Metabolic Engineering of Cupriavidus necator H16 for Sustainable Biofuels from CO2

Cupriavidus necator has a wide metabolic range and naturally creates biopolymer, poly[(R)-3 hydroxybutyrate] (PHB). Using engineering techniques, carbon flux can be directed away from PHB synthesis toward the generation of biofuels bioproducts.Researchers demonstrated production many biofuel products using C. necator, including methyl ketones, isoprenoids terpenes, isobutanol, alkanes alkenes, variety commodity chemicals CO2.Growth bioproduct electrolysis was recently demonstrated, use an artificial leaf system.While genetic remains laborious process, synthetic biology tools for this organism are being expanded with new technologies that will allow large alterations to its genome. Decelerating global warming is one predominant challenges our time require conversion CO2 usable chemicals. Of particular interest fuels, because transportation sector major source emissions. Here, we review recent technological advances in hydrogen-oxidizing bacterium H16, chemolithotroph consumes generate biomass. We discuss successes organism, implementation electrolysis/artificial photosynthesis approaches enable growth renewable electricity CO2. Last, prospects improving nonoptimal ambient concentrations Satisfying current future energy demand while allowing favorable environmental outcomes innovative solutions fuel sector. Although portions infrastructure electrified (such as automobile transport), energy-dense carbon-based fuels continue required aviation, long-distance trucking, rocketry, maritime shipping, industrial operations. Petroleum-based finite, given BP International Energy Agency (IEA) project petroleum sources likely depleted between 2050 2070 oil extraction technologiesi–iii. Climate change caused by increase (and CH4) levels Earth’s atmosphere brought cataclysmic weather events decimated ocean habitats years, trend through lifetimes emitted into at rate ?32 billion tons per year [1.Hughes T.P. et al.Global recurrent mass bleaching corals.Nature. 2017; 543: 373-377Crossref PubMed Scopus (1356) Google Scholar, 2.Frolicher T. al.Marine heatwaves under warming.Nature. 2018; 560: 360-364Crossref (312) 3.Friedlingstein P. Solomon S. Contributions past present human generations committed dioxide.Proc. Natl. Acad. Sci. U. A. 2005; 102: 10832-10836Crossref (42) 4.Smith K. al.Joint CH4 accountability warming.Proc. 2013; 110: E2865-E2874Crossref (31) 5.Stott How climate affects extreme events.Science (80-. ). 2016; 352: 1517Crossref (213) 6.Altieri Gedan dead zones.Glob. Chang. Biol. 2014; 21: 1395-1406Crossref (164) Scholar]iv. Therefore, sustainable economically viable bioconversion should realized within next decade. A small but significant mitigation anthropogenic release achieved atmospheric air feedstock produce microorganisms: if 10% aviation (accounting ~2.6% total emissions [7.Staples M. al.Aviation reductions alternative jet fuels.Energy Policy. 114: 342-354Crossref (70) Scholar]) were replaced Sustainable Aviation Fuels (SAFs) titer 1 g/l assuming 90 biomass accumulation [8.Ammann E. al.Gas consumption Hydrogenomonas eutropha continuous culture.Appl. Microbiol. 1968; 16: 822-826Crossref Scholar], ~500 000 could produced recycled (at density 0.8 kg/l). In addition, accumulated would sequester ~80 million (mitigating ?0.25% emissions). ideal it inexpensive, nontoxic, abundant starting material (?850 currently atmosphere). Furthermore, noncompetitive food supply chain. Technoeconomic analysis indicates CO2-derived US$10–250 industry 2030v. However, dilute (?0.04% volume) challenging directly nonphototrophic organisms engineered autotrophically do not grow optimally To circumvent issue, utilized engineer higher assimilation rates Alternatively, microorganisms grown elevated runoff or form syngas when bioreactor. Some companies, such LanzaTechvi, have already implemented acetogenic [9.Liew F. fermentation-a flexible platform commercial scale low-carbon-fuels waste feedstocks.Front. 7: 694Crossref (185) Scholar]. Other Climeworksvii, developed technology separate other components concentrated form. captured Climeworks system used support microbes levels. The capture coupled geothermal power plant, which H2 formate chemolithotrophic growth, system. H16 (formerly Ralstonia H16) attractive chassis most advanced systems purpose [10.Muller J. al.Engineering autotrophic heterotrophic ketones.Appl. Environ. 79: 4433-4439Crossref (82) This Gram-negative nonpathogenic ?-proteobacterium facultative (see Glossary) grows on mixtures no dependence light availability, phototroph. assimilates Calvin–Benson–Bassham (CBB) cycle. Redox chemistry largely controlled soluble hydrogenase (SH) reducing equivalents oxidation gas. O2 NO3–serves terminal electron acceptor respiration, although NO3–is poor [11.Tiemeyer al.Kinetic studies autohydrogenotrophic nitrate acceptor.Appl. Biotechnol. 2007; 76: 75-81Crossref (17) also natural biosynthetic route carbon-dense biopolymer (PHB), biodegradable plastic-like molecule stored granules during nutrient limitation titers exceed 70% dry weight (Figure 1) [12.Ishizaki al.Microbial poly-D-3-hydroxybutyrate CO2.Appl. 2001; 57: 6-12Crossref (89) Carbon diverted several cases value-added products, isoprenoids, sucrose, modified PHBs, enhancers plants, further compounds Scholar,13.Lee H. ethanol acetate eutropha.Biotechnol. Bioprocess Eng. 402-407Crossref (22) 14.Chen al.Production fatty acids beta-oxidation storage.PeerJ. 2015; 3e1468Crossref (25) 15.Nangle al.Valorization lithotrophic necator.Metab. 2020; 62: 207-220Crossref (12) 16.Liu al.Water splitting-biosynthetic reduction efficiencies exceeding photosynthesis.Science. 1210-1213Crossref (497) 17.Crepin L. al.Metabolic alka(e)ne production.Metab. 37: 92-101Crossref (47) photovoltaic-derived created water splitting indirect transfer [16.Liu Scholar,18.Li al.Integrated electromicrobial alcohols.Science. 2012; 335: 1596Crossref (428) Scholar,19.Torella al.Efficient solar-to-fuels hybrid microbial-water-splitting catalyst system.Proc. 112: 2337-2342Crossref (221) review, provide overview developments via metabolism. Subsequent evaluation inherent features provides readers insight regarding hurdles impeding commercialization bioproducts necator. guidance researchers navigate options available organism. Under conditions, fixes CBB cycle, slow (doubling ?20 h) Scholar,20.Claassens N. al.Phosphoglycolate salvage chemolithoautotroph Calvin cycle.Proc. 117: 22452-22461Crossref (10) cycle involves 11 steps key enzyme fixation type IC ribulose-1,5-bisphosphate-carboxylase/-oxygenase (rubisco) [21.Badger Bek Multiple rubisco forms proteobacteria: their functional significance relation acquisition cycle.J. Exp. Bot. 2008; 59: 1525-1541Crossref (255) With exception triose-3-phosphate isomerase (tpi) ribose-5-phosphate (rpi), all enzymes encoded cbb operon, two copies Both operon contribute autotrophy 2) [22.Li Z. Calvin-Benson-Bassham hydrogen utilization pathway improved production.Microb. Cell Factories. 19: 228Crossref (7) One copy located chromosome 2 genome encodes 14 genes. nearly identical pHG1 megaplasmid, second lacks genes chromosomal copy, LysR-type transcriptional regulator cbbR dehydrogenase-like gene, cbbB [23.Gruber al.CbbR RegA regulate transcription H16.J. 257: 78-86Crossref regulated CbbR, causes repression excessive phosphoenolpyruvate (PEP) present, occurs metabolism [24.Dangel Tabita R. master microbial dioxide fixation.J. Bacteriol. 197: 3488-3498Crossref (28) Scholar,25.Grzeszik al.Phosphoenolpyruvate single metabolite control operons eutropha.J. Mol. 2000; 2: 311-320PubMed CbbR-directed ribulose 1,5-bisphosphate (RuBP), ATP, NADPH energetically expensive, requiring net 7 moles ATP convert 3 mole pyruvate. pathway, rubisco, carboxylase oxygenase activity. Efforts rational design more efficient variants been unsuccessful, physicochemical constraints binding pocket make difficult distinguish [26.Kubis Bar-Even Synthetic photosynthesis.J. 2019; 70: 1425-1433Crossref (40) Scholar,27.Flamholz al.Revisiting trade-offs Rubisco kinetic parameters.Biochemistry. 58: 3365-3376Crossref (52) activity toxic compound 2-phosphoglycolate (2-PG), intermediate must process termed ‘phosphoglycolate salvage’, known plants photorespiration (Box [27.Flamholz organisms, Cyanobacteria some proteobacteria, mitigate weak performance utilizing CO2-concentrating mechanisms (CCMs). CCMs characteristically include inorganic (HCO3–) transporters proteinaceous housing called carboxysome, 200+ MDa icosahedral structure contains carboxylation well carbonic anhydrases, bicarbonate vicinity [28.Kerfeld Melnicki Assembly, function evolution cyanobacterial carboxysomes.Curr. Opin. Plant 31: 66-75Crossref (104) carboxysome functions concentration near active site [29.Kaplan Reinhold concentrating photosynthetic microorganisms.Annu. Rev. Physiol. 1999; 50: 539-570Crossref (556) Scholar].Box 1Phosphoglycolate Salvage necatorPhosphoglycolate expensive wasteful losing 2-PG (RuBP). Recent experiments Synechococcus elongatus C3 suggest modifying phosphoglycolate routes lead increased efficiency especially loss eliminated completely, described vitro [99.Yu al.Augmenting malyl-CoA-glycerate pathway.Nat. Commun. 9: 2008Crossref 100.South al.Synthetic glycolate pathways stimulate crop productivity field.Science. 363eaat9077Crossref (239) 101.Trudeau D. al.Design realization carbon-conserving photorespiration.Proc. 115: E11455-E11464Crossref (51) side towards RuBP oxygenation produces 2-PG, intermediate. inhibitor triose phosphate (Tpi), quickly converted metabolized maintain ability [102.Eisenhut al.The plant-like C2 bacterial-like glycerate cooperate Cyanobacteria.Plant 2006; 142: 333-342Crossref (102) Plants recycle >25% chemical least ten enzymatic Scholar,103.Tolbert C-2 oxidative cycle.Annu. 1997; 48: 1-23Crossref (111) By contrast, proceeds primarily ‘glycerate pathway’ carried out [20.Claassens Scholar,104.Eisenhut photorespiratory essential cyanobacteria might conveyed endosymbiotically plants.Proc. 105: 17199-17204Crossref (209) decarboxylated tartronic semialdehyde, then reduced glycerate, phosphorylated 3-phosphoglycerate. When removed gene knockout, relies novel ‘malate cycle’ fully oxidizes Bioinformatic suggests malate widespread chemolithotrophs Nonetheless, bacterial Phosphoglycolate lacking typical CCM 3). expresses four anhydrase-like (CA-like) obtain sufficient cytoplasm fix variant relatively high specificity [30.Satagopan RubisCO selection vigorously aerobic metabolically versatile eutropha.FEBS 283: 2869-2880Crossref (18) CA-like three different evolutionary classes Caa, Can, Can2 Cag. Three these cytoplasmic, whereas Caa localized periplasm [31.Gai al.Insights revealed characterization anhydrases H16.AMB Express. 4: 2Crossref (29) Deletion Can CO2-requiring (HCR) phenotype mixotrophic conditions Scholar,32.Kusian B. al.Carbonic anhydrase concentrations.J. 2002; 184: 5018-5026Crossref (83) types megaplasmid prominent roles generation. Hydrogenases metalloenzymes catalyze 2H+ 2e–with redox potential (Eo?) –414 mV. SH considerable biotechnology bioproduction gas introducing H2-based 2). hydrogenases unusual, they oxygentolerant. case SH, electrons reduce NAD+ NADH, providing autotrophy. hoxFUYHWI operon: HoxFU diaphorase, HoxYH [NiFe]-type hydrogenase. HoxW HoxI accessory proteins [33.Burgdorf al.[NiFe]-hydrogenases H16: modular oxygen-tolerant biological oxidation.J. 10: 181-196Crossref (165) expression HoxJ, histidine sensor kinase [34.Lenz O. Friedrich multicomponent regulatory mediates sensing Alcaligenes eutrophus.Proc. 1998; 95: 12474-12479Crossref (134) Scholar,35.Lenz al.A hydrogen-sensing regulation species.J. 179: 1655-1663Crossref requires Ni-Fe-CO-2CN–cofactor, maturase chaperone complexes HypABCD HypEF [36.Bock al.Maturation hydrogenases.Adv. Microb. 51: 1-71Crossref (287) catalytically addition membrane-bound (MBH), (RH), actinobacterial (AH) MBH [NiFe] comprising HoxK HoxG,the reduces ubiquinone, supporting respiration chain generating proton motive force drive substrate-level phosphorylation, producing RH comprises HoxB, HoxC, HoxJ subunits downstream MBH. phosphorylates HoxA factor presence molecular activate expression, directing ?54 promoters [37.Buhrke H2-sensing complex eutropha: interaction protein kinase.Mol. 2004; 1677-11689Crossref (49) Scholar,38.Schwartz al.Transcriptional eutrophus genes.J. 180: 3197-3204Crossref fourth hydrogenase, AH, characterized, (0.5 s–1) thought low [39.Cramm Genomic view 2009; 38-52Crossref (81) 40.Jugder al.An changes (Ralstonia eutropha) diauxic batch culture.Microb. 14: 42Crossref 41.Schafer al.Structure actinobacterial-type -hydrogenase reveals O2-tolerant oxidation.Structure. 24: 285-292Abstract Full Text PDF (23) 2Harnessing Power Versatility Soluble HydrogenaseThe allows oxidize (which readily passes across cellular membrane) cytosol NADH. versatile, expressed oxygen tolerant, characteristic simplifies [105.Ghosh al.Increasing capacity Escherichia coli heterologous operon.Biotech. Biofuels. 6: 122Crossref 106.Lonsdale al.H2-driven biotransformation n-octane 1-octanol recombinant Pseudomonas putida strain co-synthesizing P450 monooxygenase.Chem. 16173-16175Crossref 107.Lamont Sargent Design characterisation biohydrogen technology.Arch. 199: 495-503Crossref (8) Expression bypasses need reverse flow Q-cycle, mechanism where shuttled endothermic direction NADH I). cou