Methylotrophic pathways

Methylotrophy involved 3 major metabolic steps:

- Initial transformation of the methylotrophic compound into formaldehyde (HCHO) (not the case for chloromethane);

- Complete oxidation of the methylotrophic compound to CO2 in order to produce energy;

- Carbon assimilation for biomass production via 3 alternative pathways: the ribulose biphosphate (RuBP) pathway that utilizes CO2, the ribulose monophosphate (RuMP) pathway from formaldehyde, or the serine cycle from methylenetetrahydrofolate (CH2=H4F) and CO2

(Chistoserdova, 2011).

For most methylotrophs, the first metabolic step is the oxidation or the hydrolysis of the carbon compound into formaldehyde. The enzymes involved in this step are specific to their substrates: the soluble methane monooxygenase (sMMO) or the particular methane monooxygenase (pMMO) for methane, the methylamine deshydrogenase (MADH, periplasmic) for methylamine, the methanol deshydrogenase (Mxa, periplasmic) for methanol, or a dichloromethane deshydrogenase for dichloromethane (DcmA, cytoplasmic).

The mxaF gene has been used as a molecular marker of methanol oxidation (Neufeld et al., 2008). More widespread in the environment than the mxa genes, alternative methanol degradation systems exist such as the xox genes or the mdh gene (Kalyuzhnaya et al., 2008).

Among these genes, xoxF codes for a subunit of a methanol dehydrogenase that shares more than 50% of amino acid identity with MxaF (Schmidt et al., 2010; Taubert et al., 2015).

Several methanol oxidation pathways can be simultaneously present in an organism as found in M. extorquens (Figure 1.10, Skovran et al., 2011). Several methylamine oxidation pathways have also been found in M. extorquens strains (Vuilleumier et al., 2009; Gruffaz et al., 2014; Nayak et Marx, 2014). Halogenated compounds (chloromethane, dichloromethane) can be utilized by methylotrophic bacteria such as M. extorquens and Hyphomicrobium spp. (Gälli and Leisinger, 1985; La Roche and Leisinger, 1990; Vannelli et al., 1998; McAnulla et al., 2001). The dehalogenation of dichloromethane is catalyzed by a dichloromethane dehalogenase encoded by gene dcmA, and formaldehyde is produced.

Formaldehyde is transformed into formate by either a tetrahydrofolate-dependent (H4F) pathway or a tetrahydromethanopterin (H4MPT)-dependent pathway in Methylobacterium

strains. The first pathway is not the main one regarding the production of energy and involves inter-conversions between methylene-H4F, methenyl-H4F, formyl-H4F and formate (Marison and Attwood, 1982). This metabolic pathway might play a major role by keeping adequate concentrations of the different H4F-containing metabolites, which are essential intermediates for carbon assimilation via the serine cycle pathway (Marx et al., 2005;

Crowther et al., 2008). In the second pathway, discovered in 1998, H4MPT is condensed to formaldehyde, and successively transformed into methylene-H4F, methenyl-H4F, formyl-H4F, and finally into formate (Chistoserdova et al., 1998). This H4MPT-dependent pathway has been first described in the Archaea, and is more efficient in formaldehyde oxidation into CO2

than the H4F-dependent pathway, because its corresponding enzymes display high activities (Vorholt, 2002). Despite the fact that most methylotrophic substrates are oxidized into formaldehyde, this is the case for chloromethane. Chloromethane dehalogenation produces methylene-tetrahydrofolate (CH2=H4F), which either enters the serine cycle pathway for biomass production or is oxidized into formate for energy production (Figure 1.10). As a matter of facts, M. extorquens CM4 converts directly chloromethane into methylene-tetrahydrofolate (CH2=H4F) without formaldehyde production thanks to the activity of CmuA (methyltransferase and corrinoid-binding domain-containing protein), CmuB (methylcobalamin: H4F methyltransferase) and MetF2 (methylene-H4F reductase) (Studer et al., 1999; Studer et al., 2001; Studer et al., 2002) (see section 1.3.4.2.).

35 Figure 1.10. Scheme of different methylotrophic pathways in M. extorquens

Oxidation state is shown on the Y axis. The reactions specific for chloromethane (CH₃Cl) and dichloromethane (CH₂Cl₂) utilization as sole source of carbon and energy are shown in green and purple, respectively: CmuABC, chloromethane utilizing genes; MetF2, methylene-H₄F reductase; DcmA, dichloromethane dehalogenase. Enzymes for initial oxidation are in black:

MDH, methanol dehydrogenase; MADH, methylamine dehydrogenase. Reactions leading to complete oxidation of carbon to energy are in orange: Fae, formaldehyde activation enzyme;

MtdAB, methylene-tetrahydromethanopterin (H4MPT) dehydrogenase; Mch, methenyl- H4MPT cyclohydrolase; Fch, methylene-tetrahydrofolate (H4F) cyclohydrolase; FtfL, formyl- H4F ligase; FhcABCD, formyltransferase-hydrolase complex; Fdh, formate dehydrogenase.

Reactions for carbon assimilation to biomass are in grey: FtfL, formyl-H4F ligase; Fch, methylene- H4F cyclohydrolase; MtdA, methylene-H4MPT dehydrogenase; RuMP, ribulose monophosphate cycle; RuBP, ribulose biphosphate cycle.

More than 86 genes involved in M. extorquens C1 compound utilization have have been identified (Marx et al., 2003). These genes are involved in the methanol oxidation methylamine, formaldehyde, and formate or in the serine-cycle pathway (Figure 1.11).

Figure 1.11. Carbon assimilation in M. extorquens

Entering C1 compounds are circled in blue and main biosynthesis pathway outputs are indicated in green. Bacteria oxidize methanol (CH3OH) or dichloromethane (CH2Cl2) to formaldehyde that is condensed with a tetrahydromethanopterin (H4MPT), and further oxidized to formate (HCOOH). Formate is further converted to methylene-tetrahydrofolate (H4F) via the serine cycle or transformed in CO2. Chloromethane (CH3Cl) is assimilated via the transfer of the methyl group to H4F which is then assimilated via the serine cycle to produce biomass or oxidized to formate (HCOOH). Acetyl-CoA assimilation and glyoxylate regeneration proceed via the ethylmalonyl-CoA pathway. The tricarboxylic acid (TCA) cycle is mainly used for assimilation of multi-carbon compounds (Šmejkalová et al., 2010). Circled numbers identify distinct reactions: 1, serine hydroxymethyl transferase; 2, serine-glyoxylate aminotransferase; 3, hydroxypyruvate reductase; 4, glycerate kinase; 5, enolase; 6,

37 phosphoenolpyruvate carboxylase; 7, malate dehydrogenase; 8, malate-CoA ligase (malate thiokinase); 9, L-malyl-CoA/β-methylmalyl-CoA lyase; 10, β-cetothiolase; 11, acetoacetyl-CoA reductase; 12, crotonase (R- specific); 13, crotonyl-CoA carboxylase reductase; 14, ethylmalonyl-CoA/methylmalonyl-CoA epimerase; 15, ethylmalonyl-CoA mutase; 16, methylsuccinyl-CoA deshydrogenase; 17, mesaconyl-CoA hydratase; 18, propionyl-CoA carboxylase; 19, methylmalonyl-CoA mutase; 20, succinyl-CoA synthase; 21, succinate deshydrogenase; 22, fumarase; 23, citryl-CoA lyase; 24, aconitate hydratase; 25, NADP-dependent isocitrate dehydrogenase; 26, 2-oxoglutarate dehydrogenase complex; 27, methanol dehydrogenase; 28, dichloromethane dehalogenase; 29, formate dehydrogenase.

For a given reaction, the number of parallel arrows correspond to the number of molecules involved in the reaction. PHB, polyhydroxybutyrate; Q, quinone (adapted from Šmejkalová et al., 2010).

In Methylobacterium, for biomass production, formaldehyde incorporates the serine cycle pathway via formate and methylene-H4F. The GlyA enzyme (serine hydroxymethyltransferase) catalyzes the reaction between methylene-H4F and glycine to produce serine (Figure 1.11). Then, serine (C3 compound) is successively transformed into hydroxypyruvate, glycerate, glycerate-2-phosphate and phosphoenolpyruvate.

Phosphoenolpyruvate is subsequently converted into oxaloacetate, then malate by incorporation of CO2. Malate (C4 compound) coupled to co-enzymeA (CoA) is then cleaved in 2 compounds: glyoxylate allowing to close the cycle serine, and acetyl-CoA which is integrated in the ethylmalonyl-CoA pathway where a CO2 molecule is incorporated (Erb et al., 2008; Peyraud et al., 2009; Šmejkalová et al., 2010). This cycle generates hydroxybutyryl-CoA, a precursor of the polyhydroxybutyrate (PHB) synthesis and granule production for carbon and phosphate storage in the cell.