Trends in Biotechnology Special Issue: Bioconversion of C1 Products and Feedstocks Review Metabolic Engineering of necator H16 for Sustainable Biofuels from CO2

Justin Panich,1,*,@ Bonnie Fong,1 and Steven W. Singer 1,2,*

Decelerating global warming is one of the predominant challenges of our time and Highlights will require conversion of CO2 to usable products and commodity chemicals. Of Cupriavidus necator has a wide meta- particular interest is the production of fuels, because the transportation sector is bolic range and naturally creates a biopolymer, poly[(R)-3 hydroxybutyrate] a major source of CO2 emissions. Here, we review recent technological advances Cupriavidus necator (PHB). Using metabolic engineering in metabolic engineering of the hydrogen-oxidizing bacterium techniques, carbon flux can be directed H16, a chemolithotroph that naturally consumes CO2 to generate biomass. We away from PHB synthesis toward the discuss recent successes in biofuel production using this organism, and the imple- generation of biofuels and bioproducts. mentation of electrolysis/artificial photosynthesis approaches that enable growth C. necator Researchers demonstrated the produc- of using renewable electricity and CO2. Last, we discuss prospects of tion of many biofuel products using improving the nonoptimal growth of C. necator in ambient concentrations of CO2. C. necator, including methyl ketones, isoprenoids and terpenes, isobutanol, alkanes and alkenes, and a wide variety Mitigation of Atmospheric CO with Bioconversion 2 of commodity chemicals from CO2. Satisfying current and future global energy demand while allowing favorable environmental out- comes will require innovative solutions in the fuel sector. Although portions of the transportation Growth of C. necator and bioproduct infrastructure can be electrified (such as automobile transport), energy-dense carbon-based production using electrolysis was re- cently demonstrated, including the use fuels will continue to be required for aviation, long-distance trucking, rocketry, maritime shipping, of an artificial leaf system. and industrial operations. Petroleum-based fuels are finite, given that BP and the International Energy Agency (IEA) project that petroleum sources are likely to be depleted between 2050 While genetic engineering of C. necator and 2070 using current oil extraction technologiesi–iii. Climate change caused by an increase in remains a laborious process, synthetic biology tools for this organism are being ’ CO2 (and CH4) levels in Earth s atmosphere has brought cataclysmic weather events and has expanded with new technologies that decimated ocean habitats in recent years, a trend that will likely continue through our lifetimes will allow for large alterations to its genome. given that CO2 is being emitted into the atmosphere at a rate of ≈32 billion tons of CO2 per iv year [1–6] . Therefore, sustainable and economically viable bioconversion of CO2 to fuels and commodity chemicals should be realized within the next decade. A small but significant mitigation of anthropogenic CO2 release can be achieved by using CO2 from atmospheric air as a feedstock to produce biofuels in microorganisms: if 10% of global aviation fuel (accounting for ~2.6% of total

CO2 emissions [7]) were replaced with Sustainable Aviation Fuels (SAFs) from Cupriavidus necator at a titer of 1 g/l and assuming 90 g/l biomass accumulation [8], ~500 000 tons of sustainable fuel per year could be produced from recycled CO2 (at a carbon density of 0.8 kg/l).

In addition, the accumulated biomass would sequester ~80 million tons of CO2 per year (mitigating ≈0.25% of emissions). 1Biological Systems and Engineering CO2 is an ideal feedstock for the production of biofuels because it is an inexpensive, nontoxic, Division, Lawrence Berkeley National and abundant starting material (≈850 billion tons of CO are currently present in the atmosphere). Laboratory, Berkeley, CA 94720, USA 2 @Twitter: @DNAmanipulation (J. Panich). Furthermore, CO2 is noncompetitive with the global food supply chain. Technoeconomic analysis 2www.linkedin.com/in/steven-singer- v indicates that CO2-derived biofuel production could be a US$10–250 billion industry by 2030 . 26969912/

However, CO2 is dilute in the atmosphere (≈0.04% CO2 by volume) and is challenging to use directly because many nonphototrophic organisms that can be engineered to produce biofuels *Correspondence: autotrophically do not grow optimally in ambient CO2. To circumvent this issue, synthetic biology [email protected] (J. Panich) and approaches could be utilized to engineer higher CO2 assimilation rates at ambient concentrations [email protected] (S.W. Singer). @ of CO2. Alternatively, microorganisms could be grown using elevated CO2 concentrations from Twitter: @DNAmanipulation (J. Panich).

412 Trends in Biotechnology, April 2021, Vol. 39, No. 4 https://doi.org/10.1016/j.tibtech.2021.01.001 © 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Trends in Biotechnology OPEN ACCESS

industrial runoff or in the form of syngas when grown in a bioreactor. Some companies, such as Glossary vi LanzaTech , have already implemented such technologies using acetogenic microorganisms [9]. 2-phosphoglycolate (2-PG): awaste vii Other companies, such as Climeworks , have developed technology to separate CO2 from other product of the nonspecificactivityof rubisco towards oxygen. components in the atmosphere in a concentrated form. CO2 captured through a Climeworks system could be used to support biofuel production from microbes at elevated CO levels. The Artificial leaf: device that uses 2 photovoltaics to convert sunlight to split Climeworks CO capture technology is coupled to a geothermal power plant, which could supply 2 water, forming H2 and O2, which can renewable energy for H2 or formate production for chemolithotrophic growth, or be directly support microbial . coupled to an electrolysis system. Artificial photosynthesis: term used broadly to describe any technology that converts sunlight conversion to chemical C. necator H16 (formerly Ralstonia eutropha H16) is an attractive chassis for metabolic engineering for energy. CO2 bioconversion to fuel products and is one of the most advanced genetic systems for this pur- Autotrophic: mode of pose [10]. This organism is a Gram-negative nonpathogenic β-proteobacterium and a facultative where organisms create nutritive substances from inorganic carbon chemolithotroph (see Glossary) that grows on mixtures of H2 and CO2 or on formate with no sources. dependence on light availability, because this organism is not a phototroph. C. necator assimilates Electrolytic growth: method for

CO2 using the autotrophic Calvin–Benson–Bassham (CBB) cycle. Redox chemistry is largely growing microorganisms using an controlled in this organism by a soluble (SH) that creates reducing equivalents from electrical current. – Facultative chemolithotroph: the oxidation of H2 gas. O2 or NO3serves as the terminal electron acceptor for respiration, although organism that can grow on supplied – NO3is a poor electron acceptor in this organism [11]. C. necator also has a natural biosynthetic organic carbon sources, and can route to produce the carbon-dense biopolymer poly[(R)-3 hydroxybutyrate] (PHB), a biodegradable assimilate inorganic carbon sources when plastic-like molecule that is stored in granules during nutrient limitation with titers that can exceed organic carbon sources are not supplied. fl High CO2-requiring (HCR) 70% of total biomass by dry weight (Figure 1)[12]. Carbon ux can be diverted away from PHB syn- phenotype: phenotype requiring thesis in several cases to generate value-added products, such as isobutanol, methyl ketones, substantially higher CO2 concentrations isoprenoids, sucrose, modified PHBs, growth enhancers for plants, and a further variety of for growth, usually caused by a defect in carbon-dense compounds and commodity chemicals [10,13–17]. C. necator can also grow on aCO2-concentrating mechanism. : class of photovoltaic-derived electricity by H2 and O2 created by water splitting through indirect electron metalloenzymes that convert molecular hydrogen to protons and electrons. Indirect electron transfer: type of Figure 1. Cupriavidus necator with electrolytic growth where electrons are Poly[(R)-3 Hydroxybutyrate] (PHB)- delivered to cells by mediators, rather Containing Granules. Such granules can than used directly by . In the comprise up to 70% of the totalbiomass. case of Cupriavidus necator,electrons

Transmission electromicrograph thin- are gained through H2 gas produced section, scale bar = 0.5 μm. Reproduced, with electricity. with permission, from [110]. Isoprenoids: diverse class of chemicals that can be produced by microorganisms, and includes terpenoids. In the context of biofuels, many isoprenoids show properties that suggest they could be used for aviation and rocket fuels. Microbial electrosynthesis (MES): type of reaction where microorganisms catalyze reactions in response to an electrical current. Mixotrophic: describes metabolisms that use inorganic and organic carbon sources or energy sources. Phosphoglycolate salvage: any reaction used to recycle 2-PG that is produced by the off-target activity of rubisco; can be carried out by plants and chemolithotrophs through diverse pathways. TrendsTrends inin BiotechnologyBiotechnology Photorespiration: describes phosphoglycolate salvage, primarily used to describe the process in plants.

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transfer [16,18,19]. In this review, we provide an overview of recent developments and advances in Phototroph: an organism, such as a engineering C. necator for biofuel production via autotrophic metabolism. Subsequent evaluation of plant, that uses photosynthesis to convert sunlight to chemical energy. inherent features and CO2 bioconversion provides readers with insight regarding the major hurdles Reverse electron flow: common impeding commercialization of bioproducts from C. necator. We also provide guidance for mechanism where lithotrophic researchers to navigate the options available for engineering this organism. organisms gain reducing power for anabolic reactions from the respiratory chain. Metabolic Features of C. necator Water splitting: chemical reaction where water is converted to H and Under autotrophic growth conditions, C. necator fixes CO2 using the CBB cycle, and grows 2 in atmospheric CO concentrations at a slow rate (doubling time ≈20 h) [16,20]. The CBB cycle O2gases, which can be carried out by 2 electrolysis. involves 11 steps and the key enzyme for carbon fixation in C. necator is type IC ribulose-1,5- bisphosphate-carboxylase/-oxygenase (rubisco) [21]. With the exception of triose-3-phosphate isomerase (tpi) and ribose-5-phosphate isomerase (rpi), all of the enzymes required for the CBB cycle are encoded in the cbb operon, present in two copies in C. necator. Both copies of the cbb operon contribute to autotrophy (Figure 2)[22]. One copy of the cbb operon is located on chromosome 2 of the C. necator genome and encodes 14 genes. A nearly identical copy of the operon is also present on the pHG1 megaplasmid, although this second copy lacks two genes that are present in the chromosomal copy, including a LysR-type transcriptional regulator cbbR and a formate dehydrogenase-like gene, cbbB [23]. Both copies of the cbb operon are regulated by CbbR, which causes repression of the cbb operon when excessive concentrations of phosphoenolpyruvate (PEP) are present, which occurs during heterotrophic metabolism [24,25]. CbbR-directed transcription is also regulated by levels of ribulose 1,5-bisphosphate (RuBP), ATP, and NADPH [24].

The CBB cycle is energetically expensive, requiring net 7 moles of ATP to convert 3 moles of CO2 to 1 mole of pyruvate. The key enzyme in this pathway, rubisco, is a slow carboxylase that also

(A) (i) ChromosomeChromosome 2 σ70

cbbR cbbL cbbS cbbX cbbY cbbE cbbF cbbP cbbT cbbZ cbbG cbbK cbbA cbbB (ii) pHG1pHG1 σ70

cbbL cbbS cbbX cbbY cbbE cbbF cbbP cbbT cbbZ cbbG cbbK cbbA

Regulation Rubisco subunits Rubisco activase RuBP regeneration G3P production Formate dehydrogenase

Phosphoglycolate salvage

(B) σ54 σ70 σ70 σ70 MBHMBH andand RRHH hoxK hoxG hoxZ hoxM hoxL hoxO hoxQ hoxR hoxT hoxV hypA hypB hypF hypC hypD hypE hypX hoxA hoxB hoxC hoxJ pHG1pHG1

σ54 SHSH hoxF hoxU hoxY hoxH hoxW hoxI hypA2 hypB2 hypF2 pHG1pHG1

σ54 AHAH hofK hofG PHG066 PHG067 PHG068 PHG069 hupD hypF3 hypC2 hypD2 hypE2 hypA3 hypB3 pHG1pHG1

Regulation Hydrogenase subunits Accessory function proteins Assembly Maturation protease

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Figure 2. Key Autotrophy Operons of Cupriavidus necator. (A) The cbb operon exists in two copies in C. necator and allows for light-independent CO2 fixation. The chromosomal copy (i) encodes the regulator cbbR, and also cbbB, while the megaplasmid-encoded copy (ii) lacks those genes. (B)C. necator expresses at least four hydrogenases with differing functions (see main text for details). Abbreviation: RuBP, ribulose 1,5-bisphosphate.

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has oxygenase activity. Efforts to use rational design to engineer more efficient rubisco variants have been largely unsuccessful, because physicochemical constraints of the binding pocket make it difficult for the enzyme to distinguish between CO2 and O2 [26,27]. The oxygenase activity of rubisco forms the toxic compound 2-phosphoglycolate (2-PG), which is not a CBB interme- diate and must be recycled in a process termed ‘phosphoglycolate salvage’, known in plants as photorespiration (Box 1)[27]. Some organisms, including all Cyanobacteria and some auto- trophic , mitigate the weak performance of rubisco by utilizing CO2-concentrating – mechanisms (CCMs). CCMs characteristically include inorganic carbon (HCO3) transporters and a proteinaceous housing for rubisco, called the carboxysome, a 200+ MDa icosahedral structure that contains rubisco enzymes for carboxylation as well as carbonic anhydrases, which convert bicarbonate to CO2 in the vicinity of rubisco [28]. The carboxysome functions to increase the concentration of CO2 near the active site of rubisco, and also to sequester CO2 from O2 [29].

C. necator is lacking the typical features of a CCM (Figure 3). However, this organism expresses four carbonic anhydrase-like (CA-like) enzymes that allow it to obtain sufficient bicarbonate in the cytoplasm to allow rubisco to fixCO2. In addition, C. necator expresses a rubisco variant with a relatively high specificity for CO2 [30]. The CA-like enzymes in C. necator are from three different evolutionary classes and include the enzymes Caa, Can, Can2 and Cag. Three of these CA-like enzymes are cytoplasmic, whereas Caa is localized to the periplasm [31]. Deletion of CA-like enzymes Caa and Can causes a high CO2-requiring (HCR) phenotype under mixotrophic growth conditions [31,32].

Roles of Hydrogenases in C. necator C. necator expresses four types of hydrogenase encoded on the pHG1 megaplasmid (Figure 2) that have prominent roles in autotrophic growth and ATP generation. Hydrogenases are + – metalloenzymes that catalyze the conversion of H2 to 2H and 2e with a redox potential (Eo′)of

–414 mV. The SH is of considerable interest to biotechnology for the bioproduction of H2 gas as well as introducing H2-based metabolism in other organisms (Box 2). The hydrogenases in C. necator are unusual, because they are oxygentolerant. In the case of SH, the electrons from + H2 oxidation are used to reduce NAD to NADH, providing this organism with reducing power during autotrophy. SH is encoded by the hoxFUYHWI operon: HoxFU functions as a diaphorase, while HoxYH is a [NiFe]-type hydrogenase. HoxW and HoxI are accessory proteins [33]. SH expression is coupled to ATP levels by HoxJ, a histidine sensor kinase [34,35]. The SH also requires a Ni-Fe-CO-2CN–cofactor, which is produced by maturase and chaperone complexes HypABCD and HypEF [36]. C. necator has three catalytically active hydrogenases

Box 1. Phosphoglycolate Salvage in C. necator

Phosphoglycolate salvage is an energetically expensive and wasteful process, losing a carbon in the form of CO2 during the conversion of 2-PG to ribulose 1,5-bisphosphate (RuBP). Recent experiments in Synechococcus elongatus as well

as C3 plants suggest that modifying phosphoglycolate salvage routes would lead to increased carbon efficiency in Cupriavidus necator, especially if carbon loss can be eliminated completely, as has been described in vitro [99–101]. The side activity of rubisco towards RuBP oxygenation produces 2-PG, which must be recycled because it is not a CBB cycle intermediate. 2-PG is also an inhibitor of the CBB enzyme triose phosphate isomerase (Tpi), and must quickly

be converted or metabolized to maintain CO2 fixation ability [102]. Plants use the C2 pathway to recycle 2-PG, a pathway that consumes >25% of the chemical energy that is produced by the CBB cycle, and involves at least ten enzymatic steps [102,103]. By contrast, C. necator phosphoglycolate salvage proceeds primarily through the ‘glycerate pathway’ that is also carried out by Cyanobacteria [20,104]. In this pathway, 2-PG is decarboxylated to tartronic semialdehyde, and is then reduced to glycerate, which is then phosphorylated to form the CBB intermediate 3-phosphoglycerate. When the glycerate pathway is removed by gene knockout, C. necator relies on a novel ‘malate cycle’ that fully oxidizes 2-PG to

CO2. Bioinformatic analysis suggests that the malate cycle for 2-PG oxidation is widespread in chemolithotrophs [20]. Nonetheless, the bacterial glycerate cycle is the major route of phosphoglycolate salvage in C. necator.

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(A)Cyanobacteria (B) Cupriavidus necator Cytoplasmic membrane Thylakoid membrane Periplasm rubisco [CO2] Carbonic Central CBB anhydrase metabolism cycle Carboxysome rubisco [CO2] − [HCO3 ] [CO2] − BicA1 [HCO ] + 3 Na Putative CA-like Central − SbtA1 − transporter − − [HCO ] + [HCO ] [HCO ] [HCO ] 3 Na 3 3 3 metabolism BCT1 [HCO −] CA-like 3 ATP Caa Putative CO2 [CO ] NDH-1 transporter 2 [HCO −] 3/4 [CO ] [CO ] 3 NADPH 2 2

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Figure 3. Comparison of the Cyanobacterial CO2-Concentrating Mechanism (CCM) with the CO2-Capturing System of Cupriavidusnecator. The cyanobacterial CCM system (A) includes high-affinity inorganic carbon transporters that are membrane bound, as well as the carboxysome, which houses ribulose-1,5-bisphosphate- carboxylase/-oxygenase (rubisco) and carbonic anhydrase (CA). The CCM system allows for robust growth in ambient

CO2 in Cyanobacteria. By contrast, C. necator (B) does not have known membrane-bound inorganic carbon transporters, and instead expresses multiple CA-like enzymes along with a rubisco with a relatively high specific activity towards CO2 (465 nmol/min/mg) with an average rate (~2.5 s–1)[30]. Orange arrows represent reactions carried out by CA-like enzymes. Abbreviation: CBB, Calvin–Benson–Bassham. in addition to SH, the membrane-bound hydrogenase (MBH), a regulatory hydrogenase (RH), and an actinobacterial hydrogenase (AH) (Figure 2). The MBH is a [NiFe] hydrogenase comprising HoxK and HoxG,the activity of which reduces ubiquinone, supporting the respiration chain and generating proton motive force to drive substrate-level phosphorylation, producing ATP [33]. The RH comprises HoxB, HoxC, and HoxJ subunits and is encoded downstream from the MBH. The RH phosphorylates the HoxA transcription factor in the presence of molecular hydrogen to activate autotrophy gene expression, directing transcription of MBH and SH from σ54 promoters [37,38]. The fourth hydrogenase, AH, is not well characterized, although it –1 has a relatively slow H2 consumption rate (0.5 s ) and is thought to function in low hydrogen conditions [39–41].

Biofuel Production in C. necator: State of Technology C. necatorwas recently engineered to produce a variety of biofuel chemicals, such as branched- chain alcohols (propanol and isobutanol), alka(e)nes, fatty acid derivatives (methyl ketones and saturated hydrocarbons), as well as isoprenoids and terpenoids, largely by diverting carbon flux away from PHB synthesis (Figure 4)[10,17,42,43]. Autotrophic biomass accumulation rates of 1.1–2.47 g/l/h indicate that C. necator could be used for industrial-scale production, although commercialization of these technologies are not currently feasible due to relatively low titers of desired products [44]. Mutants that cannot biosynthesize PHB (by deletion of the phaCAB operon, the gene products of which convert acetyl CoA to PHB) secrete large amounts of pyruvate from the cell, an indication that there is an excess of disposable carbon flux that can be utilized for other purposes [45,46]. Diesel-range methyl ketones have been produced in

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Box 2. Harnessing the Power and Versatility of the Soluble Hydrogenase + The SH allows Cupriavidus necator to oxidize H2 gas (which readily passes across the cellular membrane) in the cytosol while reducing NAD to NADH. The SH is versatile, can be expressed in other organisms, and is also oxygen tolerant, a characteristic that simplifies engineering and microbial growth [105–107]. Expression of the SH bypasses the need for reverse electron flow through the Q-cycle, a mechanism used by many other lithotrophic microorganisms where electrons are shuttled in the endothermic direction to produce NADH (Figure I). This mechanism is used by autotrophic microorganisms that have membrane-bound hydrogenases coupled to cytochromes, which have a higher redox potential than NADH and, therefore, require additional electrons to produce NADH. Expressing SH in heterotrophic organisms, such as Escherichia coli [105,107], could provide the necessary reducing power to engineer hydrogen-energized autotrophic metabolism. An E. coli strain was recently described that expresses a full CBB cycle and has been evolved (~200 days of laboratory evolution) to engage in autotrophic metabolism by expression of formate dehydrogenase from a methylotroph, Pseudomonas sp. 101, to generate NADH from formate [108]. SH could be used in a similar way to transform E. coli into a hydrogen bacterium. Furthermore, the carboxysome was recently reconstituted in E. coli and allows this bacterium to grow mixotrophically at ambient concentrations of

CO2 [90]. With further laboratory-directed evolution, it is plausible that combining the CBB cycle with the carboxysome and SH in E. coli could enable autotrophy at am-

bient concentrations of CO2. In addition, a novel CO2 fixation pathway (the Gnd–Entner–Doudoroff pathway) has been uncovered in E. coli using the carboxylase Gnd, and this strain could also be used to introduce hydrogen autotrophy in E. coli [109].

(A) (B) Reverse electron flow (other ) C.necator Periplasm e_ donor + 2H+ Membrane- H bound H+ _ 2 + + e donor + H O H H 2 hydrogenase β α γ e_ Cyt c Fe-Ni Fe-S Fe Fe Q cyt A cycle cyt B cyt A

NADH NADP+

NAD+ NADPH _ H+ H+ 1/2 O2 + 2H+ H2O e + + Fe-Ni FMN NAD(P) NAD(P)H 1/2 O2 + 2H H2O H2 e_ Fe-S FMNFe-S Fe-S Fe-S Soluble hydrogenase Cytoplasm

TrendsTrends inin BiotechnologyBiotechnology Figure I. Typical Electron Flow of Lithotrophs to Produce NAD(P)H Compared with Soluble Hydrogenase (SH)-Mediated NADH Production in Cupriavidus necator. The electron flow scheme (A) often depends on reverse electron flow through the Q-cycle to produce reducing equivalents, and is common in iron, nitrite, and sulfur oxidizers, as well as hydrogen bacteria that do not express SH. The hydrogenase system found in C. necator and a handful of other hydrogen bacteria (B) is capable of oxidizing molecular hydrogen and converting electrons to NADH, which can then be converted to NADPH to drive metabolism.

C. necator by altering the β-oxidation pathway, and did not require deletion of phaCAB, although methyl ketone production required deletion of the native fadAcoupled with expression of fadB and fadMfrom Escherichia coli as well as acyl-CoA oxidase from Micrococcus luteus [10]. This strain produced >50 mg/l methyl ketone when grown heterotrophically in fructose and up to

180 mg/l in autotrophic conditions where CO2 was the sole carbon source.

Isobutanol production was achieved in ΔphaCAB mutants where the native branch-chain amino acid biosynthesis pathway was overexpressed with coupling of ketoisovalerate decar- boxylase (kivD) as well as alcohol dehydrogenase (adhE)fromLactobacillus lactis [42]. Deletion of genes that have high carbon flux in C. necator,includingilvE, bkdAB,andaceE, increased the titer to 270 mg/l isobutanol. Similarly, Li and coworkers produced 846 mg/l of isobutanol autotrophi- cally in a pH-controlled formic acid-feeding fermentor [18]. Alkane or alkene production (pentadecane, hexadecane, and heptadecane) in C. necator required the overexpression of two genes from Synechococcus elongatus: aar encoding acyl-ACP reductase and ado encoding aldehyde-deformylating oxygenase [17]. This pathway allowed production of 435 mg/lalkanes or alkenes heterotrophically and 4.4 mg/l autotrophically [17]. Production of isoprenoids, such as

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Carbon source (autotrophic)

CBB kivD adhA Cycle 2- Ketoisovalerate Isobutyraldehyde Isobutanol tes fadD β-Oxidation 13 3 Carbon source Fatty acids Methyl ketones (heterotrophic) Central Fatty acid metabolism synthesis Pyruvate Acetyl-CoA Acyl-ACP phaA AAR ADO 3 phaC phaB MVA pathway Fatty aldehydes Alka(e)nes

PHB R-HB-CoA Acetoacetyl-CoA Mevalonate Z. zerumbet derived synthase

Farnesyl pyrophosphate α-Humulene

TrendsTrends inin BiotechnologyBiotechnology Figure 4. Directing Carbon Flux Away from Poly[(R)-3 Hydroxybutyrate] (PHB) Metabolism Can Make Value-Added Products with Metabolic Engineering. Cupriavidus necator is a facultative chemolithotroph that can form biofuel products under heterotrophic and autotrophic conditions. While C. necator naturally synthesizes PHB, diversion of carbon flux away from PHB synthesis using engineered metabolic pathways and gene knockouts can allow for production of several bioproducts that could be used as fuels. Abbreviation: CBB, Calvin–Benson–Bassham.

α-humulene, is of particular interest to industry, because >20 000 known natural compounds have a similar isoprenoid backbone [47]. Isoprenoids can be produced using the heterologous mevalonate (MVA) pathway in this organism [43,48]. Many isoprenoids, such as farnesane, epi- isozizaene, and others, are promising candidates for aviation fuels directly or as a blending agent [49–51]. The isoprenoid α-pinene has been produced in E. coli and is a precursor for a type of rocket fuel, although kerosene is the current preferred fuel for this purpose [52]. Krieg and co- workers demonstrated isoprenoid production in C. necator with α-humulene production, reaching titers of 9.4 mg/l heterotrophically and up to 6.3 mg/l autotrophically in a phaCAB background by expressing the MVA pathway from Methylobacterium extorquens and α-humulene synthase derived from Zingiber zerumbet in C. necator [43].

Electrolytic Growth of Cupriavidus C. necator was first shown to grow electrolytically by water splitting in 1965 [53]. Electrolytic growth technology has the potential to be a versatile and inexpensive method for fuel production, because extensive progress has been made over the past decade regarding artificial photosyn- thesis through photovoltaic (PV) coupling to electrochemical devices [54,55]. Recently, a method to produce biofuels and bioproducts using electrolytic growth of C. necatorwas described, incorpo- rating artificial leaf technology and allowing for growth of C. necator to surprisingly high densities, even in ambient CO2 (OD600 of 1.2 after 5 days of linear growth) [16]. The artificial leaf is a microbial electrosynthesis (MES) reactor that mimics the mechanism of photosystem II found in plants by linking photovoltaics to an apparatus that splits water and allows microbial growth through indirect electron transfer (Figure 5). Indeed, the energetics associated with splitting water to produce H2 and

O2 gas using electrolysis is similar to natural photosynthesis ( G°′ of water splitting = 1.23 V; G°′ of photosynthesis = 1.24 V per mol of CO2 +H2Oto[CH2O] + O2). However, natural photosynthesis is inefficient: an experimentallyderived maximum efficiency for solar energy conversion to biomass for natural photosynthesis is ~12%, although observed efficiencies for natural photosynthesis in plants

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(A) (B) Natural photosynthesis photosynthesis

CO2 stream

H2 stream

H2O C. necator stream H2 H2O

Light Light Steel Si + CoP H i junction CoP alloy catalyst catalyst Tyr + _ + _ + _ + _ 2H O _ 2H P680 2 + 2 + _ Electron transport + _ HO + + + O + 4H + _ 4H chemiosmosis 2 + _ + _ + _ + _ + _

+ 2H2O ½O2+ 2H Photosystem II

TrendsTrends inin BiotechnoloBiotechnology Figure 5. Comparison between Natural Photosynthesis and Artificial Photosynthesis Using the Artificial Leaf Coupled with Microbial Growth. Light harvesting is carried out in plants and other phototrophs by photosystem II (A). The artificial leaf (B) produces an electric current in response to light. The artificial leaf can be used to split water, producing H2 and O2 gas, which are substrates for autotrophic growth of Cupriavidus necator.

are 1–4% [56–58]. By contrast, the artificial leaf system has a theoretical efficiency of ~18% CO2 conversion, with a measured efficiency of 10% under pure CO2, and 3–4% using ambient CO2 [55]. Modifications of the artificial leaf system have been described that overcome several obstacles associated with electrolytic growth of C. necator [16]. Initial reports detailing microbial growth with the artificial leaf utilized NiMoZn and cobalt-based catalysts that can cause the evolution of reactive oxygen species (ROS) and cobalt ion leaching from the electrodes, both of which are toxic to cells.

Evolution of ROS is thermodynamically favored over H2 evolution and occurs at <2.7 V, while water splitting requires >1.6 V of applied voltage (Eappl)[59,60]. These constraints required the use of 4.0–5.5 V of applied voltage in initial reports. However, using cobalt–phosphate alloy/cobalt– phosphate catalysts did not create substantial ROS, allowing for growth of C. necator at

Eappl =2.0–3.0 V [16]. Furthermore, these catalysts are self-healing to prevent accumulation of toxic degradation products. Isopropanol production has been demonstrated with the artifi- cial leaf, generating ~600 mg/l isopropanol [16]. The artificial leaf system also allowed for pro- duction of 220 mg/l isobutanol+2-methyl-1-butanol in C. necator [16].

The artificial leaf/C. necator system is an integrated system, without separation by a membrane between the cathode and anode, an arrangement that is associated with particular advantages. For instance, bacteria can grow directly on the cathode using the artificial leaf system, where microbial growth is thought to reduce overall energy requirements and diminish electrode overpotentials, leading to a more efficient system. Microbial growth on the cathode also increases

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the rate of H2 evolution from electrolysis [61,62]. An alternative plausible design is to uncouple electrolysis and microbial growth, and to split water using an electrolyzer, which produces high concentrations of H2 gas that is then fed into a gas bioreactor to be mixed with other gases.

The high flux of H2, along with separation of electrolysis and microbial growth in a bioreactor could overcome potential bottlenecks associated with the artificial leaf system, possibly making a separated reactor design easier to scale to industrial levels [63].

Synthetic Biology Tools for C. necator Facultative chemolithotrophy simplifies genetic engineering in C. necator relative to obligate chemolithotrophs, because genetic modification can be performed in the absence of gas substrates. Heterologous protein expression has been demonstrated in C. necator using plasmid-based expres- sion systems as well as chromosomally integrated genes [18,64]. While C. necator is the most advanced genetic system for CO2 bioconversion, genetic tools are still lacking relative to tools that are available for other microorganisms, such as E. coli. Conjugation methods are commonly used for genetic engineering in C. necator, although electroporation methods have recently been described. The low electroporation efficiency in C. necator can be circumvented in several ways. Some groups have been able to electroporate plasmids into C. necator with high-molecular-weight buffers, often containing sucrose [65]. However, Xiong and coworkers identified several restriction endonucleases encoded by the C. necator genome and created knockouts of five putative restriction systems in C. necator. Deletion of A0006,anhsdR homolog, increased electroporation efficiency by ~1600-fold relative to wild-type C. necator, and this genetic background can be used for rapid genetic engineering [66]. Synthetic vectors and expression systems in C. necator are limited, and com- mon vectors available include the broad-host pBBR1-MCS plasmid origin from the pBBR1 plasmid of Bordetella bronchiseptica [67]. Plasmids with the RP4 (RK2) origin can also be used [68]. Additional plasmids that have been used for stable gene expression in C. necator include pMOL28, RP4, RSF1010, and pSa as well as pBHR1, and modular vectors are freely available with these features through the Standard European Vector Architecture (SEVA) [65,69]. Endogenous inducible promoters can be used in certain con- texts, but are coupled to the metabolic state of the cell. For example, the phaP1 promoter is induced by inorganic phosphate accumulation [70,71]. Similarly, acetoin inducible promoters, including Pphb,PacoE, and PpdhE,are useful in some cases [72]. Synthetic biology applications require inducible and constitutive gene expression systems that are uncoupled from the meta- bolic state of the organism, and many common expression systems fail in C. necator. For example, IPTG cannot cross the cell membrane, therefore IPTG-inducible promoters are not effective in C. necator unless LacY is expressed to restore IPTG transport [73]. The lac promoter is active as a con- stitutive promoter in the absence of LacY, LacI, or the lac operator [71]. Other constitutive promoters that have been used in C. necator include Ptac and Ptrc derivatives, Pcat,andPj5, derivatives of Pj5 [71,72,74–76]. In addition, the L- arabinose-inducible (Pbad) promoter from E. coli and a synthetic anhydrotetracycline inducible promoter are functional in C. necator, [77,78].

Plasmid loss is a common problem when growing cultures at industrial-level scale. Therefore, there is much interest in expanding the capabilities of chromosomal manipulation in C. necator, which remains a challenging task in this organism. The pHG1 megaplasmid is an attractive target for integration of heterologous genes, because 17 transposes and 22 full or partial phage-like

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integrases are encoded on pHG1 [79]. The classical and most common approach to genome Outstanding Questions engineering in C. necator is allelic replacement using plasmid recombination through conjugation. Titers of products made in C. necator are This approach often includes a selection–counterselection method based on the toxicity of the currently in the mg/l range. How can sacB cassette to achieve the desired genetic manipulation, although other selection schemes titers be increased to levels that could make products from this organism can be imagined [80]. A gene-disruption system involving the retrotransposition of a type II intron more economically feasible? (referred to as ‘retrohoming’) has also been described in C. necator, although this technique is not commonly used [81]. A CRISPR/Cas9 method has also been applied in C. necator [66]. The What is the best way to grow C. necator method requires expressing guide RNAs directly onto a plasmid, which also encodes Cas9 for industrial-scale bioproduction? Is it possible to scale electrolytic growth to from Streptomyces pyogenes, followed by electroporation and 5 days of expression; resulting industrial levels? colonies are then screened for desired mutations. A multiplexed CRISPR-based interference (CRISPRi) strategy using a catalytically inactive Cas9 (dCas9) has not yet been reported in C. Which synthetic biology tools will allow for the most efficient genome necator. CRISPRi might be a powerful tool that would bypass the laborious task of knocking engineering in C. necator? How can out several genes in some instances, and has already been applied to many other bacteria high-throughput genetic engineering [82–85]. Chromosomal-engineering capabilities in C. necator could also be expanded by the methods be applied in C. necator? implementation of a recombinase system, such as the Cre/Lox system for integration, the attP integrase system, or the FLP recombinase system, none of which have been widely used in the past. Although Cre/Lox activity has been demonstrated in C. necator to remove an antibiotic resistance cassette, the full potential of this system has not been realized in this organism, because Cre/Lox recombination has been used to integrate >50 000 base pairs of DNA in the bacterial chromosome, and is functional in many undomesticated bacteria [83,86,87].

Concluding Remarks There are several approaches that might improve growth and titer yields in C. necator (see Outstanding Questions). The inefficient CBB pathway was recently replaced with a reductive glycine pathway (rGlyP) for formate fixation, demonstrating the feasibility of introducing heterologous inorganic carbon bioconversion pathways in C. necator. However, growth yield of the rGlyP- enabled strain was comparable with that of the native CBB cycle [76]. Rather than eliminat- ing the CBB cycle, it could be augmented by overexpression of rubisco or CBB enzymes, although the promiscuity of CBB enzymes and RuBP toxicity are complicating factors for such an approach [88]. Expression of alternative rubisco enzymes with more favorable kinetic parameters has been shown to increase the autotrophic growth rate of C. necator [22]. Recently, a type II rubisco variant was described from Gallionella sp.that has a rate of ~22 s–1, about tenfold faster than the rubisco present in C. necator, raising the possibility that a substantial increase in CO2 assimilation rate could be achieved in C. necator by expressing this rubisco or other newly described variants with elevated activities [89].

Heterologous expression of a CCM might improve bioconversion, and has been accomplished in E. coli [90]. The β-carboxysome from Synechococcus has been expressed heterologously in E. coli, while the α-carboxysome from Halothiobacillus neapolitanus has been expressed in E. coli and Corynebacterium glutamicum, further demonstrating the plausibility of functional expression of a CCM in C. necator [91–93]. Furthermore, the α-carboxysome from Halothiobacillus has been shown capable of encapsulating a variety of heterologous rubisco enzymes, making it a promising candidate to express modular CCM systems, including heterologous rubiscos with increased activity [89,94]. It is reasonable to suggest that transplanting a CCM in C. necator will be successful to allow growth of C. necator at low CO2 concentrations, because cyanobacterial CCMs have been shown to increase cytosolic bicarbonate by as much as 1000-fold [95–97]. Studies from Thiomicrospira crunogena, a deep-sea vent bacterium that contains a CCM, suggest that bacterial CCMs raise the internal bicarbonate concentration by a factor of ~100, and it is plausible that adding a complete CCM to C. necator could increase cytosolic bicarbonate concen- tration by 100–1000-fold [98].

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There should be an urgency to move the fuel economy to a carbon-neutral model to avoid impending environmental crises. The notable flexible metabolic capabilities of C. necator coupled with its genetic tractability make C. necator an ideal chassis for producing biofuel products while consuming CO2, an approach that will reduce anthropogenic CO2 by a modest amount. With expanded research and the implementation of scalable electrolysis, it is possible that transporta- tion fuel production from C. necator or similar microbes could become technically and economically plausible in the coming years.

Acknowledgments We thank Nikolas Dawid for help with the manuscript and Avi Flamholz for detailed feedback. This work was funded by the Energy & Biosciences Institute (EBI CW163755) and Laboratory Directed Research and Development funds from Lawrence Berkeley National Laboratory.

Declaration of Interests No interests declared.

Resources iwww.bp.com/content/dam/bp/business-sites/en/global/corporate/pdfs/energy-economics/statistical-review/bp-stats- review-2020-full-report.pdf iiwww.eia.gov/outlooks/ieo/ iiiwww.eia.gov/pressroom/presentations/sieminski_05112016.pdf ivwww.iea.org/news/decoupling-of-global-emissions-and-economic-growth-confirmed vhttps://deepblue.lib.umich.edu/handle/2027.42/150624 viwww.lanzatech.com viiwww.climeworks.com

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