Fungal methylotrophs and the MUT

Methylotrophic fungi are known since their first isolation in 1969 [Ogata et al., 1969], and several representatives are well established in biotechnological applications (e.g. single cell protein production, recombinant protein production [Wegner, 1990; Gellissen & Hollenberg, 1997; Gellissen, 2000]). They are abundant in nature and are often associated with pectin-rich (methoxy group-pectin-rich) plant compounds such as fruits, litter and wood [Craveri et al., 1976; Negruƫă et al., 2010]. It seems that methylotrophic yeast can only use methanol-derived compounds as energy and carbon source whereas methylamine might be a nitrogen source [Yurimoto et al., 2011]. Methane utilisation by some yeast strains was reported in the 1980s, but no further reports on methanotrophic fungi or putative pathways and enzymes are available [Wolf & Hanson, 1980;Anthony, 1982; Hanson & Hanson, 1996].

Most prominent are methylotrophic yeasts belonging to several genera such as Candida, Hansenula, Torulopsis, Trichosporon, Pichia, Polyporus, Poria, and Radulum, as well as the recently from Pichia separated genera Ogataea, Kuraishia, and Komagataella [Hartner &

Glieder, 2006; Kaszycki et al., 2006; Kondo et al., 2008; Limtong et al., 2008; Negruƫă et al., 2010; Yurimoto et al., 2011]. Within mould fungi methylotrophic representatives are more limited, and although some representatives possess genetic hints, methylotrophy is physiologically not proven [Kondo et al., 2008; Gvozdev et al., 2012; Kolb & Stacheter, 2013]. However, the classification and assignment of methylotrophic fungi is complicated.

For example the yeast Pichia angusta has several synonyms such as Hansenula angusta, Hansenula polymorpha, and Ogataea polymorpha [Negruƫă et al., 2010].

A B

Figure 10 Methanol metabolism in methylotrophic yeasts (A) and the crystal structure of a FAD AOx (B).

Panel A shows details of the MUT in yeast cells focussing on the peroxisomal oxidation of methanol (black lines) and the cytosolic assimilatory (dark grey dashed lines) and dissimilatory pathways (light grey dotted lines). For more details refer to the text. The amount of carbon atoms per molecule in the assimilatory pathway are indicated in grey boxes. Abbreviations: FAD AOx, FAD dependent alcohol oxidase; CTA, catalase; RR, rearrangement reactions.

Molecule abbreviations: DHA, dihydroxyacetone; FBP, fructose 1,6-bisphosphate; GAP, glyceraldehyde 3-phosphate; GSH, reduced form of glutathione; GS-CH2OH, S-hydroxymethyl glutathione; XMP, xylulose 5-phosphate. The figure is based on Yurimoto et al., 2011.

Panel B shows the homo-octameric crystal structure of the FAD AOx of the methylotrophic yeast Pichia pastoris.

Each subunit is indicated with different colours and the arrow point at one non-covalently bound FAD as prosthetic group (in total 10 FAD molecules are present). Image: http://www.rcsb.org (PDB ID: 5HSA).

All methylotrophic yeasts employ a common methanol utilisation pathway (MUT, Figure 10A) that was mainly characterized in Hansenula polymorpha (Pichia angusta) and Candida boidinii [Veenhuis et al., 1983; Tani, 1984; Large & Bamforth, 1988; Yurimoto et al., 2011].

This MUT is transcriptionally repressed by glucose and ethanol, but can be highly induced by methanol resulting in large amounts of necessary enzymes and peroxisomes [Hartner &

Glieder, 2006; Nakawaga et al., 2006; Yurimoto et al., 2011; Koch et al., 2016]. The MUT pathway is in many ways different from the pathways described for methylotrophic bacteria (see 1.4, 1.5) with the main differences being (i) the nature of the key enzyme and (ii) the compartmentation of the pathway in peroxisomes [Anthony, 1982]. Initially in peroxisomes methanol is oxidised to formaldehyde resulting in the formation of hydrogen peroxide (H₂O₂) that is subsequently removed by catalase activity [Anthony, 1982; Hartner & Glieder, 2006;

Yurimoto et al., 2011]. As for methylotrophic Bacteria formaldehyde is the branching point for dissimilation and assimilation. The cytosolic assimilatory pathway – dihydroxyacetone cycle

(DHA) or xylulose monophosphate pathway – is somehow similar to the RuMP cycle of methylotrophic Bacteria (see 1.5.1). The C1-unit (formaldehyde) is transferred to a C5-acceptor (xylulose monophosphate) resulting in the C3-units dihydroxyacetone (DHA) and glyceraldehyde phosphate (GAP) used for the biosynthesis of cell material and the rearrangement of the C5-acceptor [Anthony, 1982; Hartner & Glieder, 2006; Yurimoto et al., 2011]. The cytosolic dissimilatory pathway plays a crucial role in detoxification [Sakai et al., 1997; Lee et al., 2002; Hartner & Glieder, 2006], and formaldehyde reacts non-enzymatically with reduced glutathione (GSH) generating S-hydroxymethyl glutathione (S-HMG) that is further oxidised to CO₂ by a omnipresent GSH-dependent pathway [Harms et al., 1996;

Hartner & Glieder, 2006; Yurimoto et al., 2011].

One key enzyme for methylotrophic fungi facilitating the initial oxidation step of methanol is a flavin adenine nucleotide dependent alcohol oxidase (FAD AOx) [Hartner & Glieder, 2006;

Gvozdev et al., 2012; Koch et al., 2016], which was primarily described in a Basidiomycetes [Janssen et al., 1965; Gvozdev et al., 2012]. The FAD AOx is a homo-octameric enzyme possessing one non-covalently bound FAD as prosthetic group per monomer and is formed of two facing tetramers (Figure 10B) [Koch et al., 2016]. The FAD AOx is not restricted to methanol only, but can also oxidise other short aliphatic alcohols such as ethanol and 1-propanol [Koch et al., 2016]. Enzymatic and molecular studies have revealed two subunits of the FAD AOx differing in their amino acid residues, encoding genes (i.e., α-subunit is encoded by MOD1 (synonyms: AOX1, AUG1) and β-subunit is encoded by MOD2 (synonyms: AOX2, AUG2)) and synthesis conditions (e.g. α-subunit at low methanol concentrations; β-subunit at high (>3%) methanol concentrations) [Hartner & Glieder, 2006;

Gvozdev et al., 2012]. Thus, both subunits are active under different conditions enabling an elegant fine-tuning of the methylotrophic fungi in response to environmental conditions and resulting in up to nine different FAD AOx isoenzymes consisting of a combination of both subunits [Ito et al., 2007; Gvozdev et al., 2012]. Such FAD AOx isoenzymes are widespread among the methylotrophic yeasts, but some representatives such as the well established model yeast strain Candida boidinii and Hansenula polymorpha possess only one gene (i.e., MOD1) encoding for the FAD AOx [Hartner & Glieder, 2006; Ito et al., 2007; Negruƫă et al., 2010].

However, although methylotrophic yeast, the MUT pathway and its regulation are well understood, the role and diversity of methylotrophic fungi in the environment and especially inside microbial communities in terrestrial habitats is hardly resolved. Based on the knowledge of the methylotrophic capabilities and the metabolic versatility of fungi they might be underestimated and represent another large microbial sink of methanol besides methanol-utilising bacterial methylotrophs in forest soils.