically, but at extremely low yields27. While synthesizing long chain carbon molecules and fuels is not feasible electrocatalytically, a biological-electrocatalytic hybrid system can capitalize on the innate ability of microorganisms to form C-C bonds. Electricity can enable artificial photosynthesis in microorganisms by providing reducing power in the form of electrons to fix CO2 into complex carbon commodities28.


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The proportion of solar power converted to usable energy by photovoltaic technologies is significantly higher than that by plants or photosynthetic microorganisms29. Photovoltaic devices have a conversion efficiency of 18% while photosynthetic microorganisms currently cap around 5-7% in optimized bioreactor conditions. An electrocatalytic-biological production platform may be a more sustainable and efficient method of energy processing than either method alone. Photovoltaic devices are more efficient at energy capture but storing that energy and incorporating it into the global power grid is challenging30-31. Organisms coupled to electrocatalysis have the potential to store this energy in the form of chemical bonds or use it to generate valuable commodeties32-34. While the utility of an electrocatalytic-biological production scheme is apparent, this integrated approach is relatively new and unexplored. The main difference between the few studies on this topic is the method used to deliver electrons to the host organism. There are several ways to deliver electricity to a production host, an organic molecule such as formate, ammonia, or H2 can shuttle electrons or they can be directly transferred to an electrophilic organism from the electron source33, 35-37. Early studies utilized organic electron shuttles like neutral red, but issues such as toxicity, complicated product recovery, and cost have made them less favored33. Currently research is focused on two methods, direct electrode-to-microbe electron transfer or indirectly with electrochemically generated electron donors.

Direct microbial electrocatalysis


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The direct bio-electrochemical model requires the production host to be in immediate contact with the electrode, allowing electron transport conduit proteins to channel electrons needed to power reductive metabolism. Cathodic electron uptake dynamics in the γ-proteobacteria host Shewanella oneidensis was investigated37. In nature, Shewanella delivers electrons to extracellular terminal electron acceptors facilitating a form of anaerobic respiration. The multiheme c-type cytochromes (Mtr) respiratory pathway functions as the connection to electron acceptors beyond the outer membrane38-39. To assess the microbial electrosynthesis potential of cathode powered reductive metabolism in S. oniedensis this pathway was reversed by providing an external electron source. Multiple deletions were done to further characterize the Mtr pathway and reductive metabolism in Shewanella. Under anaerobic conditions there is only one active fumarate reductase in Shewanella that strongly favors the reductive reaction and the only source of reductive power is electrons supplied by the reverse Mtr pathway. Therefore, the conversion of fumarate to succinate was used to characterize electron flux in the cell. While this study successfully demonstrated the Mtr pathway can be reversed and characterized many of its components, significant research must still be done to incorporate the Mtr into microbial electrosynthesis. The production requirement of constant microbe-electrocatalysts contact presents significant challenges for production, costly custom electrodes and bioreactors are likely required to reach maximum host biomass. Microbial electrosynthesis of acetate was optimized by improving bioreactor design and operation including a high surface area cathode40. Converting H2 and CO2 into acetate is 12 ACS Paragon Plus Environment

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advantageous as it has been the focus of multiple previous studies and various strategies for improving yield have been described2. Instead of a pure culture, an established acetogenesis microbial electrosynthesis microbiome was used41. Strategies from previous gas fermentation studies shown to increase productivity were also incorporated, such as increasing biomass retention and media dilution rate42-43. Previous research demonstrated that increased biomass leads to higher acetate production44. This study sought to implement the same strategy during electrocatalytic acetogenesis by increasing biomass production at the cathode. By switching to a galvanostatic operation that was able to provide a higher volumetric current density, more electrons were available and accessible to the microbes. A continuous flow reactor was used which reduces nutrient limitations and alleviates product inhibition, allowing for a final acetate production of 0.78 g L-1 h-1 at a titer of 3.6 g L-1. While this study employed a variety of strategies to optimize production, there are still improvements to be made regarding product extraction and titer. An approach that could improve both areas is introducing an anion exchange membrane which compartmentalizes synthesized acetate or design a twostep production process as seen in other strategies22.

Indirect microbial electrosynthesis As discussed above, indirect microbial electrosynthesis is described as employing an organic molecule to deliver electrons from an electrode to the host powering reductive metabolism. The indirect production strategy has just recently begun to draw scientific and commercial interest owing to recent advancements in electrocatalysis and increased efficiency


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of solar devices. As such, most research is relatively new, and applications are still in their infancy. Formate is a favorable energy carrier because it is synthesized efficiently electrocatalytically and is readily soluble. Formate has a high redox potential compared to other potential donors, allowing it to support growth via the direct reduction of CO2 to other organic compounds33. Compared to other potential electron donors like ammonia or sulfide it does not require an additional electron acceptor like oxygen, which can add complexity to the production system. Formate does not have volatility safety concerns, like cultures fed with H2. A formative study to the field reported that Ralstonia eutropha produced isobutanol and 3-methyl-1-butanol (3MB) from electrochemically fixed CO245 via formate and H2 electron donors. When electrocatalytically synthesized formate accumulates at the anode it decomposes over time, reducing substrate availability. To achieve maximum yields formate must be consumed quickly after it is produced. To achieve this a one-pot CO2 to alcohol scheme was designed to electrocatalytically synthesize formate in the R. eutropha culture producing mixed alcohols. Three major obstacles were identified in this system, (i) engineering R. eutropha to produce liquid fuels, (ii) efficient electrochemical synthesis of formate from CO2 in fermentation medium, and (iii) enabling microbes to grow despite constant electrical current. Previously established pathways were introduced to R. eutropha to produce isobutanol and 3MB46-47. An in-foil cathode and a Pt anode were used to produce formate electrocatalytically in 15% CO2 media. Introducing electricity was previously shown to impede cell growth in Escherichia coli, presumably due to the generation of reactive oxygen or nitrogen species48. To determine the presence of these species, three reporter plasmids known to be induced by hydrogen peroxide, 14 ACS Paragon Plus Environment

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superoxide free radicals, and nitric oxide were employed. Electrocatalytic R. eutropha cultures were shown to be high in nitrogen oxide and superoxide free radicals while hydrogen peroxide remained at low concentrations. A porous ceramic cup was placed around the anode to provide a staunch diffusion gradient for electrochemically generated toxic intermediates. This shield ensures any reactive compounds produced are quenched before entering the fermentation media. 140 mg l-1 of isobutanol and nearly 50 mg l-1 of 3MB were produced over 100 hours. Final OD600 of R. eutropha reached 2.0, proving growth could be sustained despite exposure to constant low voltage. While this study provided insight into conducting electrocatalysis and microbial synthesis in the same bioreactor concurrently, aerobic microbial electrosynthesis has a unique set of limitations that must be overcome. Aerobic cultures are at a disadvantage compared to anaerobic fermentation in terms of production efficiency49. While aerobic cultures are efficient at generating ATP compared to anaerobic cultures, they have a tendency to utilize a large proportion of substrate to produce cell mass instead of products. An aerobic R. eutropha fermentation was supplied with H2 and formic acid at 0 voltage. H2 fermentation reached titers of ~536 mg l-1 isobutanol and ~520 mg l-1 of 3MB over 120 hours at an OD600 of ~24.0. Formic acid fermentation reached titers of ~846 mg l-1 of isobutanol and ~570 mg l-1 of 3MB at an OD600 of ~3.5. Compared to H2 and formic acid fed fermentations, titer is much lower despite a relatively comparable OD600, suggesting inhibited growth is not the only deleterious effect voltage has on the production hosts. A formate dependent microbial electrosynthesis system was established in the model organism E. coli to take advantage of its abundant genetic resources and potential to grow 15 ACS Paragon Plus Environment

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Figure 2. The reductive glycine pathway (RGP) CO2 is converted to formate electrocatalytically. Formate is then assimilated into the RGP which is composed of 3 modules. In green module 1) Formate is converted to a CH2-THF intermediate by three enzymes native to the WLP. CH2-THF is combined with CO2 via rGCS to synthesize glycine. In red module 2) An additional CH2-THF intermediate generated by module 1 is combined with glycine by GlyA to produce L-serine. In blue module 3) L-serine is deaminated to pyruvate by SdaA. Abbreviations are as follows: THF: tetrahydrofolate, rGCS: reverse glycine cleavage system, GlyA: L-serine hydroxymethyltransferase, SdaA: L-serine dehydratase, 3-PG:3-phosphoglyceric acid. anaerobically36. The genetic tractability of E. coli also allows for relatively fast and easy manipulation of production pathways, as exemplified by the multiple knockouts and alternate


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promoters introduced in this study. Pyruvate, a key intermediate in central metabolism, was synthesized by a novel pathway coined the reductive glycine pathway (RGP, Fig. 2). The RGP is theoretically the most efficient route for formate assimilation to date50. The RGP has three major advantages, (i) it is a linear pathway, (ii) its reactions are thermodynamically favorable, and (iii) no oxygen sensitive pathways are involved. The RGP can be broken into three modules. In the first module formate and CO2 are converted into glycine, which in the second module is combine with another formate to produce L-serine. In the third and last module, L-serine is converted into the pyruvate. In the first module, formate is converted into 5, 10 methylene-THF (CH2-THF) by three WLP enzymes, Fhs51, FchA52, and FolD53. In order to covert CO2 and CH2-THF into glycine, the glycine cleavage system (GCS) was utilized. The GCS is common among many organisms due to its involvement in glycine and serine catabolism54. The GCS converts glycine to CO2, NH4+, and CH2-THF (Fig. 3). Previous studies have suggested this reaction is not irreversible, allowing carbon exchange between glycine and CO2 in the presence of trace metals55. This study36 was the first to demonstrate GCS is reversible (rGCS) with the production of glycine from CO2 and CH2-THF. In the second module, another formate is converted to CH2THF then coupled with glycine to produce L-serine via native L-serine hydroxymethyltransferase (GlyA) from the Serine-Glycine cycle56. Finally, L-serine is deaminated by native L-serine dehydratase (SdaA), yielding pyruvate. Two formate, one CO2, three NAD(P)H, and two ATP molecules are required to make one pyruvate in this pathway. The RGP consumes five less ATP and two less NAD(P)H than what the CBB cycle requires to synthesize one pyruvate, making it a promising alternative to photosynthetic CO2 fixation.


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Figure 3. The glycine cleavage system (GCS) The GCS is composed of four proteins, three enzymes and a carrier protein. Two sulfhydryl groups or lipoate attached to the H protein act as intermediate shuttles. The first reaction during catabolic activity is the decarboxylation of glycine by the P-protein. The decarboxylated moiety is then further degraded by the T protein with the aid of THF. The last step is the re-oxidation of the two sulfhydryl groups to form lipoic acid generating NADH. This reaction is fully reversible as demonstrated by the functioning RGP (figure 2). Abbreviations are as follows: THF: tetrahydrofolate, P-protein: glycine dehydrogenase, Tprotein: aminomethyl transferase, L-protein: dihydrolipomide dehydrogenase Experiments on glycine and L-serine auxotrophs were conducted in conjunction with 13C labeled substrates to determine if the RGP was functioning as intended. To prove the GCS was reversible a glyA strain harboring fhs, fchA, and folD was constructed to ensure any fed bicarbonate and formate would be the main, if only, source of glycine. When both 13C bicarbonate and formate were fed into the strain the resulting GC/MS profile indicated the 18 ACS Paragon Plus Environment

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concurrent utilization of 13C labeled substrates to build glycine. A L-serine auxotrophic strain ( serA) with the entire RGP installed was used to confirm formate and CO2 were being converted into L-serine. Growth of the strain was only rescued if bicarbonate and formate were both provided, indicating the RGP functions as intended. The weak, constitutive sdaA promoter was replaced with the stronger, inducible PLlac01 promoter to encourage conversion of L-serine to pyruvate. The PLlac01:sdaA strain was able to grow solely on serine and compared to its parent strain had increased growth rate when fed both formate and bicarbonate.

13C

substrates were

again fed to this production strain to confirm that growth rescue was due to the RGP. Pyruvate is not detectable in this context, so L-alanine, a pyruvate derivative, was used as an analog to measure pyruvate synthesis through the RGP. The detection intensity profile of L-alanine generated by feeding this production strain 13C labeled formate and bicarbonate indicated that the RGP was able to produce pyruvate via an L-serine intermediate in E. coli. Electrochemical cultivation of E. coli containing the RGP was then done36. All formate provided to the culture was produced electrocatalytically in culture media from CO2 gas. An indium cathode was shown to be most efficient at converting CO2 to formate in bacterial culture. In this system 450 mg l-1 of formate was generated a day (10 mM), this rate remained constant during a 20-day experiment. A strain containing the entire RGP with glyA was grown in minimal media with CO2 introduced to the cathode operating at 2.0 V. This culture managed to reach an OD600 of 0.5 over 8 days after a long lag phase. While much lower than the positive control grown without electricity, which reached an OD600 of ~0.8 after five days, growth was achieved by providing formate synthesized from CO2 and 4 g L-1 glucose. This inhibited growth may be attributed to reactive oxygen species (ROS), as noted in previous aerobic microbial 19 ACS Paragon Plus Environment

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electrosynthesis studies45. Although pyruvate is an important central metabolite, it is not commercially valuable. However, it is a substrate for the production of many industrially relevant chemicals, easily linking the RGP with commercialization. Therefore, further engineering downstream of the RGP is required to produce any economically valuable compounds. Fortunately, there is a litany of industrially relevant, pyruvate-dependent metabolic pathways that been developed in E. coli that can be applied, a comprehensive list can be found in other reviews57-59. The RGP is also a significant consumer of NADH, resulting in a scarcity of cellular reducing power that severely limits growth. If NADH could be regenerated or another reducing source supplemented, the production rate would likely increase as a result. Coupled electrocatalysis and microbial production is still a new concept, and there are many questions that must be answered before it can become an economically viable production platform. There is no clear superior production strategy between direct and indirect electron transport as both systems have unique challenges. Direct electron transport strategies require highly specialized bioreactor conditions which may be extremely costly to scale up. Indirect electron transfer studies thus far have had problems with ROS generation and slow growth under constant voltage. ROS, such as H2O2 and superoxide, can cause oxidative enzyme and DNA damage. At high ROS concentrations, cell growth is arrested and mutagenesis accelerated60-61. Virtually all living organisms possess some way of coupling with ROS stress, typically via superoxide dismutases and peroxidases/catalases capable of degrading H2O2. Mutants lacking either these enzyme sets take damage from iron containing enzymes and cannot function in their typical growth conditions. During electrocatalysis, the concentration of ROS increases to an extent that native defensive enzymes cannot completely neutralize the 20 ACS Paragon Plus Environment

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Biochemistry

ROS’s deleterious effects. Technical considerations of bio-electrosynthesis production, such as creating a compatible biofilm-electrode interface, must also be considered. Currently, very little research has gone into exploring the effect of electrode surface composition on microbial production28, 62. Determining the most efficient electrode for electrosynthesis is often done experimentally because there is no current understanding of how the cathode and microorganism interface. Challenges like these are typical of burgeoning technologies and will be address as research into these areas continue. The described body of work is a solid foundation on which further improvements can be made. Outlook While the described acetogenic fermentation and electrosynthesis based production platforms could be transformative to industry, several challenges must be overcome first. CO2 was provided at high concentrations in all the studies mentioned above. Ironically, industrial sources such as steel mills and cement factories can supply ample CO2 in the form of syngas to feed chemical production63. While atmospheric CO2 capture technology is an ongoing field of research, there are many hurdles that must be overcome before it can efficiently provide high enough concentrations for biochemical production64. Acetogenic fermentation and electrosynthesis also require specialized production conditions and infrastructure that may prove costly. Further metabolic and process engineering must also be done to improve product specificity and production rate to make these strategies economically viable. Altering carbon flux regulation or implementing specialized microbial electrosynthesis electrodes are potential strategies to this end. A very recent study explored the theoretical economic viability of electrosynthesis production of higher alcohols with a silver based diffusion cathode65. Under 21 ACS Paragon Plus Environment

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the described conditions the alcohols produced are worth 2 fold more than the electricity required to produce them. While this analysis is encouraging, translating this premise into a working bio-electrosynthesis production plant is still years from fruition. Despite these significant hurdles, non-photosynthetic CO2 reduction remains an extremely promising production platform, especially when considering the growing emphasis on curbing fossil fuel consumption.

Acknowledgements This work was supported by the Electrofuel program of Advanced Research Projects Agency– Energy (DE-AR0000085 (S.A.)).


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