Keywords

1.1 Introduction

Yeasts prefer sugars over other carbon sources and thrive in sugar-rich environment. In addition to glucose and fructose, many yeasts assimilate α-glucosidic oligosaccharides. α-Glucosidic oligosaccharides, maltose, isomaltose, and maltotriose (Fig. 1.1), emerge from degradation of starch and glycogen by amylases (Janecek 2009). Many plants (such as sugarcane and sugar beet) and berries contain a big amount of sucrose, a disaccharide of glucose and fructose. Sucrose is also synthesized by cyanobacteria and proteobacteria (Lunn 2002). Importantly, sucrose can be converted to isomers by enzymes of many organisms including plants, yeasts, filamentous fungi, bacteria, and even insects as shown in (Lee et al. 2011) and references therein. So, turanose, palatinose, and maltulose (Fig. 1.1) present in honey are isomerization products of sucrose. Palatinose is currently enzymatically produced from sucrose at a large scale and advertised as a novel healthy sugar with low glycemic index and no cariogenic effect (Sawale et al. 2017). Melezitose (Fig. 1.1) that is also present in honey is a main constituent of aphid honeydew (Daudé et al. 2012). Fig. 1.1 shows α-glucosidic sugars assimilated by a methylotrophic yeast O. polymorpha (Viigand 2018).

Fig. 1.1
figure 1

α-Glucosidic di- and trisaccharides and the linkages between the sugar residues. (Viigand 2018)

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A question arises: Why do yeast communities have members that assimilate both methanol and disaccharides? The answer may lie in the natural habitat of these yeasts. For instance, the species of Ogataea have been isolated from spoiled orange juice, rotting plants, plant leaf surfaces and exudates, soil samples, and insect gut (Morais et al. 2004; Limtong et al. 2008; Naumov et al. 2017). These habitats certainly contain α-glucosidic sugars but may also contain methanol. In the soil, methanol is produced as a result of pectin and lignin degradation (Dorokhov et al. 2015). Furthermore, even living plant leaves emit methanol due to the recycling of pectin and lignin of the cell wall (Keppler et al. 2006). Thus, it is not surprising that yeast species metabolizing both disaccharides and methanol are found in nature.

1.2 MAL Clusters in Yeast Genomes

1.2.1 Saccharomyces cerevisiae

Metabolism of maltose has been thoroughly studied in Saccharomyces yeasts as they are commonly used in brewing – a process largely based on fermentation of maltose and maltotriose. Indeed, the beer wort produced by grain starch hydrolysis contains 50–60% of maltose and 15–20% maltotriose. During brewing, these oligosaccharides are transported into the yeast cell, hydrolyzed to glucose by maltases and isomaltases, and fermented to ethanol and CO2 (Stewart 2016). In S. cerevisiae, the genes for maltose metabolism are genomically clustered comprising five MAL clusters, MAL1, MAL2, MAL3, MAL4, and MAL6, located near the telomeres of chromosomes VII, III, II, XI, and VIII (Brown et al. 2010; Charron et al. 1986; Needleman 1991; Vanoni et al. 1989). Each MAL cluster in S. cerevisiae consists of three genes (Fig. 1.2): MALx1 (maltose permease gene), MALx2 (maltase gene), and MALx3 (MAL-activator gene) (Charron et al. 1986; Needleman 1991; Chang et al. 1988; Dubin et al. 1985). The “x” refers to the number of the cluster.

Fig. 1.2
figure 2

Composition of the S. cerevisiae MAL3 cluster. MAL31, α-glucoside permease gene; MAL32, maltase gene; MAL33, MAL-activator gene. The lower panel depicts position of the MAL cluster (a dark vertical stripe) in chromosome 2 of S. cerevisiae S288C. Subtelomeric regions (50 kbp from the chromosome end) are shown in gray. (Viigand 2018)

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It should be noted that genomic clustering of functionally related genes is quite exceptional in eukaryotes. In addition to MAL clusters, gene clusters for utilization of galactose, allantoin, and nitrate are known in yeasts and filamentous fungi (Ávila et al. 2002; Kunze et al. 2014; Slot and Rokas 2010; Wong and Wolfe 2005).

1.2.2 Ogataea polymorpha

The maltase structural gene (MAL1) of O. polymorpha CBS 4732 was isolated from a genomic library of this strain in 2001 (Liiv et al. 2001). An open reading frame of 1695 bp encoding a 564 aa protein having 58% identity with Candida albicans maltase was characterized in the library clone p51 (Liiv et al. 2001). A scheme of the genomic insert of the p51 plasmid is shown in Fig.1.3.

Fig. 1.3
figure 3

Composition of the O. polymorpha MAL locus. Genomic insert in the Génolevures library clones is shown within the frame. Respective GenBank numbers of sequences belonging to MAL genes are shown above the frames. MAL1 gene encodes a maltase (α-glucosidase), MAL2 gene encodes an α-glucoside permease, and MAL-ACT genes encode putative MAL-activators. (Modified from Viigand 2018)

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Inspection of O. polymorpha CBS 4732 genomic library clones sequenced in the Génolevures project (Blandin et al. 2000; Feldmann 2000) identified several clones (BB0AA021D05, BB0AA011B12, and BB0AA003C10) (see Fig. 1.3) that contained fragments of MAL genes (Viigand 2018; Viigand et al. 2005).

Further sequencing of the Génolevures clone inserts revealed the composition of O. polymorpha MAL locus (Fig. 1.3). Similar to S. cerevisiae, a bidirectional promoter region was identified between the MAL1 and MAL2 genes of O. polymorpha. Two hypothetical transcriptional activator genes, MAL-activator 1 (MAL-ACT 1) and MAL-activator 2 (MAL-ACT 2), were detected next to the MAL2 permease gene. Full-length sequence of the MAL locus of O. polymorpha CBS 4732 is accessible in the GenBank under number MH252366.

As the MAL loci of S. cerevisiae are subtelomeric, chromosomal location of the O. polymorpha MAL locus was inspected. The first O. polymorpha genome – of strain CBS 4732 – was sequenced in 2003 (Ramezani-Rad et al. 2003), but the sequence is not yet public. Therefore, the genome of O. polymorpha strain NCYC 495 leu1.1 (Grigoriev et al. 2014) was assayed instead. Comparison of genomic sequences of MAL clusters from O. polymorpha strains NCYC 495 leu1.1 and CBS 4732 revealed their identity, and the MAL cluster in O. polymorpha NCYC 495 leu1.1 was proven as not subtelomeric (Viigand 2018; Viigand et al. 2018).

Gene disruption experiments confirmed that MAL1 and MAL2 genes are, respectively, responsible for the hydrolysis and transport of α-glucosidic sugars in O. polymorpha (Viigand and Alamäe 2007; Viigand et al. 2016). Although hypothetical α-glucoside permease and α-glucosidase genes were recently detected outside the O. polymorpha MAL cluster, they were considered unnecessary for assimilation of α-glucosidic sugars (Viigand et al. 2018).

We conclude that O. polymorpha has a single MAL locus that consists of four genes coding for maltase MAL1 (syn. α-glucosidase, maltase-isomaltase), α-glucoside permease MAL2, and two putative MAL-activators. The functionality and role of MAL-activators have still to be clarified. However, there is indirect support of functionality for at least MAL-activator 1 gene. Namely, the promoter of this gene was regulated by carbon sources in the same way as the promoters of MAL1 and MAL2 genes (Viigand and Alamäe 2007). A four-gene non-telomeric MAL cluster similar to that of O. polymorpha was also detected in O. parapolymorpha which was formerly named O. polymorpha DL-1 (Viigand et al. 2018; Ravin et al. 2013).

1.2.3 Other Non-conventional Yeasts

Besides O. polymorpha, MAL clusters have been revealed in many yeasts such as Lodderomyces elongisporus, Torulaspora delbrueckii, Meyerozyma guilliermondii, Cyberlindnera fabianii, Debaryomyces hansenii, Lipomyces starkeyi (Viigand et al. 2018), Scheffersomyces stipitis (Jeffries and Van Vleet 2009), Kluyveromyces lactis (Fairhead and Dujon 2006; Leifso et al. 2007), and a filamentous fungus Aspergillus oryzae (Hasegawa et al. 2010). A recent genome mining study (Viigand et al. 2018) discovered the highest number of MAL clusters in L. starkeyi (Fig. 1.4), whereas no MAL clusters were found in Blastobotrys adeninivorans and Schizosaccharomyces pombe. Figure 1.4 illustrates the composition and number of MAL clusters in the genomes of non-conventional yeasts.

Fig. 1.4
figure 4

MAL loci of some non-conventional yeasts (Viigand et al. 2018). Genes (potentially) encoding α-glucosidases (AG), α-glucoside transporters (AGT), and MAL-activators (MAL-ACT) are shown with different shading. Accession numbers and annotation data of the AG and AGT genes are given in Tables S1 and S2 of (Viigand et al. 2018)

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1.3 Transport of α-Glucosidic Sugars by O. polymorpha

Permeases responsible for maltose uptake have been experimentally characterized in Saccharomyces strains (Cheng and Michels 1989; Cheng and Michels 1991), Torulaspora delbrueckii (Alves-Araújo et al. 2004), Candida utilis (Peinado et al. 1987), Schizosaccharomyces pombe (Reinders and Ward 2001), and O. polymorpha (Viigand and Alamäe 2007).

The putative maltose permease gene MAL2 from the MAL cluster of O. polymorpha was cloned in 2005. The MAL2 protein (582 amino acids) deduced from the respective gene had 51 and 57% identity to respective hypothetical maltose permeases of Candida albicans and Debaryomyces hansenii (Viigand et al. 2005). Inspection of genomes of a set of non-conventional yeasts thereafter revealed closer homologues of the MAL2: potential α-glucoside permeases of O. parapolymorpha (87% sequence identity to MAL2) and Cyberlindnera fabianii (67% identity to MAL2). Currently, the AGT1 protein of S. cerevisiae is the closest counterpart (37% sequence identity) of O. polymorpha MAL2 among experimentally studied permeases (Viigand et al. 2018). The MAL2 disruption mutant of O. polymorpha lost the ability to grow on maltose and sucrose, and complementation of the mutant with the MAL2 gene on a plasmid restored the growth on these disaccharides (Viigand and Alamäe 2007). The MAL2 of O. polymorpha was also functional in S. cerevisiae (Viigand et al. 2005).

Similar to α-glucoside transporters of other yeasts (Reinders and Ward 2001; Stambuk et al. 2000; Stambuk and de Araujo 2001), the O. polymorpha MAL2 functioned as an energy-dependent proton symport (Viigand and Alamäe 2007). In agreement with that, the transport by MAL2 was highly sensitive to protonophores and energy uncouplers and had the highest activity at pH 5.0 (Viigand and Alamäe 2007).

Most α-glucoside transporters of S. cerevisiae (e.g., MAL61 and MAL31) have a narrow substrate range – they transport just maltose and turanose. In contrast to these transporters, the AGT1 permease of S. cerevisiae is highly promiscuous – it transports sucrose, maltose, turanose, PNPG (p-nitrophenyl-α-D-glucopyranoside), trehalose, α-methylglucoside, maltotriose, isomaltose, palatinose, and melezitose (Stambuk and de Araujo 2001; Han et al. 1995; Hollatz and Stambuk 2001; Day et al. 2002a). Notably, PNPG can be used as a chromogenic substrate to measure the activity of this type of permeases (Viigand and Alamäe 2007; Hollatz and Stambuk 2001). A recent study (Kulikova-Borovikova et al. 2018) shows that PNPG hydrolysis by the cells can also be used to evaluate permeabilization status of cell membrane in the case of PNPG-transporting yeasts (Kulikova-Borovikova et al. 2018).

In the first study (Viigand and Alamäe 2007), the MAL2 permease was reported to transport maltose, sucrose, turanose, trehalose, maltotriose, and PNPG. Further assay (Viigand et al. 2016) revealed that the MAL2 permease is also required for the growth of O. polymorpha on maltulose, melezitose, palatinose, and isomaltooligosaccharides. Because of the relaxed substrate specificity, the O. polymorpha MAL2 resembles the AGT1 of S. cerevisiae (Stambuk and de Araujo 2001; Han et al. 1995; Hollatz and Stambuk 2001) and should be considered an α-glucoside transporter. Even though the MAL2 is indispensable for the growth of O. polymorpha on trehalose (an α-1,1-linked disaccharide of glucose), the α-glucosidase (MAL1) protein does not hydrolyze this sugar (Liiv et al. 2001). It was shown later that for the hydrolysis of trehalose, O. polymorpha uses a specific enzyme – trehalase (Ishchuk et al. 2009).

The MAL2 transports PNPG with high affinity (Km 0.51 mM) (Viigand and Alamäe 2007). The Km value of the AGT1 permease of S. cerevisiae for PNPG is ~3 mM (Han et al. 1995). The MALx1 transporters from S. cerevisiae do not transport PNPG (Hollatz and Stambuk 2001). Inhibition of PNPG transport by various α-glucosidic substrates was used to evaluate affinity of the MAL2 permease of O. polymorpha for these substrates (Viigand and Alamäe 2007). Sucrose, maltose, trehalose, maltotriose, turanose, and α-methylglucoside (α-MG, also methyl-α-D-glucopyranoside) competitively inhibited the PNPG transport by MAL2 in O. polymorpha (respective Ki values were between 0.23 and 1.47 mM). However, though α-MG probably binds the MAL2 permease of O. polymorpha, this yeast does not grow on this synthetic sugar (Viigand et al. 2016). The S. cerevisiae AGT1 has lower affinity for its substrates than the MAL2 permease of O. polymorpha: Km of the AGT1 for maltose is 5.1–17.8 mM (Stambuk and de Araujo 2001; Day et al. 2002b), Km for sucrose is ~ 8 mM (Stambuk et al. 2000), and Km for maltotriose is 4–18.1 mM (Stambuk et al. 2000; Day et al. 2002a). As the MAL2 permease of O. polymorpha has a considerably high affinity for α-glucosidic sugars, concentrations of these substrates are expected to be low in the habitat of O. polymorpha. Interestingly, the AGT1 protein of S. cerevisiae was also capable of glucose transport if overexpressed in a hxt1–17 gal2 deletion strain (Wieczorke et al. 1999).

The affinity of the MAL2 permease for maltose and sucrose is much higher than that of the maltase for these sugars (Viigand 2018; Liiv et al. 2001; Viigand and Alamäe 2007; Viigand et al. 2016), suggesting that these substrates must be concentrated into the cell to enable their efficient hydrolysis. The presence of intracellular substrate (e.g., maltose) is crucial for the induction of the MAL genes as reported for O. polymorpha (Viigand and Alamäe 2007) and S. cerevisiae (Wang et al. 2002). So, if a MAL2 disruptant was incubated in the medium containing maltose, no maltase induction was recorded in the cells (Viigand and Alamäe 2007).

The AGT1 permease of S. cerevisiae was recently studied by Trichez et al. using mutagenesis (Trichez et al. 2018). Given that charged residues are usually responsible for sugar and proton binding by H+-symporters, Trichez et al. selected four conserved charged residues in the predicted transmembrane α-helices 1 (Glu120, Asp123), 2 (Glu167), and 11 (Arg504) of the AGT1 permease for mutagenesis. Mutation of Glu120 and Arg504 to alanine most severely reduced the active transport of maltotriose and other α-glucosidic sugars by the yeast. In the case of Asp123 and Glu167, a double mutation (Asp123Gly/Glu167Ala) was required to reduce the transport (Trichez et al. 2018). Figure 1.5a shows alignment of O. polymorpha MAL2 transporter with AGT1 and MAL31 of S. cerevisiae in regions predicted to encode transmembrane domains (TMDs) 1, 2, and 11. Conserved charged residues proven crucial for active transport by the AGT1 are highlighted in the alignment, and predicted TMDs of MAL2 are shown overlined. Fig. 1.5b shows predicted structure of the O. polymorpha MAL2 permease. The charged residues of TMDs 1, 2, and 11 that are inferred to participate in active transport of α-glucosidic sugars are shown on dark background.

Fig. 1.5
figure 5

(a) Extract of ClustalW alignment of the O. polymorpha MAL2 with AGT1 and MAL31 of S. cerevisiae. Sequences comprising the transmembrane domains (TMDs) 1, 2, and 11 are shown overlined. (b) Predicted TMDs of the MAL2 permease of O. polymorpha. The insert highlights charged residues of TMD1 (Glu87 and Asp90), TMD2 (Glu135), and TMD11 (Arg471) of O. polymorpha MAL2 assumed crucial for active transport of α-glucosidic sugars. TMDs were predicted through the CCTOP website (Dobson et al. 2015). The MAL2 permease was visualized using the Protter program (Omasits et al. 2014)

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1.4 The MAL1 of O. polymorpha Among Yeast α-Glucosidases

The MAL1 protein of O. polymorpha was first characterized in 2001 (Liiv et al. 2001). This subchapter characterizes not only catalytic properties of the MAL1 protein but also its phylogenetic position among α-glucosidases of yeasts.

1.4.1 α-Glucosidases in CAZy

Yeast α-glucosidases belong to glycoside hydrolases (EC 3.2.1.–). Glycoside hydrolases (GHs) hydrolyze the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety (Lombard et al. 2014). For example, the α-glucosidase MAL1 of O. polymorpha also hydrolyzes an α-glycosidic bond in α-MG and PNPG liberating methanol and p-nitrophenol from respective substrates (Liiv et al. 2001). Hydrolysis of a chromogenic substrate PNPG is widely used to quantitate catalytic activity of α-glucosidases (Liiv et al. 2001; Zimmermann et al. 1977). CAZy, a protein sequence-based database of Carbohydrate-Active enZymes, classifies maltases and isomaltases to family GH13 (Lombard et al. 2014). This family also includes α-amylases, pullulanases, neopullulanases, isoamylases, trehalose synthases, trehalose-6-phosphate hydrolases, and many others. The similarity of the amino acid sequences within the GH13 family proteins is low, yet they all share highly conserved regions and a catalytic machinery – three acidic catalytic residues located in conserved regions (Yamamoto et al. 2010). In the MAL1 protein of O. polymorpha, Asp199 was predicted as catalytic nucleophile, Glu257 as acid-base catalyst, and Asp338 as transition state stabilizer. The importance of Asp199 for the catalysis was experimentally proven: the Asp199Ala (D199A) mutant of MAL1 was catalytically inactive (Viigand et al. 2016).

1.4.2 Maltases and Isomaltases of Saccharomyces cerevisiae

S. cerevisiae has two types of α-glucosidases for the hydrolysis of α-glucosidic sugars: maltases (EC 3.2.1.20) and isomaltases (EC 3.2.1.10). Isomaltases have also been referred to as oligo-1,6-glucosidases, sucrase-isomaltases, and α-methylglucosidases. Maltases (e.g., MAL12 and MAL62) degrade maltose and so-called “maltose-like” sugars, maltotriose, sucrose, turanose, and maltulose, but are not able to degrade isomaltose and “isomaltose-like” sugars such as palatinose and α-MG (Viigand 2018; Needleman et al. 1978; Krakenaĭte and Glemzha 1983; Voordeckers et al. 2012). For composition and linkages in these sugars, see Fig. 1.1. For isomaltose degradation, Saccharomyces has isomaltases IMA1 to IMA5 (Naumoff and Naumov 2010; Teste et al. 2010). In addition to isomaltose, palatinose, and α-methylglucoside, isomaltases of S. cerevisiae also hydrolyze PNPG and sucrose (Teste et al. 2010; Deng et al. 2014).

1.4.3 Substrate Specificity of the O. polymorpha MAL1

Enzymatic assay of crude extract from E. coli cells expressing the MAL1 protein revealed maltose, sucrose, and α-methylglucoside as substrates for the enzyme, whereas trehalose, melibiose, and cellobiose were not hydrolyzed, indicating that the enzyme acts on α-1,4 (as in maltose) and α-1,2 (as in sucrose) glycosidic linkages (Liiv et al. 2001). In (Viigand et al. 2016), the substrate specificity of heterologously expressed and purified MAL1 protein was addressed, and additional substrates were revealed for the enzyme. It was shown that the MAL1 can hydrolyze the following maltose-like substrates with affinities decreasing in the order: maltulose, maltotriose, sucrose, turanose, maltose, and melezitose. From isomaltose-like substrates, palatinose was the most suitable substrate, followed by isomaltose and α-MG. PNPG was the best substrate for MAL1 (Table 1.1).

Table 1.1 Substrate specificity of the O. polymorpha MAL1 (Viigand et al. 2016)
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Interestingly, the MAL1 protein could also hydrolyze fructooligosaccharides 1-kestose and 6-kestose (Viigand et al. 2016).This property has not been shown before for α-glucosidases. In the case of hydrolysis of trisaccharides melezitose and panose (see Fig. 1.1), the linkages hydrolyzed first by the enzyme were α-1,3 and α-1,6, respectively (Viigand 2018; Viigand et al. 2016). Though in early publications, the O. polymorpha MAL1 has been defined as a maltase, according to its substrate specificity, it rather belongs to maltase-isomaltases.

Phylogenetic analysis of yeast and fungal α-glucosidases revealed clustering of O. polymorpha MAL1 with α-glucosidases of O. parapolymorpha, Cyberlindnera fabianii, Scheffersomyces stipitis, Lodderomyces elongisporus, and Meyerozyma guilliermondii (Viigand et al. 2018). Of all these enzymes, only the S. stipitis MAL7, MAL8, and MAL9 (Viigand et al. 2018) and an α-glucosidase of L. elongisporus (Voordeckers et al. 2012) have been experimentally studied – these enzymes were reported to hydrolyze both maltose-like and isomaltose-like substrates. α-Glucosidases have been also studied in a phylogenetically old yeast Schizosaccharomyces pombe. The intracellular MAL1 protein of S. pombe hydrolyzed not only PNPG, maltose, and sucrose but also polymeric α-glucans dextrin and soluble starch (Chi et al. 2008). In addition to MAL1, S. pombe has an extracellular maltase AGL1, which was shown strictly specific for maltose – it did not hydrolyze other maltose-like sugars such as maltotriose and turanose (Jansen et al. 2006).

The malt extract and isomaltooligosaccharides were also substrates for O. polymorpha MAL1: the DP4 oligosaccharide was the longest substrate for the enzyme (Viigand et al. 2016). At the same time, the extracellular α-glucosidase of S. pombe used maltooligosaccharides with size up to DP7 – maltoheptaose (Okuyama et al. 2005). Some bacterial α-glucosidases also hydrolyze longer oligosaccharides (up to maltoheptaose) and in some cases also polysaccharides starch and dextrin (Egeter and Brückner 1995; Schönert et al. 1998, 1999; Cihan et al. 2011).

Similar to S. cerevisiae maltase (Kim et al. 1999), the MAL1 of O. polymorpha was very strongly inhibited by glucose and a diabetes drug acarbose. Fructose, a hydrolysis product of sucrose, turanose, maltulose, and palatinose (see Fig. 1.1), exerted much lower inhibiting power (Viigand et al. 2016). However, as in living yeast cells, glucose released from di- and trisaccharides is metabolized further; the in vivo inhibitory effect of glucose is probably lower than that recorded in vitro.

From yeast α-glucosidases, a three-dimensional structure has been resolved only for the S. cerevisiae isomaltase 1 (IMA1; PDB ID 3AJ7 and 3A4A). Structures of the IMA1 variants in complex with sugar ligands (maltose, isomaltose) have uncovered the amino acids bordering the active site (Y158, V216, G217, S218, L219, M278, Q279, D307, E411) with Val216 being crucial for selective binding of the substrate (Yamamoto et al. 2010, 2011; Deng et al. 2014). These nine positions vary between the maltases, isomaltases, and maltase-isomaltases (Voordeckers et al. 2012), and amino acids residing at these positions have been used as a signature in phylogenetic and substrate specificity analyses of α-glucosidases (Viigand et al. 2016, 2018; Voordeckers et al. 2012).

Table 1.2 illustrates signature amino acid patterns of α-glucosidases with known substrate specificity. α-Glucosidases hydrolyzing the α-1,6-glycosidic linkage have a Val residue at position equivalent to Val216 of IMA1 – a residue next to catalytic nucleophile Asp215. The corresponding residue of α-glucosidases that are able to hydrolyze maltose-like substrates is either Thr (Yamamoto et al. 2010) or in some cases Ala (Viigand et al. 2018; Tsujimoto et al. 2007). Respective residues are shown in bold in Table 1.2.

Table 1.2 Signature amino acids of α-glucosidases with known substrate specificity (Viigand et al. 2018)
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If Val216 of the S. cerevisiae IMA1 was replaced with a Thr; the enzyme gained the ability to hydrolyze maltose (Yamamoto et al. 2004). The O. polymorpha MAL1 has a Thr at a respective position (Table 1.2). Substitution of Thr200 with a Val drastically reduced the hydrolysis of maltose-like substrates by the O. polymorpha MAL1, whereas hydrolysis of isomaltose-like sugars was not affected (Fig. 1.6). Thus, it was concluded that Thr200 is required for efficient hydrolysis of maltose and maltose-like sugars. Indeed, the mutant Thr200Val became similar to isomaltases (Fig. 1.6).

Fig. 1.6
figure 6

According to catalytic efficiency kcat/Km (mM/min), a Thr200Val mutant of the O. polymorpha MAL1 protein is similar to an isomaltase. Compiled using data from Table 2 of (Viigand et al. 2016)

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1.4.4 The MAL1 of O. polymorpha Is Ancient-Like

Voordeckers et al. (Voordeckers et al. 2012) raised a hypothesis suggesting that modern maltases and isomaltases as those present in S. cerevisiae have evolved from a common promiscuous ancestor. The protein sequence of the ancestral protein (ancMalS) was predicted in silico, resurrected by heterologous synthesis in E. coli, and studied for enzymatic properties. Though the ancMalS was primarily active on maltose-like substrates, it also had a minor activity on isomaltose-like sugars (Voordeckers et al. 2012). The present-day α-glucosidases of S. cerevisiae preferentially hydrolyze either isomaltose-like sugars (IMA1, IMA2, and IMA5) or maltose-like sugars (MAL12, MAL32, MAL62) (Voordeckers et al. 2012); see Fig. 1.7 for MAL12 and IMA1).

Fig. 1.7
figure 7

Catalytic efficiencies, kcat/Km (mM/min), of the O. polymorpha (Op) maltase-isomaltase MAL1, ancestral maltase ancMALS, maltase MAL12 of S. cerevisiae (Sc), and isomaltase IMA1 of Sc. The signature amino acids of the enzymes (see Table 1.2) are presented in parentheses with the amino acid equivalent to Val216 of IMA1 shown underlined (Viigand et al. 2016)

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Intriguingly, according to substrate specificity and the signature amino acids, the O. polymorpha MAL1 is highly similar to ancMALS (Table 1.2 and Fig. 1.7). Even though it has been claimed that both maltase and isomaltase activities cannot be fully optimized in a single enzyme (Voordeckers et al. 2012), the MAL1 properties confirm that it is still possible (Fig. 1.7).

When studying evolutionary origin of α-glucosidases, Gabriško (Gabriško 2013) noted close relatedness of fungal and bacterial enzymes suggesting bacterial ancestry of fungal α-glucosidases. In agreement with this suggestion, the maltase Mal1 of an “ancient” yeast S. pombe and a bacterial maltase (from B. stearothermophilus) both have an Ala and Ile at positions corresponding to respective amino acids Val216 and Gly217 of the S. cerevisiae IMA1 protein. The S. cerevisiae maltases have Thr and Ala at respective positions (Table 1.3). Interestingly, the MAL1 gene of O. polymorpha has also a property of a bacterial gene – its promoter region is perfectly recognized in a bacterium Escherichia coli as it possesses two pairs of sigma 70-like sequences (Alamäe et al. 2003).

1.5 Regulation of the MAL Genes by Carbon Sources in O. polymorpha

1.5.1 General Features

Maltase synthesis in O. polymorpha is regulated by carbon sources in the growth medium: maltose and sucrose act as strong inducers, glucose as a repressor, and glycerol and ethanol allow moderate derepression (Alamäe and Liiv 1998). If O. polymorpha is grown on maltose or sucrose in the presence of a high concentration (2%) of glucose or fructose, induction of maltase is prevented. Interestingly, maltase activity is slightly repressed even during growth on maltose or sucrose if these substrates are provided at a high (2%) concentration. Therefore, glucose and fructose arising from the hydrolysis of maltose and sucrose in the cell most probably have some negative effect on the expression of MAL genes (Suppi et al. 2013).

Reporter gene assay of the bidirectional MAL1-MAL2 promoter showed co-regulated expression in both directions, repression by glucose and induction by maltose, whereas the basal expression was higher in the direction of the permease gene (Viigand et al. 2005). It seems reasonable, because the permease activity is first required to provide intracellular maltose that is needed for induction of the MAL genes (Viigand and Alamäe 2007). Induction of the O. polymorpha MAL1-MAL2 promoter by maltose (and sucrose) was stronger in the direction of maltase gene (Viigand and Alamäe 2007), and induced strength of the MAL1 promoter was shown to constitute up to 70% of that of the MOX promoter (Alamäe et al. 2003). This knowledge can be used in biotechnological applications. The MAL1 promoter has already been successfully used to overexpress and purify a biotechnologically relevant levansucrase protein from E. coli (Visnapuu et al. 2008).

1.5.2 Phosphorylated and Unphosphorylated Hexoses as Regulatory Signals

S. cerevisiae has three hexose kinases: hexokinase PI (HXK1) and PII (HXK2) that phosphorylate both fructose and glucose and a glucose-specific glucokinase (GLK1) (Zimmermann and Entian 1997). One of the hexokinases, the HXK2 of S. cerevisiae, has evolved to play a key role in establishing glucose repression (Gancedo 1998; Vega et al. 2016). Therefore, S. cerevisiae mutants defective in HXK2 lack glucose repression of many enzymes, including the maltase (Moreno and Herrero 2002; Zimmermann and Scheel 1977).

O. polymorpha has two hexose kinases: a hexokinase phosphorylating both glucose and fructose and a glucose-specific glucokinase (Kramarenko et al. 2000; Laht et al. 2002). Quite unexpectedly, hexokinase-negative mutants from chemical mutagenesis retained glucose repression of maltase synthesis losing only fructose repression (Kramarenko et al. 2000). Further assay of respective gene disruption mutants (Suppi et al. 2013) confirmed that in contrast to S. cerevisiae, hexokinase has no specific role in establishing glucose repression in O. polymorpha – instead the ability of the cell to phosphorylate hexoses was required for downregulation of sugar-repressed genes, including the MAL1. In this report, glucose-6-phosphate was proposed as a signaling metabolite to trigger sugar repression (Suppi et al. 2013).

Highly intriguing results regarding the regulation of MAL genes were obtained through the study of double kinase-negative mutants of O. polymorpha (Suppi et al. 2013). As these mutants have no enzymes for glucose and fructose phosphorylation, they cannot grow on sugars (glucose, fructose, sucrose, maltose, etc.), but they grow perfectly on glycerol, methanol, and ethanol. Though not growing on sugars, these mutants are capable of sugar transport (Suppi et al. 2013). Importantly, when double kinase-negative mutants were cultivated on glycerol in the presence of glucose or fructose, a very high maltase activity was recorded in the cells (Suppi et al. 2013). Thus, sugars considered to be repressors of the MAL genes promoted their activation in these mutants. Based on these results, a hypothesis was raised according to which presence of disaccharides in the environment is sensed by O. polymorpha inside the cell through a transient increase of “free” glucose and fructose (Fig. 1.8). This signal is captured by a mechanism that is yet unknown and results in initial activation of the MAL genes (Viigand 2018; Suppi et al. 2013).

Fig. 1.8
figure 8

Activation of the MAL genes in O. polymorpha by unphosphorylated glucose and fructose accumulating in the cell

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Indeed, O. polymorpha growing on non-sugar substrates (e.g., glycerol) is prepared to transport and hydrolyze maltose and sucrose – basal levels of both activities are detected in the cells (Viigand et al. 2005; Viigand and Alamäe 2007). In agreement with that, the maltase and maltose permease genes were, respectively, 89- and and 181-fold derepressed after the shift of O. polymorpha from glucose to methanol medium (van Zutphen et al. 2010). As the MAL1 protein has a very low affinity for disaccharides (Liiv et al. 2001; Viigand et al. 2016), its substrates should be concentrated in the cell by energy-dependent transport (Viigand and Alamäe 2007) to enable their efficient intracellular hydrolysis. Therefore, the hydrolysis reaction of disaccharides should produce a significant amount of free glucose (and fructose) inside the cell. As glucose- and fructose-phosphorylating activity in O. polymorpha cells growing on gluconeogenic carbon sources is low (Kramarenko et al. 2000; Parpinello et al. 1998); at least part of glucose and fructose should stay unphosphorylated and trigger initial activation of the MAL genes.

Following internalization and hydrolysis of the disaccharides, the hydrolysis products are phosphorylated and channeled to glycolysis. As accumulation of phosphorylated derivatives of glucose and fructose causes repression of the MAL genes, a fine balance should exist between intracellular concentrations of free and phosphorylated species of monosaccharides, glucose, and fructose (Fig. 1.9). We assume that glucose-6-phosphate (Glc6P) signals for both glucose and fructose repression of MAL genes (Fig. 1.9).

Fig. 1.9
figure 9

Glucose-6-phosphate as a proposed repressing metabolite in the expression of MAL genes in O. polymorpha

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Metabolic balance is also very important for S. cerevisiae growing on maltose. Therefore, if S. cerevisiae cannot manage intracellular metabolism of glucose resulting from maltose hydrolysis, excess of intracellular glucose will be exported by hexose transporters to prevent toxic effects exerted by high glucose concentration (Jansen et al. 2002).