Environmental Microbiology (2017) 19(3), 878–893 doi:10.1111/1462-2920.

13617

Minireview

Glycerol metabolism and transport in yeast and fungi:

established knowledge and ambiguities

Mathias Klein,1 Steve Swinnen,2 S. cerevisiae laboratory strains. This mini-review

Johan M. Thevelein,2,3,4 Elke Nevoigt1* summarizes what is known so far about the gene
1
Department of Life Sciences and Chemistry, Jacobs products and pathways involved in glycerol
University Bremen gGmbH, Campus Ring 1, Bremen, metabolism and transport in yeast and fungi as well
28759, Germany. as the regulation of these processes.
2
GlobalYeast NV, Kasteelpark Arenberg 31, Leuven-
Heverlee, 3001, Belgium.
3
Laboratory of Molecular Cell Biology, Institute of Botany Introduction
and Microbiology, KU Leuven, Leuven, Belgium.
4
Glycerol is an organic compound that is ubiquitous in
Department of Molecular Microbiology, VIB,
nature. It is therefore not surprising that organisms have
Kasteelpark Arenberg 31, 3001 Heverlee-Leuven,
developed ways to use this substance as a source of
Flanders, Belgium.
carbon and energy. Although it cannot be excluded that
trace amounts of glycerol in the environment could be of
geochemical origin (John Hallsworth, Personal Commu-
nication), glycerol from biotic origin likely accounts for
the largest fraction as it forms the backbone of phospho-
Summary lipids and triacylglycerols, which are common constitu-
ents of cell membranes and widespread storage lipids
There is huge variability among yeasts with regard to
respectively. Glycerol can be released from these con-
their efficiency in utilizing glycerol as the sole source
stituents into the environment through enzymatic degra-
of carbon and energy. Certain species show growth
dation by the action of microbial lipases. In modern
rates with glycerol comparable to those reached with
human society, glycerol is also generated in industrial
glucose as carbon source; others are virtually unable
processes such as the saponification and transesterifica-
to utilize glycerol, especially in synthetic medium.
tion of animal and vegetable fats and oils in soap and
Most of our current knowledge regarding glycerol
biodiesel production respectively. In fact, huge amounts
uptake and catabolic pathways has been gained from
of glycerol have been generated in recent years due to
studying laboratory strains of the model yeast
the immense growth of the biodiesel industry (Mattam
Saccharomyces cerevisiae. The growth of these
et al., 2013).
strains on glycerol is dependent on the presence of
Another biotic process of glycerol generation is its
medium supplements such as amino acids and
metabolic formation and excretion by certain (micro)or-
nucleobases. In contrast, there is only fragmentary
ganisms. Among them, mainly yeasts have been
knowledge about S. cerevisiae isolates able to grow
reported to produce relatively high quantities of glycerol
in synthetic glycerol medium without such
and, in the past, certain species have been used for the
supplements as well as about growth of non-
biotechnological production of glycerol (Wang et al.,
Saccharomyces yeast species on glycerol. Thus,
2001). The main reason why cells produce and accumu-
more research is required to understand why certain
late glycerol intracellularly is its protective properties
strains and species show superior growth
against stress, particularly hyperosmotic and thermal
performance on glycerol compared with common
stress. Moreover, the electron-dependent formation of
glycerol from the central metabolic intermediate dihy-
Received 6 September, 2016; accepted 16 November, 2016. *For
correspondence. E-mail e.nevoigt@jacobs-university.de; Tel. 149- droxyacetone phosphate (DHAP) might also serve as a
421-2003541; Fax 149-421-2003249. redox sink when regeneration of cytosolic NAD1 from
C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.
V
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.

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 concen-
tration in certain fungi and algae can reach extremely
high levels, up to 7–8 M. Such a high intracellular glyc-
erol concentration is a determinant of vigour and/or viru-
lence 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 concentra-
tions (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 concen-
trations 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 inter-
est in this topic has been driven mainly by the goal of
exploiting glycerol as a carbon source in biotechnologi-
cal 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 intra-
species 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 supple-
ments 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 pre-
vious studies with S. cerevisiae regarding growth on
glycerol were conducted with supplemented media. As
the laboratory strains used were for the most part auxo-
trophic, the supplementation with amino acids and
nucleobases could not be circumvented. The ambiguity
of the previously published data regarding the quantita-
tive performance of S. cerevisiae strains for growth on
C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,
V
Environmental Microbiology, 19, 878–893
Glycerol catabolism in yeast 879
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
(lmax) and lag phases (after shift from glucose to glycer-
ol) confirmed the significant intra-species diversity with
regard to glycerol utilization. The strain with the fastest
biomass accumulation (CBS 6412, combining a lmax of
 0.10 h21 and a relatively short lag phase of  27 h)
was selected for thorough genetic analysis. A haploid
meiotic segregant of this isolate (CBS 6412-13A) with a
lmax of  0.13 h21 was used to unravel the (poly)genic
basis of its better glycerol growth capacity in comparison
with the glycerol-growth defective, well-established labo-
ratory S. cerevisiae strain CEN.PK113-1A by genetic
mapping (Swinnen et al., 2016). Three causative genetic
determinants (UBR2CBS, SSK1CBS and GUT1CBS) 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 lmax of  0.05 h21. Swapping of all three
superior alleles in the strain CEN.PK113-1A resulted in
a lmax of  0.08 h21. 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. cere-
visiae (Finley et al., 2012). SSK1 encodes a protein that
is part of a two-component system that senses hyperos-
motic 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 syn-
thetic glycerol medium, can be evolved relatively easily
by repeated batch cultivations to obtain growth rates on
glycerol of about 0.2 h21 (Ochoa-Estopier et al., 2010;
Merico et al., 2011). These results underscore the inher-
ent potential of S. cerevisiae to utilize glycerol more effi-
ciently. The exact mutations underlying the improved
growth capacity of the evolved strains have recently
been identified (Ho et al.; submitted). The results con-
firmed 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 glyc-
erol. 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 6412-13A.
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 per-
formed (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 lmax on glycerol of 0.32 and 0.29 h21
respectively. In contrast, the S. cerevisiae strain ana-
lysed by these authors showed a lmax of 0.11 h21.
Unfortunately, this study did not include yeast species
that are favoured in current industrial applications
employing glycerol-based cultivation media such as Yar-
rowia (Candida) lipolytica, Komagataella (Pichia) pasto-
ris and Pachysolen tannophilus. For these three
species, growth rates on glycerol in the range of 0.26–
0.30 h21 have been reported in the literature (Papaniko-
laou 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 lmax observed under these conditions were
0.50, 0.42, 0.27 and 0.20 h21 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 h21 (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 ole-
aginous yeasts such as Y. lipolytica which thrive in envi-
ronments 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 break-
down, glycerol can be produced in significant amounts
by certain yeasts and fungi for osmoregulation and
redox balancing, as already mentioned in the introduc-
tion. Glycerol of such microbial origin can, in theory, be
re-utilized by the glycerol-producer itself or, after excre-
tion, by other microorganisms present in the respective
niche. We will discuss anabolism and catabolism of glyc-
erol 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 non-
tropical climates. The question on the identity of the nat-
ural niches of S. cerevisiae is still occupying researchers
intensively, as comprehensively summarized by Goddard
C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,
V
and Greig (2015). Still, when S. cerevisiae gets access
to sugar-rich environments, it will overgrow other com-
peting 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 interme-
diate, 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 FADH2 to the
respiratory chain. The second glycerol catabolic path-
way, referred to here as the ‘catabolic DHA pathway’,
starts with the oxidation of glycerol to dihydroxyacetone
(DHA) via an NAD1-dependent glycerol dehydrogenase
(GDH; EC 1.1.1.6; Table 1). DHA is subsequently phos-
phorylated to DHAP via DHA kinase (DAK) (Fig. 1Aii).
In order to understand the history of allocating a par-
ticular 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 (NAD1-depen-
dent) 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 sec-
ond group only the DHA pathway (e.g., Hansenula ofu-
naensis), and the third group both pathways (e.g.,
Candida valida). Although these early conclusions may
Environmental Microbiology, 19, 878–893
 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 respec-
tive 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: glycerolint, intracellular glycerol.

C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,
V
Environmental Microbiology, 19, 878–893
 V
882 M. Klein et al.

Table 1. Glycerol dehydrogenase activities detected in yeast and fungi

EC number Reaction pH used for in vitro Organism and gene References Experimental basis
according to predominantly enzyme assay name (if known) for the enzyme’s
BRENDA catalyzed physiological role

1.1.1.6 DHA 1 NADH 7.5–10.0 O. parapolymorpha Yamada-Onodera S. pombe gld1 deletion strain does
$ (glycerol oxidation) DL-1 gdh et al. (2002) not consume glycerol in the
Glycerol 1 NAD1 6.0 H. ofunaensis Yamada-Onodera presence of other carbon sources
(DHA reduction) S. pombe gld1 et al. (2006) (Matsuzawa et al., 2010)
Matsuzawa
et al. (2010)

1.1.1.156 DHA 1 NADPH 9.0–10.0 S. pombe Marshall et al. (1989) A. nidulans gldB
$ (glycerol oxidation) A. niger Schuurink et al. (1990) disruptant showed strongly
Glycerol 1 NADP1 6.0–7.0 A. nidulans gldB De Vries et al. (2003) reduced glycerol accumulation
(DHA reduction) A. oryzae Ruijter et al. (2004) during osmostress (De Vries
H. jecorina Liepins et al. (2006) et al., 2003)
(T. reesii) gld2

1.1.1.372/ D/L-Glyceraldehyde 9.5–10.0 N. crassa Viswanath-Reddy Assumed function in galacturonic

1.1.1.72* 1 NADPH Glycerol oxidation H. jecorina et al. (1977) acid catabolism due to transcrip-
$ The activity in the oxidizing reaction (T. reesii) gld1 Liepins et al. (2006) tional activation of A. niger gaaD
Glycerol 1 NADP1 with glycerol as substrate was A. niger gaaD Martens-Uzunova and by galacturonic acid (Martens-
Recommended name: below the detection limit (Liepins Schaap (2009) Uzunova and Schaap, 2009)
D/L-glyceraldehyde et al., 2006).
reductase 5.6–7.0
D/L-glyceraldehyde reduction

*A rationale behind having two different entries regarding this enzyme activity in BRENDA (www.brenda-enzymes.org) has not become obvious to the authors of this minireview.

Environmental Microbiology, 19, 878–893

C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,

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 func-
tionality of a metabolic pathway. More comprehensive
evidence can be delivered by showing that the enzy-
matic activity as well as the metabolic activity of glycer-
ol 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. cer-
evisiae 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 catab-
olism. The possibility for a catabolic DHA pathway in
this species has been raised by the discovery of signifi-
cant DAK activity and by the identification of the respec-
tive genes (DAK1 and DAK2) in S. cerevisiae (Norbeck
and Blomberg, 1997; Molin et al., 2003). However, no in
vitro NAD1-dependent GDH activity could ever be mea-
sured in cell extracts of this organism although attempts
have been made by several authors (Norbeck and Blom-
berg, 1997; Ford and Ellis, 2002; Nguyen and Nevoigt,
2009). The latter results make the existence of a func-
tional 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 NADP1-dependent GDH activity
of the type EC 1.1.1.372/1.1.1.72 (Table 1). However,
the contribution of this enzyme class in a glycerol cata-
bolic pathway with DHA as an intermediate is very
unlikely due to its substrate specificity, which will be fur-
ther 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 utili-
zation 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
C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,
V
Environmental Microbiology, 19, 878–893
Glycerol catabolism in yeast 883
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 car-
bon 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 NAD1-dependent GDH
activity and prevented glycerol assimilation.
An additional pathway proposed for glycerol catabo-
lism 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 NADP1-dependent GDH (EC
1.1.1.372 or 1.1.1.72; Table 1). Subsequently, two differ-
ent 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 dehydroge-
nase. D-glycerate can subsequently be phosphorylated
by a glycerate 3-kinase. The existence of the GA path-
way (including the two sub-pathways downstream of
GA) in the filamentous fungus N. crassa has been sug-
gested by Tom et al. (1978), who determined the activi-
ties 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 glycer-
ol, 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 cat-
alyzing 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 glyc-
eraldehyde has been described in the filamentous fungi
Aspergillus japonicus (Uwajima et al., 1984) and Penicil-
lium 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 1.1.3.B4) is essential for glycerol catabolism in
these organisms has not been investigated yet.
884 M. Klein et al.
Glycerol anabolic pathways abolish glycerol biosynthesis under these conditions (Fil-
linger et al., 2001). The importance of gldB for osmoreg-
The best-studied pathway for the production of glycerol
ulation in A. nidulans is also supported by the fact that
from other carbon sources starts with the NADH-
its expression is strongly induced under conditions of
dependent reduction of glycolytic DHAP to G3P by a
hyper-osmotic shock. Moreover, the NADP1-dependent
cytosolic glycerol 3-phosphate dehydrogenase
GDH in A. nidulans as well as its orthologs in other fun-
(cG3PDH), after which G3P is dephosphorylated to glyc-
gal species (Table 1) have indeed strong preference for
erol by a glycerol 3-phosphatase (GPP) (Fig. 1Bi). This
the reduction of DHA under physiological conditions
‘anabolic G3P pathway’ is the major (or exclusive) path-
(i.e., at neutral pH). Although GDH activity has been
way for glycerol production in S. cerevisiae during osmo-
clearly demonstrated, this anabolic DHA pathway also
regulation and anaerobic redox balancing (Nevoigt and
requires a ‘DHAPase’ activity, which has to the best of
Stahl, 1997). In S. cerevisiae, cG3PDH activity is
our knowledge not yet been identified in any yeast or fil-
encoded by the two isogenes GPD1 and GPD2, while
amentous fungus. One might postulate that the glycerol
GPP activity is encoded by the isogenes GPP1 and
3-phosphatases Gpp1 and Gpp2 can also accept DHAP
GPP2. Genes encoding NAD1-dependent cG3PDH
as a substrate. However, a genetic screen specifically
have also been characterized in several other yeast spe-
developed for identifying genes whose gene products
cies and filamentous fungi, such as Candida magnoliae
dephosphorylate DHAP in S. cerevisiae did not result in
(Lee et al., 2008), Candida glycerinogenes (Chen et al.,
any candidate gene (E. Boles, Personal Communica-
2008), Debaryomyces hansenii (Thome, 2004) and A.
tion). Up to now, there are only two organisms (Neisse-
nidulans (Fillinger et al., 2001). In addition, cG3PDH
ria meningitidis and Plasmodium falciparum) listed in
activity using NADPH instead of NADH as the cofactor
BRENDA (www.brenda-enzymes.org) that exhibit sugar
(EC 1.1.1.94) has been identified in certain yeast spe-
phosphatase activity (EC 3.1.3.23) with DHAP as sub-
cies, such as Candida versatilis (Watanabe et al., 2008).
strate (Lee and Sowokinos, 1967; Guggisberg et al.,
A second pathway for glycerol production using DHA
2014).
as an intermediate has been proposed in filamentous
In Fig. 1Biii, we propose a third theoretical pathway for
fungi (referred to here as the ‘anabolic DHA pathway’,
glycerol formation. The existence of this pathway has not
Fig. 1Bii). The first step in this pathway is assumed to
been indicated by any molecular data so far nor even
be catalyzed by a so far uncharacterized enzyme that
been suggested in the literature. However, enzymes
dephosphorylates DHAP to DHA (see below). The sec-
converting D-glyceraldehyde to glycerol (EC-number
ond step is catalyzed by an NADP1-dependent GDH of
1.1.1.372 or 1.1.1.72; Table 1) have been described in fila-
the type EC 1.1.1.156 (Table 1). In contrast to the
mentous fungi such as N. crassa (Viswanath-Reddy et al.,
above-mentioned dehydrogenases belonging to EC
1977), H. jecorina (Liepins et al., 2006) and A. niger
1.1.1.372 or 1.1.1.72, enzymes from EC 1.1.1.156 are
(Martens-Uzunova and Schaap, 2009). As this enzymatic
able to reduce DHA in vitro but have only very low activi-
activity was induced in A. niger in the presence of galac-
ty (if at all) with glyceraldehyde. This anabolic DHA path-
turonic acid, a role of these enzymes in galacturonic acid
way has been postulated based on the fact that NADP-
dependent GDH activities converting DHA to glycerol catabolism has been suggested (Martens-Uzunova and
(EC 1.1.1.156) have been identified in S. pombe (Mar- Schaap, 2009). However, in principle the enzyme could
shall et al., 1989), A. nidulans (Redkar et al., 1995; De also be involved in an anabolic GA pathway for glycerol
Vries et al., 2003), A. niger (Schuurink et al., 1990), A. formation provided that an enzyme is present with sugar
oryzae (Ruijter et al., 2004) and H. jecorina (Liepins phosphatase activity, catalyzing the first step of the path-
et al., 2006) as also summarized in Table 1. To our way (EC 3.1.3.23 converting glyceraldehyde 3-phosphate
knowledge, studies using mutant strains for allocation of to GA).
this anabolic DHA pathway have only been conducted in
The dubious endogenous ‘DHA pathway’ in
A. nidulans (Fillinger et al., 2001; De Vries et al., 2003).
S. cerevisiae
These studies suggested that the anabolic DHA pathway
is the major pathway responsible for glycerol accumula- The first considerations concerning the possible exis-
tion during osmoregulation in this organism, while the tence of a catabolic DHA pathway in S. cerevisiae date
alternative anabolic G3P pathway did not seem to play a back to the study of Norbeck and Blomberg (1997).
significant role. In fact, the disruption of gldB encoding These authors analysed salt-induced changes in gene
NADPH-dependent GDH led to significantly reduced expression in S. cerevisiae at the proteome level and
intracellular glycerol levels and a severe growth defect in detected a significant increase in the abundance of
medium containing 1 M NaCl (De Vries et al., 2003), Dak1 (EC 2.7.1.29). Inspired by this finding, the authors
while the deletion of gfdA encoding cG3PDH did not attempted to identify the corresponding GDH but were
C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,
V
Environmental Microbiology, 19, 878–893

unable to detect any in vitro GDH activity although differ-
ent pH values, substrates (DHA and glycerol) and co-
factors (NADH, NADPH, NAD1 and NADP1) were
tested.
At the time when Norbeck and Blomberg (1997) con-
ducted their study, no protein sequence of an NAD1-
dependent GDH was available, which would have
allowed a search for genes encoding homologous pro-
teins 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 NADP1-depen-
dent GDH of A. niger (allocated to EC 1.1.1.72). 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
NADP1-dependent A. niger enzyme. The observation
that Gcy1 expression is strongly increased upon osmo-
stress 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 charac-
terize 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 low-
er. 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 kcat 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 accu-
mulation further increased when GCY1 was overex-
pressed, indicating that Gcy1 has indeed a weak
glycerol oxidizing activity. However, it cannot be exclud-
ed that the glycerol oxidation activity apparently present
under these conditions is just a moonlighting activity that
has become significant only because of the artificially
C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,
V
Environmental Microbiology, 19, 878–893
Glycerol catabolism in yeast 885
imposed conditions (Dak1/2 abolishment/Gcy1 overex-
pression) 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 evi-
dence 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 cat-
abolic 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 NAD1-dependent GDH from O. parapolymorpha
(EC 1.1.1.6; Table 1) and overexpression of the endoge-
nous DAK1 gene in a gut1 deletion mutant in several
S. cerevisiae genetic backgrounds. Surprisingly, this path-
way replacement together with the expression of a heter-
ologous glycerol facilitator (see section ‘Glycerol transport
mechanisms’) even abolished the growth deficit in syn-
thetic 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 sur-
plus of cytosolic NAD1 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 filamen-
tous 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 comprehen-
sively summarized by Neves et al. (2004). In fact, pas-
sive 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/H1-symporter encoded by STL1
(Ferreira et al., 2005). The existence of such an active
886 M. Klein et al.
transport system in S. cerevisiae had already been pre-
dicted by Sutherland et al. (1997) based on the demon-
stration of simultaneous uptake of glycerol and protons
accompanied by intracellular glycerol accumulation. Fer-
reira 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 auxo-
trophic laboratory strains, but the same deletion in the
wild-type S. cerevisiae isolate CBS 6412-13A also
resulted in inability to grow in synthetic glycerol medium
(Swinnen et al., 2013).
Similar active glycerol uptake systems using H1- but
also Na1-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. cerevi-
siae since transcription of STL1 is transiently induced by
osmotic shock during exponential growth in glucose-
based 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 res-
cued in this strain by supplementation of the growth
medium with small amounts of glycerol, which apparent-
ly 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 media-
tor 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,
C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,
V
additional deletion of GUP1 in a gpd1 gpd2 deletion
mutant compromises the rescuing effect of glycerol sup-
plementation in hyperosmotic media suggesting a role of
Gup1 in glycerol import (Holst et al., 2000). However,
even before the glycerol/H1-symporter encoded by
STL1 had been identified, studies in which the expres-
sion patterns of GUP1 and GUP2 were compared with
glycerol uptake activity failed to support the concept that
these genes encode glycerol transporters in S. cerevi-
siae (Oliveira and Lucas, 2004). While GUP1 and GUP2
expression was found to be constitutive, glycerol uptake
activity is clearly affected by carbon catabolite repres-
sion (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/
H1 symport under salt stress (Neves et al., 2004). In
addition, deletion of GUP1 in the wild-type isolate CBS
6412-13A 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 previ-
ously described and extensively studied glycerol facilita-
tor 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 initial-
ly assumed also to contribute significantly to glycerol
uptake in this organism. Early experimental studies dem-
onstrated 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 respec-
tive 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 inter-
fered 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 find-
ing 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
Environmental Microbiology, 19, 878–893

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 glyc-
erol facilitator with regard to glycerol uptake at all as it
does not exhibit uptake properties (Vmax values) that
would be in agreement with the definition of a facilitated
diffusion type of transport.
Oliveira et al. (2003) also questioned the hitherto gen-
erally accepted concept that glycerol is able to cross the
lipid bilayer of the plasma membrane by passive diffu-
sion 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 dele-
tion mutants are unable to grow in synthetic glycerol
medium as mentioned above. Theoretical considerations
based on comparison of the glycerol olive oil/water parti-
tion coefficient (logP) with that of other molecules also
support the view that glycerol is unlikely to show signifi-
cant 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 non-
conventional 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 demonstrat-
ed that glycerol uptake (described as membrane perme-
ability) in the S. cerevisiae strain studied was  105 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 pos-
sessing four distinct glycerol/H1-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 gen-
eration 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 con-
trast 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
C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,
V
Environmental Microbiology, 19, 878–893
Glycerol catabolism in yeast 887
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 glycerol-
based 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 dif-
ferent 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) demon-
strated 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 6412-
13A of PtFPS2 and similar FPS1 homologs from differ-
ent non-conventional yeast species (C. jadinii, P. pastoris
and Y. lipolytica) with superior growth performance on
glycerol compared with S. cerevisiae, resulted in a sig-
nificantly improved lmax from  0.13 h21 (for wild-type
CBS 6412-13A) to  0.18 h21 (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 reverse-
engineered S. cerevisiae strain of CEN.PK113-1A, car-
rying allele replacements for UBR2, GUT1 and SSK1
from CBS 6412-13A, further improved the strain’s
growth performance on glycerol (Swinnen et al., 2016).
However, the glycerol facilitators from the aforemen-
tioned 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 analy-
sis of the different transporters in the original species
might provide more insight into the molecular basis of
the superior glycerol uptake capability of many non-
conventional yeast species in comparison to S. cerevi-
siae. In particular, the fact that many of these yeast spe-
cies carry several STL1 and/or FPS1 isogenes and the
888 M. Klein et al.
different molecular structure of the Fps homologs (see
above) stands in sharp contrast with the genetic consti-
tution of S. cerevisiae in this respect.
The improvement of growth on glycerol achieved by
the replacement of S. cerevisiae’s native glycerol/H1-
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 rate-
controlling factor could for example reside in an ineffi-
cient glycerol catabolic pathway as elaborated in the
section ‘The dubious catabolic DHA pathway in S. cere-
visiae’. 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 organ-
isms is glucose and its presence often excludes the utili-
zation of alternative carbon sources such as glycerol.
This repressive effect often mediated by multiple inter-
linked regulatory interactions and signalling pathways is
generally referred to as carbon catabolite repression.
The shift to an alternative carbon source is character-
ized by a transition period with delayed cellular growth
(designated as diauxic shift) and the underlying molecu-
lar 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 glu-
cose to full respiratory metabolism of the previously pro-
duced 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 dem-
onstrated by many studies of this phenomenon, includ-
ing 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
C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,
V
expression of genes encoding proteins related to mito-
chondrial function and energy metabolism as well as
that of genes encoding enzymes involved in gluconeo-
genesis, the TCA and glyoxalate cycles, and in carbohy-
drate 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 ribo-
some biogenesis and assembly, was significantly down-
regulated 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 sam-
ples around the diauxic shift after glucose depletion.
The direct comparison of the results allowed the identifi-
cation of carbon source specific regulatory patterns
(Roberts and Hudson, 2006). For example, abundance
of STL1 transcripts encoding the glycerol/H1-symporter
for glycerol uptake was strongly increased after glucose
depletion and reached a level that was several hundred-
fold higher during continuous growth on glycerol as com-
pared with glucose. Likewise, the GUT1 and GUT2
genes encoding the enzymes catalyzing the initial glyc-
erol catabolic G3P pathway, showed a strong transcrip-
tional up-regulation in glycerol-based medium.
Surprisingly, derepression of these genes was also
observed to a certain extent in medium containing etha-
nol 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 NADP1-
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 per-
formed in media containing complex medium compo-
nents 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 labo-
ratory 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
€ller, 2003; Turcotte et al.,
this field for more details (Schu
2010; Kayikci and Nielsen, 2015). Although it is clear
Environmental Microbiology, 19, 878–893

that glucose depletion has a major impact on the initia-
tion of the regulatory cascades resulting in growth on
alternative respiratory carbon sources, the information
specifically related to the utilization of glycerol is rather
fragmentary. For example, it has been shown that the
transcriptional activator Cat8 seems to affect the level of
STL1 derepression during the diauxic shift (Haurie et al.,
2001). Cat8 is essential for growth on non-fermentable
carbon sources (Hedges et al., 1995) and derepresses
target genes by binding to their upstream carbon source
responsive elements (CSREs) (Young et al., 2003; Roth
et al., 2004). Another transcription factor important for
glycerol utilization is Adr1 that (together with Ino2 and
Ino4) is required for the derepression of GUT1 (Graus-
lund et al., 1999). The derepression of GUT2 is depen-
dent on the activity of the protein kinase Snf1 as well as
on the heteromeric protein complex Hap2-Hap5, which
activates the transcription of many nuclear genes encod-
ing proteins with a mitochondrial function (Grauslund
and Ronnow, 2000). Both GUT1 and GUT2 are
repressed by the negative regulator Opi1 during growth
on glucose (Grauslund et al., 1999; Grauslund and Ron-
now, 2000). Several studies have identified other factors
with a crucial function for growth of S. cerevisiae on
glycerol, such as Rsf1 (Lu et al., 2003; Roberts and
Hudson, 2009) and Rsf2 (Zms1) (Lu et al., 2005). Dele-
tion mutants of RSF1 showed an imbalanced expression
of genes encoding enzymes for glycerol catabolic and
anabolic pathways, decreased transcription of HAP4
(whose gene product is part of the aforementioned
Hap2-Hap5 protein complex) as well as an increased
transcript level of genes whose products function during
stress responses (Roberts and Hudson, 2009).
In comparison to S. cerevisiae, even less is known
about the regulation of glycerol utilization in other yeast
species. It would be particularly interesting to under-
stand the respective aspects of regulation in those yeast
species that exhibit a superior growth capability on glyc-
erol. The fact that, for example, Y. lipolytica consumes
and actually prefers glycerol even if glucose is present
(Workman et al., 2013) implies regulatory mechanisms
in this species which are distinct from common carbon
catabolite repression. Future investigations of such regu-
latory principles might deliver more detailed explanations
for the superior glycerol utilization of certain non-
conventional yeast species.
In many fungal species, enzymes and transporters for
a glycerol catabolic pathway often coexist with those
required for the formation of glycerol. For example, glyc-
erol is a usual by-product of sugar catabolism in S. cere-
visiae where it serves as the major redox sink for
reducing equivalents produced in biosynthetic pathways
(biomass production) particularly under anaerobic condi-
tions (Bakker et al., 2001). Moreover, glycerol production
C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,
V
Environmental Microbiology, 19, 878–893
Glycerol catabolism in yeast 889
from sugar and intracellular accumulation of glycerol is
increased during hyperosmotic stress (Nevoigt and Stahl,
1997; Hohmann, 2002; 2015). Therefore, during growth
on glycerol as the sole carbon source, catabolic and ana-
bolic pathways should be more or less strictly regulated
in order to avoid futile cycling. Indeed, transcriptome pro-
filing of S. cerevisiae cells continuously growing on glycer-
ol revealed a reduction of transcript levels for genes
involved in glycerol anabolic pathways, such as GPD2,
GPP1 and GPP2 (Roberts and Hudson, 2006). Interest-
ingly, transcription of GPD1 was found to be slightly up-
regulated on glycerol- as compared with glucose-based
medium probably reflecting an increased activity of the
G3P shuttle (see Larsson et al., 1998) under these
conditions.
Concluding remarks
Most of our knowledge on glycerol metabolism in yeasts
and filamentous fungi has been derived originally from
research on laboratory strains of the yeast S. cerevisiae.
Although this has led to elucidation of the glycerol
uptake mechanisms and the backbone of all major path-
ways of glycerol metabolism, there are still some con-
spicuous gaps left in our knowledge of the genes
encoding enzymes supposed to be active in specific
steps of these pathways. Detailed insight has also been
gained for the regulation of glycerol metabolism in
S. cerevisiae, in particular in the mechanism of carbon
catabolite repression for the utilization of non-
fermentable carbon sources such as glycerol. Progress
has also been made in understanding the striking differ-
ences between S. cerevisiae strains in their capacity to
use glycerol as a sole source of carbon and this may
lead to the generation of industrial yeast strains with
greatly improved capacity to utilize crude glycerol as a
carbon source. In spite of this, other yeast species still
display much higher growth rates on glycerol and more
insight is needed into the molecular basis of this superi-
or growth capacity in order to be able to use it as a tool
for further improvement of glycerol catabolic capacity in
S. cerevisiae.
References
Babel, W., and Hofmann, K.H. (1982) The relation between
the assimilation of methanol and glycerol in yeasts. Arch
Microbiol 132: 179–184.
Bakker, B.M., Overkamp, K.M., van Maris, A.J., Kotter, P.,
Luttik, M.A., van Dijken, J.P., and Pronk, J.T. (2001) Stoichi-
ometry and compartmentation of NADH metabolism in Sac-
charomyces cerevisiae. FEMS Microbiol Rev 25: 15–37.
Blomberg, A. (2000) Metabolic surprises in Saccharomyces
cerevisiae during adaptation to saline conditions: ques-
tions, some answers and a model. FEMS Microbiol Lett
182: 1–8.
890 M. Klein et al.
Bosson, R., Jaquenoud, M., and Conzelmann, A. (2006)
GUP1 of Saccharomyces cerevisiae encodes an O-
acyltransferase involved in remodeling of the GPI anchor.
Mol Biol Cell 17: 2636–2645.
Brauer, M.J., Saldanha, A.J., Dolinski, K., and Botstein, D.
(2005) Homeostatic adjustment and metabolic remodeling in
glucose-limited yeast cultures. Mol Biol Cell 16: 2503–2517.
Chang, Q., Griest, T.A., Harter, T.M., and Petrash, J.M.
(2007) Functional studies of aldo-keto reductases in Sac-
charomyces cerevisiae. Biochim Biophys Acta 1773:
321–329.
Chen, X., Fang, H., Rao, Z., Shen, W., Zhuge, B., Wang,
Z., and Zhuge, J. (2008) Cloning and characterization of
a NAD1-dependent glycerol-3-phosphate dehydrogenase
gene from Candida glycerinogenes, an industrial glycerol
producer. FEMS Yeast Res 8: 725–734.
Cray, J.A., Stevenson, A., Ball, P., Bankar, S.B., Eleutherio,
E.C., Ezeji, T.C., et al. (2015) Chaotropicity: a key factor
in product tolerance of biofuel-producing microorganisms.
Curr Opin Biotechnol 33: 228–259.
De Jong, J.C., McCormack, B.J., Smirnoff, N., and Talbot,
N.J. (1997) Glycerol generates turgor in rice blast. Nature
389: 244–244.
De Vries, R.P., Flitter, S.J., van de Vondervoort, P.J.,
Chaveroche, M.K., Fontaine, T., Fillinger, S., et al. (2003)
Glycerol dehydrogenase, encoded by gldB is essential for
osmotolerance in Aspergillus nidulans. Mol Microbiol 49:
131–141.
DeRisi, J.L., Iyer, V.R., and Brown, P.O. (1997) Exploring
the metabolic and genetic control of gene expression on
a genomic scale. Science 278: 680–686.
Dujon, B., Sherman, D., Fischer, G., Durrens, P.,
Casaregola, S., Lafontaine, I., et al. (2004) Genome evo-
lution in yeasts. Nature 430: 35–44.
Falb, M., Muller, K., Konigsmaier, L., Oberwinkler, T., Horn,
P., von Gronau, S., et al. (2008) Metabolism of halophilic
archaea. Extremophiles 12: 177–196.
Ferreira, C., van Voorst, F., Martins, A., Neves, L., Oliveira,
R., Kielland-Brandt, M.C., et al. (2005) A member of the
sugar transporter family, Stl1p is the glycerol/H1 sym-
porter in Saccharomyces cerevisiae. Mol Biol Cell 16:
2068–2076.
Fillinger, S., Ruijter, G., Tamas, M.J., Visser, J., Thevelein,
J.M., and D’enfert, C. (2001) Molecular and physiological
characterization of the NAD-dependent glycerol 3-
phosphate dehydrogenase in the filamentous fungus
Aspergillus nidulans. Mol Microbiol 39: 145–157.
Finley, D., Ulrich, H.D., Sommer, T., and Kaiser, P. (2012)
The ubiquitin-proteasome system of Saccharomyces cer-
evisiae. Genetics 192: 319–360.
Ford, G., and Ellis, E.M. (2002) Characterization of Ypr1p
from Saccharomyces cerevisiae as a 2-methylbutyraldehyde
reductase. Yeast 19: 1087–1096.
Gancedo, C., Gancedo, J.M., and Sols, A. (1968) Glycerol
metabolism in yeasts. Pathways of utilization and produc-
tion. Eur J Biochem 5: 165–172.
Goddard, M.R., and Greig, D. (2015) Saccharomyces cere-
visiae: a nomadic yeast with no niche? FEMS Yeast Res
15: 9.
Grauslund, M., and Ronnow, B. (2000) Carbon source-
dependent transcriptional regulation of the mitochondrial
C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,
V
glycerol-3-phosphate dehydrogenase gene, GUT2, from
Saccharomyces cerevisiae. Can J Microbiol 46: 1096–
1100.
Grauslund, M., Lopes, J.M., and Ronnow, B. (1999) Expres-
sion of GUT1, which encodes glycerol kinase in Saccha-
romyces cerevisiae, is controlled by the positive
regulators Adr1p, Ino2p and Ino4p and the negative regu-
lator Opi1p in a carbon source-dependent fashion.
Nucleic Acids Res 27: 4391–4398.
Guggisberg, A.M., Park, J., Edwards, R.L., Kelly, M.L.,
Hodge, D.M., Tolia, N.H., and Odom, A.R. (2014) A sugar
phosphatase regulates the methylerythritol phosphate
(MEP) pathway in malaria parasites. Nat Commun 5:
4467.
Hallsworth, J.E., and Magan, N. (1995) Manipulation of
intracellular glycerol and erythritol enhances germination
of conidia at low water availability. Microbiology 141 (Pt
5): 1109–1115.
Haurie, V., Perrot, M., Mini, T., Jeno, P., Sagliocco, F., and
Boucherie, H. (2001) The transcriptional activator Cat8p
provides a major contribution to the reprogramming of
carbon metabolism during the diauxic shift in Saccharo-
myces cerevisiae. J Biol Chem 276: 76–85.
Hedges, D., Proft, M., and Entian, K.D. (1995) CAT8, a new
zinc cluster-encoding gene necessary for derepression of
gluconeogenic enzymes in the yeast Saccharomyces cer-
evisiae. Mol Cell Biol 15: 1915–1922.
Heller, K.B., Lin, E.C., and Wilson, T.H. (1980) Substrate
specificity and transport properties of the glycerol facilita-
tor of Escherichia coli. J Bacteriol 144: 274–278.
Heredia, C.F., Sols, A., and DelaFuente, G. (1968) Specific-
ity of the constitutive hexose transport in yeast. Eur J
Biochem 5: 321–329.
Hibuse, T., Maeda, N., Nagasawa, A., and Funahashi, T.
(2006) Aquaporins and glycerol metabolism. Biochim Bio-
phys Acta 1758: 1004–1011.
Hohmann, S. (2002) Osmotic stress signaling and osmoa-
daptation in yeasts. Microbiol Mol Biol Rev 66: 300–372.
Hohmann, S. (2015) An integrated view on a eukaryotic
osmoregulation system. Curr Genet 61: 373–382.
Holst, B., Lunde, C., Lages, F., Oliveira, R., Lucas, C., and
Kielland-Brandt, M.C. (2000) GUP1 and its close homo-
logue GUP2, encoding multimembrane-spanning proteins
involved in active glycerol uptake in Saccharomyces cere-
visiae. Mol Microbiol 37: 108–124.
Jung, J.Y., Kim, T.Y., Ng, C.Y., and Oh, M.K. (2012) Charac-
terization of GCY1 in Saccharomyces cerevisiae by meta-
bolic profiling. J Appl Microbiol 113: 1468–1478.
Kayikci, O., and Nielsen, J. (2015) Glucose repression in
Saccharomyces cerevisiae. FEMS Yeast Res 15: 68.
Klein, M., Carrillo, M., Xiberras, J., Islam, Z.U., Swinnen,
S., and Nevoigt, E. (2016a) Towards the exploitation of
glycerol’s high reducing power in Saccharomyces cerevi-
siae-based bioprocesses. Metab Eng 38: 464–472.
Klein, M., Islam, Z., Knudsen, P.B., Carrillo, M., Swinnen,
S., Workman, M., and Nevoigt, E. (2016b) The expres-
sion of glycerol facilitators from various yeast species
improves growth on glycerol of Saccharomyces cerevi-
siae. Metab Eng Commun 3: 252–257.
Kurtzman, C.P., Fell, J.W., and Boekhout, T. (eds) (2011)
The Yeasts - a Taxonomic Study. New York: Elsevier.
Environmental Microbiology, 19, 878–893

Lages, F., and Lucas, C. (1995) Characterization of a glyc-
erol/H1 symport in the halotolerant yeast Pichia sorbito-
phila. Yeast 11: 111–119.
Lages, F., Silva-Graca, M., and Lucas, C. (1999) Active
glycerol uptake is a mechanism underlying halotolerance
in yeasts: a study of 42 species. Microbiology 145 (Pt 9):
2577–2585.
Larsson, C., Pahlman, I.L., Ansell, R., Rigoulet, M., Adler,
L., and Gustafsson, L. (1998) The importance of the glyc-
erol 3-phosphate shuttle during aerobic growth of Sac-
charomyces cerevisiae. Yeast 14: 347–357.
Lee, Y.P., and Sowokinos, J.R. (1967) Sugar phosphate
phosphohydrolase. I. Substrate specificity, intracellular
localization, and purification from Neisseria meningitidis.
J Biol Chem 242: 2264–2271.
Lee, D.H., Kim, M.D., Ryu, Y.W., and Seo, J.H. (2008) Cloning
and characterization of CmGPD1, the Candida magnoliae
homologue of glycerol-3-phosphate dehydrogenase. FEMS
Yeast Res 8: 1324–1333.
Liepins, J., Kuorelahti, S., Penttila, M., and Richard, P.
(2006) Enzymes for the NADPH-dependent reduction of
dihydroxyacetone and D-glyceraldehyde and L-
glyceraldehyde in the mould Hypocrea jecorina. Febs J
273: 4229–4235.
Lin, S.F., Chiou, C.M., and Tsai, Y.C. (1996) Purification
and characterization of a glycerol oxidase from Penicilli-
um sp. TS-622. Enzyme Microb Technol 18: 383–387.
Liu, X., Jensen, P.R., and Workman, M. (2012) Bioconver-
sion of crude glycerol feedstocks into ethanol by Pachy-
solen tannophilus. Bioresour Technol 104: 579–586.
Liu, X., Mortensen, U.H., and Workman, M. (2013) Expres-
sion and functional studies of genes involved in transport
and metabolism of glycerol in Pachysolen tannophilus.
Microb Cell Fact 12: 27.
Lu, L., Roberts, G., Simon, K., Yu, J., and Hudson, A.P.
(2003) Rsf1p, a protein required for respiratory growth of
Saccharomyces cerevisiae. Curr Genet 43: 263–272.
Lu, L., Roberts, G.G., Oszust, C., and Hudson, A.P. (2005)
The YJR127C/ZMS1 gene product is involved in glycerol-
based respiratory growth of the yeast Saccharomyces
cerevisiae. Curr Genet 48: 235–246.
Lucas, C., Da Costa, M., and van Uden, N. (1990) Osmo-
regulatory Active Sodium-Glycerol Co-transport in the
Halotolerant Yeast Debaryomyces hansenii. Yeast 6:
187–191.
Luyten, K., Albertyn, J., Skibbe, W.F., Prior, B.A., Ramos,
J., Thevelein, J.M., and Hohmann, S. (1995) Fps1, a
yeast member of the MIP family of channel proteins, is a
facilitator for glycerol uptake and efflux and is inactive
under osmotic stress. Embo J 14: 1360–1371.
Maeda, T., Wurgler-Murphy, S.M., and Saito, H. (1994) A
two-component system that regulates an osmosensing
MAP kinase cascade in yeast. Nature 369: 242–245.
Marshall, J.H., Kong, Y.C., Sloan, J., and May, J.W. (1989)
Purification and properties of glycerol:NADP1 2-
oxidoreductase from Schizosaccharomyces pombe.
J Gen Microbiol 135: 697–701.
Martens-Uzunova, E.S., and Schaap, P.J. (2009) Assess-
ment of the pectin degrading enzyme network of Asper-
gillus niger by functional genomics. Fungal Genet Biol 46
Suppl 1: S170–S179.
C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,
V
Environmental Microbiology, 19, 878–893
Glycerol catabolism in yeast 891
Matsuzawa, T., Ohashi, T., Hosomi, A., Tanaka, N., Tohda,
H., and Takegawa, K. (2010) The gld1 gene encoding
glycerol dehydrogenase is required for glycerol metabo-
lism in Schizosaccharomyces pombe. Appl Microbiol Bio-
technol 87: 715–727.
Mattam, A.J., Clomburg, J.M., Gonzalez, R., and Yazdani,
S.S. (2013) Fermentation of glycerol and production of
valuable chemical and biofuel molecules. Biotechnol Lett
35: 831–842.
Mattanovich, D., Graf, A., Stadlmann, J., Dragosits, M.,
Redl, A., Maurer, M., et al. (2009) Genome, secretome
and glucose transport highlight unique features of the
protein production host Pichia pastoris. Microb Cell Fact
8: 29.
Maurel, C., Reizer, J., Schroeder, J.I., Chrispeels, M.J., and
Saier, M.H. Jr. (1994) Functional characterization of the
Escherichia coli glycerol facilitator, GlpF, in Xenopus
oocytes. J Biol Chem 269: 11869–11872.
Merico, A., Ragni, E., Galafassi, S., Popolo, L., and
Compagno, C. (2011) Generation of an evolved Saccha-
romyces cerevisiae strain with a high freeze tolerance
and an improved ability to grow on glycerol. J Ind Micro-
biol Biotechnol 38: 1037–1044.
Molin, M., Norbeck, J., and Blomberg, A. (2003) Dihydroxy-
acetone kinases in Saccharomyces cerevisiae are
involved in detoxification of dihydroxyacetone. J Biol
Chem 278: 1415–1423.
Neves, L., Lages, F., and Lucas, C. (2004) New insights on
glycerol transport in Saccharomyces cerevisiae. FEBS
Lett 565: 160–162.
Nevoigt, E. (2008) Progress in metabolic engineering of
Saccharomyces cerevisiae. Microbiol Mol Biol Rev 72:
379–412.
Nevoigt, E., and Stahl, U. (1997) Osmoregulation and glyc-
erol metabolism in the yeast Saccharomyces cerevisiae.
FEMS Microbiol Rev 21: 231–241.
Nguyen, H.T., and Nevoigt, E. (2009) Engineering of Sac-
charomyces cerevisiae for the production of dihydroxyac-
etone (DHA) from sugars: a proof of concept. Metab Eng
11: 335–346.
Norbeck, J., and Blomberg, A. (1997) Metabolic and regula-
tory changes associated with growth of Saccharomyces
cerevisiae in 1.4 M NaCl. Evidence for osmotic induction
of glycerol dissimilation via the dihydroxyacetone path-
way. J Biol Chem 272: 5544–5554.
Ochoa-Estopier, A., Lesage, J., Gorret, N., and Guillouet,
S.E. (2010) Kinetic analysis of a Saccharomyces cerevi-
siae strain adapted for improved growth on glycerol:
implications for the development of yeast bioprocesses
on glycerol. Bioresour Technol 102: 1521–1577.
Oliveira, R., and Lucas, C. (2004) Expression studies of
GUP1 and GUP2, genes involved in glycerol active trans-
port in Saccharomyces cerevisiae, using semi-
quantitative RT-PCR. Curr Genet 46: 140–146.
Oliveira, R., Lages, F., Silva-Graca, M., and Lucas, C.
(2003) Fps1p channel is the mediator of the major part of
glycerol passive diffusion in Saccharomyces cerevisiae:
artefacts and re-definitions. Biochim Biophys Acta 1613:
57–71.
Papanikolaou, S., Muniglia, L., Chevalot, I., Aggelis, G., and
Marc, I. (2002) Yarrowia lipolytica as a potential producer
892 M. Klein et al.
of citric acid from raw glycerol. J Appl Microbiol 92: 737–
744.
Pavlik, P., Simon, M., Schuster, T., and Ruis, H. (1993)
The glycerol kinase (GUT1) gene of Saccharomyces
cerevisiae: cloning and characterization. Curr Genet 24:
21–25.
Pfeiffer, T., Schuster, S., and Bonhoeffer, S. (2001) Cooper-
ation and competition in the evolution of ATP-producing
pathways. Science 292: 504–507.
Piskur, J., Rozpedowska, E., Polakova, S., Merico, A., and
Compagno, C. (2006) How did Saccharomyces evolve to
become a good brewer?. Trends Genet 22: 183–186.
Posas, F., Chambers, J.R., Heyman, J.A., Hoeffler, J.P., de
Nadal, E., and Arino, J. (2000) The transcriptional
response of yeast to saline stress. J Biol Chem 275:
17249–17255.
Redkar, R.J., Locy, R.D., and Singh, N.K. (1995) Biosyn-
thetic pathways of glycerol accumulation under salt stress
in Aspergillus nidulans. Exp Mycol 19: 241–246.
Rep, M., Krantz, M., Thevelein, J.M., and Hohmann, S.
(2000) The transcriptional response of Saccharomyces
cerevisiae to osmotic shock. Hot1p and Msn2p/Msn4p
are required for the induction of subsets of high osmolari-
ty glycerol pathway-dependent genes. J Biol Chem 275:
8290–8300.
Roberts, G.G., and Hudson, A.P. (2006) Transcriptome pro-
filing of Saccharomyces cerevisiae during a transition
from fermentative to glycerol-based respiratory growth
reveals extensive metabolic and structural remodeling.
Mol Genet Genom 276: 170–186.
Roberts, G.G., 3rd, and Hudson, A.P. (2009) Rsf1p is
required for an efficient metabolic shift from fermentative
to glycerol-based respiratory growth in S. cerevisiae.
Yeast 26: 95–110.
Romano, A.H. (1986) Microbial sugar transport systems
and their importance in biotechnology. Trends Biotechnol
4: 207–213.
Roth, S., Kumme, J., and Schuller, H.J. (2004) Transcrip-
tional activators Cat8 and Sip4 discriminate between
sequence variants of the carbon source-responsive pro-
moter element in the yeast Saccharomyces cerevisiae.
Curr Genet 45: 121–128.
Ruijter, G.J., Visser, J., and Rinzema, A. (2004) Polyol
accumulation by Aspergillus oryzae at low water activity
in solid-state fermentation. Microbiology 150: 1095–
1101.
Schu €ller, H.J. (2003) Transcriptional control of nonfermenta-
tive metabolism in the yeast Saccharomyces cerevisiae.
Curr Genet 43: 139–160.
Schuurink, R., Busink, R., Hondmann, D.H., Witteveen,
C.F., and Visser, J. (1990) Purification and properties of
NADP(1)-dependent glycerol dehydrogenases from
Aspergillus nidulans and A. niger. J Gen Microbiol 136:
1043–1050.
Sformo, T., Walters, K., Jeannet, K., Wowk, B., Fahy,
G.M., Barnes, B.M., and Duman, J.G. (2010) Deep
supercooling, vitrification and limited survival to
2100{degrees}C in the Alaskan beetle Cucujus clavipes
puniceus (Coleoptera: Cucujidae) larvae. J Exp Biol
213: 502–509.
C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,
V
Sprague, G.F., and Cronan, J.E. (1977) Isolation and char-
acterization of Saccharomyces cerevisiae mutants defec-
tive in glycerol catabolism. J Bacteriol 129: 1335–1342.
Stevenson, A., Hamill, P.G., Medina, A., Kminek, G.,
Rummel, J.D., Dijksterhuis, J., et al. (2016) Glycerol
enhances fungal germination at the water-activity limit for
life. Environ Microbiol. doi: 10.1111/1462-2920.13530.
[Epub ahead of print]
Sutherland, F.C., Lages, F., Lucas, C., Luyten, K., Albertyn,
J., Hohmann, S., et al. (1997) Characteristics of Fps1-
dependent and -independent glycerol transport in Sac-
charomyces cerevisiae. J Bacteriol 179: 7790–7795.
Sweet, G., Gandor, C., Voegele, R., Wittekindt, N., Beuerle,
J., Truniger, V., et al. (1990) Glycerol facilitator of Escheri-
chia coli: cloning of glpF and identification of the glpF
product. J Bacteriol 172: 424–430.
Swinnen, S., Klein, M., Carrillo, M., McInnes, J., Nguyen,
H.T., and Nevoigt, E. (2013) Re-evaluation of glycerol uti-
lization in Saccharomyces cerevisiae: characterization of
an isolate that grows on glycerol without supporting sup-
plements. Biotechnol Biofuels 6: 157.
Swinnen, S., Ho, P.W., Klein, M., and Nevoigt, E. (2016)
Genetic determinants for enhanced glycerol growth of
Saccharomyces cerevisiae. Metab Eng 36: 68–79.
Tamas, M.J., Luyten, K., Sutherland, F.C., Hernandez, A.,
Albertyn, J., Valadi, H., et al. (1999) Fps1p controls the accu-
mulation and release of the compatible solute glycerol in
yeast osmoregulation. Mol Microbiol 31: 1087–1104.
Tani, T., and Yamada, K. (1987) Glycerol metabolism in
methylotrophic yeasts. Agric Biol Chem 51: 1927–1933.
Thome, P.E. (2004) Isolation of a GPD gene from Debaryo-
myces hansenii encoding a glycerol 3-phosphate dehy-
drogenase (NAD1). Yeast 21: 119–126.
Tom, G.D., Viswanath-Reddy, M., and Howe, H.B. Jr.
(1978) Effect of carbon source on enzymes involved in
glycerol metabolism in Neurospora crassa. Arch Microbiol
117: 259–263.
Turcotte, B., Liang, X.B., Robert, F., and Soontorngun, N.
(2010) Transcriptional regulation of nonfermentable carbon
utilization in budding yeast. FEMS Yeast Res 10: 2–13.
Uwajima, T., Shimizu, Y., and Terada, O. (1984) Glycerol
oxidase, a novel copper hemoprotein from Aspergillus
japonicus. Molecular and catalytic properties of the
enzyme and its application to the analysis of serum trigly-
cerides. J Biol Chem 259: 2748–2753.
Van Zyl, P.J., Kilian, S.G., and Prior, B.A. (1990) The role of
an active transport mechanism in glycerol accumulation
during osmoregulation by Zygosaccharomyces rouxii.
Appl Microbiol Biotechnol 34: 231–235.
Vasiliadis, G.E., Sloan, J., Marshall, J.H., and May, J.W.
(1987) Glycerol and dihydroxyacetone metabolizing
enzymes in fission yeasts of the genus Schizosaccharo-
myces. Arch Microbiol 147: 263–267.
Viswanath-Reddy, M., Bennett, S.N., and Branch Howe,
H.J. (1977) Characterization of glycerol nonutilizing and
protoperithecial mutants of Neurospora. Mol Gen Genet
153: 29–38.
Wang, Z.X., Zhuge, J., Fang, H., and Prior, B.A. (2001)
Glycerol production by microbial fermentation: a review.
Biotechnol Adv 19: 201–223.
Environmental Microbiology, 19, 878–893

Watanabe, Y., Nagayama, K., and Tamai, Y. (2008) Expression
of glycerol 3-phosphate dehydrogenase gene (CvGPD1) in
salt-tolerant yeast Candida versatilis is stimulated by high
concentrations of NaCl. Yeast 25: 107–116.
Workman, M., Holt, P., and Thykaer, J. (2013) Comparing cellular
performance of Yarrowia lipolytica during growth on glucose
and glycerol in submerged cultivations. AMB Express 3: 58.
Yale, J., and Bohnert, H.J. (2001) Transcript expression in
Saccharomyces cerevisiae at high salinity. J Biol Chem
276: 15996–16007.
Yamada-Onodera, K., Yamamoto, H., Emoto, E., Kawahara,
N., and Tani, Y. (2002) Characterisation of glycerol
C 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd,
V
Environmental Microbiology, 19, 878–893
Glycerol catabolism in yeast 893
dehydrogenase from a methylotrophic yeast, Hansenula
polymorpha DL-1, and its gene cloning. Acta Biotechnol
22: 337–353.
Yamada-Onodera, K., Nakajima, A., and Tani, Y. (2006)
Purification, characterization, and gene cloning of glycerol
dehydrogenase from Hansenula ofunaensis, and its
expression for production of optically active diol. J Biosci
Bioeng 102: 545–551.
Young, E.T., Dombek, K.M., Tachibana, C., and Ideker, T.
(2003) Multiple pathways are co-regulated by the protein
kinase Snf1 and the transcription factors Adr1 and Cat8.
J Biol Chem 278: 26146–26158.