Glycerol metabolism and transport in yeast and fungi: established knowledge and ambiguities

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1 Environmental Microbiology (2017) 19(3), doi: / Minireview Glycerol metabolism and transport in yeast and fungi: established knowledge and ambiguities Mathias Klein, 1 Steve Swinnen, 2 Johan M. Thevelein, 2,3,4 Elke Nevoigt 1 * 1 Department of Life Sciences and Chemistry, Jacobs University Bremen ggmbh, Campus Ring 1, Bremen, 28759, Germany. 2 GlobalYeast NV, Kasteelpark Arenberg 31, Leuven- Heverlee, 3001, Belgium. 3 Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven, Belgium. 4 Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, 3001 Heverlee-Leuven, Flanders, Belgium. Summary There is huge variability among yeasts with regard to their efficiency in utilizing glycerol as the sole source of carbon and energy. Certain species show growth rates with glycerol comparable to those reached with glucose as carbon source; others are virtually unable to utilize glycerol, especially in synthetic medium. Most of our current knowledge regarding glycerol uptake and catabolic pathways has been gained from studying laboratory strains of the model yeast Saccharomyces cerevisiae. The growth of these strains on glycerol is dependent on the presence of medium supplements such as amino acids and nucleobases. In contrast, there is only fragmentary knowledge about S. cerevisiae isolates able to grow in synthetic glycerol medium without such supplements as well as about growth of non- Saccharomyces yeast species on glycerol. Thus, more research is required to understand why certain strains and species show superior growth performance on glycerol compared with common Received 6 September, 2016; accepted 16 November, *For correspondence. e.nevoigt@jacobs-university.de; Tel ; Fax S. cerevisiae laboratory strains. This mini-review summarizes what is known so far about the gene products and pathways involved in glycerol metabolism and transport in yeast and fungi as well as the regulation of these processes. Introduction Glycerol is an organic compound that is ubiquitous in nature. It is therefore not surprising that organisms have developed ways to use this substance as a source of carbon and energy. Although it cannot be excluded that trace amounts of glycerol in the environment could be of geochemical origin (John Hallsworth, Personal Communication), glycerol from biotic origin likely accounts for the largest fraction as it forms the backbone of phospholipids and triacylglycerols, which are common constituents of cell membranes and widespread storage lipids respectively. Glycerol can be released from these constituents into the environment through enzymatic degradation by the action of microbial lipases. In modern human society, glycerol is also generated in industrial processes such as the saponification and transesterification of animal and vegetable fats and oils in soap and biodiesel production respectively. In fact, huge amounts of glycerol have been generated in recent years due to the immense growth of the biodiesel industry (Mattam et al., 2013). Another biotic process of glycerol generation is its metabolic formation and excretion by certain (micro)organisms. Among them, mainly yeasts have been reported to produce relatively high quantities of glycerol and, in the past, certain species have been used for the biotechnological production of glycerol (Wang et al., 2001). The main reason why cells produce and accumulate glycerol intracellularly is its protective properties against stress, particularly hyperosmotic and thermal stress. Moreover, the electron-dependent formation of glycerol from the central metabolic intermediate dihydroxyacetone phosphate (DHAP) might also serve as a redox sink when regeneration of cytosolic NAD 1 from VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

2 Glycerol catabolism in yeast 879 NADH through electron transport to oxygen via the respiratory chain is insufficient. These two functions of glycerol formation have been very well studied in the yeast S. cerevisiae, and summarized in several reviews (Nevoigt and Stahl, 1997; Bakker et al., 2001; Hohmann, 2002; 2015). Notably, the intracellular glycerol concentration in certain fungi and algae can reach extremely high levels, up to 7 8 M. Such a high intracellular glycerol concentration is a determinant of vigour and/or virulence of insect- and plant-pathogenic fungi (Hallsworth and Magan, 1995; De Jong et al., 1997). In addition, the insect haemolymph, in which entomopathogenic fungi proliferate, can also contain glycerol at molar concentrations (Sformo et al., 2010). It has been demonstrated that such high glycerol concentrations might act as a stress factor in yeasts and fungi, particularly at concentrations higher than 3 4 M (Cray et al., 2015; Stevenson et al., 2016). The ability to catabolize environmentally available glycerol as a carbon and energy source is widespread from archeabacteria (Falb et al., 2008) to human cells (Hibuse et al., 2006). This minireview deals with glycerol metabolism in fungal microorganisms with a particular focus on the catabolism of glycerol in yeasts. Our interest in this topic has been driven mainly by the goal of exploiting glycerol as a carbon source in biotechnological applications, in particular to valorize the large excess of glycerol from biodiesel production. However, there are many experimental findings worth to be investigated in more detail from a fundamental point of view, particularly with regard to the molecular basis of metabolic diversity. Glycerol utilization by yeasts: high inter- and intraspecies diversity It is clear from the literature that different yeast species, as well as different strains from the same species, show high diversity with regard to growth on glycerol as the sole source of carbon (Kurtzman et al., 2011). However, some of the reported differences might be caused by the growth medium used, instead of strain differences, as already elaborated by Vasiliadis et al. (1987). In fact, it has been shown (at least for S. cerevisiae) that the addition of amino acids, nucleobases or complex supplements such as peptone or yeast extract positively affects the capacity to grow on glycerol (Merico et al., 2011; Swinnen et al., 2013). Indeed, almost all remaining previous studies with S. cerevisiae regarding growth on glycerol were conducted with supplemented media. As the laboratory strains used were for the most part auxotrophic, the supplementation with amino acids and nucleobases could not be circumvented. The ambiguity of the previously published data regarding the quantitative performance of S. cerevisiae strains for growth on glycerol encouraged us to screen 52 different isolates including commonly used laboratory and industrial strains as well as natural isolates in synthetic glycerol medium, thereby explicitly avoiding the addition of any of the above-mentioned supplements (Swinnen et al., 2013). The observed maximum specific growth rates (l max ) and lag phases (after shift from glucose to glycerol) confirmed the significant intra-species diversity with regard to glycerol utilization. The strain with the fastest biomass accumulation (CBS 6412, combining a l max of 0.10 h 21 and a relatively short lag phase of 27 h) was selected for thorough genetic analysis. A haploid meiotic segregant of this isolate (CBS A) with a l max of 0.13 h 21 was used to unravel the (poly)genic basis of its better glycerol growth capacity in comparison with the glycerol-growth defective, well-established laboratory S. cerevisiae strain CEN.PK113-1A by genetic mapping (Swinnen et al., 2016). Three causative genetic determinants (UBR2 CBS, SSK1 CBS and GUT1 CBS ) were identified. Among these three genes, allele swapping of UBR2 in the genetic background of CEN.PK113-1A had the largest impact, establishing growth on glycerol in this strain with a l max of 0.05 h 21. Swapping of all three superior alleles in the strain CEN.PK113-1A resulted in a l max of 0.08 h 21. While the function of GUT1 with regard to glycerol utilization is obvious (GUT1 encodes a glycerol kinase), the relevance of UBR2 and SSK1 remains unknown. UBR2 encodes a ubiquitin ligase that functions in the ubiquitin proteasome system of S. cerevisiae (Finley et al., 2012). SSK1 encodes a protein that is part of a two-component system that senses hyperosmotic stress and transduces the signal to the Hog1 MAPK cascade to increase intracellular glycerol levels (Maeda et al., 1994). Further studies are necessary to unravel the action mechanism of the superior alleles in improving growth on glycerol. Notably, two independent studies have reported that wild-type S. cerevisiae strains not able to grow in synthetic glycerol medium, can be evolved relatively easily by repeated batch cultivations to obtain growth rates on glycerol of about 0.2 h 21 (Ochoa-Estopier et al., 2010; Merico et al., 2011). These results underscore the inherent potential of S. cerevisiae to utilize glycerol more efficiently. The exact mutations underlying the improved growth capacity of the evolved strains have recently been identified (Ho et al.; submitted). The results confirmed our above-mentioned finding that UBR2 and GUT1 are the most important targets for improving the capacity of the strain CEN.PK113-1A for growth on glycerol. The latter study identified GUT1 alleles (when expressed in CEN.PK113-1A and combined with UBR2 allele swapping) that resulted in an even higher growth rate than the GUT1 allele previously identified in the strain CBS A. VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,

3 880 M. Klein et al. Literature surveys for studies on glycerol utilization in different yeast species have revealed that there are not much comparative data available. To the best of our knowledge, only one comprehensive study of 42 yeast species in synthetic glycerol medium has been performed (Lages et al., 1999). In this work, the yeasts Cyberlindnera jadinii (formerly known as Candida utilis and later as Pichia jadinii) and Pichia anomala showed the highest l max on glycerol of 0.32 and 0.29 h 21 respectively. In contrast, the S. cerevisiae strain analysed by these authors showed a l max of 0.11 h 21. Unfortunately, this study did not include yeast species that are favoured in current industrial applications employing glycerol-based cultivation media such as Yarrowia (Candida) lipolytica, Komagataella (Pichia) pastoris and Pachysolen tannophilus. For these three species, growth rates on glycerol in the range of h 21 have been reported in the literature (Papanikolaou et al., 2002; Mattanovich et al., 2009; Liu et al., 2012). In order to directly compare these yeast species to each other and to S. cerevisiae, we recently performed a growth analysis in synthetic medium containing 6% (v/v) glycerol. One representative strain of Y. lipolytica, C. jadinii, P. tannophilus and K. pastoris was tested, and the values for l max observed under these conditions were 0.50, 0.42, 0.27 and 0.20 h 21 respectively (Klein et al., 2016b). Under the same conditions, the best performing wild-type isolates of the species S. cerevisiae showed growth rates on glycerol of up to 0.15 h 21 (Swinnen et al., 2013). One could assume that the ability of efficient glycerol utilization by certain yeast species is connected to their prevalence in habitats where significant quantities of glycerol are found. Such a connection is obvious for oleaginous yeasts such as Y. lipolytica which thrive in environments rich in lipids. However, up to now there has not been any dedicated work investigating this putative correlation in a comprehensive way for yeast and fungal species. In addition to its release in fat and lipid breakdown, glycerol can be produced in significant amounts by certain yeasts and fungi for osmoregulation and redox balancing, as already mentioned in the introduction. Glycerol of such microbial origin can, in theory, be re-utilized by the glycerol-producer itself or, after excretion, by other microorganisms present in the respective niche. We will discuss anabolism and catabolism of glycerol in S. cerevisiae in this context. It is well known that S. cerevisiae outperforms any other microorganism in sugar-rich media. However, such environments rich in sugar, as found for instance in damaged fruits, are rare in nature and, moreover, not available all year in nontropical climates. The question on the identity of the natural niches of S. cerevisiae is still occupying researchers intensively, as comprehensively summarized by Goddard and Greig (2015). Still, when S. cerevisiae gets access to sugar-rich environments, it will overgrow other competing microorganisms due to the fact that it liberates the energy from sugar much faster via its fermentative metabolism caused by the strong-crabtree effect (Pfeiffer et al., 2001). In addition, the rapid accumulation of ethanol inhibits the growth of other microorganisms present in the same niche. Hence, yeast species with a strong Crabtree effect, such as S. cerevisiae, may not have developed or may have lost during evolution the capability of efficiently utilizing glycerol. In fact, potential competitors for the consumption of the fermentation products ethanol and glycerol during the post-diauxic growth phase have already been outcompeted during the sugar-consuming phase. This supports the so-called make-accumulate-consume strategy of S. cerevisiae when growing in sugar-rich environments (Piskur et al., 2006). Glycerol catabolic pathways Similar to bacteria, two pathways for the dissimilation of glycerol have been reported in yeasts (Fig. 1A). The phosphorylative glycerol catabolic pathway, in which L-glycerol 3-phosphate (G3P) is formed as the intermediate, is widespread among fungal microorganisms (Fig. 1Ai). This pathway, referred to here as the catabolic G3P pathway, involves a glycerol kinase (GK) and an FAD-dependent glycerol 3-phosphate dehydrogenase located at the outer surface of the inner mitochondrial membrane (FAD-dependent mg3pdh). The latter enzyme directly transfers the electrons via FADH 2 to the respiratory chain. The second glycerol catabolic pathway, referred to here as the catabolic DHA pathway, starts with the oxidation of glycerol to dihydroxyacetone (DHA) via an NAD 1 -dependent glycerol dehydrogenase (GDH; EC ; Table 1). DHA is subsequently phosphorylated to DHAP via DHA kinase (DAK) (Fig. 1Aii). In order to understand the history of allocating a particular glycerol catabolic pathway to a certain yeast species or filamentous fungus, it has to be noted that in early studies conclusions were solely based on the measured in vitro activities of the pathways key enzymes. The most comprehensive investigations have been delivered by Babel and Hoffmann (1982) and Tani and Yamada (1987). Based on measuring GK as an indicator for the G3P pathway and GDH (NAD 1 -dependent) for the DHA pathway, Tani and Yamada (1987) divided the yeast species investigated into three groups. The first group was assumed to exclusively use the G3P pathway (e.g., Candida boidinii), the second group only the DHA pathway (e.g., Hansenula ofunaensis), and the third group both pathways (e.g., Candida valida). Although these early conclusions may VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,

4 Glycerol catabolism in yeast 881 Fig. 1. Proposed pathways for glycerol catabolism (A) and anabolism (B) in yeasts via (i) L-glycerol 3-phosphate (G3P), (ii) dihydroxyacetone (DHA) and (iii) glyceraldehyde (GA) as intermediate respectively. When known, the names of the S. cerevisiae genes allocated to the respective enzyme activities are indicated in italics. The asterisk (*) indicates those enzymes whose involvement in the pathway has been confirmed by analysing deletion mutants of the respective genes in at least one fungal organism. Abbreviation: glycerol int, intracellular glycerol. VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,

5 882 M. Klein et al. Table 1. Glycerol dehydrogenase activities detected in yeast and fungi EC number according to BRENDA Reaction predominantly catalyzed ph used for in vitro enzyme assay Organism and gene name (if known) References Experimental basis for the enzyme s physiological role DHA 1 NADH $ Glycerol 1 NAD (glycerol oxidation) 6.0 (DHA reduction) O. parapolymorpha DL-1 gdh H. ofunaensis S. pombe gld1 Yamada-Onodera et al. (2002) Yamada-Onodera et al. (2006) Matsuzawa et al. (2010) S. pombe gld1 deletion strain does not consume glycerol in the presence of other carbon sources (Matsuzawa et al., 2010) DHA 1 NADPH $ Glycerol 1 NADP (glycerol oxidation) (DHA reduction) S. pombe A. niger A. nidulans gldb A. oryzae H. jecorina (T. reesii) gld2 Marshall et al. (1989) Schuurink et al. (1990) De Vries et al. (2003) Ruijter et al. (2004) Liepins et al. (2006) A. nidulans gldb disruptant showed strongly reduced glycerol accumulation during osmostress (De Vries et al., 2003) / * D/L-Glyceraldehyde 1 NADPH $ Glycerol 1 NADP 1 Recommended name: D/L-glyceraldehyde reductase Glycerol oxidation The activity in the oxidizing reaction with glycerol as substrate was below the detection limit (Liepins et al., 2006) D/L-glyceraldehyde reduction N. crassa H. jecorina (T. reesii) gld1 A. niger gaad Viswanath-Reddy et al. (1977) Liepins et al. (2006) Martens-Uzunova and Schaap (2009) Assumed function in galacturonic acid catabolism due to transcriptional activation of A. niger gaad by galacturonic acid (Martens- Uzunova and Schaap, 2009) *A rationale behind having two different entries regarding this enzyme activity in BRENDA ( has not become obvious to the authors of this minireview. VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,

6 Glycerol catabolism in yeast 883 correctly reflect the glycerol catabolic pathways that are active in vivo, the sole measurement of a single enzymatic activity (or the presence of a homologous gene) is, in principle, not sufficient to assess the functionality of a metabolic pathway. More comprehensive evidence can be delivered by showing that the enzymatic activity as well as the metabolic activity of glycerol production or the capacity to grow on glycerol is reduced, abolished or improved, by mutating, deleting or overexpressing, respectively, specific pathway gene(s). In general, studies on the pathways of glycerol catabolism using mutant strains have been conducted extensively in the model yeast S. cerevisiae, but are rare in other yeast species or filamentous fungi. Such studies with regard to the G3P pathway showed that the deletion of either GUT1 (encoding GK) or GUT2 (encoding FAD-dependent mg3pdh) in different S. cerevisiae strains led to the complete abolishment of growth on glycerol (Sprague and Cronan, 1977; Pavlik et al., 1993; Swinnen et al., 2013), strongly suggesting that this pathway is the main route for glycerol utilization in S. cerevisiae. These experimental findings confirmed (at least for S. cerevisiae) the assumption of Gancedo et al. (1968) that the G3P pathway is used in C. utilis (nowadays classified as C. jadinii) and S. cerevisiae based on enzyme activity measurements in vitro. Several authors have stated that S. cerevisiae might also use the DHA pathway (Fig. 1Aii) for glycerol catabolism. The possibility for a catabolic DHA pathway in this species has been raised by the discovery of significant DAK activity and by the identification of the respective genes (DAK1 and DAK2) in S. cerevisiae (Norbeck and Blomberg, 1997; Molin et al., 2003). However, no in vitro NAD 1 -dependent GDH activity could ever be measured in cell extracts of this organism although attempts have been made by several authors (Norbeck and Blomberg, 1997; Ford and Ellis, 2002; Nguyen and Nevoigt, 2009). The latter results make the existence of a functional endogenous glycerol catabolic DHA pathway in S. cerevisiae very unlikely. Still, S. cerevisiae expresses proteins such as Gcy1 and Ypr1 which are homologous to fungal proteins with NADP 1 -dependent GDH activity of the type EC / (Table 1). However, the contribution of this enzyme class in a glycerol catabolic pathway with DHA as an intermediate is very unlikely due to its substrate specificity, which will be further scrutinized in the section The dubious endogenous DHA pathway in S. cerevisiae. With regard to the DHA pathway (Fig. 1Aii), there is only one single study providing experimental evidence that this pathway would indeed be used for glycerol utilization in yeast (Matsuzawa et al., 2010). The studied yeast species was Schizosaccharomyces pombe and the strain used in this study did notably not grow in media containing glycerol as the sole carbon source (at least in the medium and conditions used) and glycerol assimilation only occurred in the presence of other carbon sources, such as galactose and ethanol. In spite of this, the authors identified the gene gld1 whose product is homologous to bacterial GDH enzymes and whose deletion caused a reduction in NAD 1 -dependent GDH activity and prevented glycerol assimilation. An additional pathway proposed for glycerol catabolism with D-glyceraldehyde (GA) as the intermediate (here referred to as the catabolic GA pathway ) is shown in Fig. 1Aiii. In the proposed pathway, glycerol is first oxidized to GA by an NADP 1 -dependent GDH (EC or ; Table 1). Subsequently, two different pathways have been proposed through which GA could be channeled into central carbon metabolism (Fig. 1Aiii). In the first pathway, GA is phosphorylated to glyceraldehyde 3-phosphate by a glyceraldehyde kinase, while in the second pathway, GA is converted into D-glycerate via the action of an aldehyde dehydrogenase. D-glycerate can subsequently be phosphorylated by a glycerate 3-kinase. The existence of the GA pathway (including the two sub-pathways downstream of GA) in the filamentous fungus N. crassa has been suggested by Tom et al. (1978), who determined the activities of eight enzymes possibly involved in glycerol metabolism. In crude cell extracts of a wild-type strain and two mutant strains with improved growth on glycerol, a low but significant level of glyceraldehyde kinase activity was detected in the mutants, but not in the wild type. Moreover, the level of glycerate 3-kinase was about twofold higher in the mutants compared with the wild type. As glycerol kinase activity is also detectable in N. crassa indicating the presence of the classical G3P pathway (Viswanath-Reddy et al., 1977), it has been suggested that the catabolic GA pathway, including the two sub-pathways for the second part, might represent another route for glycerol catabolism in this organism. Although the GA catabolic pathway has not been described in yeasts so far, homologs of the enzyme catalyzing the first step in this pathway have been reported in S. cerevisiae (Gcy1 and Ypr1) as already mentioned above. For the sake of completeness, it has to be mentioned that another enzyme activity converting glycerol to glyceraldehyde has been described in the filamentous fungi Aspergillus japonicus (Uwajima et al., 1984) and Penicillium sp. (Lin et al., 1996). This enzyme catalyzes the oxidation of glycerol with the consumption of oxygen to form glyceraldehyde and hydrogen peroxide. Whether this enzyme activity (preliminary BRENDA-supplied EC number B4) is essential for glycerol catabolism in these organisms has not been investigated yet. VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,

7 884 M. Klein et al. Glycerol anabolic pathways The best-studied pathway for the production of glycerol from other carbon sources starts with the NADHdependent reduction of glycolytic DHAP to G3P by a cytosolic glycerol 3-phosphate dehydrogenase (cg3pdh), after which G3P is dephosphorylated to glycerol by a glycerol 3-phosphatase (GPP) (Fig. 1Bi). This anabolic G3P pathway is the major (or exclusive) pathway for glycerol production in S. cerevisiae during osmoregulation and anaerobic redox balancing (Nevoigt and Stahl, 1997). In S. cerevisiae, cg3pdh activity is encoded by the two isogenes GPD1 and GPD2, while GPP activity is encoded by the isogenes GPP1 and GPP2. Genes encoding NAD 1 -dependent cg3pdh have also been characterized in several other yeast species and filamentous fungi, such as Candida magnoliae (Lee et al., 2008), Candida glycerinogenes (Chen et al., 2008), Debaryomyces hansenii (Thome, 2004) and A. nidulans (Fillinger et al., 2001). In addition, cg3pdh activity using NADPH instead of NADH as the cofactor (EC ) has been identified in certain yeast species, such as Candida versatilis (Watanabe et al., 2008). A second pathway for glycerol production using DHA as an intermediate has been proposed in filamentous fungi (referred to here as the anabolic DHA pathway, Fig. 1Bii). The first step in this pathway is assumed to be catalyzed by a so far uncharacterized enzyme that dephosphorylates DHAP to DHA (see below). The second step is catalyzed by an NADP 1 -dependent GDH of the type EC (Table 1). In contrast to the above-mentioned dehydrogenases belonging to EC or , enzymes from EC are able to reduce DHA in vitro but have only very low activity (if at all) with glyceraldehyde. This anabolic DHA pathway has been postulated based on the fact that NADPdependent GDH activities converting DHA to glycerol (EC ) have been identified in S. pombe (Marshall et al., 1989), A. nidulans (Redkar et al., 1995; De Vries et al., 2003), A. niger (Schuurink et al., 1990), A. oryzae (Ruijter et al., 2004) and H. jecorina (Liepins et al., 2006) as also summarized in Table 1. To our knowledge, studies using mutant strains for allocation of this anabolic DHA pathway have only been conducted in A. nidulans (Fillinger et al., 2001; De Vries et al., 2003). These studies suggested that the anabolic DHA pathway is the major pathway responsible for glycerol accumulation during osmoregulation in this organism, while the alternative anabolic G3P pathway did not seem to play a significant role. In fact, the disruption of gldb encoding NADPH-dependent GDH led to significantly reduced intracellular glycerol levels and a severe growth defect in medium containing 1 M NaCl (De Vries et al., 2003), while the deletion of gfda encoding cg3pdh did not abolish glycerol biosynthesis under these conditions (Fillinger et al., 2001). The importance of gldb for osmoregulation in A. nidulans is also supported by the fact that its expression is strongly induced under conditions of hyper-osmotic shock. Moreover, the NADP 1 -dependent GDH in A. nidulans as well as its orthologs in other fungal species (Table 1) have indeed strong preference for the reduction of DHA under physiological conditions (i.e., at neutral ph). Although GDH activity has been clearly demonstrated, this anabolic DHA pathway also requires a DHAPase activity, which has to the best of our knowledge not yet been identified in any yeast or filamentous fungus. One might postulate that the glycerol 3-phosphatases Gpp1 and Gpp2 can also accept DHAP as a substrate. However, a genetic screen specifically developed for identifying genes whose gene products dephosphorylate DHAP in S. cerevisiae did not result in any candidate gene (E. Boles, Personal Communication). Up to now, there are only two organisms (Neisseria meningitidis and Plasmodium falciparum) listed in BRENDA ( that exhibit sugar phosphatase activity (EC ) with DHAP as substrate (Lee and Sowokinos, 1967; Guggisberg et al., 2014). In Fig. 1Biii, we propose a third theoretical pathway for glycerol formation. The existence of this pathway has not been indicated by any molecular data so far nor even been suggested in the literature. However, enzymes converting D-glyceraldehyde to glycerol (EC-number or ; Table 1) have been described in filamentous fungi such as N. crassa (Viswanath-Reddy et al., 1977), H. jecorina (Liepins et al., 2006) and A. niger (Martens-Uzunova and Schaap, 2009). As this enzymatic activity was induced in A. niger in the presence of galacturonic acid, a role of these enzymes in galacturonic acid catabolism has been suggested (Martens-Uzunova and Schaap, 2009). However, in principle the enzyme could also be involved in an anabolic GA pathway for glycerol formation provided that an enzyme is present with sugar phosphatase activity, catalyzing the first step of the pathway (EC converting glyceraldehyde 3-phosphate to GA). The dubious endogenous DHA pathway in S. cerevisiae The first considerations concerning the possible existence of a catabolic DHA pathway in S. cerevisiae date back to the study of Norbeck and Blomberg (1997). These authors analysed salt-induced changes in gene expression in S. cerevisiae at the proteome level and detected a significant increase in the abundance of Dak1 (EC ). Inspired by this finding, the authors attempted to identify the corresponding GDH but were VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,

8 Glycerol catabolism in yeast 885 unable to detect any in vitro GDH activity although different ph values, substrates (DHA and glycerol) and cofactors (NADH, NADPH, NAD 1 and NADP 1 ) were tested. At the time when Norbeck and Blomberg (1997) conducted their study, no protein sequence of an NAD 1 - dependent GDH was available, which would have allowed a search for genes encoding homologous proteins in the S. cerevisiae genomic DNA sequence that had just become publically available at that time. Instead, a search was conducted using a partial protein sequence of a commercially available NADP 1 -dependent GDH of A. niger (allocated to EC ). The authors identified the S. cerevisiae genes YPR1 and GCY1 as encoding proteins with a sequence showing the highest homology to the peptide fragment of the NADP 1 -dependent A. niger enzyme. The observation that Gcy1 expression is strongly increased upon osmostress prompted (Blomberg, 2000) to speculate that Gcy1 might function as a GDH for conversion of glycerol to DHA in a glycerol catabolic pathway. In addition, he proposed that increased activity of a catabolic DHA pathway during osmostress would lead to an ATP futile cycle that might help to avoid substrate accelerated cell death. However, so far no experimental proof has been provided supporting this hypothesis. The S. cerevisiae gene YPR1, of which the product shows 65% sequence similarity to that of the GCY1 gene, has been expressed in Escherichia coli to characterize the enzyme in more detail. It was demonstrated that Ypr1 uses NADPH as a cofactor and preferably reduces D/L-glyceraldehyde to glycerol. The activity of Ypr1 using DHA as a substrate was about 100-fold lower. Ypr1 also oxidized glycerol, however, this activity was about 4,000-times lower than the activity in the reducing direction with D/L-glyceraldehyde as substrate (Ford and Ellis, 2002). The gene GCY1 has also been expressed in E. coli and the activity of the gene product tested with several substrates. D/L-glyceraldehyde was the substrate with the highest k cat and NADPH was shown to be the co-factor (Chang et al., 2007). These results suggest that neither Ypr1 nor Gcy1 significantly contribute to in vivo DHA formation from glycerol in S. cerevisiae. It has to be mentioned that Jung et al. (2012) have shown that, at least under microaerobic conditions, a small amount of DHA is accumulated in a dak1 dak2 double deletion strain of S. cerevisiae. This DHA accumulation further increased when GCY1 was overexpressed, indicating that Gcy1 has indeed a weak glycerol oxidizing activity. However, it cannot be excluded that the glycerol oxidation activity apparently present under these conditions is just a moonlighting activity that has become significant only because of the artificially imposed conditions (Dak1/2 abolishment/gcy1 overexpression) and that it is not relevant in wild-type cells. To our knowledge, the major physiological functions for Gcy1 and Ypr1 gene products in S. cerevisiae have not yet been identified. Although there does not seem to be convincing evidence for the existence of a native catabolic DHA pathway in S. cerevisiae, it is possible to engineer S. cerevisiae strains for growth on glycerol using exclusively a synthetic DHA pathway. We have shown recently that the native catabolic G3P pathway of S. cerevisiae can be replaced by an artificial catabolic DHA pathway (Klein et al., 2016a). This was achieved by simultaneous expression of the NAD 1 -dependent GDH from O. parapolymorpha (EC ; Table 1) and overexpression of the endogenous DAK1 gene in a gut1 deletion mutant in several S. cerevisiae genetic backgrounds. Surprisingly, this pathway replacement together with the expression of a heterologous glycerol facilitator (see section Glycerol transport mechanisms ) even abolished the growth deficit in synthetic glycerol medium of several of the S. cerevisiae strains used, indicating that the growth deficit may be due to insufficient activity or aberrant regulation of the native G3P pathway. Our results demonstrate that S. cerevisiae naturally contains all prerequisites to utilize glycerol via the DHA pathway (such as the ability to re-oxidize the surplus of cytosolic NAD 1 efficiently). Obviously, generation of highly efficient utilization pathways for glycerol has not been a priority during the evolutionary development of S. cerevisiae as already speculated in the section Glycerol utilization by yeasts: high inter- and intra-species diversity. Glycerol transport mechanisms In order to be metabolized, glycerol must first enter the cell. Among fungi, the molecular details of the glycerol uptake mechanisms have been studied in greatest detail in the yeast S. cerevisiae. Only a few studies have been conducted in non-saccharomyces yeasts and in filamentous fungi as elaborated below. The question of which type of transport proteins are responsible for glycerol uptake in S. cerevisiae during growth on glycerol has been a matter of dispute for a long time, as comprehensively summarized by Neves et al. (2004). In fact, passive and facilitated diffusion as well as active transport have all been considered in the last decades. We will summarize below the experimental findings in favour or against the different hypotheses, which culminated in the currently accepted model of active transport. It is now generally accepted that during growth of S. cerevisiae on glycerol the uptake of glycerol is solely driven by a glycerol/h 1 -symporter encoded by STL1 (Ferreira et al., 2005). The existence of such an active VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,

9 886 M. Klein et al. transport system in S. cerevisiae had already been predicted by Sutherland et al. (1997) based on the demonstration of simultaneous uptake of glycerol and protons accompanied by intracellular glycerol accumulation. Ferreira et al. (2005) finally revealed the crucial function of Stl1 by showing that the deletion of STL1 completely abolished active glycerol transport as well as growth on glycerol. These results were originally obtained in auxotrophic laboratory strains, but the same deletion in the wild-type S. cerevisiae isolate CBS A also resulted in inability to grow in synthetic glycerol medium (Swinnen et al., 2013). Similar active glycerol uptake systems using H 1 - but also Na 1 -symport have been described for halotolerant yeast species such as Debaryomyces hansenii (Lucas et al., 1990), Pichia sorbitophila (Lages and Lucas, 1995) and Zygosaccharomyces rouxii (Van Zyl et al., 1990). In these highly osmotolerant species, the active glycerol uptake has been studied mainly in the context of establishing and maintaining a glycerol gradient across the cytoplasmic membrane in the presence of high salt concentrations. It seems that uptake of glycerol in response to osmotic stress also occurs in S. cerevisiae since transcription of STL1 is transiently induced by osmotic shock during exponential growth in glucosebased media (Posas et al., 2000; Rep et al., 2000; Yale and Bohnert, 2001; Ferreira et al., 2005). However, this osmostress-induced active glycerol uptake seems to be replaced subsequently by an increase in cellular glycerol production (and its intracellular retention due to closing of the Fps1 channel preventing glycerol efflux) as Stl1 abundance and transport activity started to decrease gradually 1.5 h after the shift to hyperosmotic medium. Interestingly, downregulation of STL1 expression and degradation of Stl1 protein did not occur in a gpd1 gpd2 double deletion mutant (Ferreira et al., 2005). Such a strain cannot synthesize any glycerol and is therefore sensitive to hyperosmotic shock. However, growth in the presence of increased salt concentrations can be rescued in this strain by supplementation of the growth medium with small amounts of glycerol, which apparently allows the persisting Stl1 activity to compensate at least partially for the absent glycerol anabolic pathway. Prior to the demonstration that Stl1 is the main mediator of glycerol uptake in S. cerevisiae, GUP1 and its paralog GUP2 had been suggested to encode proteins responsible for glycerol active transport (Holst et al., 2000). GUP1 and GUP2 encode membrane proteins with multiple predicted transmembrane domains. These authors showed that the GUP1 gene product is essential for efficient growth in media containing glycerol as the sole carbon source and found that it also seems to play an important role in the alleviation of the osmotic-stress induced cell death of gpd1 gpd2 mutants. In fact, additional deletion of GUP1 in a gpd1 gpd2 deletion mutant compromises the rescuing effect of glycerol supplementation in hyperosmotic media suggesting a role of Gup1 in glycerol import (Holst et al., 2000). However, even before the glycerol/h 1 -symporter encoded by STL1 had been identified, studies in which the expression patterns of GUP1 and GUP2 were compared with glycerol uptake activity failed to support the concept that these genes encode glycerol transporters in S. cerevisiae (Oliveira and Lucas, 2004). While GUP1 and GUP2 expression was found to be constitutive, glycerol uptake activity is clearly affected by carbon catabolite repression (see below) as well as during growth under salt stress. Moreover, mutants carrying deletions of gup1 and gup2 still show active glycerol uptake by glycerol/ H 1 symport under salt stress (Neves et al., 2004). In addition, deletion of GUP1 in the wild-type isolate CBS A did not impair the strain s natural ability to grow in synthetic glycerol medium (our unpublished results). Therefore, the actual function of Gup1 and Gup2 in glycerol uptake remains to be resolved. As GUP1 was later shown to encode an O-acyltransferase involved in remodelling of GPI anchors (Bosson et al., 2006) the gene product might affect glycerol uptake indirectly. It is well-known that S. cerevisiae expresses a glycerol facilitator, Fps1, a member of the major intrinsic protein (MIP) family of channel proteins (Luyten et al., 1995). This protein shows high sequence similarity to the previously described and extensively studied glycerol facilitator GlpF from E. coli (Heller et al., 1980; Sweet et al., 1990; Maurel et al., 1994). Notably, channel-mediated diffusion via GlpF is the sole glycerol uptake system in E. coli. The constitutively expressed glycerol facilitator, Fps1, in S. cerevisiae was shown to control glycerol efflux as a function of medium osmolarity and was initially assumed also to contribute significantly to glycerol uptake in this organism. Early experimental studies demonstrated a saturable glycerol uptake component and thus seemed to confirm this hypothesis (Luyten et al., 1995; Sutherland et al., 1997). However, the results later turned out to be caused by an artefact since the respective studies used glucose-grown S. cerevisiae cells or cells harvested during the diauxic shift (Oliveira et al., 2003). Such cells contain considerable Gut1 activity (Grauslund et al., 1999; Oliveira et al., 2003) that interfered with the experimental set-up causing the saturable glycerol uptake kinetics. Indeed, Oliveira et al. (2003) demonstrated that the latter was abolished by deletion of GUT1. These results are also consistent with the finding of Luyten et al. (1995) that growth on glycerol as sole carbon source was unaffected in fps1d mutants. Further studies confirmed that the main function of Fps1 in S. cerevisiae is the control of glycerol export VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,

10 Glycerol catabolism in yeast 887 during osmoregulation rather than glycerol uptake (Tamas et al., 1999). Oliveira et al. (2003) even claimed that S. cerevisiae Fps1 should not be considered a glycerol facilitator with regard to glycerol uptake at all as it does not exhibit uptake properties (V max values) that would be in agreement with the definition of a facilitated diffusion type of transport. Oliveira et al. (2003) also questioned the hitherto generally accepted concept that glycerol is able to cross the lipid bilayer of the plasma membrane by passive diffusion solely driven by its concentration gradient (Gancedo et al., 1968; Heredia et al., 1968; Romano, 1986). Based on their experimental data, they hypothesized that the S. cerevisiae plasma membrane is almost impermeable to glycerol and that any detectable passive diffusion into the cell is actually mediated via the Fps1 channel. Transport components different from active transport can anyway only be very low since stl1 deletion mutants are unable to grow in synthetic glycerol medium as mentioned above. Theoretical considerations based on comparison of the glycerol olive oil/water partition coefficient (logp) with that of other molecules also support the view that glycerol is unlikely to show significant diffusion through biological membranes (Oliveira et al., 2003). Still, the same authors claim that some passive diffusion via the lipid bilayer cannot be completely excluded as cytoplasmic membranes are highly flexible and dynamic systems. As described in the section Glycerol utilization by yeasts: high inter-species diversity several nonconventional yeast species have been reported to show growth rates on glycerol significantly higher than that of S. cerevisiae. The molecular basis for the more efficient glycerol utilization in these yeast species as compared to S. cerevisiae is not clear, although Gancedo et al. (1968) provided indications that glycerol uptake might constitute a major limitation in S. cerevisiae. This study demonstrated that glycerol uptake (described as membrane permeability) in the S. cerevisiae strain studied was 10 5 fold lower as compared with that in C. jadinii. In several yeast genome sequencing projects, homologs of STL1 have been identified, such as in Y. lipolytica (Dujon et al., 2004), P. tannophilus as well as K. pastoris, the latter even possessing four distinct glycerol/h 1 -symporters (Mattanovich et al., 2009). Blast searches with FPS1 and genome sequence comparisons also resulted in the identification of several homologs in various yeast species and the generation of unrooted phylogenetic trees showed that many of them, such as Fps1 homologs in P. tannophilus, K. pastoris and Y. lipolytica, cluster in a branch separate from S. cerevisiae Fps1 (ScFps1) (Liu et al., 2013). In contrast to S. cerevisiae the contribution of Stl1 and Fps1 homologs to glycerol utilization in other yeast species have not yet been comprehensively studied. At least, Liu et al. (2013) showed that FPS2 from the P. tannophilus strain CBS 4044 (PtFPS2) was among the most strongly upregulated genes upon switch from glucose- to glycerolbased medium. Interestingly, expression of the predicted glycerol facilitator was able to suppress the growth defect on glycerol of a S. cerevisiae stl1 deletion mutant, which was in remarkable contrast to overexpression of the endogenous S. cerevisiae FPS1. These results imply different functions for the two partially conserved facilitator proteins with respect to glycerol transport (Liu et al., 2013). The apparently different physiological function of the ScFps1 and PtFps2 orthologs is also reflected at the molecular level. The overall sequence similarity between the two proteins is 32% but the sequence similarity is largely restricted to the core section of ScFps1 with its six putative trans-membrane domains (TMDs). ScFps1 has 669 amino acid residues, which makes it much longer than PtFps2 (323 residues). Tamas et al. (1999) demonstrated that the domains at the N- and C-terminus are important for controlling the specific ScFps1 function of osmoregulated glycerol export. More specifically, the N- terminus was found to control the closing of the channel thereby restricting glycerol efflux through ScFps1 during osmotic stress. This property would be dispensable in case the channel was solely used during glycerol assimilation. Overexpression in the S. cerevisiae strain CBS A of PtFPS2 and similar FPS1 homologs from different non-conventional yeast species (C. jadinii, P. pastoris and Y. lipolytica) with superior growth performance on glycerol compared with S. cerevisiae, resulted in a significantly improved l max from 0.13 h 21 (for wild-type CBS A) to 0.18 h 21 (for the engineered strains) (Klein et al., 2016b). The high growth rates were retained even after deletion of the endogenous STL1 demonstrating that the heterologous facilitators are able to fully replace the active transport system without loss of the superior growth capacity. Similarly, overexpression of FPS1 from C. jadinii (CjFPS1) in a reverseengineered S. cerevisiae strain of CEN.PK113-1A, carrying allele replacements for UBR2, GUT1 and SSK1 from CBS A, further improved the strain s growth performance on glycerol (Swinnen et al., 2016). However, the glycerol facilitators from the aforementioned non-conventional yeast species should also be analysed by functional studies in the original organisms in order to learn more about their actual contribution to glycerol uptake in these species. A more detailed analysis of the different transporters in the original species might provide more insight into the molecular basis of the superior glycerol uptake capability of many nonconventional yeast species in comparison to S. cerevisiae. In particular, the fact that many of these yeast species carry several STL1 and/or FPS1 isogenes and the VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,

11 888 M. Klein et al. different molecular structure of the Fps homologs (see above) stands in sharp contrast with the genetic constitution of S. cerevisiae in this respect. The improvement of growth on glycerol achieved by the replacement of S. cerevisiae s native glycerol/h 1 - symport system by a heterologous glycerol facilitator confirmed that glycerol uptake is a the rate-limiting step for growth on glycerol in this yeast species. However, many yeast species still show even higher growth rates on glycerol than the engineered S. cerevisiae variants implying that other factors still restrict more efficient glycerol utilization in S. cerevisiae. Such a ratecontrolling factor could for example reside in an inefficient glycerol catabolic pathway as elaborated in the section The dubious catabolic DHA pathway in S. cerevisiae. In any case, the alleviation of glycerol transport limitation now accomplished in S. cerevisiae paves the way for identification of further rate-controlling steps in glycerol utilization and ultimately for further improvement of the capacity of S. cerevisiae to grow on glycerol. Carbon source regulation of glycerol transport and catabolic pathways The preferred carbon source of many unicellular organisms is glucose and its presence often excludes the utilization of alternative carbon sources such as glycerol. This repressive effect often mediated by multiple interlinked regulatory interactions and signalling pathways is generally referred to as carbon catabolite repression. The shift to an alternative carbon source is characterized by a transition period with delayed cellular growth (designated as diauxic shift) and the underlying molecular remodelling has been most thoroughly investigated in the yeast S. cerevisiae. Being a Crabtree-positive yeast species (exhibiting fermentative metabolism even under aerobic conditions as soon as the glucose concentration exceeds a certain threshold concentration), S. cerevisiae naturally displays an extreme case of such a diauxic shift, when it switches from fermentative growth on glucose to full respiratory metabolism of the previously produced fermentation products, mainly ethanol and to a smaller extent glycerol. It is evident that such a drastic shift from utilizing a fermentable to a respiratory carbon source requires a dramatic reprogramming of the cellular machinery. In S. cerevisiae, the latter is mainly (but not exclusively) exerted at the transcriptional level as demonstrated by many studies of this phenomenon, including microarray-based transcriptome analyses at a global scale (DeRisi et al., 1997; Brauer et al., 2005; Roberts and Hudson, 2006). Evaluation of the respective data revealed many adaptations to respiratory growth that are common to both carbon sources glycerol and ethanol. For instance all three studies showed that the expression of genes encoding proteins related to mitochondrial function and energy metabolism as well as that of genes encoding enzymes involved in gluconeogenesis, the TCA and glyoxalate cycles, and in carbohydrate storage was highly up-regulated. Interestingly, the expression of genes encoding products associated with stress response was also found to be up-regulated. In contrast, the expression of genes encoding proteins required for biosynthesis, such as transcription by the RNA polymerases I and III, DNA replication and ribosome biogenesis and assembly, was significantly downregulated reflecting the generally lower growth rate of S. cerevisiae on respiratory carbon sources (DeRisi et al., 1997; Brauer et al., 2005; Roberts and Hudson, 2006). However, one has to emphasize that only Roberts and Hudson (2006) investigated the transcriptome of cells exponentially growing on either glycerol or ethanol, while the other two studies solely focused on analysing samples around the diauxic shift after glucose depletion. The direct comparison of the results allowed the identification of carbon source specific regulatory patterns (Roberts and Hudson, 2006). For example, abundance of STL1 transcripts encoding the glycerol/h 1 -symporter for glycerol uptake was strongly increased after glucose depletion and reached a level that was several hundredfold higher during continuous growth on glycerol as compared with glucose. Likewise, the GUT1 and GUT2 genes encoding the enzymes catalyzing the initial glycerol catabolic G3P pathway, showed a strong transcriptional up-regulation in glycerol-based medium. Surprisingly, derepression of these genes was also observed to a certain extent in medium containing ethanol as sole carbon source (Roberts and Hudson, 2006). Interestingly, the expression of GCY1 also showed a strong up-regulation in medium containing glycerol, but not ethanol, as sole source of carbon. As discussed in the section The dubious endogenous DHA pathway in S. cerevisiae, Gcy1 is a homolog of the fungal NADP 1 - dependent GDH but its real function in S. cerevisiae s physiology has remained rather unclear. With regard to the above-mentioned transcriptome studies, one has to keep in mind, that all three were performed in media containing complex medium components and that data for exponentially growing S. cerevisiae cells in synthetic media are still lacking. This evidently (at least for glycerol) can be attributed to the fact that (as elaborated above) commonly used laboratory strains of S. cerevisiae do not grow in synthetic glycerol medium at all. The molecular basis for carbon catabolite repression has been studied in great detail in S. cerevisiae, and the reader is referred to a number of excellent reviews in this field for more details (Sch uller, 2003; Turcotte et al., 2010; Kayikci and Nielsen, 2015). Although it is clear VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,