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• W3127738948 abstract "Cupriavidus necator has a wide metabolic range and naturally creates a biopolymer, poly[(R)-3 hydroxybutyrate] (PHB). Using metabolic engineering techniques, carbon flux can be directed away from PHB synthesis toward the generation of biofuels and bioproducts.Researchers demonstrated the production of many biofuel products using C. necator, including methyl ketones, isoprenoids and terpenes, isobutanol, alkanes and alkenes, and a wide variety of commodity chemicals from CO2.Growth of C. necator and bioproduct production using electrolysis was recently demonstrated, including the use of an artificial leaf system.While genetic engineering of C. necator remains a laborious process, synthetic biology tools for this organism are being expanded with new technologies that will allow for large alterations to its genome. Decelerating global warming is one of the predominant challenges of our time and will require conversion of CO2 to usable products and commodity chemicals. Of particular interest is the production of fuels, because the transportation sector is a major source of CO2 emissions. Here, we review recent technological advances in metabolic engineering of the hydrogen-oxidizing bacterium Cupriavidus necator H16, a chemolithotroph that naturally consumes CO2 to generate biomass. We discuss recent successes in biofuel production using this organism, and the implementation of electrolysis/artificial photosynthesis approaches that enable growth of C. necator using renewable electricity and CO2. Last, we discuss prospects of improving the nonoptimal growth of C. necator in ambient concentrations of CO2. Decelerating global warming is one of the predominant challenges of our time and will require conversion of CO2 to usable products and commodity chemicals. Of particular interest is the production of fuels, because the transportation sector is a major source of CO2 emissions. Here, we review recent technological advances in metabolic engineering of the hydrogen-oxidizing bacterium Cupriavidus necator H16, a chemolithotroph that naturally consumes CO2 to generate biomass. We discuss recent successes in biofuel production using this organism, and the implementation of electrolysis/artificial photosynthesis approaches that enable growth of C. necator using renewable electricity and CO2. Last, we discuss prospects of improving the nonoptimal growth of C. necator in ambient concentrations of CO2. Satisfying current and future global energy demand while allowing favorable environmental outcomes will require innovative solutions in the fuel sector. Although portions of the transportation infrastructure can be electrified (such as automobile transport), energy-dense carbon-based fuels will continue to be required for aviation, long-distance trucking, rocketry, maritime shipping, 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 and 2070 using current oil extraction technologiesi–iii. Climate change caused by an increase in CO2 (and CH4) levels in Earth’s atmosphere has brought cataclysmic weather events and has decimated ocean habitats in recent years, a trend that will likely continue through our lifetimes given that CO2 is being emitted into the atmosphere at a rate of ≈32 billion tons of CO2 per year [1.Hughes T.P. et al.Global warming and recurrent mass bleaching of corals.Nature. 2017; 543: 373-377Crossref PubMed Scopus (1356) Google Scholar, 2.Frolicher T. et al.Marine heatwaves under global warming.Nature. 2018; 560: 360-364Crossref PubMed Scopus (312) Google Scholar, 3.Friedlingstein P. Solomon S. Contributions of past and present human generations to committed warming caused by carbon dioxide.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10832-10836Crossref PubMed Scopus (42) Google Scholar, 4.Smith K. et al.Joint CO2 and CH4 accountability for global warming.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E2865-E2874Crossref PubMed Scopus (31) Google Scholar, 5.Stott P. How climate change affects extreme weather events.Science (80-. ). 2016; 352: 1517Crossref PubMed Scopus (213) Google Scholar, 6.Altieri A. Gedan K. Climate change and dead zones.Glob. Chang. Biol. 2014; 21: 1395-1406Crossref PubMed Scopus (164) Google Scholar]iv. 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.Staples M. et al.Aviation CO2 emissions reductions from the use of alternative jet fuels.Energy Policy. 2018; 114: 342-354Crossref Scopus (70) Google Scholar]) 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.Ammann E. et al.Gas consumption and growth rate of Hydrogenomonas eutropha in continuous culture.Appl. Microbiol. 1968; 16: 822-826Crossref PubMed Google Scholar], ~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). CO2 is an ideal feedstock for the production of biofuels because it is an inexpensive, nontoxic, and abundant starting material (≈850 billion tons of CO2 are currently present in the atmosphere). Furthermore, CO2 is noncompetitive with the global food supply chain. Technoeconomic analysis indicates that CO2-derived biofuel production could be a US$10–250 billion industry by 2030v. 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 autotrophically do not grow optimally in ambient CO2. To circumvent this issue, synthetic biology approaches could be utilized to engineer higher CO2 assimilation rates at ambient concentrations of CO2. Alternatively, microorganisms could be grown using elevated CO2 concentrations from industrial runoff or in the form of syngas when grown in a bioreactor. Some companies, such as LanzaTechvi, have already implemented such technologies using acetogenic microorganisms [9.Liew F. et al.Gas fermentation-a flexible platform for commercial scale production of low-carbon-fuels and chemicals from waste and renewable feedstocks.Front. Microbiol. 2016; 7: 694Crossref PubMed Scopus (185) Google Scholar]. Other companies, such as Climeworksvii, have developed technology to separate CO2 from other 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 CO2 levels. The Climeworks CO2 capture technology is coupled to a geothermal power plant, which could supply renewable energy for H2 or formate production for chemolithotrophic growth, or be directly coupled to an electrolysis system. C. necator H16 (formerly Ralstonia eutropha H16) is an attractive chassis for metabolic engineering for CO2 bioconversion to fuel products and is one of the most advanced genetic systems for this purpose [10.Muller J. et al.Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones.Appl. Environ. Microbiol. 2013; 79: 4433-4439Crossref PubMed Scopus (82) Google Scholar]. This organism is a Gram-negative nonpathogenic β-proteobacterium and a facultative chemolithotroph (see Glossary) that grows on mixtures of H2 and CO2 or on formate with no dependence on light availability, because this organism is not a phototroph. C. necator assimilates CO2 using the autotrophic Calvin–Benson–Bassham (CBB) cycle. Redox chemistry is largely controlled in this organism by a soluble hydrogenase (SH) that creates reducing equivalents from the oxidation of H2 gas. O2 or NO3–serves as the terminal electron acceptor for respiration, although NO3–is a poor electron acceptor in this organism [11.Tiemeyer A. et al.Kinetic studies on autohydrogenotrophic growth of Ralstonia eutropha with nitrate as terminal electron acceptor.Appl. Microbiol. Biotechnol. 2007; 76: 75-81Crossref PubMed Scopus (17) Google Scholar]. C. necator also has a natural biosynthetic route to produce the carbon-dense biopolymer poly[(R)-3 hydroxybutyrate] (PHB), a biodegradable plastic-like molecule that is stored in granules during nutrient limitation with titers that can exceed 70% of total biomass by dry weight (Figure 1) [12.Ishizaki A. et al.Microbial production of poly-D-3-hydroxybutyrate from CO2.Appl. Microbiol. Biotechnol. 2001; 57: 6-12Crossref PubMed Scopus (89) Google Scholar]. Carbon flux can be diverted away from PHB synthesis in several cases to generate value-added products, such as isobutanol, methyl ketones, isoprenoids, sucrose, modified PHBs, growth enhancers for plants, and a further variety of carbon-dense compounds and commodity chemicals [10.Muller J. et al.Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones.Appl. Environ. Microbiol. 2013; 79: 4433-4439Crossref PubMed Scopus (82) Google Scholar,13.Lee H. et al.Microbial production of ethanol from acetate by engineered Ralstonia eutropha.Biotechnol. Bioprocess Eng. 2016; 21: 402-407Crossref Scopus (22) Google Scholar, 14.Chen J. et al.Production of fatty acids in Ralstonia eutropha H16 by engineering beta-oxidation and carbon storage.PeerJ. 2015; 3e1468Crossref PubMed Scopus (25) Google Scholar, 15.Nangle S. et al.Valorization of CO2 through lithotrophic production of sustainable chemicals in Cupriavidus necator.Metab. Eng. 2020; 62: 207-220Crossref PubMed Scopus (12) Google Scholar, 16.Liu C. et al.Water splitting-biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis.Science. 2016; 352: 1210-1213Crossref PubMed Scopus (497) Google Scholar, 17.Crepin L. et al.Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production.Metab. Eng. 2016; 37: 92-101Crossref PubMed Scopus (47) Google Scholar]. C. necator can also grow on photovoltaic-derived electricity by H2 and O2 created by water splitting through indirect electron transfer [16.Liu C. et al.Water splitting-biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis.Science. 2016; 352: 1210-1213Crossref PubMed Scopus (497) Google Scholar,18.Li H. et al.Integrated electromicrobial conversion of CO2 to higher alcohols.Science. 2012; 335: 1596Crossref PubMed Scopus (428) Google Scholar,19.Torella J. et al.Efficient solar-to-fuels production from a hybrid microbial-water-splitting catalyst system.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 2337-2342Crossref PubMed Scopus (221) Google Scholar]. In this review, we provide an overview of recent developments and advances in engineering C. necator for biofuel production via autotrophic metabolism. Subsequent evaluation of inherent features and CO2 bioconversion provides readers with insight regarding the major hurdles impeding commercialization of bioproducts from C. necator. We also provide guidance for researchers to navigate the options available for engineering this organism. Under autotrophic growth conditions, C. necator fixes CO2 using the CBB cycle, and grows in atmospheric CO2 concentrations at a slow rate (doubling time ≈20 h) [16.Liu C. et al.Water splitting-biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis.Science. 2016; 352: 1210-1213Crossref PubMed Scopus (497) Google Scholar,20.Claassens N. et al.Phosphoglycolate salvage in a chemolithoautotroph using the Calvin cycle.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 22452-22461Crossref PubMed Scopus (10) Google Scholar]. The CBB cycle involves 11 steps and the key enzyme for carbon fixation in C. necator is type IC ribulose-1,5-bisphosphate-carboxylase/-oxygenase (rubisco) [21.Badger M. Bek E. Multiple rubisco forms in proteobacteria: their functional significance in relation to CO2 acquisition by the CBB cycle.J. Exp. Bot. 2008; 59: 1525-1541Crossref PubMed Scopus (255) Google Scholar]. 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.Li Z. et al.Engineering the Calvin-Benson-Bassham cycle and hydrogen utilization pathway of Ralstonia eutropha H16 for improved autotrophic growth and PHB production.Microb. Cell Factories. 2020; 19: 228Crossref PubMed Scopus (7) Google Scholar]. 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.Gruber S. et al.CbbR and RegA regulate cbb operon transcription in Ralstonia eutropha H16.J. Biotechnol. 2017; 257: 78-86Crossref PubMed Scopus (7) Google Scholar]. 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.Dangel A. Tabita R. CbbR, the master regulator for microbial carbon dioxide fixation.J. Bacteriol. 2015; 197: 3488-3498Crossref PubMed Scopus (28) Google Scholar,25.Grzeszik C. et al.Phosphoenolpyruvate is a single metabolite in transcriptional control of the cbb CO2 fixation operons in Ralstonia eutropha.J. Mol. Microbiol. Biotechnol. 2000; 2: 311-320PubMed Google Scholar]. CbbR-directed transcription is also regulated by levels of ribulose 1,5-bisphosphate (RuBP), ATP, and NADPH [24.Dangel A. Tabita R. CbbR, the master regulator for microbial carbon dioxide fixation.J. Bacteriol. 2015; 197: 3488-3498Crossref PubMed Scopus (28) Google Scholar]. 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 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.Kubis A. Bar-Even A. Synthetic biology approaches for improving photosynthesis.J. Exp. Bot. 2019; 70: 1425-1433Crossref PubMed Scopus (40) Google Scholar,27.Flamholz A. et al.Revisiting trade-offs between Rubisco kinetic parameters.Biochemistry. 2019; 58: 3365-3376Crossref PubMed Scopus (52) Google Scholar]. The oxygenase activity of rubisco forms the toxic compound 2-phosphoglycolate (2-PG), which is not a CBB intermediate and must be recycled in a process termed ‘phosphoglycolate salvage’, known in plants as photorespiration (Box 1) [27.Flamholz A. et al.Revisiting trade-offs between Rubisco kinetic parameters.Biochemistry. 2019; 58: 3365-3376Crossref PubMed Scopus (52) Google Scholar]. Some organisms, including all Cyanobacteria and some autotrophic proteobacteria, 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.Kerfeld C. Melnicki M. Assembly, function and evolution of cyanobacterial carboxysomes.Curr. Opin. Plant Biol. 2016; 31: 66-75Crossref PubMed Scopus (104) Google Scholar]. The carboxysome functions to increase the concentration of CO2 near the active site of rubisco, and also to sequester CO2 from O2 [29.Kaplan A. Reinhold L. The CO2 concentrating mechanisms in photosynthetic microorganisms.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 539-570Crossref PubMed Scopus (556) Google Scholar].Box 1Phosphoglycolate Salvage in C. necatorPhosphoglycolate 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.Yu H. et al.Augmenting the Calvin-Benson-Bassham cycle by a synthetic malyl-CoA-glycerate carbon fixation pathway.Nat. Commun. 2018; 9: 2008Crossref PubMed Scopus (40) Google Scholar, 100.South P. et al.Synthetic glycolate metabolism pathways stimulate crop growth and productivity in the field.Science. 2019; 363eaat9077Crossref PubMed Scopus (239) Google Scholar, 101.Trudeau D. et al.Design and in vitro realization of carbon-conserving photorespiration.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E11455-E11464Crossref PubMed Scopus (51) Google Scholar]. 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.Eisenhut M. et al.The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in Cyanobacteria.Plant Physiol. 2006; 142: 333-342Crossref PubMed Scopus (102) Google Scholar]. 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.Eisenhut M. et al.The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in Cyanobacteria.Plant Physiol. 2006; 142: 333-342Crossref PubMed Scopus (102) Google Scholar,103.Tolbert N. The C-2 oxidative photosynthetic carbon cycle.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 1-23Crossref PubMed Scopus (111) Google Scholar]. By contrast, C. necator phosphoglycolate salvage proceeds primarily through the ‘glycerate pathway’ that is also carried out by Cyanobacteria [20.Claassens N. et al.Phosphoglycolate salvage in a chemolithoautotroph using the Calvin cycle.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 22452-22461Crossref PubMed Scopus (10) Google Scholar,104.Eisenhut M. et al.The photorespiratory glycolate metabolism is essential for cyanobacteria and might have been conveyed endosymbiotically to plants.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 17199-17204Crossref PubMed Scopus (209) Google Scholar]. 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.Claassens N. et al.Phosphoglycolate salvage in a chemolithoautotroph using the Calvin cycle.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 22452-22461Crossref PubMed Scopus (10) Google Scholar]. Nonetheless, the bacterial glycerate cycle is the major route of 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.Yu H. et al.Augmenting the Calvin-Benson-Bassham cycle by a synthetic malyl-CoA-glycerate carbon fixation pathway.Nat. Commun. 2018; 9: 2008Crossref PubMed Scopus (40) Google Scholar, 100.South P. et al.Synthetic glycolate metabolism pathways stimulate crop growth and productivity in the field.Science. 2019; 363eaat9077Crossref PubMed Scopus (239) Google Scholar, 101.Trudeau D. et al.Design and in vitro realization of carbon-conserving photorespiration.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E11455-E11464Crossref PubMed Scopus (51) Google Scholar]. 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.Eisenhut M. et al.The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in Cyanobacteria.Plant Physiol. 2006; 142: 333-342Crossref PubMed Scopus (102) Google Scholar]. 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.Eisenhut M. et al.The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in Cyanobacteria.Plant Physiol. 2006; 142: 333-342Crossref PubMed Scopus (102) Google Scholar,103.Tolbert N. The C-2 oxidative photosynthetic carbon cycle.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 1-23Crossref PubMed Scopus (111) Google Scholar]. By contrast, C. necator phosphoglycolate salvage proceeds primarily through the ‘glycerate pathway’ that is also carried out by Cyanobacteria [20.Claassens N. et al.Phosphoglycolate salvage in a chemolithoautotroph using the Calvin cycle.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 22452-22461Crossref PubMed Scopus (10) Google Scholar,104.Eisenhut M. et al.The photorespiratory glycolate metabolism is essential for cyanobacteria and might have been conveyed endosymbiotically to plants.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 17199-17204Crossref PubMed Scopus (209) Google Scholar]. 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.Claassens N. et al.Phosphoglycolate salvage in a chemolithoautotroph using the Calvin cycle.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 22452-22461Crossref PubMed Scopus (10) Google Scholar]. Nonetheless, the bacterial glycerate cycle is the major route of phosphoglycolate salvage in C. necator. 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 fix CO2. In addition, C. necator expresses a rubisco variant with a relatively high specificity for CO2 [30.Satagopan S. Tabita F. RubisCO selection using the vigorously aerobic and metabolically versatile bacterium Ralstonia eutropha.FEBS J. 2016; 283: 2869-2880Crossref PubMed Scopus (18) Google Scholar]. 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.Gai C. et al.Insights into bacterial CO2 metabolism revealed by the characterization of four carbonic anhydrases in Ralstonia eutropha H16.AMB Express. 2014; 4: 2Crossref PubMed Scopus (29) Google Scholar]. Deletion of CA-like enzymes Caa and Can causes a high CO2-requiring (HCR) phenotype under mixotrophic growth conditions [31.Gai C. et al.Insights into bacterial CO2 metabolism revealed by the characterization of four carbonic anhydrases in Ralstonia eutropha H16.AMB Express. 2014; 4: 2Crossref PubMed Scopus (29) Google Scholar,32.Kusian B. et al.Carbonic anhydrase is essential for growth of Ralstonia eutropha at ambient CO2 concentrations.J. Bacteriol. 2002; 184: 5018-5026Crossref PubMed Scopus (83) Google Scholar]. 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.Burgdorf T. et al.[NiFe]-hydrogenases of Ralstonia eutropha H16: modular enzymes for oxygen-tolerant biological hydrogen oxidation.J. Mol. Microbiol. Biotechnol. 2005; 10: 181-196Crossref PubMed Scopus (165) Google Scholar]. SH expression is coupled to ATP levels by HoxJ, a histidine sensor kinase [34.Lenz O. Friedrich B. A novel multicomponent regulatory system mediates H2 sensing in Alcaligenes eutrophus.Proc. Natl. Acad. Sci. 1998; 95: 12474-12479Crossref PubMed Scopus (134) Google Scholar,35.Lenz O. et al.A hydrogen-sensing system in transcriptional regulation of hydrogenase gene expression in Alcaligenes species.J. Bacteriol. 1997; 179: 1655-1663Crossref PubMed Google Scholar]. The SH also requires a Ni-Fe-CO-2CN–cofactor, which is produced by maturase and chaperone complexes HypABCD and HypEF [36.Bock A. et al.Maturation of hydrogenases.Adv. Microb. Physiol. 2006; 51: 1-71Crossref PubMed Scopus (287) Google Scholar]. C. necator has three catalytically active hydrogenases 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.Burgdorf T. et al.[NiFe]-hydrogenases of Ralstonia eutropha H16: modular enzymes for oxygen-tolerant biological hydrogen oxidation.J. Mol. Microbiol. Biotechnol. 2005; 10: 181-196Crossref PubMed Scopus (165) Google Scholar]. 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.Buhrke T. et al.The H2-sensing complex of Ralstonia eutropha: interaction between a regulatory [NiFe] hydrogenase and a histidine protein kinase.Mol. Microbiol. 2004; 51: 1677-11689Crossref PubMed Scopus (49) Google Scholar,38.Schwartz E. et al.Transcriptional regulation of Alcaligenes eutrophus hydrogenase genes.J. Bacteriol. 1998; 180: 3197-3204Crossref PubMed Google Scholar]. The fourth hydrogenase, AH, is not well characterized, although it has a relatively slow H2 consumption rate (0.5 s–1) and is thought to function in low hydrogen conditions [39.Cramm R. Genomic view of energy metabolism in Ralstonia eutropha H16.J. Mol. Microbiol. Biotechnol. 2009; 16: 38-52Crossref PubMed Scopus (81) Google Scholar, 40.Jugder B. et al.An analysis of the changes in soluble hydrogenase and global gene expression in Cupriavidus necator (Ralstonia eutropha) H16 grown in heterotrophic diauxic batch culture.Microb. Cell Factories. 2015; 14: 42Crossref PubMed Scopus (12) Google Scholar, 41.Schafer C. et al.Structure of an actinobacterial-type -hydrogenase reveals insight into O2-tolerant H2 oxidation.Structure. 2016; 24: 285-292Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar].Box 2Harnessing the Power and Versatility of the Soluble HydrogenaseThe 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.Ghosh D. et al.Increasing the metabolic capacity of Escherichia coli for hydrogen production through heterologous expression of the Ralstonia eutropha SH operon.Biotech. Biofuels. 2013; 6: 122Crossref PubMed Scopus (17) Google Scholar, 106.Lonsdale T. et al.H2-driven biotransformation of n-octane to 1-octanol by a recombinant Pseudomonas putida strain co-synthesizing an O2-tolerant hydrogenase and a P450 monooxygenase.Chem. Commun. 2015; 51: 16173-16175Crossref PubMed Google Scholar, 107.Lamont C. Sargent F. Design and characterisation of synthetic operons for biohydrogen technology.Arch. Microbiol. 2017; 199: 495-503Crossref PubMed Scopus (8) Google Scholar]. 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 cou" @default.
• W3127738948 created "2021-02-15" @default.
• W3127738948 creator A5007745033 @default.
• W3127738948 creator A5009087228 @default.
• W3127738948 creator A5022026178 @default.
• W3127738948 date "2021-04-01" @default.
• W3127738948 modified "2023-10-18" @default.
• W3127738948 title "Metabolic Engineering of Cupriavidus necator H16 for Sustainable Biofuels from CO2" @default.
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