Methylotroph

Methylotrophs use the electron transport chain to conserve energy produced from the oxidation of ${\displaystyle {\ce {C1}}}$ compounds. An additional activation step is required in methanotrophic metabolism to allow degradation of chemically-stable methane. This oxidation to methanol is catalyzed by MMO, which incorporates one oxygen atom from ${\displaystyle {\ce {O2}}}$ into methane and reduces the other oxygen atom to water, requiring two equivalents of reducing power.[4][5] Methanol is then oxidized to formaldehyde through the action of methanol dehydrogenase (MDH) in bacteria,[12] or a non-specific alcohol oxidase in yeast.[13] Electrons from methanol oxidation are passed to a membrane-associated quinone of the electron transport chain to produce ${\displaystyle {\ce {ATP}}}$.[14]

In dissimilatory processes, formaldehyde is completely oxidized to ${\displaystyle {\ce {CO2}}}$ and excreted. Formaldehyde is oxidized to formate via the action of Formaldehyde dehydrogenase (FALDH), which provides electrons directly to a membrane associated quinone of the electron transport chain, usually cytochrome b or c.[2][5] In the case of ${\displaystyle {\ce {NAD+}}}$ associated dehydrogenases, ${\displaystyle {\ce {NADH}}}$ is produced.[7]

Finally, formate is oxidized to ${\displaystyle {\ce {CO2}}}$ by cytoplasmic or membrane-bound Formate dehydrogenase (FDH), producing ${\displaystyle {\ce {NADH}}}$[15] and ${\displaystyle {\ce {CO2}}}$.

The main metabolic challenge for methylotrophs is the assimilation of single carbon units into biomass. Through de novo synthesis, methylotrophs must form carbon-carbon bonds between 1-Carbon (${\displaystyle {\ce {C1}}}$) molecules. This is an energy intensive process, which facultative methylotrophs avoid by using a range of larger organic compounds.[16] However, obligate methylotrophs must assimilate ${\displaystyle {\ce {C1}}}$ molecules.[2][5] There are four distinct assimilation pathways with the common theme of generating one ${\displaystyle {\ce {C3}}}$ molecule.[2] Bacteria use three of these pathways[7][11] while Fungi use one.[17] All four pathways incorporate 3 ${\displaystyle {\ce {C1}}}$ molecules into multi-carbon intermediates, then cleave one intermediate into a new ${\displaystyle {\ce {C3}}}$ molecule. The remaining intermediates are rearranged to regenerate the original multi-carbon intermediates.

Each species of methylotrophic bacteria has a single dominant assimilation pathway.[5] The three characterized pathways for carbon assimilation are the ribulose monophosphate (RuMP) and serine pathways of formaldehyde assimilation as well as the ribulose bisphosphate (RuBP) pathway of CO2 assimilation.[2][7][11][18]

Unlike the other assimilatory pathways, bacteria using the RuBP pathway derive all of their organic carbon from ${\displaystyle {\ce {CO2}}}$ assimilation.[5][19] This pathway was first elucidated in photosynthetic autotrophs and is better known as the Calvin Cycle.[19][20] Shortly thereafter, methylotrophic bacteria who could grow on reduced ${\displaystyle {\ce {C1}}}$ compounds were found using this pathway.[21]

First, 3 molecules of ribulose 5-phosphate are phosphorylated to ribulose 1,5-bisphosphate (RuBP). The enzyme ribulose bisphosphate carboxylase (RuBisCO) carboxylates these RuBP molecules which produces 6 molecules of 3-phosphoglycerate (PGA). The enzyme phosphoglycerate kinase phosphorylates PGA into 1,3-diphosphoglycerate (DPGA). Reduction of 6 DPGA by the enzyme glyceraldehyde phosphate dehydrogenase generates 6 molecules of the ${\displaystyle {\ce {C3}}}$ compound glyceraldehyde-3-phosphate (GAP). One GAP molecule is diverted towards biomass while the other 5 molecules regenerate the 3 molecules of ribulose 5-phosphate.[7][20]

RuMP pathway in type I methanotrophs

A new pathway was suspected when RuBisCO was not found in the methanotroph Methylmonas methanica.[22] Through radio-labelling experiments, it was shown that M. methanica used the Ribulose monophate (RuMP) pathway. This has led researchers to propose that the RuMP cycle may have preceded the RuBP cycle.[5]

Like the RuBP cycle, this cycle begins with 3 molecules of ribulose-5-phosphate. However, instead of phosphorylating ribulose-5-phosphate, 3 molecules of formaldehyde form a C-C bond through an aldol condensation, producing 3 ${\displaystyle {\ce {C6}}}$ molecules of 3-hexulose 6-phosphate (hexulose phosphate). One of these molecules of hexulose phosphate is converted into GAP and either pyruvate or dihydroxyacetone phosphate (DHAP). The pyruvate or DHAP is used towards biomass while the other 2 hexulose phosphate molecules and the molecule of GAP are used to regenerate the 3 molecules of ribulose-5-phosphate.[6][22]

Unlike the other assimilatory pathways, the serine cycle uses carboxylic acids and amino acids as intermediates instead of carbohydrates.[5][23] First, 2 molecules of formaldehyde are added to 2 molecules of the amino acid glycine. This produces two molecules of the amino acid serine, the key intermediate of this pathway. These serine molecules eventually produce 2 molecules of 2-phosphoglycerate, with one ${\displaystyle {\ce {C3}}}$ molecule going towards biomass and the other being used to regenerate glycine. Notably, the regeneration of glycine requires a molecule of ${\displaystyle {\ce {CO2}}}$ as well, therefore the Serine pathway also differs from the other 3 pathways by its requirement of both formaldehyde and ${\displaystyle {\ce {CO2}}}$.[22][23]

Methylotrophic yeast metabolism differs from bacteria primarily on the basis of the enzymes used and the carbon assimilation pathway. Unlike bacteria which use bacterial MDH, methylotrophic yeasts oxidize methanol in their peroxisomes with a non-specific alcohol oxidase. This produces formaldehyde as well as hydrogen peroxide.[24][25] Compartmentalization of this reaction in peroxisomes likely sequesters the hydrogen peroxide produced. Catalase is produced in the peroxisomes to deal with this harmful by-product.[17][24]

The dihydroxyacetone (DHA) pathway, also known as the xylulose monophosphate (XuMP) pathway, is found exclusively in yeast.[24][26] This pathway assimilates three molecules of formaldehyde into 1 molecule of DHAP using 3 molecules of xylulose 5-phosphate as the key intermediate.

DHA synthase acts as a transferase (transketolase) to transfer part of xylulose 5-phosphate to DHA. Then these 3 molecules of DHA are phosphorylated to DHAP by triokinase. Like the other cycles, 3 ${\displaystyle {\ce {C3}}}$ molecules are produced with 1 molecule being directed for use as cell material. The other 2 molecules are used to regenerate xylulose 5-phosphate.[27]