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Home , Glucokinase, Glycerate dehydrogenase, Methanotroph, Methylocapsa acidiphila, Methylococcaceae, Methylococcus capsulatus

Metabolic Features of Aerobic : News and Views Valentina N. Khmelenina*, Sergey Y. But, Olga N. Rozova and Yuri A. Trotsenko

G.K. Skryabin Institute of and of Microorganisms, Russian Academy of Sciences, Pushchino, Russia. *Correspondence: [email protected] htps://doi.org/10.21775/cimb.033.085

Abstract halotolerant representatives are especially Tis review is focused on recent studies of promising for industrial applications, as they are in aerobic methanotrophs that spe- genetically tractable due to the availability of a cifcally addressed the properties, distribution and variety of genetic manipulation tools (Kalyuzh- phylogeny of some of the key involved naya et al., 2015; Mustakhimov et al., 2015; Fu and in assimilation of carbon from . Tese Lidstrom, 2017; Garg et al., 2018). Te adaptation include enzymes involved in synthesis and of halo- and thermotolerant methanotrophs to cleavage, conversion of intermediates of the tricar- extreme environmental conditions includes the boxylic acid cycle, as well as in osmoadaptation in acquisition of the specifc mechanisms for syn- halotolerant methanotrophs. thesis and reutilization of the compatible solutes. Tis review focuses on the recent studies of carbon metabolism in three model species of the aerobic Introduction methanotrophs Methylomicrobium alcaliphilum Methanotrophs inhabit a variety of ecosystems 20Z, trichosporium OB3b, and Methy- including soils, fresh and marine waters and sedi- lococcus capsulatus Bath. ments, saline and alkaline lakes, hot springs, rice paddies, peatlands, and tissues of higher organisms. Tey play a major role in both global carbon and The carbon assimilation global cycles (Hanson and Hanson, 1996; pathways McDonald et al., 2008, Etwig et al., 2010; Khadem Methanotrophs obtain for growth pre- et al., 2010). Te metabolic potential of methano- dominantly via oxidation of methane to CO2 and trophs as industrial platforms for bioconversion of assimilate carbon at the level of , for- methane into value added compounds is currently mate and/or CO2 via three biochemical pathways under intense investigation, aimed at organisms that have been originally deciphered by Professor that are best suited for such applications. Te recent J. R. Quayle and colleagues in the middle of the genomic and biochemical studies demonstrate high 20th century. For more details, we refer the reader metabolic fexibility of methanotrophs, and high to the recent reviews (Bowmann, 2006; Kelly et specialization of the enzymes of their central meta- al., 2014; Webb et al., 2014). Te known aerobic bolic pathways towards methane utilization and methanotrophs belong to the phyla Proteobacte- also towards extreme condition adaptations. From ria, , and the yet unnamed NC10 the multitude of the characterized methanotrophs, phylum. Te proteobacterial methanotrophs are

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classifed as type I or type II, according to whether methanotrophs possess non-homologous enzymes they belong to the Alpha- or which can perform PEP carboxylation, one pos- class. Te consensus is that gammaproteobacterial sibility being a PPi-dependent PEP carboxykinase methanotrophs, represented by the families Methy- (Chiba et al., 2015; Khmelenina et al., 2018). Among lococcaceae, Methylothermaceae and Candidatus the methanotrophs, the glyoxylate regeneration family Crenotrichaceae, assimilate formaldehyde cycle associated with the poly-β-hydroxybutyrate via the ribulose monophosphate (RuMP) pathway, (PHB) pathway is present only in members of the where hexosephosphates are the early products family, whereas in the Beiyerincki- formed by the condensation of formaldehyde and aceae family, this function can be fulflled by the ribulose-5-phosphate. Te alphaproteobacterial glyoxylate shunt (Chen et al., 2010; Matsen et al., methanotrophs of the families Methylocystaceae and 2013). However, gammaproteobacterial methano- assimilate carbon from methane via trophs presumably lack this pathway. the serine pathway, where the serine is Two key serine cycle enzymes, hydroxypyruvate frstly formed. Te methanotrophic representatives reductase (Hpr) and serine-glyoxylate ami- of the Verrucomicrobia (family Methylacidiphi- notransferase (Sga), have been purifed from three laceae) and the NC10 phyla use methane as the methanotrophic : Mm. alcaliphilum 20Z, Mc.

energy source and fx carbon at the CO2 level, via capsulatus Bath and Ms. trichosporium OB3b (But et the Calvin–Benson–Bassham (CBB) cycle (Dun- al., 2017, 2018a). Te biochemical properties of feld et al., 2007; Islam et al., 2008; Khadem et al., methanotrophic Hprs difered from those of other 2011; van Teeseling et al., 2014; Rasigraf et al., organisms. Unlike plants or non-methanotrophic 2014). In this chapter we will concentrate on the bacteria (Chistoserdova et al., 1991; Ho et al., 1999; heterotrophic methanotrophs that use reasonably Ali et al., 2003), the three methanotrophic Hprs efcient RuMP or serine cycles. catalysed the irreversible NAD(P)H-dependent reduction of hydroxypyruvate and glyoxylate, but The serine cycle were unable to oxidize of glycerate and glycolate Genomic analysis revealed that the encod- (But et al., 2017). Such peculiarity, along with high ing the enzymes of the serine cycle occur in both activity and afnity to hydroxypyruvate, suggest type I and type II methanotrophs, though the that these enzymes must be of primary importance functionality and the role of the serine cycle in for C1 assimilation. Like other Hprs characterized type I methanotrophs remain not fully under- to date, Hprs from Mm. alcaliphilum 20Z and Mc. stood (But et al., 2017). In both alpha- and capsulatus Bath are homodimeric, whereas the Ms. gammaproteobacterial methanotrophs, genes trichosporium Hpr is tetrameric (But et al., 2017). encoding hydroxypyruvate reductase (hpr), Te three enzymes displayed similar pH optima serine-glyoxylate aminotransferase (sga), glycerate- in vitro, while these strains are either neutrophilic 2- (gck2), malate thiokinase (mtkAB), (strains OB3b and Bath) or alkaliphilic (strain malyl-CoA- (mcl), 20Z). Hpr from Ms. trichosporium OB3b displayed (mdh), serine-transhydroxymethylase (glyA), higher activity and afnity than the enzymes from as well as methylene tetrahydromethanopterin/ Mm. alcaliphilum 20Z and Mc. capsulatus Bath. Hpr

methylene tetrahydrofolate (H4F) dehydroge- from Mm. alcaliphilum 20Z was shown to be a sub- nase (mtdA) and formate-tetrahydrofolate lygase ject to , its activity increasing in (ffL) are adjacently located in a single, two or the presence of the intermediates of and several clusters (Fig. 4.1), thus corroborating the the TCA cycle, serine, ADP, ATP and inorganic ‘module’ organization of the serine cycle genes in , but decreasing in the presence of (Chistoserdova, 2011). In all type Pi, pyruvate, acetyl-CoA, phosphoenolpyruvate II methanotrophs, the cluster additionally contains and ribulose-5-phosphate. Te capacity of the genes encoding phosphoenolpyruvate carboxylase to reduce glyoxylate into glycolate, in com-

(ppc) and methylene H4F cyclohydrolase (fch), bination with FAD-dependent glycolate oxidase whereas among type I genomes, only (ADVE02_v2_14253-14256; MCA1499-150 and the species harbour the ppc genes. MALCv4_1676-1678) (Ward et al., 2004; Stain et It is possible that other representatives of type I al., 2010; Vuilleumier et al., 2012), suggests that

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Methylomicrobium alcaliphilum, mtkB mtkA mcl sga hpr mdh mtdA gck2 glyA ftfL Methylomicrobium kenyense, Methylomarinum vadi, Methylomicrobium agile, Methylosarcina lacus, Methylobacter luteus, Methylobacter whittenburyi, Methylobacter marinus mtkB mtkA mcl sga hpr mdh mtdA gck2 glyA ftfL Methylosarcina fibrata

Methylomonas denitrificans, mtkB mtkA mcl sga hpr mdh mtdA gck2 glyA ftfL ppc , Methylomonas sp. MK1, Methylomonas sp. 11B, Methylovulum miyokonese mtkB mtkA mcl sga hpr mdh mtdA gck2 glyA ftfL

Methylomicrobium album

mtkB mtkA mcl sga hpr gck3 mdh mtdA glyA ftfL Methycaldum szegediense, Methylohalobus crimeensis, 73a,

mtkB mtkA mcl sga hpr mdh mtdA gck2 glyA ftfL

Methyloglobus morosus

glyA ftfL sga hpr mtdA fch mtkB mtkA ppc mcl gck2 Methylosinus sp. PW1, Methylosinus sp. LW3, Methylosinus sp. LW4, Methylosinus sp. LW5, Methylosinus trichosporium, Methylocystis parvus, Methylocystis sp. ATCC 49242, Methylocystis sp. SC2, Methylocystis rosea, Methylocystis SB2

glyA ftfL sga hpr gck2 mtdA fch mtkB mtkA ppc mcl Methylocapsa acidiphila

mtkA mtkB mcl sga hpr gck2 glyA ftfL ppc Methylferula stellata

sga hpr gck2 mdh glyA Candidatus “Methylomirabilis oxyfera”

Figure 4.1 Organization of clusters encoding enzymes of the serine cycle in alpha- and gammaproteobacterial methanotrophs. Hydroxypyruvate reductase (hpr), serine-glyoxylate aminotransferase (sga), glycerate-2-kinase (gck2), glycerate-3-kinase (gck3), malate thiokinase (mtkAB), malyl-CoA-lyase (mcl), malate dehydrogenase (mdh), serine-transhydroxymethylase (glyA), tetrahydromethanopterine/ methylentetrahydrofolate dehydrogenase (mtdA), formate-tetrahydrofolate (ftfL), phosphoenolpyruvate

carboxylase (ppc), methylene H4F cyclohydrolase (fch).

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Hpr could be involved in the regulation of the with diferent activities (172 and 22 U/mg) and

reduced equivalents content (But et al., 2017). Phy- afnities to formaldehyde (Km 0.98 mM versus logenetically, methanotrophic Hpr’s can be divided 0.64 mM). AMP and ADP were shown to be pow- into two divergent groups, and these share low erful inhibitors for both enzymes. According to the

identities with enzymes from non-methanotrophic kcat/Km ratio, HPS had a higher catalytic efciency heterotrophs (37–43%), algae (32–37%), plants compared with HPS-PHI. Unexpectedly, the HPS- (35–65%) or animals (26–46%). Te phylogenetic PHI fused enzyme lacked the activity. tree of the methanotrophic Hprs implies a vertical HPS but not the fused enzyme is prerequisite for evolution of the enzyme in the methanotrophs, carbon assimilation, since the insertion of a kana- with none or few instances of horizontal gene trans- mycin cassete into the hps-phi fused gene in Mm. fers (But et al., 2017). alcaliphilum 20Z did not alter growth rate of the Another key enzyme of the serine cycle is serine- mutant strain, whereas atempts to obtain a viable glyoxylate aminotransferase (Sga, EC 2.6.1.45), strain missing either hps or phi were unsuccessful and these were also obtained from Mm. alcaliphilum (Rozova et al., 2017). Although HPS-PHI was inac- 20Z, Ms. trichosporium OB3b and Mc. capsulatus tive as the isomerase, when expressed separately, Bath (But et al., 2018a). Te three enzymes showed both domains of the fused enzyme possessed the similar biochemical properties, and those were also respective synthase and isomerase activities. Te similar to the properties of Sga enzymes from other absence of the isomerase activity in the dimeric microbes and from plants (Kendziorek et al., 2008; HPS-PHI could be due to the inactive enzyme Kameya et al., 2010). Te methanotrophic Sgas confguration, since only tetrameric PHI have can also transfer the amino group from serine to been characterized so far as active enzymes, whose pyruvate, but display the highest catalytic efcien- catalytic centres were composed of four monomers cies in the reaction between serine and glyoxylate, (Orita et al., 2005).

which would be consistent with a role in C1 assimi- Te unique membrane-bound hexulosephos- lation. Te enzymes from strains 20Z and Bath phate synthase purifed from Mc. capsulatus Bath also transferred the amino group from serine to by traditional chromatographic methods displayed α-ketoglutarate and from to glyoxylate. Dis- very high activity and afnity to formaldehyde ruption of sga or simultaneously sga and hpr genes (Ferenci et al., 1974). Its high molecular mass caused retardation of growth of Mm. alcaliphilum (310 kDa) implied that the bi-domain, hexameric and an increase in lag-phase duration afer the pas- enzyme was characterized. Importantly, besides the sage from methane to . Te mutant strain hps-phi fused gene, two complete hps/phi operons was shown to accumulate formaldehyde in the are present in the genome of Mc. capsulatus Bath , suggesting that the serine cycle (Ward et al., 2004). plays a role in type I methanotrophs, serving as one Surprisingly, none of the methanotroph of the formaldehyde detoxifcation pathways (But genomes sequenced so far harbour the hps-phi et al., 2018a). fused gene alone. In contrast, none of the methylo- trophic bacteria unable to use methane as a growth The RuMP cycle encode the HPS-PHI fused enzyme. In the of gammaproteobacteral Interestingly, the genome of the model methano- methanotrophs, there are up to three copies of troph Methylomicrobium buryatense possesses the hps/phi operons coding for hexulosephosphate hps-phi fused gene along with three copies of hps/ synthase (HPS) and phosphohexulose isomerase phi operons. It remains to be demonstrated whether (PHI), key enzymes of the RuMP pathway. About there is any efect of HPS multiplication on the half of the known type I methanotroph genomes methanotrophic growth (Rozova et al., 2017). High additionally contain the fused gene hps-phi coding between HPS and the synthase domain for bi-domain HPS-PHI protein (Rozova et al., of the fused enzyme in the same methanotroph 2017). HPS and HPS-PHI purifed from Mm. alcal- (73% identity in Mm. alcaliphilum 20Z) imply iphilum 20Z are homodimeric enzymes (2 × 20 kDa relatively recent division of HPS-PHI into separate and 2 × 40 kDa, respectively) catalysing condensa- enzymes (Rozova et al., 2017). Te sequences tion of formaldehyde with ribulose-5-phosphate of the methanotrophic HPS comprise a tight

Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb Metabolic Features of Aerobic Methanotrophs | 89 phylogenetic cluster, distant from those of deciphering the respiratory mechanisms in metha- (sharing 38–40% identities), in which this enzyme notrophs (Tonge et al., 1977; Chetina et al., 1986). functions in synthesis of pentose phosphates from In addition, glycolysis and fermentation have been

C6-phosphosugars (Yurimoto et al., 2002; Mitsui et proven to be essential sources of energy in at least al., 2003; Orita et al., 2005, 2006). Te bi-domain gammaproteobacterial methanotrophs. Tese organization of the enzymes in thermophilic methanotrophs operate a modifed version of the archaea is considered to provide enhanced stability Embden–Meyerhof–Parnas pathway, where PPi to the enzyme against thermal inactivation (Orita et instead of ATP serves as the phosphate donor in al., 2005). However, such a property would not be an energy consuming reaction of -6-phos- very relevant in mesophilic bacteria. phate . Along the PPi-dependent 6- (PPi-PFK, EC 2.7.1.90), PPi-dependent glycolysis they possess another glycolytic enzyme, pyruvate At least three biochemical routes for sugar phosphate dikinase (PPDK), catalysing the PPi- cleavage can operate in gammaproteobacterial dependent reversible interconversion between methanotrophs: the Entner–Doudorof pathway, phosphoenolpyruvate (PEP) and pyruvate, accom- the glycolysis and the phosphoketolase pathway panied by interconversion between ATP and AMP. (Fig. 4.2). Te cause and efect of excessive meta- Tese substitutions make the glycolytic pathway bolic fexibility in methanotrophs is not clear. in these organisms more efcient with respect to Methanotrophs obtain energy for growth predomi- ATP production, compared with the classical ver- nantly from oxidation of C1 substrates to CO2 and sion of the pathway. Owing to the PPi-dependent can acquire ATP from oxidative phosphorylation, reactions, the glycolysis in methanotrophs is based on several experimental studies devoted to fully reversible. It therefore can operate as the

CH4 sMMO pMMO

CH OH 3 Pentose Ribulose-5P phosphate HCHO pathway

H4MPT

Hexulose-6P Erythrose-4P CO2 HCOOH ATP

H4F NADPH Acetyl-P Acetate Fructose-6P C -H F Glycine 1 4 PPi Pi -6P Glucose-1P UDP-glucose Sucrose Serine Fructose-1,6P 2 ADP-glucose + NH 4 KDPG DHAP Hydroxypyruvate + NH4 Fructose NAD(P)H GAP ATP Pyruvate Alanine P-Glycerate Glyoxylate AMP PPi ATP CO2 Fructose-6P Acetyl-CoA ADP Phosphoenolpyruvate NADH Acetyl-CoA Ectoine Citrate

Isocitrate Malyl-CoA Pi, CO2 Oxaloacetate CO PPi 2 α-Ketoglutarate Glutamate Malate CO2 CoA Fumarate Succinyl-CoA

Succinate Succinic semialdehyde

Figure 4.2 Pathways of carbon metabolism in gammaproteobacterial (Type I) methanotrophs. Modifed from

Rozova et al. (2015). H4MPT, tetrahydromethanopterin; THF, tetrahydrofolate.

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energy-inert gluconeogenic route where the energy feature implies an evolutionary link between meth- contained in ATP can be saved in PPi molecules. In anotrophy and autotrophy and corroborates the Mm. alcaliphilum 20Z, due to the presence pyruvate recently uncovered essential regulatory functions kinase, the glycolytic direction is prevailing (Kalyu- of and RuBP in methylotro- zhnaya et al., 2013). Considering the presence, phy (Ochsner et al., 2017). in addition, of gluconeogenic enzymes, fructose Among the methanotrophs, the representatives bisphosphatase and PEP synthase, of the Beijerenkiaceae family and the phylum Verru-

metabolism in type I methanotrophs is exceedingly comicrobia lack PPi-PFK (Khmelenina et al. 2018). fexible (Fig. 4.2). In contrast, PPi-PFK is essential for methanotrophs PPi-PFKs of methanotrophs catalysing the possessing the intracytoplasmic membranes reversible reaction of phosphorylation of fructose- (ICM), suggesting a special function for PPi in 6-phosphate (Fr6P) somewhat vary in their their bioenergetics. Te two -consuming biochemical properties. PPi-PFK from Methylo- processes, methane oxidation by particulate meth- monas methanica (2x45 kDa) and Mm. alcaliphilum ane monooxygenase (pMMO) and oxidative 20Z (4x45 kDa) are very active enzymes that are not phosphorylation, are both associated with the regulated allosterically (Reshetnikov et al., 2008; ICMs, suggesting complex regulation. At the same Rozova et al., 2010). PPi-PFK from Mс. capsulatus time, the reactive oxygen species generated by the Bath is highly active as a sedoheptulose-7-phos- electron transfer to pMMO (Murrell et al., 2000; phate (S7P) kinase, phosphorylating S7P with Kamachi and Okura, 2018) and the oxygen radicals much higher activity than fructose-6-phosphate as generated from the respiratory chain may disturb a substrate. It can also phosphorylate Ru5P. In Mс. membrane integrity and therefore hamper ATP syn- capsulatus Bath, which, in addition to the RuMP thesis or methane oxidation. Te use of PPi instead pathway and the serine cycle, encodes reactions of of ATP as a phosphoryl donor in some cytoplasmic the Calvin cycle, PPi-PFK is involved in synthesis reactions can mitigate this bio-energetic problem.

of RuBP, a primary acceptor for CO2. Owing to Analogous problem is faced by chemoautotrophs + modulations of activity of the ribulosebisphosphate oxidizing NH4 via ammonia monooxygenase, carboxylase/oxygenase (RuBisCO) in response to which is structurally and functionally similar to temperature fuctuations (Eshinimaev et al., 2004), pMMO. Tis corroborates the supposition that the

an intracellular content of RuBP can be occasion- PPi-PFK is specifc to organisms that are energeti- ally increased. In the case of temperature decrease cally constrained (Mertens, 1991). Te exceptions

or CO2 defciency, RuBP can be returned to the from this rule are acidophilic methanotrophs basic metabolism, retaining metabolic energy. having pMMO but not possessing PPi-PFK-encod- In at least several methanotrophs, the gene ing genes, the Methylacidiphilum species that do not is co-transcribed with the gene coding for a V-type appear to form ICMs and only possess ATP-PFK H+-pyrophosphatase (H+-PPi-ase, EC 3.6.1.1). (Fig. 4.3), and Methylocapsa acidiphila that forms Terefore, direct involvement of PPi in membrane ICMs but does not harbour a phosphofructokinase. energization and ATP synthesis has been proposed Te anaerobic methanotroph Methylomirabilis (Reshetnikov et al., 2008; Khmelenina et al., 2018). oxyfera possesses both pMMO and a very divergent In alphaproteobacterial methanotrophs, the PPi-PFK (Fig. 4.3), while apparently lacking ICMs gluconeogenic function of PPi-PFK is obvious. (Wu et al., 2012a). In this polygon-shaped bacte- Te hexameric PPi-PFK from Ms. trichosporium rium able to couple anaerobic methane oxidation to OB3b (6 × 45 kDa) performs dephosphorylation denitrifcation, pMMO appears to be located in the of FBP with higher efciency in comparison to cytoplasmic membrane (Wu et al., 2012a,b). Fur- phosphorylation of Fr6P (Rozova et al., 2012). ther studies are needed to unravel the evolutionary Unexpectedly, Ms. trichosporium PPi-PFK also history and the physiological roles of PPi and PPi- phosphorylates S7P and Ru5P, although with much dependent enzymes in the methanotrophs. lower activities and afnities that are displayed with Phylogenetically, methanotrophic PPi-PFKs Fr6P. Tis bacterium also encodes ATP-dependent belong to several divergent groups, and these share phosphoribulokinase (ID 2507407232), while it identities with the enzymes from non-methano- lacks the RuBisCO encoding genes. Tis metabolic trophic heterotrophs and the protozoans (Fig. 4.3).

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25. Nakamurella multipartite (ACV78222)

Metabolic Features of Aerobic Methanotrophs | 91

26. Propionibacterium acnes (AAT82837) 24. Mastigamoeba balamuthi (AAF70463) 27. Beutenbergia cavernae (ACQ80161) 23. Rhodopirellula baltica (CAD78962) 28. Sinorhizobium medicae (ABR60939) 22. Opitutus terrae (ACB73441) 29. Methylobacterium sp. 4-46 (ACA16250) 3. Prevotella saccharolytica (EKY02337) 21. Methylomagnum ishizawai (SMF93536) 30. Methylobacterium nodulans (ACL55713) 2. Treponema azotonutricium (AEF82681) 20. Methylovulum psychrotolerans (POZ49993) 1. Entamoeba histolytica (EAL47787) 19. Methylovulum miyakonense (WP_019866931) 18. Methylocucumis oryzae (KJV05300) Rhodopirellula baltica (ATP; CAD75440) Candidatus Methylomirabilis oxyfera (CBE67765) 17. Methylomicrobium buryatense (WP_017840709) 16. Methylomicrobium alcaliphilum (CCE24967) 3. Entamoeba histolytica (ATP; CAA57659) 15. Methylomicrobium kenyense (MKEN v1 12973) 2. Nakamurella multipartite (ATP; ACV76558) 14. Methylomarinum vadi (WP 031435725) 1. Amycolatopsis methanolica (ATP, AF298119) 13. Crenothrix polyspora (SJM91314) 12. Methylobacter tundripaludum (EGW21280) Planctomyces maris (ATP; EDL59313) 11. Methyloglobulus morosus (ESS72592) 7. Methylobacter tundripaludum (ATP; EGW21181) 10. Methylosarcina fibrata (WP_026223761) 6. Methylovulum miyakonense (WP_040572823) 9. Methylomicrobium agile (WP 005370363) 5. Magnetococcus sp. MC-1 (ATP; ABK45515) 8. Methylomicrobium album (EIC28912) 7. Methylosarcina lacus (25172486926) 4. Clostridium difficile (ATP; CAJ70298) 6. Methylogaea oryzae (WP_054773052) 3. Escherichia coli (ATP; POA796) 5. Methylomonas denitrificans (AMK76130) 2. Propionibacterium acnes (ATP; EFD02377) 4. Methylomonas lenta (OAI16061) 1. Nakamurella multipartite (ATP; ACV78222) 3. Methylomonas methanica (AEF99237) 8. Propionibacterium acnes (ATP; EFD04141) 2. Methylomonas koyamae (ATG88991) 7. Gallionella capsiferriformans (ATP; ADL55590) 1. Methylomonas methanica (AAY28468) 6. Planctomyces maris (ATP; EDL56936) 5. Amycolatopsis methanolica (AAB01683) 4. Rhodospirillum rubrum (ATP; ABC21848) 1. Nitrosomonas communis (WP 074667755) 3. Methylacidiphilum infernorum (ATP; ACD84176) 2. Thiothrix lacustris (WP 028489681) 2. Methylacidiphilum kamchatkense (JQNX01_v1_100047) 3. Methyloterricola oryzae (WP 045226056) 1. Methylacidiphilum fumariolicum (Mfumv2_0253) 4. Methylococcus capsulatus (AAU92465) 5. Methylocaldum szegediense (WP 026611520) 6. Methylocaldum marinum (BBA36313) 13. Methylocystis sp. SB2 (WP 029648267) 7. Methylobacter luteus (WP 027160171) 12. Methylocystis rosea (2517404262) 8. Methylobacter sp. BBA5.1 (WP 036248461) 11. Methylocystis sp. SC2 (CCJ06220) 9. Methylobacter whittenburyi (WP 036292928) 10. Methylocystis parvus (WP 016919815) 10. Methylobacter marinus (WP 020159343) 9. Methylocystis sp. ATCC 49242 (EFY00463) 11. Methylibium petroleiphilum (ABM95730) 8. Methylocystis bryophila (ARN81253) 12. Marinobacter aquaeolei (ABM19478) 7. Methylosinus sporium (PWB93550) 13. Xanthomonas oryzae (BAE67633) 1. Mesorhizobium loti (BAB51545)

14. Saccharophagus degradans (ATP, ABD80197) 2. Gallionella capsiferriformans (ADL55266) 6. Methylosinus trichosporium 17. Opitutus terrae (ACB74686) 3. Magnetococcus sp. MC-1 (ABK44425) 5. Methylosinus sp. LW4 (WP 018266557) 18. Clostridium difficile (CAJ70002) 4. Rhodospirillum rubrum (ABC24201)

Figure 4.3 The phylogenetic tree of PPi- and ATP-dependent 6-. The phosphate donor (ATP) is indicated for ATP-PFKs in the brackets. Monophyletic groups are named according to the classifcation (Müller0.1 et al., 2001; Bapteste et al. 2003) as ‘B2’ (dark-blue), ‘BI’ (orange), ‘III’ (green), ‘X’ (brown), ‘LONG’ (blue), and ‘P’ (red). Amino acid sequences were obtained from the NCBI database (http://www.ncbi.nlm.nih. gov), the Microbial Genome Annotation and Analysis Platform (www.genoscope.cns.fr), and from the Integrated Microbial Genomes and Microbiomes system (img.jgi.doe.gov) by BLAST. The tree was constructed using the MEGA 4 program (computed from 100 independent trials).

Such distribution on the phylogenetic tree cor- glycolysis and pyruvate decarboxylation where the roborates hypothesis of ‘random’ lateral transfer of C–C bond would be cleaved. Te PKT pathway can genes coding PPi-PFKs in microorganisms (Bapt- produce ATP, as is co-expressed with este et al., 2003). Nevertheless, some correlations PKT (Rozova et al., 2015a). Acetate kinase cataly- between the phylogenetic position and the type of ses the reversible reaction of dephosphorylation of carbon metabolism could also be observed. acetyl-P into acetate with higher catalytic efciency in the direction of ATP formation, as compared The phosphoketolase pathway to acetate phosphorylation. Moreover, PKT in Phosphoketolase (PKT, EC 4.1.2.9/EC 4.1.2.22) Mm. buryatense has proven to be involved in the catalyses the cleavage of xylulose-5-phosphate and/ global regulatory alterations of the methanotrophic or fructose-6-phosphate into acetyl-phosphate and metabolism (Henard et al., 2017). PKT pathway glyceraldehyde-3-phosphate or erythrose-4-phos- engineering has been successfully used to increase phate (Sánchez et al., 2010). PKT-encoding genes yields of an array of acetyl-CoA-derived products, occurs in all gammaproteobacterial methanotrophs, in diverse microbial biocatalysts. Owing to overex- and at least two PKT isoforms are present in the pression of the PKT isoform, Mm. buryatense has genomes of halotolerant methanotrophs (Rozova et an increased biomass yields and the lipid content al., 2015). Te PKT pathway produces acetyl-CoA, (Herand and Guarnieri, 2018). which is the precursor of the compatible solute In the methanotrophs, the PKT-shunted ectoine that is accumulated by halotolerant metha- glycolytic pathway operates along with the PPi- notrophs in response to increased medium salinity. dependent glycolysis, another energy efcient Te methanotroph M. buryatense expresses all the pathway, thus providing the metabolic fexibility and PKT pathway components (Henard et al., 2017). In allowing the gammaproteobacterial methanotrophs this pathway, the C2 compounds synthesized bypass to survive in micro-aerobic ecosystems. Numerous

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environmental studies indicate that methanotrophs in the serine pathway, where malate activation to thrive at oxic–anoxic interfaces. Te discovery of malyl-CoA and its further cleavage into acetyl-CoA the ATP-producing assimilatory routes raised the and glyoxylate are key reactions in glycine regen- possibility of fermentation in methanotrophs, as eration. Acetyl-CoA enters the ethylmalonyl-CoA

a new mode of methane utilization at low O2 ten- (EMC) cycle, where it is transformed to glyoxylate sions, where formate, acetate, succinate, lactate, (Chistoserdova et al., 2009) or is directed to the hydroxybutyrate and hydrogen can be the end syntheses of faty acids and the storage compound products (Kalyuzhnaya et al., 2013). Tis discovery poly-β-hydroxybutyrate (PHB). Te main source challenges our understanding of methanotrophy as of OAA for the MDH reaction is carboxylation of linked solely to respiration, PEP by PEP carboxylase, which is a highly active and it has major implications for the indispensa- enzyme in type II methanotrophs. Some metha- ble role of methanotrophic bacteria in removing notrophs, such as Ms. trichosporium OB3b, possess

the greenhouse gas methane in O2-limited envi- two PEP carboxylases, sharing 33% identity at the ronments, including landfelds. In such specifc amino acid level (Matsen et al., 2013). environments, methanotrophs drive the conver- Alternatively, MDH from Mm. alcaliphilum sion of methane to excreted organic products and 20Z shows preference for malate oxidation (OAA hydrogen, which are then used and transformed by formation) versus malate synthesis, as follows from

non-methanotrophs. respective kcat/Km ratios (Rozova et al., 2015b). Such enzyme features are in agreement with the The TCA cycle high demand for aspartate in this organism, as a pre- In methanotrophs generating metabolic energy by cursor of the osmoprotectant ectoine (Reshetnikov

the direct oxidation of reduced C1 compounds or et al., 2011). by formaldehyde fermentation, the predominantly Te malic enzyme (MaE) is another enzyme

anabolic role of the tricarboxylic acid (TCA) that is thought to be involved in anaplerotic CO2 cycle is evident. While no or negligible activity of fxation and malate biosynthesis. However, MaE 2-oxoglutarate dehydrogenase has been found in from Ms. trichosporium OB3b (EC 1.1.1.40) pre- methanotrophs where tested, the complete set of dominantly catalyses NADP+-dependent reaction the genes coding for this enzyme have been iden- of malate decarboxylation, while it has much lower tifed in the genomes, and the functionality of the activity and afnity to pyruvate (Rozova et al., oxidative TCA cycle has been further proven in 2019). NADP+-dependency and high efciency in some methanotrophs (Matsen et al., 2013; Fu et the direction of decarboxylation suggest that MaE al., 2017). Based on the genomic evidence, in type is the ‘lipogenic enzyme’ important for the produc-

I methanotrophs, additional routes may bypass tion of NADPH2 that is needed for biosynthesis of the need for the classic 2-oxoglutarate dehydroge- long-chain faty acids and/or steroids (Fig. 4.4). nase complex, such as 2-oxoglutarate ferredoxin In contrast, Mm. alcaliphilum MaE (EC 1.1.1.38) , catalysing the reversible oxidative catalyses non-reversible NAD+-dependent reac- decarboxylation of 2-oxoglutarate to succinyl-CoA, tion of malate decarboxylation into pyruvate

as well as 2-oxoglutarate oxidase + succinate semial- but is unable to fx CO2. Evidently, the source of dehyde dehydrogenase (Fu et al., 2017) (Fig. 4.2). malate for this reaction could be the reactions of In Mm. buryatense 5GB1, genes for both of these the TCA cycle, which, in turn, can be replenished

enzymes are expressed at the levels similar to the by CO2 fxation. Although type I methanotrophs ones of other TCA cycle genes. lack the PEP carboxylase (with an exception of the Malate dehydrogenases (MDH, l-malate: NAD Methylomonas species), those may possess unusual oxidoreductase, EC 1.1.1.37) purifed from Ms. PPi-dependent PEP-carboxykinases catalysing the

trichosporium OB3b and Mm. alcaliphilum 20Z dis- reversible reaction of CO2 fxation. However, the played properties refecting their roles in respective functionality and the roles of such enzymes remain primary (Rozova et al., 2015b). Te to be proven. As seen in Fig. 4.5, reactions catalysed high catalytic efciency of MDH from Ms. trichos- by PEP carboxykinase, MDH, MaE and PPDK can porium OB3b in the direction of malate synthesis produce two PPi molecules, to trigger the modifed would be refective of the central position of malate the Emden-Meyerhof-Parnas glycolysis.

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CH4

CH4 Methylene-THF

Glycine CH2O L-Serine + NH4 Hydroxypyruvate 6-Phosphosugars

Phosphoglycerate Triose-P Pi, CO2 PPi

CO2 Phospho- PPi, AMP enolpyruvate Phosphoenolpyruvate Oxaloacetate

AMP, PPi, Pi, ATP NADH Oxaloacetate Pyruvate CO2 ATP, Pi

CO2 Pyruvate Malate

NADPH NADH Malate NADH, CO 2 2 Acetyl-CoA Malyl-CoA

Glyoxylate Acetyl-CoA

TCA Lipids TCA cycle cycle

Fig. 3

Figure 4.4 Pathways for interconversionFig. 4 of Figure 4.5 Pathways for conversion of phosphoenolpyruvate and pyruvate in Type II phosphoenolpyruvate to pyruvate in Type I methanotrophs and the role of the malic enzyme in methanotrophs and the role of the malic enzyme in NADPH production. TCA, tricarboxylic acid (modifed the synthesis of PPi. TCA, tricarboxylic acid. from Rozova et al., 2019).

Sucrose metabolism phosphorylated by the respective ATP-dependent Te ability to synthesize sucrose is inherent in photo- , to enter into central metabolic pathways (But et al., 2013, 2015). Te from Mm. trophic bacteria and plants, and it has recently been revealed in non-photosynthetic bacteria, including alcaliphilum 20Z (EC 2.7.1.1) also phosphorylates aerobic methylotrophs that assimilate carbon via (Mustakhimov et al., 2017), whereas the RuMP pathway (But ., 2013, 2015). In FruK (EC 2.7.1.4) is highly specifc to fructose and et al the methylotrophs, the biochemical pathway for ATP (But et al., 2012). Two molecules of nucleo- sucrose synthesis from fructose-6-phosphate and tide triphosphate (NTP) are needed for ataching UDP-glucose is rather similar to the one in other the glucosyl residue to the glycogen primer by Ams, pro- and eukaryotic organisms, being composed of and only one molecule of NTP is required for elon- two enzymes, sucrose phosphate synthase (Sps, EC gation of glycogen via glucose pyrophosphorylase 2.4.1.14) and sucrose phosphate phosphatase (Spp, (GlgC) and (GlgA). Terefore, EC 3.1.3.24), with sucrose-6-phosphate as an inter- the sucrose cycle, where glycogen synthesis involves mediate. Tese enzymes are encoded by the operon sucrose as an intermediate, could be envisaged as a sps–spp–fuK–ams, which also encodes, in addition dynamic mechanism that balances the intracellular to Sps and Spp, enzymes for sucrose metabolism levels of ATP. amylosucrase (Ams, EC 2.4.1.4) and Sucrose synthesis can provide some advantages (FruK, EC 2.7.1.4). Amylosucrase transfers the gly- to the methylotrophs. Being a carbon storage com- cosyl residue from sucrose onto glycogen primer, pound, sucrose also serves as a compatible solute, and it also catalyses cleavage of sucrose to fructose contributing to turgor maintenance at elevated and glucose. Glucose and fructose need to be salinity. However, sucrose accumulation ensures

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only moderate salt tolerance by methylotrophic pathway encoded by the ectABC operon, which, bacteria (But et al., 2013). Of note, the genomes in Mm. alcaliphilum 20Z, also includes a gene for of most gammaproteobacterial methanotrophs negative transcriptional regulator EctR, of the isolated from soils possess the sucrose biosynthe- MarR family, and a gene for an additional aspar- sis genes, whereas methanotrophs obtained from tokinase. DABA aminotransferase (EctB) from freshwater or sewage systems lack these genes Mm. alcaliphilum 20Z is a hexameric pyridoxal- (But et al., 2015). Te halotolerant methylotrophs 5′-phosphate-dependent enzyme (300 kDa) are known to tolerate extraordinarily high con- synthesizing diaminobutyric acid (DABA) from centrations of methanol, being able to grow at 7% d,l-aspartyl semialdehyde using l-glutamate as a methanol (v/v; Eshinimaev et al., 2002). Since donor of an amino group. It requires К+ for activity sucrose synthesis starts with early products of the and stability, being more active in the presence of RuMP pathway, high rates of formaldehyde outfow 0.01–0.5 М KCl (Reshetnikov et al., 2011). may be responsible for the methanol tolerance. DABA acetyltransferase (EctA) catalyses the In the thermophilic Md. szegediense O12, sucrose acetylation of DABA, yielding γ-N-acetyl-α,γ- synthase (Sus) is involved in metabolism of sucrose diaminobutyric acid. Te properties of EctA from in place of Ams, cleaving disaccharide into fructose Mm. alcaliphilum 20Z and non-methanotrophic and ADP-/or UDP-glucose (But et al., 2018b). Te methylotrophs Methylophaga alcalica ATCC 35842 clear preference of Sus for ADP implies a connection and Methylophaga thalassica ATCC 33146 represent between sucrose and glycogen metabolism, since striking examples of bacterial eco-physiological ADP-glucose is a substrate for glycogen synthesis. adaptation at the enzyme level (Mustakhimov et al., So far, no genes for sucrose metabolism have been 2008). Te EctA from the neutrophilic M. thalas- identifed in methanotrophs assimilating methane sica has optimal pH between 8.2 or 9.0, whereas via the serine pathway (phylum Alphaproteobacte- the enzymes from the alkaliphilic Mm. alcaliphilum ria) or through the Calvin cycle (Verrucomicrobia 20Z and M. alcalica have pH optima at ≥ 9.5. Activi- and NC10 phyla) (But et al., 2015). ties of EctA from M. alcalica and Mm. alcaliphilum 20Z, which have been isolated from soda lakes, Adaptation of methanotrophs to were increased in the presence of carbonate ions, saline environments whereas EctA from a marine isolate M. thalassica Methanotrophs isolated from soda and hyper- was considerably inhibited by carbonates. Copper saline lakes, marine waters and soils are ofen ions completely inhibited the enzyme activity from halotolerant or halophilic, and these species belong M. alcalica but had no efect on the enzyme from to the genera Methylomicrobium, Methylobacter Mm. alcaliphilum 20Z. Ectoine synthase (35 kDa) and Methylohalobius. All halotolerant/halophilic catalyses the cyclization of N-acetyl-DABA into methanotrophs accumulate in their cells ectoine, ectoine. Te methylotrophs synthesizing ectoine sucrose and glutamate, which all participate in also encode a pathway for ectoine degradation, balancing osmotic pressure between frst proposed for a non-methylotrophic bacterium and the cell surroundings. Te cyclic imino acid Halomonas elongata (Schwibbert et al., 2011). ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine Methylohalobius crimeensis 10Ki displays the ) is the major compatible solute highest halotolerance recognized among the halo- accumulating at concentration of up to ≈1 mol per philic methanotrophs, and it is able to grow at 12% 1 l of intracellular water content. Te halophilic/tol- NaCl (Sharp et al., 2015). It possesses an array of erant methanotrophs are the promising producers mechanisms for osmoadaptation. In addition to the of ectoine, which is a multifunctional bioprotect- ectABCD gene cluster, it encodes ectoine hydroxy- ant useful in pharmaceutical applications (Graf et lase (EctD), an enzyme responsible for biosynthesis al., 2008). Te biochemical pathway for ectoine of hydroxyectoine. It also possesses genes for a biosynthesis in methanotrophs is almost identi- high-afnity importer of choline/glycine betaine, cal to that in other halophilic . Tree driven by a sodium-motive force. It also harbours specifc enzymes, diaminobutyric acid (DABA) three copies of a gene for aminotransferase (EctB), DABA acetyltransferase and a gene encoding betaine aldehyde dehydroge- (EctA, and ectoine synthase (EctC) compose this nase, indicating possible glycine betaine synthesis

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from choline. In addition, it encodes a pathway for Bowman, J. (2006). Te methanotrophs: the families Methylococcaceae and Methylocystaceae. In Te sucrose synthesis and degradation/reutilization, Prokaryotes, Dworkin, M., ed. (Springer, New York, which includes Sps, Spp, FruK and sucrose synthase NY), pp. 266–289. instead of amylosucrase characteristic of other But, S.Y., Rozova, O.N., Khmelenina, V.N., Reshetnikov, A.S., halotolerant methanotrophs. Te use of a sodium- and Trotsenko, Y.A. (2012). Properties of recombinant + ATP-dependent fructokinase from halotolerant motive force and Na export have been suggested methanotroph Methylomicrobium alcaliphilum 20Z. by the presence of the genes encoding a putative Biochem. Mosc. 77, 372–377. htps://doi.org/10.1134/ Na+/H+ antiporter localized within the gene cluster S0006297912040086 encoding an ATP synthase, and a complete nqr gene But, S.Y., Khmelenina, V.N., Reshetnikov, A.S., and + Trotsenko, Y.A. (2013). 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