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Metabolic Features of Aerobic Methanotrophs: News and Views Valentina N Metabolic Features of Aerobic Methanotrophs: News and Views Valentina N. Khmelenina*, Sergey Y. But, Olga N. Rozova and Yuri A. Trotsenko G.K. Skryabin Institute of Biochemistry and Physiology 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 carbon promising for industrial applications, as they are metabolism 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 enzymes involved naya et al., 2015; Mustakhimov et al., 2015; Fu and in assimilation of carbon from methane. Tese Lidstrom, 2017; Garg et al., 2018). Te adaptation include enzymes involved in sugar 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, Methylosinus 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 nitrogen cycles (Hanson and Hanson, 1996; pathways McDonald et al., 2008, Etwig et al., 2010; Khadem Methanotrophs obtain energy 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 formaldehyde, 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, Verrucomicrobia, and the yet unnamed NC10 the multitude of the characterized methanotrophs, phylum. Te proteobacterial methanotrophs are Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb 86 | Khmelenina et al. classifed as type I or type II, according to whether methanotrophs possess non-homologous enzymes they belong to the Alpha- or Gammaproteobacteria 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 Methylocystaceae 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- Beijerinckiaceae assimilate carbon from methane via trophs presumably lack this pathway. the serine pathway, where the amino acid 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 bacteria: 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 genes 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-kinase (gck2), malate thiokinase (mtkAB), (strains OB3b and Bath) or alkaliphilic (strain malyl-CoA-lyase (mcl), malate dehydrogenase 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 allosteric regulation, its activity increasing in (ffL) are adjacently located in a single, two or the presence of the intermediates of glycolysis 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 pyrophosphate, but decreasing in the presence of methylotrophs (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 enzyme to reduce glyoxylate into glycolate, in com- (ppc) and methylene H4F cyclohydrolase (fch), bination with FAD-dependent glycolate oxidase whereas among type I methanotroph genomes, only (ADVE02_v2_14253-14256; MCA1499-150 and the Methylomonas 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 Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb Metabolic Features of Aerobic Methanotrophs | 87 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 methanica, 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, Methylococcaceae 73a, Methylococcus capsulatus 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 gene 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),
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