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Methylotrophic Yeasts: Current Understanding of Their C1-Metabolism and Its Regulation by Sensing Methanol for Survival on Plant Leaves

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Methylotrophic Yeasts: Current Understanding of Their C1-Metabolism and Its Regulation by Sensing Methanol for Survival on Plant Leaves Methylotrophic Yeasts: Current Understanding of Their C1-Metabolism and its Regulation by Sensing Methanol for Survival on Plant Leaves Hiroya Yurimoto and Yasuyoshi Sakai* Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, Japan. *Correspondence: [email protected] htps://doi.org/10.21775/cimb.033.197 Abstract Tese yeasts belong to a restricted number of Methylotrophic yeasts, which are able to utilize genera, including Komagataella, Ogataea, Kuraishia, methanol as the sole carbon and energy source, and Candida (Kurtzman, 2005; Péter et al., 2005; have been intensively studied in terms of physi- Suh et al., 2006; Limtong et al., 2008), while methy- ological function and practical applications. When lotrophic bacteria belong to diverse genera and these yeasts grow on methanol, the genes encod- subclasses (Kolb, 2009). Methylotrophic yeasts ing enzymes and proteins involved in methanol can also use another one-carbon (C1) compound, metabolism are strongly induced. Simultaneously, methylamine, as a nitrogen source, but not as the peroxisomes, organelles that contain the key sole carbon source. enzymes for methanol metabolism, massively Since the frst isolation of the methylotrophic proliferate. Tese characteristics have made methy- yeast Kloeckera sp. (later identifed as Candida lotrophic yeasts efcient hosts for heterologous boidinii) in 1969 (Ogata et al., 1969), both their protein production using strong and methanol- physiological functions and their applications have inducible gene promoters and also model organisms been intensively studied. During the 1970s, the for the study of peroxisome dynamics. Much aten- metabolic pathways for methanol assimilation and tion has been paid to the interaction between dissimilation were elucidated mainly with Candida methylotrophic microorganisms and plants. In this boidinii and Hansenula polymorpha (reclassifed as chapter, we describe how methylotrophic yeasts Ogataea polymorpha), and methylotrophic yeasts proliferate and survive on plant leaves, focusing were revealed to have a common methanol-utilizing on their physiological functions and lifestyle in pathway (Anthony, 1982; Veenhuis et al., 1983; Tani, the phyllosphere. Our current understanding of 1984; Yurimoto et al., 2002). One of the diferences the molecular basis of methanol-inducible gene between bacterial and yeast methanol metabolism expression, including methanol-sensing and its is in the initial oxidation reaction of methanol. applications, is also summarized. While methylotrophic bacteria oxidize methanol to formaldehyde by using a pyrroloquinoline qui- none (PQQ)- or NAD+-dependent dehydrogenase, Introduction methylotrophic yeasts oxidize methanol with an Methylotrophic yeasts are capable of utilizing alcohol oxidase (AOD) using molecular oxygen methanol as the sole source of carbon and energy. as an electron acceptor. When methylotrophic Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb 198 | Yurimoto and Sakai yeasts grow on methanol, genes encoding a number methylotrophic yeasts utilize carbon and nitrogen of enzymes and proteins involved in methanol sources in the phyllosphere by regulating their metabolism are strongly induced. Tis strong cellular metabolism. Ten the recently revealed inducibility by methanol together with the ability molecular basis of methanol-inducible gene expres- to grow to extremely high cell density have enabled sion, including the methanol-sensing machinery is methylotrophic yeasts to become promising hosts summarized. for high-level heterologous protein production. During the mid-1980s to early 1990s, heterologous gene expression systems driven by strong meth- Methanol metabolism in the anol-inducible gene promoters were developed methylotrophic yeasts in various methylotrophic yeast strains, including Fig. 11.1 summarizes the pathway of methanol Pichia pastoris (reclassifed as Komagataella phaf- metabolism, which so far is common to all isolated fi), H. polymorpha, and C. boidinii (Cregg et al., methylotrophic yeasts (Yurimoto et al., 2002, 2000; Gellissen, 2000; Yurimoto, 2009). Another 2011). Methanol metabolism begins with the oxi- characteristic of yeast methanol metabolism is that dation of methanol to formaldehyde by AOD. AOD methylotrophic growth is accompanied by massive is a favoprotein that contains FAD and uses O2 as development of a membrane-bound organelle, the an electron acceptor to yield formaldehyde and peroxisome, in which AOD and other key metha- H2O2, both of which are extremely toxic to cells. nol-assimilating enzymes are compartmentalized. H2O2 is broken down by catalase (CTA) and per- Terefore, methylotrophic yeasts have also been oxiredoxin (Pmp20) (Horiguchi et al., 2001a,b). used as model organisms to reveal the molecular Formaldehyde, which is situated at the branch point machineries and mechanisms of peroxisomal pro- between assimilation and dissimilation pathways, tein import as well as peroxisome degradation by is a key central intermediate in methanol metabo- selective autophagy (pexophagy) (van der Klei et lism (Yurimoto et al., 2005). In the assimilation al., 2006; Oku et al., 2010). pathway, formaldehyde (C1-compound) is fxed Methanol is a volatile atmospheric carbon to xylulose 5-phosphate (Xu5P; C5 compound) by compound, and its annual emission is estimated dihydroxyacetone synthase (DAS) to generate two to be 100 Tg (Galbally et al., 2002). Methanol, C3 compounds, dihydroxyacetone (DHA) and which originates from methylesters in the plant glyceraldehyde 3-phosphate (GAP). DHA is phos- cell wall constituent pectin, is emited from plant phorylated by dihydroxyacetone kinase (DAK) leaves during plant cell elongation and division to yield dihydroxyacetone phosphate (DHAP). (Nemecek-Marshall et al., 1995; Fall et al., 1996). GAP and DHAP are used for the synthesis of cell Te phyllosphere, defned as the aerial portion constituents and the regeneration of Xu5P afer of plants, has been recognized as a habitat for rearrangement reactions. methylotrophic microorganisms. Phyllospheric In the cells of methylotrophic yeasts, per- methylotrophic bacteria were identifed in the oxisomes, the organelles specialized for methanol 1980s (Corpe et al., 1982), and since then, the metabolism, massively proliferate during growth symbiotic relationship between plants and methy- on methanol. Te peroxisomal localization of lotrophic bacteria, Methylobacterium spp., which AOD, CTA, Pmp20, and DAS has been described can promote plant growth, has been intensively in detail. Recently, proteomic analysis of the per- investigated (Vorholt, 2012; Dourado et al., 2015). oxisomal fraction of methanol-grown P. pastoris Similarly, a number of methylotrophic yeast strains cells revealed that further assimilatory metabolism have been isolated from plant resources, e.g. forest catalysed by DAK and the sugar phosphate rear- soils, fallen leaves, and the skins of olives and grapes rangement reactions are also localized within (Limtong et al., 2008; Péter et al., 2009). However, peroxisomes (Russmayer et al., 2015). the ecology and physiology of methylotrophic In the dissimilation pathway, formaldehyde is yeasts in the phyllosphere have not been studied in oxidized to CO2 by the glutathione-dependent detail. formaldehyde oxidation pathway. Formaldehyde In this chapter, afer outlining methanol metab- reacts non-enzymatically with the reduced form olism in methylotrophic yeasts, we describe how of glutathione (GSH) to form S-hydroxymethyl Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb Yeast Methylotrophy on Plant Leaves | 199 CH3OH Cytosol CH3OH O NADH NADH CTA 2 + + AOD NAD H2O NAD H O Pmp20 2 2 HCHO GS-CH OH GS-CHO HCOOH CO 2 FLD FGH FDH 2 GSH GSH DAS Xu5P GAP Rearrangement reactions + DAK Cell DHA DHAP FBP constituents ATP Peroxisome ADP Figure 11.1 Methanol metabolism in methylotrophic yeasts. AOD, alcohol oxidase; CTA, catalase; DAK, dihydroxyacetone kinase; DAS, dihydroxyacetone synthase; FDH, formate dehydrogenase; FGH, S-formylglutathione hydrolase; FLD, formaldehyde dehydrogenase; Pmp20, peroxiredoxin (glutathione peroxidase). Abbreviations: DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; FBP, fructose 1,6-bisphosphate; GAP, glyceraldehyde 3-phosphate; GS-CH2OH, S-hydroxymethyl glutathione; GS-CHO, S-formylglutathione; GSH, reduced form of glutathione; Xu5P, xylulose 5-phosphate. Reactions occurring within peroxisomes are highlighted in yellow. glutathione (S-HMG). Since GSH was shown to of plants, termed the phyllosphere, is one of the be present in peroxisomes, S-HMG can be formed main habitats of methylotrophic microorganisms. within peroxisomes and then be exported to the Indeed, methylotrophic yeasts can ofen be isolated cytosol (Horiguchi et al., 2001b; Yurimoto et al., from plant material (Limtong et al., 2008; Péter et 2003). S-HMG is oxidized to S-formylglutathione al., 2009). However, while the symbiotic relation- (S-FG) by NAD+-linked and GSH-dependent ship between plants and methylotrophic bacteria formaldehyde dehydrogenase (FLD). S-FG is has been well characterized (Vorholt, 2012), it was then hydrolysed to formate and GSH by S-formyl- not known whether methylotrophic yeasts can sur- glutathione hydrolase (FGH). Finally, formate is vive and proliferate in the phyllosphere. + oxidized to CO2 by NAD -linked formate dehy- Methanol emission from the phyllosphere was drogenase (FDH). Terefore, in methylotrophic frst reported by Nemecek-Marshall et al. (1995). yeasts, the
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