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Solar-to-chemical and solar-to-fuel production from CO2 by metabolically engineered microorganisms
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ScienceDirect Solar-to-chemical and solar-to-fuel production from CO2 by metabolically engineered microorganisms Han Min Woo Recent development of carbon capture utilization (CCU) for reduction of greenhouse gas emission are reviewed. In the case of CO2 utilization, I describe development of solar-to-chemical and solar-to-fuel technology that refers to the use of solar energy to convert CO2 to desired chemicals and fuels. Photoautotrophic cyanobacterial platforms have been extensively developed on this principle, producing a diverse range of alcohols, organic acids, and isoprenoids directly from CO2. Recent breakthroughs in the metabolic engineering of cyanobacteria were reviewed. In addition, adoption of the light harvesting mechanisms from nature, photovoltaics-derived water splitting technologies have recently been integrated with microbial biotechnology to produce desired chemicals. Studies on the integration of electrode material with next-generation microbes are showcased for alternative solar-to-chemical and solar-to-fuel platforms. Address Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea Corresponding author: Woo, Han Min ([email protected])
Current Opinion in Biotechnology 2017, 45:1–7 This review comes from a themed issue on Energy biotechnology Edited by Scott Banta and Brian Pfleger
http://dx.doi.org/10.1016/j.copbio.2016.11.017 0958-1669/Published by Elsevier Ltd.
Introduction The direct conversion of carbon dioxide to chemicals and fuels presents a sustainable solution for reducing greenhouse gas emissions and sustaining our supply of energy [1]. Ultimately, solar energy must be used for CO2 reduction and conversions to provide a sustainable system, and this system is now available in the forms of solar-tochemical (S2C) and solar-to-fuels (S2F) technologies. Thus, the S2C and S2F technology must be developed to capture and convert the essential feedstocks using only three inputs (CO2, H2O, and solar energy) to produce the desired value-added chemicals and fuels (Figure 1). In the post-genomic era, photosynthetic organisms (including cyanobacteria) have been engineered to produce www.sciencedirect.com
value-added chemicals, providing a number of promising S2C and S2F platforms. In addition to engineering photosynthetic organisms, improving natural systems of capturing solar energy and converting CO2 has motivated the development of inorganic-based S2C and S2F technologies. Electro-catalytic conversion of CO2 has been shown to produce methane and methanol [2] and efficient solar water-splitting (hydrolysis) using a photocatalyst has also been developed [3]. However, catalyst-based S2C and S2F technology only, has proven inadequate to complete biological CO2 conversion systems with carbon-carbon bond formation, high specificity, and low-cost materials. Moreover, such systems lack the properties of self-replication and self-repair. Thus, hybrid systems comprising an electrochemical in situ hydrogen-evolution reaction at the electrode and the biological CO2 fixation using autotrophic bacteria have been suggested as an alternative S2C and S2F platform. The purpose of this review is to summarize the recent literature on S2C and S2F technology to produce desired products from CO2 and to describe their potential role in bioenergy applications using next-generation microbebased technologies. The details on the platforms for S2C and S2F are shown in Figure 2.
Metabolic engineering of photoautotrophs for solar-to-chemicals (S2C) Photoautotrophic bacteria (and cyanobacteria) are promising microbial platforms for continuous production of biochemicals and biofuels from CO2 and light (carbon and energy sources, respectively). This is because the practical maximum efficiency of the direct CO2 conversion process with cyanobacteria is seven-fold higher that of an algal open pond in terms of photon loss [4]. Furthermore, metabolic engineering of cyanobacteria has been focused for direct production, product secretion, and process optimization. Reviews on several types of engineered cyanobacteria have been described. The focus has been on chemical and fuel production by improving native pathways (e.g., CO2 fixation) and by introducing heterologous pathways [5,6]. A discussion from a systems biology perspective [7] has been provided as well. Recently, cyanobacteria subjected to pathway engineering have enabled increases in the overall productivity of 2,3-butanediol after feeding with glucose or xylose under diurnal conditions [8]. To do so, heterologous expressions of galactose transporter (GalP) or xylose transporter (XylE), xylose isomerase (XylA), and xylulokinase (XylB) Current Opinion in Biotechnology 2017, 45:1–7
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Figure 1
Prism of Solar-to-Chemical & -Fuels
hυ
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(a), (b), (c), (d), (e) Platforms Current Opinion in Biotechnology
Scheme of a solar-to-chemical and solar-to-fuel platform as a prism that disperses light: The solar-to-chemical and solar-to-fuel technologies enable next-generation microbe-based biological systems or hybrid systems (integration of material with biology) capturing and converting the essential feedstocks (using only three inputs: CO2, H2O, and solar energy) to produce desired value-added chemicals and fuels. The details on solar-to-chemical and solar-to-fuel technologies are listed as (a), (b), (c), (d), (e) in Figure 2.
from Escherichia coli to a 2,3-butanediol-producing species of cyanobacterium were necessary and resulted in continuous production of 2,3-butanediol in the dark phase (after a light phase). In this case, sixty-seven percent carbon was derived from glucose utilization, based on 13 C-U-labled glucose analysis. In other work, photomixotrophic production of D-lactic acid (2.17 g/L) [9] and ethylene (821 52 mL/L/hour) [10], in engineered cyanobacteria under constant light, were increased 1.9-fold and 1.6-fold (respectively), compared to autotrophic production of D-lactic acid and of ethylene. According to both 13 C-labled bicarbonate and 12C-U-labled xylose, 50% of the carbon was derived from xylose utilization, in the tested mixotrophic condition. The productivities and titers of interest were increased with additional sugar carbon sources, but life cycle assessment of the mixotrophic growth of cyanobacteria and their production could be analyzed in terms of net reduction rate of CO2 emissions. Because the cellular activities in phototropic cyanobacteria and target-chemical product-formation are strongly affected by the presence and absence of light, metabolic and genetic regulatory aspects of cyanobacteria under diurnal conditions have been intensively studied [11,12]. This knowledge will be useful for engineering carbon and energy metabolism adjustments in cyanobacteria to improve the cyanobacterial S2C and S2F platform [13]. Importantly, many details of cyanobacterial carbon metabolism have been elucidated to reveal their complexity and plasticity, but the functions of many genes remain unknown. The presence of 2-oxoglutarate decarboxylase and succinate semialdehyde dehydrogenase activities have been proven to complete the tricarboxylic acid cycle (TCA) [14], and the presence of the Entner– Doudoroff pathway [15], glyoxylate cycle [16], and Current Opinion in Biotechnology 2017, 45:1–7
gamma-aminobutyric acid shunt [17] have been validated in cyanobacteria. With this knowledge, further metabolic engineering to reroute carbon flux via synthetic pathways, blocking flux of competing pathways, and reinforcing the native flux can be provided to re-direct the flow of fixed carbon to formation of the desired product. For instance, ethylene productivity from CO2 (10% of fixed carbons) was enhanced 3.9-fold over production (specific ethylene productivity at 718 19 mL/L/hour/OD730) by the baseline strain by rewiring the bifurcated TCA cycle using expression of the efe gene to create a cyclic TCA cycle [18]. The remodeled TCA cycle in engineered cyanobacteria showed remarkable metabolic flexibility for carbon metabolism, redirecting carbon flux from 13% total fixed carbon to 37% total fixed carbon, and 10% of the fixed carbon portion to ethylene [18]. In addition, a functional phosphoketolase (PHK) pathway added to cyanobacteria has been characterized and the PHK pathway confers flexibility in energy and carbon metabolism [19]. Furthermore, engineering cyanobacteria with the PHK pathway enhanced the photosynthetic production of n-butanol [20] and acetone [21] by increasing the levels of acetyl-coA. Besides pathway engineering, protein engineering combined with metabolic engineering of cyanobacteria has allowed substantial increase in production of isoprenederived chemicals from CO2. Overexpression of the phycocyanin b-subunit-fused to b-phellandrene synthase (CpcBPHLS) under the control of the native cpc promoter in Synechocystis sp. PCC 6803, led to accumulation of recombinant fusion proteins up to 20% of total cell protein and enhanced the production levels of b-phellandrene (3.2 mg/gDCW in 48 hour) by 100-fold, compared to sole expression of PHLS under the same promoter [22]. High levels of PHLS with slow catalytic turnover could be helpful for competing with other GPP-utilizing native enzymes. In addition, co-expression of the heterologous mevalonate pathway enzyme and geranyl diphosphate synthase with the fusion protein, significantly improved b-phellandrene production (10 mg/gDCW in 48 hour) from CO2 by favoring the isopentenyl diphosphate pathway over the methylerythritol 4-phosphate (MEP) pathway, resulting in a 1% carbon-partitioning ratio (product: biomass) [23]. Although regulation of the native MEP pathway in cyanobacteria remains unknown, the MEP pathway is an energetically balanced and efficient pathway for microbial production of isoprenoids [24]. Recent engineering of the MEP pathway and protein engineering in cyanobacteria resulted in substantial levels of isoprene production (1.26 g/L) in 21 days and calculations indicated about 40% of the photosynthetically fixed carbon flux flowed into the isoprene production pathway [25] since photosynthetic isoprene production has been first reported in engineered Synechocystis [26]. Based on the kinetic flux profiling analysis, overexpression of the fused enzyme of Saccharomyces cerevisiae IDI www.sciencedirect.com
Solar-to-chemical and solar-to-fuel production from CO2 Woo 3
Figure 2
(a) Engineered photoautotroph CO2
Input hv CO2 H 2O
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(b) Engineered electroautotroph Potentially engineered Moorella thermoacetica
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(e) Solar-Microbial electrosynthesis with bacteria CO2
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e
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Current Opinion in Biotechnology
Summary of current solar-to-chemical and solar-to-fuel platforms that convert CO2 with solar energy (hn) to produce desired value-added chemicals and fuels: Blue symbols are essential inputs and outputs. Red arrows indicate synthetic biological pathways to produce desired products in nextgeneration microbes. Inset: Essential three inputs (solar energy, CO2, and water) are converted to outputs (desired product) via synthetic biological www.sciencedirect.com
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with Populus alba IspS, increased the DMAPP/IPP ratio and the rate of IPP conversion to isoprene through substrate channeling. Simultaneously, another overexpression of Dxs and IspG of Thermosynechococcus elongatus allowed optimization of the MEP pathway and improved isoprene production. However, the scientific community needs to verify the work for the substantial amount of isoprene production from CO2. In addition, overexpression of IspA enzyme also has relieved a bottleneck in the MEP pathway for the production of farnesyl diphosphatederived isoprenoid (19.8 mg/L amorpha-4,11-diene or 4.98 mg/L/OD730 squalene) in cyanobacteria [27]. In order to construct cyanobacterial platforms that make S2C production economically feasible, metabolic engineering of photoautotrophic cyanobacteria needs to be developed such that production exceeds the one-digit g/L scale [28]. Tight and tunable gene expression systems are necessary to construct efficient cyanobacterial platforms. Recent studies on synthetic biology of cyanobacteria have been reviewed [29,30]. In addition, dynamic-TetR-regulated promoters [31], a controllable synthetic promoter library [32], a green-light inducible gene expression systems [33], a theophylline-dependent riboswitch [34], a trans-acting small RNA-based gene expression system [35], and oxygen-responsive genetic circuits [36] have been developed extensively and could be applied to additional metabolic engineering. Moreover, the CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease) has been adapted for targeted gene editing [37] and controlling [38,39] in cyanobacteria. Expression of Cas9, and a synthetic guide RNA with editing template, improved gene deletion efficiency because 100% successful mutants in the first patch (using conjugation transformation and transient expression of toxic Cas9) [37]. Subsequently, the CRISRPR-Cas9 system allowed efficient marker-less genome editing in cyanobacteria. For multiple gene repression in cyanobacteria, customized CRISPR interference systems resulted in inducible and reversible repression of target genes at 50–95% repression, and also enabled multiple repression for different target genes simultaneously, without losing the repression capabilities [38]. In addition to the CRISPR/Cas9 system, identification of the essential gene set of
cyanobacteria and its transposon mutants [40] could be knowledge useful for understanding the core biological processes and metabolic engineering of cyanobacteria for development of the S2C and S2F platform.
Integrated bio-electrochemical systems with engineered chemolithoautotrophs for solarto-chemicals (S2C) Chemolithoautotrophic bacteria are non-phototrophic, CO2-utilizing microorganisms that oxidize dihydrogen (H2) or metabolically accept electrons for reduction. Gas fermentation and metabolic engineering of the chemolithoautotrophic strains have been discussed in terms of C1-carbon sources [41] and production of fuels and chemicals [42], as will be discussed in more detail below. The role of CO2 fixation through various metabolic pathways in chemolithoautotrophs can be substituted into the ‘dark reactions’ of the photoautotrophs. In order to complete the S2C and S2F platform, the other side, the ‘light reactions’, must also be developed to provide for reduction in the absence of photosynthesis. Electricity from renewable energy sources (e.g., wind, hydroelectric) can be directly used as an energy source for electron-utilizing autotrophic acetogen strains. However, metabolic engineering of the acetogenic bacteria and microbial nanowires must be further developed to produce desired electro-biocommodities from CO2 [43]. Recent development of a hybrid system based upon a self-photosensitizing, non-photosynthetic form of the bacterium Moorella thermoacetica, has produced acetate from CO2 via the Wood–Ljundgdahl pathway with reduction equivalent. In this case, a reducing equivalent, [H] was generated by electron through the absorption of a photon by CdS [44]. To overcome the limitations of engineering CO2-utilizing hosts in a hybrid system, a secondary engineered host has been introduced to produce value-added chemicals via synthetic metabolic pathways [45]. Interaction of light-absorbing Si nanowire arrays (semiconductor nanodevices) with an anaerobic bacterium (acetogenic Sporomusa ovata) produced acetate at a high reaction rate of CO2 reduction, and with selective control of mass transport within the nano-wire, under exhaust-gas or open-air operating conditions. Electrosynthetic acetate was used as a common biosynthetic
(Figure 2 Legend Continued) pathways. (a) Metabolic engineering of photoautotrophs (including cyanobacteria) has allowed CO2 fixation via the Calvin cycle and production of desired chemicals via synthetic pathways [5,28]. (b) The Wood–Ljungdahl pathway (WLP)-mediated CO2-fixing bacterium (Moorella thermoacetica) with semiconductor nanoparticle (Cadmium sulfide: CdS) has been able to produce acetate via direct photoelectron transfer [44]. Further metabolic engineering of electrotrophs could produce desired products from CO2. (c) A hybrid bio-inorganic approach has allowed mimicking natural photosynthesis in which solar energy drives production of hydrogen by water splitting at the cathode, and the anaerobic bacterium Methanosarcina barkeri produces methane from H2 and CO2 via the CH4 biosynthetic pathway in a separate cathode chamber [46]. Further metabolic engineering of anaerobic CO2-fixing bacteria could be applied to produce value-added chemicals from H2 and CO2 in the cathode chamber. (d) Another hybrid approach of solar-to-chemicals/fuels in a one-pot reactor has produced various alcohols and bio-polymers from H2 and CO2 [47,48]. Co–P alloy allowed hydrogen production via water-splitting and biocompatible cell growth. Metabolically-engineered chemolithoautotrophs (e.g., Ralstonia eutropha) have been used to produce various desired products in a hybrid microbial-catalyst system. (e) Nanowire-bacteria hybrids have been used to produce various desired chemicals [45]. The acetogenic bacterium Sporomusa ovata, converts CO2 via direct electron transfer and produces acetate. Electrosynthetic acetate was used as carbon and energy source for engineered chemoheterotrophs where various desired products were produced via synthetic pathways. Current Opinion in Biotechnology 2017, 45:1–7
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feedstock for engineered E. coli that produced n-butanol, PHB polymer, and isoprenoid (including amorpha-4,-11diene). On the other hand, a solar-driven photo(electro)chemical hydrogen evolution reaction can generate hydrogen in a sustainable reduction process to a microorganism for CO2 fixation and conversion. Without external electrical bias and/or sacrificial chemical quenchers, a hybrid bioinorganic system that comprises a biocompatible hydrogen evolution reaction electrocatalyst (nanoparticulate nickel sulfide; a-NiS) and conversion of CO2 by Methanosarcina barkeri, allowed production of methane (110 mL) with 86% overall Faradaic efficiency for over 7 days [46]. Yet, metabolic engineering of M. barkeri and M. thermoacetica has not yet been developed for production of target chemicals. Recently, a scalable bio-electrochemical (artificial photosynthetic) system has been developed based on watersplitting (hydrolysis) catalyst system integrated with an engineered microbial system to capture solar energy and to convert CO2 (along with H2 and O2) into desired alcohols or other substances [47,48]. Optimization of inorganic materials at anode (cobalt phosphate, CoPi) and cathode (nickel molybdenum zinc, NiMoZn; or stainlesssteel, SS) allowed bacterial cell growth at Ecell = 2.3–3.0 V electric potential in a single-cell configuration by oxidizing H2 and fixing CO2 in a chloride-free medium at pH 7.0 with 36 mM phosphate [47]. Subsequently, the bioelectrochemical cell (Ecell = 2.7 V, 2.6 mA/cm2 for SS cathode) with engineered chemolithoautotrophic Ralstonia eutropha that was genetically modified by expressing genes for a ketothiolase (PhaA), acetoacetyl-coA transferase (Ctf), acetoacetate decarboxylase (Adc), and alcohol dehydrogenase (Adh) were able to produce 216 mg/L isopropanol in 5 days from CO2. However, the ROS toxicity (mainly H2O2 that is produced at the cathode via oxidation of oxygen), and leaching of Ni from the NiMoZn in the bio-electrochemical system, caused critical growth inhibition. In addition, a hybrid hydrolysisbacterial system was further optimized to develop a biocompatible catalyst system by replacing a NiMoZn cathode with a ROS resistant cobalt-phosphorus (Co–P) alloy, resulting in minimal accumulation of H2O2 while H2 evolution reactions (HER) occur [48] at lower Ecell = 2.2 than previously (Ecell = 2.7). Combined with metabolic engineering of R. eutropha, this hybrid system achieved a CO2 reduction energy efficiency of over 10% for biomass, bio-plastic (poly(3-hydroxybutyrate), PHB), and fusel alcohols, which was calculated to exceed the production of natural photosynthetic systems. Also, this Co–P|CoPi|R. eutropha hybrid system was shown to behave as artificial cyanobacteria for S2C from CO2. It was intrinsically compatible with diurnal conditions, depending on H2 generation in the day time (photovoltaics requires solar energy). Metabolic engineering of www.sciencedirect.com
R. eutropha has progressed to produce methyl ketone [49] and 2-hdyroxyisobutyric acid [50] from CO2 and H2, which can be integrated to a hybrid hydrolytic-bacterial system. In addition to hydrogen, photosynthetic formate from CO2 can be used as another electron donor and carbon source for the S2C and S2F platform. A CuFeO2 and CuO mixed p-type catalyst at the cathode has generated photosynthetic formate with 1% energy efficiency for 1 week without any external bias [51]. Similarly, an integrated electromicrobial system using engineered R. eutropha has produced a variety of higher alcohols from electrosynthetic formate that was generated at the Indium foil cathode [52]. Further applications of integrated microbial systems will be developed with electrosynthetic formate as sole carbon and energy source.
Conclusions In this paper, I review the current status of solar-tochemical and solar-to-fuels platforms for production of value-added chemicals from CO2, focusing on engineering of photosynthetic organisms and developing microbewater splitting catalyst systems. Synthetic biologyinspired metabolic engineering of next-generation microbes will be established to accommodate more efficient S2C and S2F platforms. Beyond the proof-of-concept work on these platforms, further development for industrial scale-up with input from engineering disciplines will also be necessary to succeed in creating a post-oil-refinery society in a sustainable and economic manner.
Acknowledgements I would like to thank Prof. Dr. Sang Jun Sim at Korea University, Dr. Byoung Koun Min, Dr. Youngsoon Um, Dr. Yun Jeong Hwang, and Dr. Sun-Mi Lee at Korea Institute of Science and Technology (KIST) for helpful discussions. This work was supported by Korea CCS R&D Center (KCRC) (Grant No. 2014M1A8A1049277) funded by the Korean Government (Ministry of Science, Information and Communications Technology (ICT) & Future Planning).
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38. Yao L, Cengic I, Anfelt J, Hudson EP: Multiple gene repression in cyanobacteria using CRISPRi. ACS Synth Biol 2016, 5:207-212. In this work, multiple gene repression using CRISPR interference technology was achieved by repressing the target genes at 50–90% levels in Synechocystis sp. PCC 6803. This paper also showed that tightly repressed promoters allowed for tunable and reversible gene expression. 39. Gordon GC, Korosh TC, Cameron JC, Markley AL, Begemann MB, Pfleger BF: CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. Metab Eng 2016, 38:170-179. 40. Rubin BE, Wetmore KM, Price MN, Diamond S, Shultzaberger RK, Lowe LC, Curtin G, Arkin AP, Deutschbauer A, Golden SS: The essential gene set of a photosynthetic organism. Proc Natl Acad Sci U S A 2015, 112:E6634-E6643. 41. Durre P, Eikmanns BJ: C1-carbon sources for chemical and fuel production by microbial gas fermentation. Curr Opin Biotechnol 2015, 35:63-72. 42. Nybo SE, Khan NE, Woolston BM, Curtis WR: Metabolic engineering in chemolithoautotrophic hosts for the production of fuels and chemicals. Metab Eng 2015, 30:105-120. 43. Guo K, Prevoteau A, Patil SA, Rabaey K: Engineering electrodes for microbial electrocatalysis. Curr Opin Biotechnol 2015, 33:149-156. 44. Sakimoto KK, Wong AB, Yang P: Self-photosensitization of nonphotosynthetic bacteria for solar-to-chemical production. Science 2016, 351:74-77. 45. Liu C, Gallagher JJ, Sakimoto KK, Nichols EM, Chang CJ, Chang MC, Yang P: Nanowire-bacteria hybrids for unassisted solar carbon dioxide fixation to value-added chemicals. Nano Lett 2015, 15:3634-3639. 46. Nichols EM, Gallagher JJ, Liu C, Su Y, Resasco J, Yu Y, Sun Y, Yang P, Chang MC, Chang CJ: Hybrid bioinorganic approach to solar-to-chemical conversion. Proc Natl Acad Sci U S A 2015, 112:11461-11466.
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In this work, a novel approach to solar-to-fuel was suggested to produce methane from CO2 using a biocompatible inorganic component and a methanotroph with 86% overall faradaic efficiency. The artificial photosynthesis system produced 4.3 mmol of methane over 7 days. 47. Torella JP, Gagliardi CJ, Chen JS, Bediako DK, Colon B, Way JC, Silver PA, Nocera DG: Efficient solar-to-fuels production from a hybrid microbial-water-splitting catalyst system. Proc Natl Acad Sci U S A 2015, 112:2337-2342. This paper demonstrated the first hybrid application of a microbial-watersplitting catalyst system that enabled generation of hydrogen from water splitting and to produce various fusel alcohols from CO2 using metabolically-engineered Ralstonia eutropha in a bioelectrochemical reactor. The integrated set-up resulted in a solar-to-biomass efficiency of 3.2% that exceeded the efficiency of natural systems. 48. Liu C, Colon BC, Ziesack M, Silver PA, Nocera DG: Water splitting-biosynthetic system with CO(2) reduction efficiencies exceeding photosynthesis. Science 2016, 352:1210-1213. 49. Muller J, MacEachran D, Burd H, Sathitsuksanoh N, Bi C, Yeh YC, Lee TS, Hillson NJ, Chhabra SR, Singer SW et al.: Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones. Appl Environ Microbiol 2013, 79:4433-4439. 50. Przybylski D, Rohwerder T, Dilssner C, Maskow T, Harms H, Muller RH: Exploiting mixtures of H2, CO2, and O2 for improved production of methacrylate precursor 2-hydroxyisobutyric acid by engineered Cupriavidus necator strains. Appl Microbiol Biotechnol 2015, 99:2131-2145. 51. Kang U, Choi SK, Ham DJ, Ji SM, Choi W, Han DS, AbdelWahabe A, Park H: Photosynthesis of formate from CO2 and water at 1% energy efficiency via copper iron oxide catalysis. Energy Environ Sci 2015, 8:2638-2643. 52. Li H, Opgenorth PH, Wernick DG, Rogers S, Wu TY, Higashide W, Malati P, Huo YX, Cho KM, Liao JC: Integrated electromicrobial conversion of CO2 to higher alcohols. Science 2012, 335: 1596.
Current Opinion in Biotechnology 2017, 45:1–7
ScienceDirect Solar-to-chemical and solar-to-fuel production from CO2 by metabolically engineered microorganisms Han Min Woo Recent development of carbon capture utilization (CCU) for reduction of greenhouse gas emission are reviewed. In the case of CO2 utilization, I describe development of solar-to-chemical and solar-to-fuel technology that refers to the use of solar energy to convert CO2 to desired chemicals and fuels. Photoautotrophic cyanobacterial platforms have been extensively developed on this principle, producing a diverse range of alcohols, organic acids, and isoprenoids directly from CO2. Recent breakthroughs in the metabolic engineering of cyanobacteria were reviewed. In addition, adoption of the light harvesting mechanisms from nature, photovoltaics-derived water splitting technologies have recently been integrated with microbial biotechnology to produce desired chemicals. Studies on the integration of electrode material with next-generation microbes are showcased for alternative solar-to-chemical and solar-to-fuel platforms. Address Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea Corresponding author: Woo, Han Min ([email protected])
Current Opinion in Biotechnology 2017, 45:1–7 This review comes from a themed issue on Energy biotechnology Edited by Scott Banta and Brian Pfleger
http://dx.doi.org/10.1016/j.copbio.2016.11.017 0958-1669/Published by Elsevier Ltd.
Introduction The direct conversion of carbon dioxide to chemicals and fuels presents a sustainable solution for reducing greenhouse gas emissions and sustaining our supply of energy [1]. Ultimately, solar energy must be used for CO2 reduction and conversions to provide a sustainable system, and this system is now available in the forms of solar-tochemical (S2C) and solar-to-fuels (S2F) technologies. Thus, the S2C and S2F technology must be developed to capture and convert the essential feedstocks using only three inputs (CO2, H2O, and solar energy) to produce the desired value-added chemicals and fuels (Figure 1). In the post-genomic era, photosynthetic organisms (including cyanobacteria) have been engineered to produce www.sciencedirect.com
value-added chemicals, providing a number of promising S2C and S2F platforms. In addition to engineering photosynthetic organisms, improving natural systems of capturing solar energy and converting CO2 has motivated the development of inorganic-based S2C and S2F technologies. Electro-catalytic conversion of CO2 has been shown to produce methane and methanol [2] and efficient solar water-splitting (hydrolysis) using a photocatalyst has also been developed [3]. However, catalyst-based S2C and S2F technology only, has proven inadequate to complete biological CO2 conversion systems with carbon-carbon bond formation, high specificity, and low-cost materials. Moreover, such systems lack the properties of self-replication and self-repair. Thus, hybrid systems comprising an electrochemical in situ hydrogen-evolution reaction at the electrode and the biological CO2 fixation using autotrophic bacteria have been suggested as an alternative S2C and S2F platform. The purpose of this review is to summarize the recent literature on S2C and S2F technology to produce desired products from CO2 and to describe their potential role in bioenergy applications using next-generation microbebased technologies. The details on the platforms for S2C and S2F are shown in Figure 2.
Metabolic engineering of photoautotrophs for solar-to-chemicals (S2C) Photoautotrophic bacteria (and cyanobacteria) are promising microbial platforms for continuous production of biochemicals and biofuels from CO2 and light (carbon and energy sources, respectively). This is because the practical maximum efficiency of the direct CO2 conversion process with cyanobacteria is seven-fold higher that of an algal open pond in terms of photon loss [4]. Furthermore, metabolic engineering of cyanobacteria has been focused for direct production, product secretion, and process optimization. Reviews on several types of engineered cyanobacteria have been described. The focus has been on chemical and fuel production by improving native pathways (e.g., CO2 fixation) and by introducing heterologous pathways [5,6]. A discussion from a systems biology perspective [7] has been provided as well. Recently, cyanobacteria subjected to pathway engineering have enabled increases in the overall productivity of 2,3-butanediol after feeding with glucose or xylose under diurnal conditions [8]. To do so, heterologous expressions of galactose transporter (GalP) or xylose transporter (XylE), xylose isomerase (XylA), and xylulokinase (XylB) Current Opinion in Biotechnology 2017, 45:1–7
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Figure 1
Prism of Solar-to-Chemical & -Fuels
hυ
Spectrum of products
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(a), (b), (c), (d), (e) Platforms Current Opinion in Biotechnology
Scheme of a solar-to-chemical and solar-to-fuel platform as a prism that disperses light: The solar-to-chemical and solar-to-fuel technologies enable next-generation microbe-based biological systems or hybrid systems (integration of material with biology) capturing and converting the essential feedstocks (using only three inputs: CO2, H2O, and solar energy) to produce desired value-added chemicals and fuels. The details on solar-to-chemical and solar-to-fuel technologies are listed as (a), (b), (c), (d), (e) in Figure 2.
from Escherichia coli to a 2,3-butanediol-producing species of cyanobacterium were necessary and resulted in continuous production of 2,3-butanediol in the dark phase (after a light phase). In this case, sixty-seven percent carbon was derived from glucose utilization, based on 13 C-U-labled glucose analysis. In other work, photomixotrophic production of D-lactic acid (2.17 g/L) [9] and ethylene (821 52 mL/L/hour) [10], in engineered cyanobacteria under constant light, were increased 1.9-fold and 1.6-fold (respectively), compared to autotrophic production of D-lactic acid and of ethylene. According to both 13 C-labled bicarbonate and 12C-U-labled xylose, 50% of the carbon was derived from xylose utilization, in the tested mixotrophic condition. The productivities and titers of interest were increased with additional sugar carbon sources, but life cycle assessment of the mixotrophic growth of cyanobacteria and their production could be analyzed in terms of net reduction rate of CO2 emissions. Because the cellular activities in phototropic cyanobacteria and target-chemical product-formation are strongly affected by the presence and absence of light, metabolic and genetic regulatory aspects of cyanobacteria under diurnal conditions have been intensively studied [11,12]. This knowledge will be useful for engineering carbon and energy metabolism adjustments in cyanobacteria to improve the cyanobacterial S2C and S2F platform [13]. Importantly, many details of cyanobacterial carbon metabolism have been elucidated to reveal their complexity and plasticity, but the functions of many genes remain unknown. The presence of 2-oxoglutarate decarboxylase and succinate semialdehyde dehydrogenase activities have been proven to complete the tricarboxylic acid cycle (TCA) [14], and the presence of the Entner– Doudoroff pathway [15], glyoxylate cycle [16], and Current Opinion in Biotechnology 2017, 45:1–7
gamma-aminobutyric acid shunt [17] have been validated in cyanobacteria. With this knowledge, further metabolic engineering to reroute carbon flux via synthetic pathways, blocking flux of competing pathways, and reinforcing the native flux can be provided to re-direct the flow of fixed carbon to formation of the desired product. For instance, ethylene productivity from CO2 (10% of fixed carbons) was enhanced 3.9-fold over production (specific ethylene productivity at 718 19 mL/L/hour/OD730) by the baseline strain by rewiring the bifurcated TCA cycle using expression of the efe gene to create a cyclic TCA cycle [18]. The remodeled TCA cycle in engineered cyanobacteria showed remarkable metabolic flexibility for carbon metabolism, redirecting carbon flux from 13% total fixed carbon to 37% total fixed carbon, and 10% of the fixed carbon portion to ethylene [18]. In addition, a functional phosphoketolase (PHK) pathway added to cyanobacteria has been characterized and the PHK pathway confers flexibility in energy and carbon metabolism [19]. Furthermore, engineering cyanobacteria with the PHK pathway enhanced the photosynthetic production of n-butanol [20] and acetone [21] by increasing the levels of acetyl-coA. Besides pathway engineering, protein engineering combined with metabolic engineering of cyanobacteria has allowed substantial increase in production of isoprenederived chemicals from CO2. Overexpression of the phycocyanin b-subunit-fused to b-phellandrene synthase (CpcBPHLS) under the control of the native cpc promoter in Synechocystis sp. PCC 6803, led to accumulation of recombinant fusion proteins up to 20% of total cell protein and enhanced the production levels of b-phellandrene (3.2 mg/gDCW in 48 hour) by 100-fold, compared to sole expression of PHLS under the same promoter [22]. High levels of PHLS with slow catalytic turnover could be helpful for competing with other GPP-utilizing native enzymes. In addition, co-expression of the heterologous mevalonate pathway enzyme and geranyl diphosphate synthase with the fusion protein, significantly improved b-phellandrene production (10 mg/gDCW in 48 hour) from CO2 by favoring the isopentenyl diphosphate pathway over the methylerythritol 4-phosphate (MEP) pathway, resulting in a 1% carbon-partitioning ratio (product: biomass) [23]. Although regulation of the native MEP pathway in cyanobacteria remains unknown, the MEP pathway is an energetically balanced and efficient pathway for microbial production of isoprenoids [24]. Recent engineering of the MEP pathway and protein engineering in cyanobacteria resulted in substantial levels of isoprene production (1.26 g/L) in 21 days and calculations indicated about 40% of the photosynthetically fixed carbon flux flowed into the isoprene production pathway [25] since photosynthetic isoprene production has been first reported in engineered Synechocystis [26]. Based on the kinetic flux profiling analysis, overexpression of the fused enzyme of Saccharomyces cerevisiae IDI www.sciencedirect.com
Solar-to-chemical and solar-to-fuel production from CO2 Woo 3
Figure 2
(a) Engineered photoautotroph CO2
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Current Opinion in Biotechnology
Summary of current solar-to-chemical and solar-to-fuel platforms that convert CO2 with solar energy (hn) to produce desired value-added chemicals and fuels: Blue symbols are essential inputs and outputs. Red arrows indicate synthetic biological pathways to produce desired products in nextgeneration microbes. Inset: Essential three inputs (solar energy, CO2, and water) are converted to outputs (desired product) via synthetic biological www.sciencedirect.com
Current Opinion in Biotechnology 2017, 45:1–7
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with Populus alba IspS, increased the DMAPP/IPP ratio and the rate of IPP conversion to isoprene through substrate channeling. Simultaneously, another overexpression of Dxs and IspG of Thermosynechococcus elongatus allowed optimization of the MEP pathway and improved isoprene production. However, the scientific community needs to verify the work for the substantial amount of isoprene production from CO2. In addition, overexpression of IspA enzyme also has relieved a bottleneck in the MEP pathway for the production of farnesyl diphosphatederived isoprenoid (19.8 mg/L amorpha-4,11-diene or 4.98 mg/L/OD730 squalene) in cyanobacteria [27]. In order to construct cyanobacterial platforms that make S2C production economically feasible, metabolic engineering of photoautotrophic cyanobacteria needs to be developed such that production exceeds the one-digit g/L scale [28]. Tight and tunable gene expression systems are necessary to construct efficient cyanobacterial platforms. Recent studies on synthetic biology of cyanobacteria have been reviewed [29,30]. In addition, dynamic-TetR-regulated promoters [31], a controllable synthetic promoter library [32], a green-light inducible gene expression systems [33], a theophylline-dependent riboswitch [34], a trans-acting small RNA-based gene expression system [35], and oxygen-responsive genetic circuits [36] have been developed extensively and could be applied to additional metabolic engineering. Moreover, the CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease) has been adapted for targeted gene editing [37] and controlling [38,39] in cyanobacteria. Expression of Cas9, and a synthetic guide RNA with editing template, improved gene deletion efficiency because 100% successful mutants in the first patch (using conjugation transformation and transient expression of toxic Cas9) [37]. Subsequently, the CRISRPR-Cas9 system allowed efficient marker-less genome editing in cyanobacteria. For multiple gene repression in cyanobacteria, customized CRISPR interference systems resulted in inducible and reversible repression of target genes at 50–95% repression, and also enabled multiple repression for different target genes simultaneously, without losing the repression capabilities [38]. In addition to the CRISPR/Cas9 system, identification of the essential gene set of
cyanobacteria and its transposon mutants [40] could be knowledge useful for understanding the core biological processes and metabolic engineering of cyanobacteria for development of the S2C and S2F platform.
Integrated bio-electrochemical systems with engineered chemolithoautotrophs for solarto-chemicals (S2C) Chemolithoautotrophic bacteria are non-phototrophic, CO2-utilizing microorganisms that oxidize dihydrogen (H2) or metabolically accept electrons for reduction. Gas fermentation and metabolic engineering of the chemolithoautotrophic strains have been discussed in terms of C1-carbon sources [41] and production of fuels and chemicals [42], as will be discussed in more detail below. The role of CO2 fixation through various metabolic pathways in chemolithoautotrophs can be substituted into the ‘dark reactions’ of the photoautotrophs. In order to complete the S2C and S2F platform, the other side, the ‘light reactions’, must also be developed to provide for reduction in the absence of photosynthesis. Electricity from renewable energy sources (e.g., wind, hydroelectric) can be directly used as an energy source for electron-utilizing autotrophic acetogen strains. However, metabolic engineering of the acetogenic bacteria and microbial nanowires must be further developed to produce desired electro-biocommodities from CO2 [43]. Recent development of a hybrid system based upon a self-photosensitizing, non-photosynthetic form of the bacterium Moorella thermoacetica, has produced acetate from CO2 via the Wood–Ljundgdahl pathway with reduction equivalent. In this case, a reducing equivalent, [H] was generated by electron through the absorption of a photon by CdS [44]. To overcome the limitations of engineering CO2-utilizing hosts in a hybrid system, a secondary engineered host has been introduced to produce value-added chemicals via synthetic metabolic pathways [45]. Interaction of light-absorbing Si nanowire arrays (semiconductor nanodevices) with an anaerobic bacterium (acetogenic Sporomusa ovata) produced acetate at a high reaction rate of CO2 reduction, and with selective control of mass transport within the nano-wire, under exhaust-gas or open-air operating conditions. Electrosynthetic acetate was used as a common biosynthetic
(Figure 2 Legend Continued) pathways. (a) Metabolic engineering of photoautotrophs (including cyanobacteria) has allowed CO2 fixation via the Calvin cycle and production of desired chemicals via synthetic pathways [5,28]. (b) The Wood–Ljungdahl pathway (WLP)-mediated CO2-fixing bacterium (Moorella thermoacetica) with semiconductor nanoparticle (Cadmium sulfide: CdS) has been able to produce acetate via direct photoelectron transfer [44]. Further metabolic engineering of electrotrophs could produce desired products from CO2. (c) A hybrid bio-inorganic approach has allowed mimicking natural photosynthesis in which solar energy drives production of hydrogen by water splitting at the cathode, and the anaerobic bacterium Methanosarcina barkeri produces methane from H2 and CO2 via the CH4 biosynthetic pathway in a separate cathode chamber [46]. Further metabolic engineering of anaerobic CO2-fixing bacteria could be applied to produce value-added chemicals from H2 and CO2 in the cathode chamber. (d) Another hybrid approach of solar-to-chemicals/fuels in a one-pot reactor has produced various alcohols and bio-polymers from H2 and CO2 [47,48]. Co–P alloy allowed hydrogen production via water-splitting and biocompatible cell growth. Metabolically-engineered chemolithoautotrophs (e.g., Ralstonia eutropha) have been used to produce various desired products in a hybrid microbial-catalyst system. (e) Nanowire-bacteria hybrids have been used to produce various desired chemicals [45]. The acetogenic bacterium Sporomusa ovata, converts CO2 via direct electron transfer and produces acetate. Electrosynthetic acetate was used as carbon and energy source for engineered chemoheterotrophs where various desired products were produced via synthetic pathways. Current Opinion in Biotechnology 2017, 45:1–7
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Solar-to-chemical and solar-to-fuel production from CO2 Woo 5
feedstock for engineered E. coli that produced n-butanol, PHB polymer, and isoprenoid (including amorpha-4,-11diene). On the other hand, a solar-driven photo(electro)chemical hydrogen evolution reaction can generate hydrogen in a sustainable reduction process to a microorganism for CO2 fixation and conversion. Without external electrical bias and/or sacrificial chemical quenchers, a hybrid bioinorganic system that comprises a biocompatible hydrogen evolution reaction electrocatalyst (nanoparticulate nickel sulfide; a-NiS) and conversion of CO2 by Methanosarcina barkeri, allowed production of methane (110 mL) with 86% overall Faradaic efficiency for over 7 days [46]. Yet, metabolic engineering of M. barkeri and M. thermoacetica has not yet been developed for production of target chemicals. Recently, a scalable bio-electrochemical (artificial photosynthetic) system has been developed based on watersplitting (hydrolysis) catalyst system integrated with an engineered microbial system to capture solar energy and to convert CO2 (along with H2 and O2) into desired alcohols or other substances [47,48]. Optimization of inorganic materials at anode (cobalt phosphate, CoPi) and cathode (nickel molybdenum zinc, NiMoZn; or stainlesssteel, SS) allowed bacterial cell growth at Ecell = 2.3–3.0 V electric potential in a single-cell configuration by oxidizing H2 and fixing CO2 in a chloride-free medium at pH 7.0 with 36 mM phosphate [47]. Subsequently, the bioelectrochemical cell (Ecell = 2.7 V, 2.6 mA/cm2 for SS cathode) with engineered chemolithoautotrophic Ralstonia eutropha that was genetically modified by expressing genes for a ketothiolase (PhaA), acetoacetyl-coA transferase (Ctf), acetoacetate decarboxylase (Adc), and alcohol dehydrogenase (Adh) were able to produce 216 mg/L isopropanol in 5 days from CO2. However, the ROS toxicity (mainly H2O2 that is produced at the cathode via oxidation of oxygen), and leaching of Ni from the NiMoZn in the bio-electrochemical system, caused critical growth inhibition. In addition, a hybrid hydrolysisbacterial system was further optimized to develop a biocompatible catalyst system by replacing a NiMoZn cathode with a ROS resistant cobalt-phosphorus (Co–P) alloy, resulting in minimal accumulation of H2O2 while H2 evolution reactions (HER) occur [48] at lower Ecell = 2.2 than previously (Ecell = 2.7). Combined with metabolic engineering of R. eutropha, this hybrid system achieved a CO2 reduction energy efficiency of over 10% for biomass, bio-plastic (poly(3-hydroxybutyrate), PHB), and fusel alcohols, which was calculated to exceed the production of natural photosynthetic systems. Also, this Co–P|CoPi|R. eutropha hybrid system was shown to behave as artificial cyanobacteria for S2C from CO2. It was intrinsically compatible with diurnal conditions, depending on H2 generation in the day time (photovoltaics requires solar energy). Metabolic engineering of www.sciencedirect.com
R. eutropha has progressed to produce methyl ketone [49] and 2-hdyroxyisobutyric acid [50] from CO2 and H2, which can be integrated to a hybrid hydrolytic-bacterial system. In addition to hydrogen, photosynthetic formate from CO2 can be used as another electron donor and carbon source for the S2C and S2F platform. A CuFeO2 and CuO mixed p-type catalyst at the cathode has generated photosynthetic formate with 1% energy efficiency for 1 week without any external bias [51]. Similarly, an integrated electromicrobial system using engineered R. eutropha has produced a variety of higher alcohols from electrosynthetic formate that was generated at the Indium foil cathode [52]. Further applications of integrated microbial systems will be developed with electrosynthetic formate as sole carbon and energy source.
Conclusions In this paper, I review the current status of solar-tochemical and solar-to-fuels platforms for production of value-added chemicals from CO2, focusing on engineering of photosynthetic organisms and developing microbewater splitting catalyst systems. Synthetic biologyinspired metabolic engineering of next-generation microbes will be established to accommodate more efficient S2C and S2F platforms. Beyond the proof-of-concept work on these platforms, further development for industrial scale-up with input from engineering disciplines will also be necessary to succeed in creating a post-oil-refinery society in a sustainable and economic manner.
Acknowledgements I would like to thank Prof. Dr. Sang Jun Sim at Korea University, Dr. Byoung Koun Min, Dr. Youngsoon Um, Dr. Yun Jeong Hwang, and Dr. Sun-Mi Lee at Korea Institute of Science and Technology (KIST) for helpful discussions. This work was supported by Korea CCS R&D Center (KCRC) (Grant No. 2014M1A8A1049277) funded by the Korean Government (Ministry of Science, Information and Communications Technology (ICT) & Future Planning).
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Solar-to-chemical and solar-to-fuel production from CO2 Woo 7
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In this work, a novel approach to solar-to-fuel was suggested to produce methane from CO2 using a biocompatible inorganic component and a methanotroph with 86% overall faradaic efficiency. The artificial photosynthesis system produced 4.3 mmol of methane over 7 days. 47. Torella JP, Gagliardi CJ, Chen JS, Bediako DK, Colon B, Way JC, Silver PA, Nocera DG: Efficient solar-to-fuels production from a hybrid microbial-water-splitting catalyst system. Proc Natl Acad Sci U S A 2015, 112:2337-2342. This paper demonstrated the first hybrid application of a microbial-watersplitting catalyst system that enabled generation of hydrogen from water splitting and to produce various fusel alcohols from CO2 using metabolically-engineered Ralstonia eutropha in a bioelectrochemical reactor. The integrated set-up resulted in a solar-to-biomass efficiency of 3.2% that exceeded the efficiency of natural systems. 48. Liu C, Colon BC, Ziesack M, Silver PA, Nocera DG: Water splitting-biosynthetic system with CO(2) reduction efficiencies exceeding photosynthesis. Science 2016, 352:1210-1213. 49. Muller J, MacEachran D, Burd H, Sathitsuksanoh N, Bi C, Yeh YC, Lee TS, Hillson NJ, Chhabra SR, Singer SW et al.: Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones. Appl Environ Microbiol 2013, 79:4433-4439. 50. Przybylski D, Rohwerder T, Dilssner C, Maskow T, Harms H, Muller RH: Exploiting mixtures of H2, CO2, and O2 for improved production of methacrylate precursor 2-hydroxyisobutyric acid by engineered Cupriavidus necator strains. Appl Microbiol Biotechnol 2015, 99:2131-2145. 51. Kang U, Choi SK, Ham DJ, Ji SM, Choi W, Han DS, AbdelWahabe A, Park H: Photosynthesis of formate from CO2 and water at 1% energy efficiency via copper iron oxide catalysis. Energy Environ Sci 2015, 8:2638-2643. 52. Li H, Opgenorth PH, Wernick DG, Rogers S, Wu TY, Higashide W, Malati P, Huo YX, Cho KM, Liao JC: Integrated electromicrobial conversion of CO2 to higher alcohols. Science 2012, 335: 1596.
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