The formate bio-economy

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ScienceDirect The formate bio-economy Oren Yishai, Steffen N Lindner, Jorge Gonzalez de la Cruz, Hezi Tenenboim and Arren Bar-Even In this review we discuss the concept of the formate bioeconomy: formate can be produced efficiently from various available resources and can be consumed by microbes as the sole carbon source for the production of value-added chemicals, directly addressing major challenges in energy storage and chemical production. We show that the formate assimilation pathways utilized by natural formatotrophs are either inefficient or are constrained to organisms that are difficult to cultivate and engineer. Instead, adapting model industrial organisms to formatotrophic growth using synthetic, specially tailored formate-assimilation routes could prove an advantageous strategy. Several studies have started to tackle this challenge, but a fully active synthetic pathway has yet to be established, leaving room for future undertakings. Address Max Planck Institute of Molecular Plant Physiology, Am Mu¨hlenberg 1, 14476 Potsdam-Golm, Germany Corresponding author: Bar-Even, Arren ([email protected])

Current Opinion in Chemical Biology 2016, 35:1–9 This review comes from a themed issue on Energy Edited by Wenjun Zhang and David F Savage

http://dx.doi.org/10.1016/j.cbpa.2016.07.005 1367-5931/# 2016 Elsevier Ltd. All rights reserved.

Introduction Storing excess electricity, produced at off-peak hours from renewable and intermittent sources, is a central aim of the energy industry and an essential step towards increasing the share of these sources in electricity production [1]. Similarly, the production of value-added chemicals currently depends almost entirely on fossil carbons or simple sugars, whose utilization directly competes with human consumption and thereby undermines food security [2]. Hence, there is an urgent need to develop methods to utilize available and cheap resources as feedstock for the production of chemicals [3]. Notably, these two challenges may be interwoven: energy might be efficiently stored within stable chemicals, for example, fuels; and, complementarily, available electricity and CO2 may serve as promising feedstocks for the production of www.sciencedirect.com

value-added chemicals [4–6]. Such a process can be entirely physicochemical. For example, electrochemical reduction of CO2 to carbon monoxide alongside hydrogen production via water splitting or water-gas shift [7] can support a downstream Fischer–Tropsch process, producing various hydrocarbons [8]. However, such purely physicochemical processes usually necessitate large infrastructure, tend to require extreme conditions (e.g., high temperature and pressure), and in most cases are not product-specific, leading to the formation of a mixture of compounds [8]. On the other hand, microbial production of chemicals tends to be product-specific, can be carried out under ambient conditions, and can be run economically using a medium-sized apparatus [9]. A sustainable and efficient approach for converting energy into fuels and other chemicals should therefore preferably mix physicochemical and biological strategies [10]. Here, we put forward the case for formate as a mediator between the physicochemical and biological realms, that is, formate can be synthesized efficiently using excess energy and then serve as the sole source for microbial growth (Figure 1). Formatotrophic microbes can then be used to convert formate into a myriad of products, such as fuels, other value-added chemicals (e.g., solvents, plastic monomers, pigments), and even protein meal for animal and human consumption [11] (Figure 1).

Formate production: electrosynthesis and other promising methods As electricity serves as our main energy currency, electrochemical reduction of CO2 will likely prove to be the most sustainable way to produce formate. In recent years, the electrochemical reduction of CO2 to various compounds has gained considerable attention [12]. Yet, out of the wide array of compounds that were shown to be generated by this approach, only carbon monoxide and formate can be produced efficiently [12]. As both compounds require only a two-electron reduction of CO2, finding a good catalyst for their production is less challenging than for multi-electron products, such as methane, methanol, ethylene, oxalate and acetate [13]. Carbon monoxide is a highly toxic gas of low solubility and low mass transfer rate, which makes its downstream utilization challenging, especially with respect to microbial cultivation [14]. On the other hand, formate is a highly soluble compound that can be handled rather easily, making it a suitable mediator between electricity and microbial cultivation. Supporting this approach, recent studies have demonstrated multiple strategies for the electrochemical reduction of CO2 to formate — with current Faradaic efficiency of well above 90% — and put Current Opinion in Chemical Biology 2016, 35:1–9

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Figure 1

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A schematic representation of the formate bio-economy concept. Multiple approaches could support the synthesis of formic acid/formate from available sources. Formate could be then consumed by natural formatotrophic microbes or by microbes engineered to efficiently assimilate formate for the production of fuels, value-added chemicals, and protein meal for animal or human consumption. Source: All icons are taken from www.icons8.com.

forward the feasibility of an industrial scale-up in the nearfuture [15,16,17,18,19,20]. Electrochemical reduction of CO2 to formate circumvents several major shortcomings of other proposed strategies for electricity-dependent microbial cultivation: (1) Direct electron transfer from an electrode to living cells [21] is restricted to a small number of organisms and to low current-density. Conversely, formate can be produced at considerably higher rates and could potentially be assimilated by many biotechnologically engineered organisms (see below); (2) unlike H2, which can be produced even more efficiently from electricity [22] and serve as a microbial feedstock [23,24,25], formate is completely soluble and not explosive. Furthermore, unlike H2, formate can be assimilated directly, rather than being completely oxidized for reducing power; such direct metabolism can support considerably higher biomass and product yields [26]. Current Opinion in Chemical Biology 2016, 35:1–9

A comprehensive economic analysis has put the price of electrochemical production of formate at $500 [15]. This price changes considerably depending on the cost of electricity, the consumables required for the process, the cost of concentrated CO2, the efficiency of CO2 reduction (Faradaic efficiency as well as energetic efficiency [22]), the rate of formate production, and the composition and durability of the electrode. A recent study has demonstrated that, from an industrial perspective, current technologies display satisfactory Faradaic efficiencies and quite acceptable current densities; however, the overpotentials currently applied are still too high and the durability and cost of the electrodes are far from optimal [24]. Improving these factors could substantially reduce the cost of large-scale CO2 reduction. Even with current technologies, the use of cheap electricity produced at off-peak hours and concentrated CO2 from power plants or other industrial factories could www.sciencedirect.com

The formate bio-economy Yishai et al.

significantly reduce of cost of formate production to $200/ ton or even lower [15]. Still, as the market price of glucose is $300–400/ton, and since glucose is a much better feedstock than formate (a ton of glucose contains three times more available electrons for oxidation than a ton of formate), formate cannot directly outcompete glucose as a feedstock. However, agricultural production of glucose has a limited capacity and cannot displace fossil carbons as the prime feedstock for the chemical industry [27]. Only a feedstock with an almost unlimited capacity and scalability might truly displace fossil carbons. Electricity and CO2 are the only sources close to meeting this requirement. Hence, a fast-growing bio-refinery sector, with ever-increasing demand for feedstock, could benefit greatly by replacing depletable glucose with electricallyproduced formate. Alternative methods may also present a great promise for large-scale and sustainable production of formate (Figure 1). For example, CO2 can be efficiently reacted with molecular hydrogen to produce formate [28], providing a promising strategy for hydrogen storage that could alleviate the technical difficulties associated with its handling and transport [29]. The source of the molecular hydrogen can be non-renewable, e.g., steam reforming of fossil carbons, or renewable, e.g., electrolysis of water supported by electricity-generating wind turbines or photovoltaic cells. Formate can also be produced by photoreduction of CO2 at high turnover and product selectivity [30], providing a more direct route from renewable energy to formate. Another interesting approach for sustainable production of formate is the use of recyclable, low-cost catalysts to support the selective oxidation of biomass with molecular oxygen to give formate [31,32]. This method is quite insensitive to the exact composition of the biomass, which can constitute cellulose, lignin, waste paper, or even living organisms [33]. Furthermore, the process approaches a yield of 100% in converting some biomass compositions to formate (and CO2 as a completely oxidized waste-product) [32]. This strategy, followed by downstream formate-dependent microbial growth and production of value-added chemicals, could replace the current biomass processing methods — fermentation, gasification, and pyrolysis — which require costly pretreatment, product separation and/or extreme conditions (e.g., high temperature) [34]. Finally, it is worth mentioning that formate can also be produced by partial oxidation of natural gas [35] or from the hydration of SynGas (i.e., carbon monoxide) [36] that originates, for example, from biomass gasification.

Formate assimilation: native formatotrophs As a carbon source, formate has an advantage over other one-carbon compounds, for example, methane and methanol, as its reduction potential is low enough www.sciencedirect.com

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(430  E80  380 mV at 6  pH  8 and 0  I  0.25 M [37], where I corresponds to ionic strength) to efficiently donate its electrons to NAD(P)H (physiological 370  E0  280 mV), thus directly providing the cell with reducing power. However, formate presents a challenge that more traditional carbon sources, such as glucose, avoid: formate is toxic at medium to high concentrations. This toxicity can be partly attributed to the diffusion of the protonated acid across the cell membrane, which acidifies the cytoplasm and reduces the proton motive force [38], and to formate inhibiting the respiratory cytochromes [39]. The sensitivity of organisms to formate varies greatly and at least partially depends on the activity of formate dehydrogenase. For example, Escherichia coli, in which only weak formate dehydrogenase activity is present [40], shows severe growth impairment at formate concentration higher than 100 mM [41]. Other organisms, such as Saccharomyces cerevisiae, in which formate dehydrogenase activity is high, can tolerate and even benefit from a formate concentration in the hundreds of mM [42,43]. In fact, formate has been previously tested in multiple organisms and under various conditions as an auxiliary substrate to support increased growth and product yields [44], indicating that it can be tolerated at relatively high concentrations by at least some organisms. Furthermore, as previously demonstrated for inhibitory feedstock compounds (e.g., acetate, furans) or fermentation products (e.g., various carboxylic acids and alcohols), evolutionary and rational engineering can substantially improve microbial tolerance towards a toxic compound [45]. A similar adaptation could serve to decrease the sensitivity of formate-utilizing microbes towards high concentration of this feedstock. The most straightforward approach of sustaining growth on formate is to employ organisms that can naturally use formate as a sole carbon source. The only current study that has demonstrated a simultaneous electrochemical formate production and biological formate conversion to useful chemicals (higher alcohols in this case) was indeed established using a natural formatotroph: Cupriavidus necator (Ralstonia eutropha) [46]. This integrated process bypasses the need for costly and energy-intensive formate separation and further avoids formate accumulation, which can harm the microbe and also lead to formate decomposition at the anode. The study has further presented a clever way to use a porous ceramic cup to shield the anode, avoiding anode-produced reactive oxygen and nitrogen species from inhibiting microbial metabolism [46]. Yet, the utilization of natural formatotrophs has its disadvantages. Most of them are not as suitable to be used in the bio-industry as highly optimized industrial strains of S. cerevisiae, E. coli, C. glutamicum and other model microbes. As compared to these industrial strains, natural formatotrophs may require costlier cultivation media, Current Opinion in Chemical Biology 2016, 35:1–9

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[52], limiting their potential industrial application. For example, the production of acetyl-CoA, a precursor for many downstream products [53], necessitates 11 formate molecules (4 for NAD(P)H regeneration and 7 for ATP production).

reach lower densities, grow slower, and/or present higher sensitivity to environmental conditions (e.g., oxygen level). Furthermore, as the metabolic networks of these microbes are not fully known and genetic tools for their manipulation are not fully available, it can be challenging to engineer them for the efficient production of a desired compound. Most of these difficulties can be addressed by careful cell physiology studies and by applying strain optimization techniques. However, there is one major disadvantage of natural formatotrophs that is difficult to bypass: the formate assimilation pathways they utilize are far from ideal for biotechnological applications.

Many methylotrophs assimilate reduced one-carbon compounds, including formate, via the serine pathway, and were previously suggested as potential industrial strains [54,55] (Figure 2b). However, while the serine pathway directly assimilates formate, it is still rather ATP-inefficient and hence can support only a moderate growth yield [26]. Production of acetyl-CoA by this pathway requires 7 formate molecules (1 is assimilated, 3 provide NAD(P)H, and 3 for ATP generation). On the other hand, the reductive acetyl-CoA pathway provides the energetically most efficient approach for formate assimilation [56,57] (Figure 2c,d): the production of acetyl-CoA costs only 4 formate molecules (1 is assimilated, 3 provide NAD(P)H) and produces ATP rather than consume it. Indeed, many acetogens and methanogens can assimilate formate as sole carbon and energy source via the reductive acetyl-CoA pathway (e.g., [56,58]). These organisms were further shown to be useful for biotechnological applications (e.g., [59,60]). Hence, it is tempting to suggest that acetogens and methanogens could be used as ideal hosts for the conversion of formate into value-added chemicals. However, these organisms are substantially more difficult to cultivate, manipulate and engineer than model industrial microbes [56], and their product spectrum remains limited [34,59]. In addition, the proteomics of the reductive

Natural formatotrophs use one of three pathways to assimilate formate: the reductive pentose phosphate cycle (i.e., Calvin–Benson–Bassham cycle), the serine pathway, or the reductive acetyl-CoA pathway (i.e., Wood–Ljungdahl pathway), as shown in Figure 2. In the first of these formate-assimilation strategies, employed also by C. necator [46], formate is oxidized to CO2, providing reducing power to run carbon fixation via the reductive pentose phosphate cycle (Figure 2a, e.g., [47–49]). This approach is, however, inefficient: (i) as compared to direct formate assimilation, the complete oxidation of formate to provide reducing power for carbon fixation is a wasteful strategy, as it dissipates the energy released by the electron transport from formate to the cellular redox carriers [26]; (ii) the reductive pentose phosphate cycle is one of the least ATP-efficient carbon-fixation pathways [50,51]. As a result, the growth rate as well as biomass and product yields of formatotrophs employing this pathway are quite low Figure 2

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Metabolic pathways supporting growth on formate in natural formatotrophic microbes. A red star marks reactions that consume redox equivalents or ATP, the production of both is dependent on formate metabolism. (a) The reductive pentose phosphate cycle (i.e., Calvin–Benson–Bassham cycle). (b) The serine pathway (encircled in orange). (c) The reductive acetyl-CoA pathway (i.e., Wood–Ljungdahl pathway) as operating in acetogens (encircled in cyan). (d) The reductive acetyl-CoA pathway as operating in methanogens. Current Opinion in Chemical Biology 2016, 35:1–9

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The formate bio-economy Yishai et al.

acetyl-CoA pathway is highly complicated, requiring multiple auxiliary enzymes, some of them oxygen-sensitive and some are yet to be identified. Hence, it is unlikely that this pathway could be functionally expressed in a foreign, industrial strain.

Formate assimilation: synthetic pathways and enzymes An alternative approach to using natural formatotrophs or naturally employed formate-assimilation pathways is to adapt model industrial microbes to grow on formate using synthetic, specially tailored routes. As the endogenous metabolic networks of different organisms are not equally suitable for the synthesis of particular products, it is not surprising that the choice of a host organism can dramatically affect biosynthetic efficiency [61–63]. Furthermore, different microbes display different levels of tolerance for different environmental conditions as well as for the accumulation of different products and byproducts. For example, only few known industrial organisms can tolerate low pH; yet, low pH is beneficial for the industrial production of acids, as it keeps them protonated and reduces the cost of product separation. Therefore, for formate assimilation to become a useful bio-industrial process, it is crucial to design efficient formate-assimilation routes that could be easily implemented in various model organisms and support the production of a wide variety of products. Importantly, these synthetic pathways for formate assimilation could be directly engineered in strains that were already optimized for industrial applications, for example, requiring only cheap cultivation media and sustaining high growth rate and cellular density. Even if such strains are natively sensitive to formate, it is expected that the establishment of a strong sink for formate, that is, a synthetic assimilation pathway, will reduced formate toxicity substantially (as it is the case in organisms that display high formate dehydrogenase activity). Considering the thousands of metabolic reactions whose catalyzing enzymes are known [64], it is possible to systematically search and identify multitudes of routes that could potentially perform a given metabolic task [65]. Although the components of such routes exist in nature, the pathways themselves are mostly novel, as they integrate enzymes from different organisms and organelles. This approach was applied to identify synthetic formate-assimilation pathways [26]. The candidate pathways were then compared according to various physicochemical properties that relate to their resourceusage efficiency, thermodynamic profile, kinetic capacity, and connectivity to the endogenous metabolic network of model microbes such as E. coli and S. cerevisiae [51,65,66]. This analysis resulted in the identification of the reductive glycine pathway (Figure 3a) as a promising route to support efficient aerobic formate assimilation [26]. www.sciencedirect.com

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The reductive glycine pathway is similar in structure to the reductive acetyl-CoA pathway: both attach formate to tetrahydrofolate (THF), reduces it, and use an enzyme complex to condense it with another CO2, giving rise to a C2-compound (glycine and acetyl-CoA, respectively, Figures 2c and 3a). However, unlike the reductive acetyl-CoA pathway, the reductive glycine pathway avoids oxygen-sensitive components and utilizes only common enzymes that can be found in almost all living organisms. The pathway is expected to be more efficient than the reductive pentose phosphate cycle or the serine pathway; production of acetyl-CoA costs only 6 formate molecules (1 is assimilated, 3 provide NAD(P)H, and 2 for ATP generation). More importantly, the overlap between the reductive glycine pathway and central metabolism is quite limited, avoiding the need for complicated flux regulation. Conversely, the serine pathway is completely intertwined with glycolysis and the TCA cycle, making its implementation within a foreign host extremely challenging in terms of flux control. The major challenge in establishing formate assimilation via the reductive glycine pathway is sustaining high flux of glycine synthesis via the reversible glycine cleavage system. Although the system was shown to catalyze glycine production in vitro [67,68] and in vivo [69–71] and was suggested to serve as the sole source of glycine in numerous prokaryotic lineages [72], the rate of glycine synthesis might be too low to support efficient growth on formate. To sustain high activity of the reductive glycine pathway it might therefore be preferable to express a foreign glycine cleavage system from organisms that were evolved to carry high reductive flux via the system. For example, purine-fermenting bacteria reduce CO2 to glycine in substantial amounts — via the THF and glycine cleavage systems — in order to dissipate excess reducing power [73–75]. If one relaxes the constraint of using only existing enzymes and considers also reactions that require enzyme engineering, other promising solutions for formate assimilation emerge. A recent study put forward such a synthetic pathway in which formate is first reduced to formaldehyde, which is then condensed to form dihydroxyacetone (Figure 3b) [76]. To catalyze the key reaction of condensing three formaldehyde molecules, a formolase enzyme was computationally designed, engineered, and was found to be active in vitro and in vivo [76]. The resulting formate assimilation pathway should be at least as efficient as the reductive glycine pathway in converting formate to biomass and downstream products, and its main advantage over this latter pathway is that it operates under very high thermodynamic motive force. Such high motive force minimizes backward flux [66] and hence could support a high rate of formate metabolism. Furthermore, the pathway is completely linear and its intermediates do not interact with central metabolism, Current Opinion in Chemical Biology 2016, 35:1–9

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Proposed synthetic formate assimilation pathways. (a) The reductive glycine pathway, composed of only existing enzymes. The glycine cleavage system is shown in blue. THF corresponds to tetrahydrofolate and LP to lipoprotein, that is, H-protein of the glycine cleavage system. (b) The formose–dihydroxyacetone pathway, harboring an evolved enzyme that condenses three formaldehyde molecules to dihydroxyacetone. (c) The formose–glycoladlehyde pathway, in which the condensation of formaldehyde lead to the formation of glycolaldehyde.

making it easy to implement in vivo without considerable regulatory barriers. Unfortunately, the formolase enzyme is currently too slow to support sustainable formate assimilation. Hopefully, the catalytic efficiency of the enzyme can be improved using in vitro optimization cycles or in vivo selection techniques. A more troubling issue is that formaldehyde, its essential intermediate, is a toxic compound that can inhibit cell growth even at low, sub-mM concentrations [77]. Aggravating this problem is the fact that the kinetics of the formolase reaction has triple dependence on the concentration of formaldehyde (owing to the three reacting formaldehydes), suggesting that to support a sustainable rate of formaldehyde condensation, high concentrations will be required. In fact, it was found that at low, more physiologically-reasonable concentration of formaldehyde (0.2 mM), the main product of formaldehyde condensation is glycolaldehyde, whose assimilation to central metabolism is less efficient than that of dihydroxyacetone (requiring 6 ATP molecules, instead of four, for the production of triose phosphate, Figure 3c) [78]. Current Opinion in Chemical Biology 2016, 35:1–9

Confining the enzymes of the formolase-dependent pathway into a synthetically engineered microcompartment could potentially address the challenges associated with its activity [79,80]. Specifically, formate could diffuse into such a microcompartment to be reduced internally to formaldehyde, thereby protecting the surrounding cellular milieu from the toxicity of this intermediate (as is the case with microcompartments in which propionaldehyde is metabolized [81]). In parallel, production of formaldehyde in such close proximity to the formose enzyme could result in high local concentration of this substrate and thus facilitate the formaldehyde condensation reaction. Alternatively, a synthetic protein scaffold that keeps the pathway enzymes next to each other, thereby increasing the effective concentration of each intermediate, could serve to support higher pathway activity [82].

Conclusions Renewable energy is increasingly taking a larger share of the energy market and bio-refineries make up an everincreasing part in the production of value-added chemicals. Accordingly, the problems of efficient energy storage and sustainable microbial feedstock become more acute. www.sciencedirect.com

The formate bio-economy Yishai et al.

The formate bio-economy addresses these challenges by suggesting that excess energy can be utilized to generate formate which could then serve as a sole feedstock for cultivation of microbes and production of value-added chemicals. Yet, for this approach to become efficient and flexible in its product spectrum, model industrial organisms, such as E. coli and S. cerevisiae, need to be engineered to grow on formate using synthetic, specially tailored pathways. Although at least two such pathways were previously suggested and partially analyzed, a completely functional synthetic formate-assimilation route has yet to be constructed. Indeed, it is more than possible that the most promising strategies for formate assimilation are yet to be discovered. It is our hope that this review will serve to attract enzyme and metabolic engineers alike to address the challenge of microbial formate assimilation.

Acknowledgements The authors thank Avi Flamholz and Elad Noor for productive discussions and critique reading of the manuscript. A.B.-E. is funded by the Max Planck Society.

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