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    Highly Efficient Methane Biocatalysis Revealed in A Methanotrophic Bacterium - Enhanced Reader

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    Bilal Kazmi

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    © All Rights Reserved
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    nature COMMUNICATIONS ARTICLE Received 21 May 2013 | Accepted 16 Oct 2013 | Published 3 Dec 2013 Highly efficient methane biocatalysis revealed in a methanotrophic bacterium MG. Kalyuzhnaya!, S. Yang, O.N. Rozova3, N.E. Smalley, J. Clubb2, A. Lamb?, G.A. Nagana Gowda‘, D Raftery’, Y. Fu2, F. Bringel®, S. Vuilleumier®, D.A.C. Beck2®, Y.A. Trotsenko’, VN. Khmelenina? & ME. Lidstrom!? Methane is an essential component of the global carbon cycle and one of the most powerful -reenhouse gases, yet it is also a promising alternative source of carbon for the biological production of value-added chemicals. Aerobic methane-consuming bacteria (methanatraphs) represent a potential biological platform for methane-based biocatalysis. Here we use a mult- pronged systems-level approach to reassess the metabolic functions for methane utilization in a promising bacterial biocatalyst. We demonstrate that methane assimilation is coupled with a highly efficent pyrophosphate-mediated glycolytic pathway, which under oxygen limitation participates in a novel form of fermentation-based methanotrophy. This surprising discovery suggests a novel made of methane utilization in oxygen-limited environments, and ‘opens new opportunities for a modular approach towards producing a variety of excreted ‘chemical products using methane as a feedstock. "Department of Microbiology, University of Washington, Gox 355014, Seate, Washington 98195, USA. 2 Department of Chemical Engineering University of Washington, Box 355014, Seale, Washington 98195, USA. GK. Skryabin Inttte of Biochemistry and Physiology of Microorganisms, Russian Academy of Scences, Pushchino 142290, Russia. # Northwest Metabolomics Research Center, Anesthesiology a Pain Medicine, University of Washington 8 Republican Stcct, Seattle, Washington 98108, USA. °Equpe Adaptations ot Itoractios Microbionnes dans rwcernement, UMR 7156 UdS ~ CNRS Génétique Molculite, Genomique, Mircbiolog, Université de Stasbourg 67083 Strasbourg Cedex, France. eScience Insitute, University of Washington, ox 35501, Seatle, Washington 98195, USA, t Present addresses: Shandong Province Key Laboratory of Applied Mycology, Schoolot Lie Soences, Qingdao Asricultural University, Changeheng Road 700, Chengyare District, Qingdao 266108, China, and Key Laboratory of Systems Bioengineering, Ministry ol Education Tanjn University, Tianjin 300072, China Corespendence and request fr materials should be ackessed to MLGX (nal: matnaalyuzhaagrailcom), NATURE COMMUNICATIONS] 42785] 00% 101036/neamme37B5 [wu atrecon/nattecommuricons 1 ©2013 Macmillan Publishers Linited. Al rights rosorved ARTICLE resource: natural gas, relatively abundant today but still a non-renewable fossil fuel, and renewable biogas, a byproduct of modern society that is often wasted!" Interest in new technologies for effective conversion of flared/waste sources, ‘of methane into chemical compounds, including next-generation fuels, continues to increase®. The use of microbial cells and, enzymes as catalysts for methane conversion represents an appealing approach in this context’~"". The benefits of methane biotechnology include a sef-sustainable component, as any biomass generated could be used as. single cell protein or converted back to methane via anaerobic digestion. However, besides single cell protein and polyhydroxybutyrate, exploitation, ‘of methane-based catalysis for the production of chemicals and fuels has not yet proven successful at the commercial level ‘Gammaproteobacterial methanotrophs with the ribulose mono- phosphate (RuMP) pathway are among the most promising microbial systems for methane-based biotechnology. Reconstruc: tion of the methane utilization network in these methanotrophs, thas been based on a number of biochemical studies that pointed towards the Entner-Doudorolf (EDD)-variant of the RuMP pathway as the major route for single carbon (C,)assiila tion!2“F, The major biochemical evidence that favoured the EDD. \ariant of the RuMP cycle included relatively high activities of two key enzymes of the pathway (6-phosphogluconate dehydratase and 2-keto-3-deoxy-6:phosphogluconate aldolase) and multiple enzymatic lesions. inthe Embden-Meyerhof-Parnas (EMP) pathway!®4®, Activity of pyruvate kinase has not been detected previously in any gammaproteobacterial methanotroph!™"”, The Presence of a reversible pyrophosphate _(PP!)- 3 PGA+3NADH + 3H" (9) 9CH,O + 3 NAD! +2 ADP +5P, 3 PGA+3NADH+ 3H" 2ATP. Figure 1 | Methane oxidation and formaldehyde assimilation. (8) Predicted positions of incorporated C (indicted in ed) for the EDD (dark red arows) and the EMP (blue arrows) variants. Dashed lines inicate multistep reactions. The pentese-phosphate pathway variant for regeneration of ribulose S-phosphateis indicated by orange dashed arcows. Enzyme activites (in nmol in~ per mg protein) in cll ee extract of M.alcaliphilum 202 were as follows: methane monosygenase (Nim), 705: PQQ-dependent methanol dehydrogenase (Mh), 230+ 12; NAD-dependent formate dehydrogenase (Fan), 130 £7; hexulose phosphate synthase/hexulose phosphate isomerase (Hos/Hp)), 600+30; glucose phosphate isomerase (Gpi), 325; NADP. dependent glucose 6-phosphate dehycrogenase, 34:42 (Gp); NAD-dependent glucose 6-phosphate dehydrogenase, 2342 (Gdo); NADP-dependent ‘6 phosphogluconate dehydrogenase (Edd), 32 +2; KDPG aldolase (Eda), 60 +4; fructose bisphosphate aldolase (Fea), 35+ 2: Pi phosphofrucokinase (i, 70+ 4, (b) Summary equations for poduction of 3-phosphoslycerae from formaldehyde va the RUMP pathway forthe EDD variant 1) or the EMP variant with either the ATP-dependent EMP pathway (2) oF the PPr-dependent EMP pathway (2). Abbreviations: RUBP, ribulose S-phosphate: HesP, “-hevulese & phosphate FP, ructose 6-phosphate; KOPG, 2-Lto-3-deoxy 6-phasphogluconate; FP, fructose 16-bsphosphate; DAP, hydrxyacetone phosphate: GAP, glyceraldehyde 3-phosphate; PGA, 3-chosphoslycerate; Pi, inorganic phosphate, ‘Table 1 | Kinetic characteristics of pyruvate kinase 2 from ‘M. alcaliphilum 202. Substrates Vos (Umg of Kho)" Ca) PEP inthe presence of 25 mM base SP 1a3.654.443 384006" 25mM frctoce-cP 179142559. (0.122008) 251M glicose-6P 200101160 (017 2002" Mach! 1992017" ADP 13542945 (0160.03 pet 82662197 0162001 cor’ 13295: 6.02 0392005 opt 998021850 {Niwarome were teen these 2S tee hope should be labelled in position 3 (Fig. 1a). As shown in Fig. 2, only ‘a small fraction of pyruvate was labelled in position 1 during the ‘course of the experiment. The rate of C incorporation into position 3 of pyruvate was at least six-fold higher than the rate of incorporation into position 1. These experiments confirmed that the major fraction of cellular pyruvate comes fom the EMP pathway during growth of the methanotrophic culture on methane. A similar carbon isotopic distribution in pyruvate was, observed for Methlomonas sp. LW13, a typical representative of gammaproteobacterial methanotrophic bacteria (Supplementary Fig, $2). ‘Methane utilization via fermentation. The new arrangement of the methanotrophic network opens up a possibility for ferme tation, Methanotrophs require O> for the oxidation of methane, so experiments were carried outwith cells grown in bioreactors in which air was provided at low levels and the dissolved O, con- centrations were kept at undetectable to 0.1%. In a continuous bioreactor culture, M. alcaliphilum 202. grew slowly, with a doubling time of 23h, Transcriptomic profiles of batch bioreactor cultures grown at low O2 showed that similar to what is observed {or aerobic growth, relative expression of EMP genes is high. The ‘most notable changes in the transcriptome were the down- regulation of genes for NADH¢ubiquinone oxidoreductase and cytochrome ¢ oxidase, and the upregulation of genes for the O3 carrier bacteriohemerythrin and for the pathways for mixed-acid fermentation and H; production (Supplementary Table SI and NATURE COMMUNICATIONS] 42785] 00% 101036/neamme37B5 [wu atrecon/nattecommuricons a ©2013 Macmillan Publishers Linited. Al rights rosorved ARTICLE Supplementary Fig. $3). In our experiments, the expression prolile of bacteriohemerythrin, shown to be essential for high in vitro activity of particulate methane monooxygenase in 'M, capsulatus Bath, indicated that it most likely contributes to ‘Oy-scavenging/partitioning in M. alealiphilum 202. Genes predicted to encode fermentation pathway enzymes with Increased expression under mictooxic conditions included a putative acetate kinase, 3-ketoacyl-CoA thiolase, 3-hydroxyacyl CoA dehydrogenase, malate dehydrogenase, fumarase, succinate dehydrogenase and lactate dehydrogenase. Intriguingly, upregulation of genes encoding a NAD-reducing hycrogenase was observed (Supplementary Table S1). These changes suggested production of a set of possible fermentation products, including formate, acetate, succinate, lactate, 3-hydroxybutyrate and Hs. Significantly, in bioreactor cultures acetate, succinate, lactate and H, were detected, but only in medium from batch and chemostat cultures grown at low O: tension, whereas formate increased about threefold (Table 2). Extracellular concentrations of these ‘acids increased markedly alter incubation of low O: bioreactor samples in closed vials flushed with Na. Low amounts of S-hydroxybutyrate also accumulated. Furthermore, significant ‘accumulation of H, was detected in closed vial experiments (Table 2). Similar incubations supplied with !C-methane confirmed that formic and acetic acids were produced from methane (Supplementary Table $4). ‘The rate of methane ‘consumption in the closed vial experiments was exceptionally low (1.75041 nmol min~! per mgprotein~'); however 3% and 15% of the added methane was consumed in 12 and 60b, respectively. The total amount of produced extracellular carbon, Relate abundance (%) mer (Crabotog) mM (C34aboled) wM2ne Figure 2| Pyruvate™C-abeling patterns in M. acaliphitum 202. Intracellar pyruvate was resolved by multiple reactions monitoring scan ‘mode on mass spectrometry. Green, "™C-doubly and tpl labelled pyruvate; red, PC-pyruvate labelled in poston t; Blue, C- pyruvate Isbell in poston 3 NATURE COMMUNICATIONS | DOE: 10.1038/ncomms3785 mostly acetate and formate, was equivalent to 40-50% of the total ‘methane carbon consumed. These data suggest that in the presence of sufficient O, to drive methane oxidation, 1M. alealiphilum 202 is capable of fermentation from methane derived formaldehyde, and that methane utilization at low O; tension involves switching to a novel fermentation mode leading to the formation of formate, acetate, succinate, lactate and hydroxybutyrate as end products, with ttle biomass synthesis (Fig. 3). The presence of putative fermentation genes in the genome of gammaproteobacterial ethanotrophs suggests that this type of metabolism is likely widespread (Supplementary Table $5). Discussion ‘The experiments presented here change our understanding of ethane assimilation through the RuMP. pathway in obligate methanotrophic bacteria in. some fundamental aspects. Tits, Ulliation of the PPi-mediated EMP pathway significantly increases the predicted efxiency of one-carbon assimilation. Genes encoding a membrane-bound. proton-translocating pyrophosphatase show significant expression, suggesting that this Enuyme could be ‘one of the possible candidates’ for the regeneration of PPI from ATP. The predicted ratio of ATP Ihydolysi/PPs formation for this lass of enzymes is 13 (ref. 23). Therefore, not only does the assimilation af 9mol. of formaldehyde by this metabolic scheme to generate three carbon intermediates require no additional energy. it actually produces three moles of reducing power (NADI) and two moles OF ATP (Fig. 1b). Second, the surprising discovery of this ATP-producing assimilatory route Taised the possibilty of fermentation asa new mode of methane ulation. The ability of methanotrophic cultures to convert methane into excreted organic compounds has. previously been described and it has been suggested. to support denitrification by’ wastewater treatment comms. nities", However, metabolic pathways for production of ‘organic compounds from methane were unknown, and excreted compounds were often describe asthe product of cll ysis or a Starvation response?"=”, Our results suggested the possibility that donganic acide could be produced as a result of fermentation, This discovery challenges our understanding of methanotrophy, as ‘nicrobial metabolism linked to respiration (O3, NO; of SO) tnd has “major implications for’ the environmental role of methanotrophic bacteria in removing this greenhouse gis in" Oylimited environments, ‘Numerous environmental Studies” indicate that ~gammaproteabacterial_ methanotrophs thrive at oxic-anoxic interfaces". If fermentation is the ‘major metabolic mode of methane cycling under O,-limiting conditions, then our understanding ofthe role of methanottophic bacteria in supporting the lobal carbon cycle may be in need of ‘Table 2 | Accumulation of extracellular metabolites (jumol (gDCW~")). compound ‘Aerobic bioreactor 49-54% 403 Micro-aerobic bioreactor (0-0.1% d0,) Vial incubations Formate 687275 15324272 1872 # 649 (339 +184)" Acetate ‘race 2046022 504 #13 (484 213) Succinate (0252003 6562078 (631407) Lactate 381087 1o2+13 (eau 3-Hydronbutyrate - e 0.424003 (0.42 40.03)" 70x03 2237 238 Methane consumed No. ND. 2902468 {ON ay a nag na arn 0» Sed Os vot one 4 NATURE COMMUNICATIONS 42785] 0% 1028/1 me |wawnaturecom/atrecomaricatore ©2013 Macmillan Publishers Linited. Al rights rsorved NATURE COMMUNICATIONS | DO! 10.1038/ncomens3785 ARTICLE a ‘Aerob growth b Ortinited rowan on, o HO mate Pyrvate Puck fate at ‘BIOMASS, 60, Acetate, lacie, Formate succinate, Hy Figure 3 | Proposed schemes for methane utilization in M.alcalphlum 202. (a) Methane utiizaton at O» saturation (b) Methane utlization at ‘Ortimiting conltions revision in some respects ~ not so much in terms of carbon being mineralized into COs, but of the proportion of carbon ending in biomass (Fig. 3). Most notably, in our experiments, only a smal fraction of the oxidized methane was converted to biomass, so methanotrophic bacteria may represent only a minor part of the ‘overall microbial community involved in the coaversion of methane into biomass. Rather, our results suggest the possibilty that in. Oplimiting environments, methanotrophs drive the conversion of methane to excreted products and hydrogen, ‘which are then used and transformed by noa-methanotrophs. If confizmed by future work, this bas implications for microbial ‘community strueture and functioning in environments where the methane cycle is prominent. “The surprising discovery of glycolyss-hased methane assimila- tion and production of hydrogen in strain M. alcaliphilum 202, may open new opportunities to producing a variety of products using methane as a feedstock, provided this pathway may be ‘operated in a suficiently efficient way inthe laboratory. Virtually all biosynthetic modules forthe production of a wide variety of chemicals developed for glucose-based catalysis in E. coli could ‘aso be implemented in cultures containing this EMP variant of the RuMP pathway. This, our demonstration of methane-based fermentation suggests new approaches for the commercial conversion of methane to hydrogen and excreted products using mmicrooxic production conditions. Methods Cell eultvation and growth parameters slip 202 sls were grown ‘sings mine alt etiam’ nether coed vals 30 ml clare in. 250 Wl ‘ea shaking a 20 rp) or brractor clare (il atch or cherosa. ‘working volume in «1 bench top Bs 110 medklarbeeactor, New Brunswick ‘Scie. Edison, N}, USA). Cs were grown a 28°C. Optical density of cl ‘ces nae mensied on 2 Beckman DU G1 spscrophotomster in psi 5 Inlcietter eth» Tem path length Chemostat clare msintind # as ate pees deny 30 nm (OD) of ~ 20202. The dition rate wan D2 oe Stobart gus mature ~ 20% CH,20% Os Ny, dncredO: Tens wat 49-54) apd MOSH fr low: ears (auent gas mtu 20% (Ci 0275 SN dssled , tension was non detectable 0.1%. pH (0) was ‘Srl bythe atoms addition of 1X NaOL. pation sas hep conta at 1Oa0 ep Sample ofl and ton ones were elt daly in apts for gs analyse The rates of methane canton and Hy, production were

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