Báo cáo khoa học: Relationships between the ethanol utilization (alc ) pathway and unrelated catabolic pathways in Aspergillus nidulans pptx

Relationships between the ethanol utilization (
alc
) pathway
and unrelated catabolic pathways in
Aspergillus nidulans
Michel Flipphi, Janina Kocialkowska and Be
´
atrice Felenbok
Institut de Ge
´
ne
´
tique et Microbiologie, CNRS UMR 8621, Universite
´
Paris-Sud XI, Centre d’Orsay, Orsay, France
The ethanol utilization pathway in Aspergillus nidulans is a
model system, which has been thoroughly elucidated at the
biochemical, genetic and molecular levels. Three main ele-
ments are involved: (a) high level expression of the positively
autoregulated activator AlcR; (b) the strong promoters of
the structural genes for alcohol dehydrogenase (alcA)and
aldehyde dehydrogenase (aldA); and (c) powerful activa-
tion of AlcR by the physiological inducer, acetaldehyde,
produced from growth substrates such as ethanol and
L
-threonine. We have previously characterized the chemical
features of direct inducers of the alc regulon. These studies
allowed us to predict which type of carbonyl compounds
might induce the system. In this study we have determined
that catabolism of different amino acids, such as
L

-valine,
L
-isoleucine,
L
-arginine and
L
-proline, produces aldehydes
that are either not accumulated or fail to induce the alc
system. On the other hand, catabolism of
D
-galacturonic
acid and putrescine, during which aldehydes are transiently
accumulated, gives rise to induction of the alc genes. We
show that the formation of a direct inducer from carboxylic
esters does not depend on alcA-encoded alcohol dehydro-
genase I or on AlcR, and suggest that a cytochrome P450
might be responsible for the initial formation of a physio-
logical aldehyde inducer.
Keywords: Aspergillus nidulans; activation of transcription;
alc genes; aldehydes; carboxylic esters.
The saprophytic hyphal fungus Aspergillus nidulans can
utilize a wide range of organic compounds as sole sources
of carbon and nitrogen. One such alternative nutrient
is ethanol. The pathway-specific transcriptional activator
AlcR is essential for the ethanol-induced expression of the
two structural genes necessary for the utilization of the
alcohol, alcA and aldA (reviewed in [1]). These genes
encode, respectively, alcohol dehydrogenase I (ADHI),
which oxidizes ethanol into acetaldehyde, and aldehyde
dehydrogenase (ALDH), converting acetaldehyde into

acetate. Acetate is further metabolized into acetyl-CoA by
an acetyl-CoA synthetase encoded by the facA gene [2–4],
which is not subject to ethanol-specific control.
The molecular means by which transcriptional regulation
of ethanol utilization is achieved have been studied exten-
sively [1]. The functional characteristics of the AlcR
binuclear zinc cluster activator, its DNA-binding specificity,
its three-dimensional structure and its nuclear localization
sequence, have been elucidated [5–15]. The alcA, alcR
and aldA promoters are subject to different powerful
mechanisms of transcriptional activation [9,13]. These
properties have been exploited in the use of the A. nidulans
alc system as a strongly inducible tool for heterologous
expression [1,16]. When a rich carbon source such as glucose
is present, expression of the alc system is repressed by the
action of CreA, the DNA-binding protein mediating carbon
catabolite repression in A. nidulans. Several mechanisms
account for the direct repression of the alcR and alcA genes
while the aldA gene is subject to indirect CreA control via
direct repression of alcR [7,12,13,17,18]. A subtle interplay
between induction and repression allows A. nidulans to
adapt rapidly to changing nutritional conditions in the
environment.
The degradation pathways of small aliphatic primary
alcohols and monoamines as well as that of the amino acid
L
-threonine, converge on a common catabolic intermediate,
an aliphatic aldehyde [1,13,19]. The three principal alc genes,
alcA, aldA and alcR, are essential for the use of ethylamine
and

L
-threonine as sole sources of carbon (but not for their
utilization as sources of nitrogen). Recently, it has been
demonstrated that ethanol, ethylamine and
L
-threonine do
not induce the alc system directly but that these growth
substrates have to be converted into acetaldehyde, which is
the physiological inducer of the alc system [13,19].
However, the alc system was found to be inducible by a
range of carbonyl compounds and, interestingly, some of
these provoke substantially higher levels of expression than
the maximal level obtainable with acetaldehyde [19]. Fur-
thermore, transcription of three genes of yet unknown
function, alcO, alcM and alcS (clustered along with the alcR
and alcA genes on chromosome VII) is subject to strict AlcR
control [20]. Despite being coordinately expressed with the
alcR, alcA and aldA genes, these three additional alc genes
Correspondence to M. Flipphi, Consejo Superior de Investigaciones
Cientı
´
ficas (CSIC), Instituto de Agroquı
´
mica y Tecnologı
´
a de Ali-
mentos (IATA), Apartado de Correos 73, 46100 Burjassot, Valencia,
Spain. Fax: + 34 96 363 63 01, Tel.: + 34 96 390 00 22,
E-mail: fl
Abbreviations: ADHI, alcohol dehydrogenase I; ALDH, aldehyde

dehydogenase; GABA, c-aminobutyric acid; P450, cytochrome P450.
Enzymes: ADHI, alcohol dehydrogenase I (EC 1.1.1.1); ALDH,
aldehyde dehydogenase (EC 1.2.1.5); P450, cytochrome P450
(EC 1.14.14.1).
(Received 15 May 2003, revised 26 June 2003, accepted 2 July 2003)
Eur. J. Biochem. 270, 3555–3564 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03738.x
are dispensable for growth on ethanol, ethylamine and
L
-threonine (S. Fillinger, M. Flipphi & B. Felenbok,
unpublished results). This raises the question as to whether
the alc system contributes to the nutritional versatility of
A. nidulans beyond providing the fungus with the ability
to utilize ethanol, ethylamine and
L
-threonine as growth
substrates. A whole range of structurally diverse alternative
carbon and nitrogen sources are catabolized via aldehyde
intermediates, some of which might serve as in vivo
substrates for ADHI and/or ALDH under physiologically
relevant conditions. In this study, we have addressed the
possible involvement of the alc genes in the conversion of
a number of nutrients, i.e.
D
-galacturonate, glycerol, six
amino acids, putrescine, c-aminobutyric acid (GABA) and
small carboxylic esters.
Materials and methods
Strains, media and growth conditions
Aspergillus nidulans strains used in this study are listed in
Table 1. The references refer to the mutations relevant to

this work; all other markers are in standard use [21]. Media
composition, supplements and basic growth conditions at
37 °C were as described by Cove [22], using diammonium
tartrate (5 m
M
) as nitrogen source with the carbon source
present at 1% (w/v or v/v), unless stated otherwise.
Mycelia for transcript analysis and esterase expression
were grown for 20–24 h in minimal medium with lactose
(3% w/v) as the carbon source and urea (5 m
M
)asthe
nitrogen source. Figure 1 shows that this growth medium is
perfectly neutral with respect to induction by ethanol. alc
gene transcript levels in lactose-grown and ethanol-induced
mycelia are essentially the same as those observed in
ethanol-grown biomass and notably 10- to 20-fold higher
than those obtained in ethanol-induced mycelia grown on
0.1% (w/v)
D
-fructose. Regardless of the growth substrate,
the gratuitous inducer 2-butanone always elicites more
powerful induction than the convertible compound ethanol.
Furthermore, the response of the acetyl-CoA synthetase
gene (facA) showed that inducing amounts of acetate had
been formed from the ethanol applied to lactose-grown
biomass during the induction period as well as in the
ethanol-grown mycelia. The use of lactose as the growth
substrate allows accurate analysis of alc gene transcription
upon addition of different compounds to principally

nonrepressed and noninduced mycelia in all genetic back-
grounds, and even monitoring catabolism of certain effector
compounds beyond the first aldehyde intermediate.
Induction was achieved by addition of the effector
compounds to 50 m
M
(final concentration), unless stated
otherwise. Cultures were harvested after a further 2.5 h of
incubation (inducing conditions) for Northern analysis and
after 4–6 h for esterase expression. Where necessary, the
effector compound was added from a concentrated solution
Table 1. A. nidulans strains and transformants used in this study.
Strain Genotype References for characterized mutation or strain
BF054 yA2 pabaA1
BF064 yA2 pabaA1; alc500 [13,20]
BF107 yA2 pabaA1; aldA67 [13]
BF129 yA2 pabaA1; alcR125; aldA67 [13,55] M F. Cochet & B. Felenbok (unpublished results)
G277 biA1; punA11 [47]
H1269–12.1 yA2 biA1;(riboB2) D alcC::riboB
a
[43]
TgpdA::alcR yA2;(argB2); (alcR125) pantoB100 TargB/gpdA::alcR
b
[13]
TgpdA::aldA pabaA1;(argB2); (aldA67)TargB/gpdA::aldA
b
[13]
alc500 TalcR yA2 pabaA1;(argB2); alc500 TargB/alcR
c
This work (see Materials and methods)

Corresponding phenotype:
a
riboflavin prototroph;
b
arginine prototroph, ethanol utilizing;
c
arginine prototroph, ethanol nonutilizing.
Fig. 1. Lactose is a neutral growth substrate with respect to induction of
the alc genes by ethanol. Wild type mycelia were grown on either
D
-fructose or lactose (noninduced conditions, NI) and cultures were
induced by addition of ethanol (E) or the gratuitous inducer 2-butanone
(2B) to a final concentration of 50 m
M
, as detailed in Materials and
method. Ethanol-grown biomass is considered to be induced from the
start of cultivation (E*). Culture growth conditions, RNA isolation
andNorthernblotswereasdescribedinMaterialsandmethods.
Membranes were hybridized with
32
P-labelled probes specific for the
alcA, alcR, facA, facB and c-actin (acnA) genes. Hybridization with a
probe specific for the 18S rRNA species provided a control of the
quantity of total RNA in each lane that correlates well with the con-
comitant expression of the regulatory facB gene (encodes a transacti-
vator of acetate catabolism [2,29]), under the various growth
conditions. Note that transcription of the c-actin gene was much
higher in
D
-fructose-grown mycelia than in biomass generated on

lactose or ethanol. The c-actin gene cannot therefore be used as an
internal control to normalize the amounts of mRNA when comparing
D
-fructose-grown mycelia with lactose- or ethanol-grown mycelia.
However, this gene still provided a reliable control among different
cultures grown on one particular carbon source, and also for com-
paring lactose- with ethanol-grown cultures.
3556 M. Flipphi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
of pH 6.8. In those cases where the effector compound
constitutes a poor nitrogen source for the fungus mycelia
were also grown in the presence of the effector (25 m
M
)as
the sole nitrogen source. Such cultures were supplemented
with
D
-biotin to enhance uptake of these compounds [23].
Chemicals were purchased either from Sigma-Aldrich or
Merck-Eurolab.
Generation of a mutant strain lacking the ADHI-encoding
alcA
gene
The best characterized alc deletion mutant isolated upon
selection for resistance to allyl alcohol is alc500 [24,25]. This
mutant lacks all five AlcR-controlled genes comprizing the
alc gene cluster [20] (J. Kocialkowska, B. Felenbok & M.
Flipphi, unpublished results). The unlinked aldA gene is no
longer inducible but is constitutively expressed at a low level
[13]. To monitor pathway-specific induction of the aldA gene
in the absence of ADHI-activity, a single, functional copy of

the alcR gene was introduced into an
L
-arginine-auxotroph
argB2 alc500 double mutant strain by cotransformation with
the plasmids pBSalcRSal (carrying the alcR gene [26]) and
pFB39 (carrying the A. nidulans argB gene [27]), essentially
as described by Flipphi et al.[13].
L
-Arginine-prototrophs
were screened for the presence of the alcR gene by dot-blot
analysis. A single copy cotransformant was selected by
Southern blot analysis (not shown). This transformant was
back-crossed with an alc500 mutant and offspring carrying a
functional alcR gene were screened via their ability to be
crossfed by a leaky aldA15 mutant strain, which accumulates
and secretes acetaldehyde on plates containing 1% ethanol
as sole carbon source and 10 m
M
sodium nitrate as the
nitrogen source [13]. The alcR-complemented alc500 strains
behave as alcA loss-of-function mutants. In one of the
crossfed strains, alc500 TalcR, proper inducibility of alcR
and aldA was demonstrated by Northern blot analysis.
Isolation of RNA and Northern blot analysis
Transcript analysis was carried out as described by Flipphi
et al. [13] using
32
P-labelled probes corresponding to
fragments of the cloned A. nidulans genes alcA [9], alcR
[26], aldA [28], facA [4], facB [29], c-actin (acnA)[30]and

prnD (GenBankÒ AJ223459). To monitor gabA transcrip-
tion, a probe was made from gabA cDNA [31]. 18S
ribosomal RNA was detected with a probe for horseradish
18S rDNA [32]. Autoradiographs were exposed for various
times to avoid saturation of the film. In lactose-grown
mycelia the c-actin gene is constitutively expressed and was
used as an internal control for the amounts of mRNA
loaded. All induction experiments were repeated at least
once.
Qualitative zymogram analysis of carboxyl-/
acetylesterase activity
For the analysis of intracellular esterase activity cell-free
extracts were prepared from about 250 mg mycelial powder,
obtained by grinding freshly harvested mycelia in liquid
nitrogen. The mycelial powder was quickly suspended in
500 lL of ice-cold extraction buffer (10 m
M
sodium phos-
phate pH 6.5, 2 m
M
dithiothreitol). The suspension was
subsequently centrifuged in an Eppendorf centrifuge for
5min at 10 000 g at 4 °C and the supernatant was
recovered and put on ice. For the analysis of extracellular
esterase activity, media samples ( pH 6.8) were taken
directly from cultures. After centrifugation as above, the
supernatant was recovered and put on ice. Protein concen-
tration was determined with Bradford’s method using
bovine serum albumin as the standard.
Proteins were separated in native 7.5% polyacrylamide

(29 : 1 acrylamide/bisacrylamide, v/v) minigels buffered
with 10 m
M
Tris, 76 m
M
glycine, pH 8.5. Electrophoresis
was performed at 4 °C. For cell-free extracts, samples
containing 25 lg protein were mixed with 1/5 volume of
loading buffer (10 m
M
sodium phosphate pH 7.0, 50%
(v/v) glycerol, 2 per thousand (w/v) bromophenol blue). For
extracellular samples, 20 lL of culture medium (containing
3–5 lgproteinÆmL
)1
) were applied to gels. The samples
were allowed to migrate into the gel at 1 VÆcm
)1
,afterwhich
the gel was run at 4 VÆcm
)1
for 3–4 h. After electrophoresis,
the gel was immersed in 100 mL of assay buffer (25 m
M
potassium phosphate pH 7.0, 1 m
M
EDTA, 1 per thousand
(v/v) 2-mercaptoethanol) at room temperature for 2 min.
4-Methylumbelliferyl acetate was gradually added from a
1

M
solution in dimethylsulfoxide to a final concentration of
500 l
M
. The fluorogenic ester is a substrate for both
carboxyl- and acetylesterases (EC 3.1.1.1/3.1.1.6). Forma-
tion of 4-methylumbelliferone could be detected within
minutes upon illumination with UV light (312 nm).
Results and discussion
D
-Galacturonic acid provokes an induction response
but glycerol does not
Induction of the alc system in lactose-grown mycelia by
D
-galacturonic acid is very strong at 50 m
M
(Fig. 2A, left
panel) but hardly significant at 10 m
M
(not shown).
D
-Galacturonic acid is a good growth substrate for A. nidu-
lans that is catabolized into pyruvate and
D
-glyceraldehyde
[33], and the observed induction should be attributable to
the latter compound.
D
-Glyceraldehyde appears to be a
relatively weak inducer when added to pregrown mycelia

(Fig. 2B) but this could be due to inefficient uptake.
D
-Glyceraldehyde is converted into the glycolytic inter-
mediate
D
-glyceraldehyde-3-phosphate via glycerol and
glycerol-3-phosphate [34]. Glycerol is noninducing (Fig. 2B)
even in the strongly derepressed mutant creA
d
30 or in a
strain carrying a derepressed alcA gene in addition to a
constitutively expressed alcR gene (results not shown).
Dihydroxyacetone, a noninducing double hydroxyl-
substituted a-ketone [19], is also catabolized via glycerol.
In A. nidulans, reduction of
D
-glyceraldehyde is achieved by
NADPH-dependent reductases, one of which is constitu-
tively expressed but substrate-specific [35] while the second
is specifically induced on
D
-galacturonate [36]. It is therefore
not surprising that mutants in alc grow normally on both
D
-galacturonic acid and glycerol [34]. The alc gene induction
observed upon addition of the higher concentration of the
uronic acid is probably due to a transient accumulation of
D
-glyceraldehyde, which ceases upon full expression of the
inducible reductase. In mycelia grown on

D
-galacturonic
acid, expression of the alc genes is repressed (Fig. 2A, right
Ó FEBS 2003 Activation of alc genes in Aspergillus nidulans (Eur. J. Biochem. 270) 3557
panel). However, induction does not cease in mycelia that
are grown on ethanol, or on lactose with either ethylamine
or
L
-threonine as the nitrogen source (results not shown).
Catabolism of
L
-proline and
L
-arginine does not
lead to induction of the
alc
genes
L
-Proline and
L
-arginine can serve as sole carbon and
nitrogen sources for A. nidulans and both are catabolized
via
L
-D
1
-pyrroline-5-carboxylate, the internal Schiff base of
L
-glutamic semialdehyde with which it is in continuous
equilibrium [37,38]. The Schiff base intermediate can be

reduced by PrnD (
L
-D
1
-pyrroline-5-carboxylate reductase/
L
-proline oxidase EC 1.5.1.2) to yield
L
-proline, and oxi-
dized by PrnC (
L
-D
1
-pyrroline-5-carboxylate dehydrogenase
EC 1.5.1.12) to produce
L
-glutamate. These three amino
acids do not induce the alc genes while, as expected,
L
-proline and
L
-arginine do induce the prnD gene. This was
also the case in a strain constitutively overexpressing the
alcR gene, TgpdA::alcR (Fig. 3). In this genetic background,
alcA transcription is more sensitive to minor inducing
signals because the expression of its activator, AlcR, is no
longer limiting [13]. Thus, and in agreement with previous
predictions [19], the semialdehyde does not induce the alc
genes as it carries a carboxy substituent. Loss-of-function
aldA mutants are reported to grow more slowly on

L
-proline
than wild type strains [37]. This could be a pleiotropic effect
due to accumulation of acetaldehyde [13] in addition to that
of
L
-glutamic semialdehyde in these mutants. However, we
were unable to detect a phenotype characteristic for the
aldA67 mutation on plates with
L
-proline or
L
-arginine as
sole carbon and nitrogen sources, distinguishing it from
alc mutations (results not shown).
Catabolism of
L
-valine,
L
-isoleucine and
L
-tryptophan
does not induce the
alc
system
The branched-chain aliphatic amino acids
L
-valine,
L
-leucine and

L
-isoleucine can serve as (poor) growth
substrates for A. nidulans [39,40]. The first catabolic step is
a transamination to yield the corresponding 2-oxo acids. In
Saccharomyces cerevisiae these latter compounds enter the
Ehrlich pathway where they undergo decarboxylation to
form the corresponding aldehydes, which are subsequently
detoxified by constitutive alcohol dehydrogenase activity
[41].
2-Methylbutyraldehyde emerged previously as the most
effective alc gene inducer [19]. In A. nidulans,thisaldehyde
could be formed as a physiological intermediate when
L
-isoleucine is catabolized via the Ehrlich pathway.
However, transcript analyses in wild type (Fig. 4) and an
alcA-deletion strain (not shown) grown on either of the
branched-chain aliphatic amino acids as sole nitrogen
source showed that the alc system was not induced.
Therefore, the expected aldehyde intermediates are either
not accumulated or not formed at all. This result can be
anticipated for
L
-leucine as this amino acid is not catabo-
lized via the Ehrlich pathway in A. nidulans [40]. Even if
L
-isoleucine and
L
-valine are degraded in A. nidulans as they
are in yeast, then the formation and conversion of the
Fig. 2.

D
-Galacturonic acid provokes induction of the alc genes.
(A) Northern blot analysis of induction upon addition of
D
-galact-
uronic acid (
D
-GAA) to lactose-grown wildtype mycelia (left panel)
andupongrowthon
D
-galacturonic acid (
D
-GAA*)(rightpanel).
(B) Northern blot analysis of the response of the alc genes to addition
of
D
-glyceraldehyde and glycerol. Ethanol served as the reference for
induction. Induction was achieved by adding effector compounds
to uninduced cultures to 50 m
M
(final concentration) except for
D
-glyceraldehyde (*), which was added to 4 m
M
.Whentheeffector
compound served as the carbon source for growth, its initial concen-
tration in culture was 50 m
M
. Experimental details and abbreviations
were as described in the legend to Fig. 1.

Fig. 3. The amino acids
L
-proline and
L
-arginine do not induce the alc
system. Northern blot analysis was done in gpdA::alcR,astraincon-
stitutively overexpressing the alcR gene (alcR*). The prnD (
L
-proline
oxidase) and alcA genes were used to monitor the responses to the two
amino acids. Experimental details and abbreviations were as described
in the legends to Figs 1 and 2.
3558 M. Flipphi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
aldehyde intermediates clearly does not require activation of
the ethanol utilization pathway. Interestingly both
L
-isoleu-
cine and
L
-valine modestly induce the alc genes in a strain
deleted for another alcohol dehydrogenase, ADHIII, pre-
sumably due to some aldehyde accumulation, while
L
-leucine does not (results not shown). Expression of the
ADHIII-encoding alcC gene is not under the control of
AlcR and is irrelevant for ethanol catabolism [1,42,43].
L
-Tryptophan can also serve as a nitrogen source for
A. nidulans [39]. Multiple routes are known in bacteria,
fungi and plants to convert this amino acid into indole-

3-acetic acid, an important plant hormone (in bacteria,
reviewed by [44]; in fungi [45,46]). The pathways via indole-
3-pyruvate and tryptamine produce indole-3-acetaldehyde
as an intermediate, which is further oxidized by an alcohol-
inducible ALDH in the fungus Ustilago maydis [45]. In
A. nidulans it has been observed that tryptamine is inducing
for the alc genes at relatively low (nontoxic) concentrations
(M. Flipphi, J. Kocialkowska & B. Felenbok, unpublished
results), probably by conversion into indole-3-acetaldehyde.
This reaction would be similar to that of the deamination of
unbranched aliphatic monoamines; these latter compounds
have been shown previously to be inducers of the alc system
[19]. However,
L
-tryptophan itself is noninducing even in
mycelia grown on lactose with this amino acid as sole
nitrogen source (results not shown). Therefore indole-3-
acetaldehyde is apparently not accumulated upon
L
-trypto-
phan degradation in A. nidulans.
Putrescine provokes an induction of the
alc
system
but GABA does not
A. nidulans is capable of using putrescine (1,4-diamino-
butane) as a sole source of nitrogen but not as a carbon
source [47]. Breakdown of this diamine into the tricarboxylic
acid-cycle intermediate succinate (Fig. 5) involves two
different aldehyde intermediates, c-aminobutyraldehyde

and succinic semialdehyde [37,48–50]. Both have a back-
bone of four carbon atoms, the optimal length for the
aliphatic tail of an alc-inducing aldehyde. Succinic semi-
aldehyde is predicted to be noninducing due to its terminal
carboxy group [19] but c-aminobutyraldehyde permits
analysis of the effect of an amino substituent on the
inductive capacity of a physiologically relevant aldehyde.
Transcript analysis shows that putrescine is indeed able to
moderately induce the alc genes when added to pregrown
mycelia (Fig. 6A). As expected, no induction is observed
upon addition of c-aminobutyric acid (GABA). The forma-
tion of GABA from putrescine can be monitored with the
expression of the GABA permease gene (gabA) [31,49].
Using the leaky punA11 mutation, which partially impairs
the utilization of putrescine [47], we could confirm that the
diamine needs to be converted into the corresponding
aldehyde to induce the alc genes; putrescine does not induce
the alc system in this background, while the gabA gene is only
very modestly expressed (Fig. 6B). Moreover, induction
by putrescine is titratable in vivo with semicarbazide, an
aldehyde scavenger (see References [13,19]; results not
shown). Thus, it can be concluded that putrescinebreakdown
results in the accumulation of a weakly inducing compound,
c-aminobutyraldehyde, and that an amino substituent
strongly limits the inducing capacity of an aldehyde.
As is the case for
D
-galacturonic acid, no alc gene
induction could be observed in mycelia grown on lactose/
putrescine although the gabA gene is expressed under these

growth conditions (results not shown). Expression of the alc
gene is thus likely to respond to a transient accumulation of
c-aminobutyraldehyde when putrescine is added to a non-
induced culture. Interestingly, GABA is also produced from
putrescine in the stringent loss-of-function aldA67 mutant as
well as in an aldA67 alcR125 double mutant, which is unable
to induce the alc genes (Fig. 6A). This indicates that
Fig. 4. The branched-chain aliphatic amino acids
L
-valine and
L
-isoleu-
cine do not induce the alc system. Transcript analysis was performed in
a wild-type strain. Induced mycelia were grown in the presence of
either amino acid (25 m
M
) as the sole nitrogen source instead of urea.
The aldehyde that was presumed to be formed upon
L
-isoleucine
turnover, 2-methylbutyraldehyde (2MB), served as an additional
control of induction and was added to urea-grown mycelia at 2 m
M
[19]. Further experimental details and abbreviations were as described
in the legends to Figs 1 and 2. Similar results were obtained for
L
-leucine (results not shown).
Fig. 5. Catabolism of putrescine and GABA in A. nidulans. Putrescine
is first deaminated to form c-aminobutyraldehyde, which is further
oxidized by an unknown ALDH to yield c-amino butyric acid

(GABA). The second amino group is liberated by GABA amino
transferase (gatA gene [48]) to produce succinic semialdehyde, which is
finally converted into succinate by a substrate-specific ALDH specified
by the ssuA locus [37,49,50].
Ó FEBS 2003 Activation of alc genes in Aspergillus nidulans (Eur. J. Biochem. 270) 3559
c-aminobutyraldehyde is oxidized by another ALDH
enzyme not dependent upon AlcR for its expression. In
mammals, a specialized ALDH is involved in the conversion
of this aminoaldehyde [51]. However, its expression does not
seem to be as rapid as that of the alc genes. Interestingly,
constitutive ALDH overexpression in the TgpdA::aldA
transformant not only decreases alc gene induction by
putrescine but also results in reduced gabA expression
(Fig. 6A). Overall, the data suggest that c-aminobutyralde-
hyde can serve as a (poor) in vivo substrate of both aldA-
encoded ALDH and alcA-encoded ADHI. Moreover, it
appears that expression of the putrescine pathway-specific
ALDH is inducible by its aminoaldehyde substrate.
Interestingly, the level of pseudo-constitutive expression
of the alc genes in the loss-of-function aldA67 mutant [13]
is reduced upon addition of either putrescine or GABA
(Fig. 6A). This could be explained on the basis that the
accumulated aldehyde is partly neutralized as an inducer in
the presence of these primary amines, suggesting that Schiff
base interactions between aldehydes and primary amines
occur in vivo. The formation of a Schiff base is a possible
mechanism by which the DNA-binding regulator AlcR is
activated by direct binding of the inducer compound [19].
An alternative hypothesis is that the aldehydes formed from
putrescine and GABA (c-aminobutyraldehyde and succinic

semialdehyde, respectively) are able to compete in some way
with the aldehyde accumulated from general metabolism in
aldA67, leading to decreased pseudo-constitutive alc gene
expression. This second hypothesis implies that a non-
inducing aldehyde, succinic semialdehyde, competes with
the inducer for binding to AlcR, although it is unable to
effect activation.
Some small carboxylic esters induce the
alc
system
but lactones do not
Carboxylic esters are interesting compounds with respect to
alc gene induction. They are structurally related to ketones,
one class of direct inducers of the alc system, and their
hydrolysis results in alcohols that upon oxidation yield
aldehydes, a second class of direct inducers. We have shown
previously that the small methyl ketones 2-butanone and
2-pentanone induce the alc genes [19]. Circular ketones such
as cyclopentanone and cyclohexanone also induce but the
smallest linear b-ketone, 3-pentanone, does not. We have
therefore investigated whether or not the esters and lactones
(intramolecular esters) corresponding to the above ketones
induce the alc genes.
Transcript analyses show that whereas the ester ethyl-
acetate (which corresponds to the inducer 2-pentanone)
indeed provokes a strong induction of the alc genes,
methylpropionate (which corresponds to the inert ketone
3-pentanone) fails to induce (Fig. 7A). By contrast, none of
the lactones tested (c-butyrolactone, d-valerolactone and
e-caprolactone) provoked induction (Fig. 7B). Moreover,

although 2-butanone is the most powerful of all the ketone
inducers, its structurally related ester methylacetate elicits
only a marginal induction of alcA while alcR and aldA
expression do not exceed their basal, noninduced levels
(results not shown).
These data thus show that there is no correlation between
the inducing capabilities of a ketone and the structurally
related ester or lactone. It rather appears that esters induce
the alc genes when their degradation results in the produc-
tion of an inducing aldehyde derived from the ester’s alcohol
moiety. Acetaldehyde would therefore be responsible for the
induction observed on ethylacetate. Methylpropionate does
not induce as formaldehyde is inert while hydrolysis of the
lactones tested would principally yield noninducing carb-
oxy-substituted alcohols and aldehydes, in agreement with
previous predictions [19].
We have verified these deductions using the unbranched
aliphatic esters propylacetate and ethylpropionate. Upon
hydrolysis, ethylpropionate would yield acetaldehyde while
propylacetate would yield propionaldehyde, both direct
Fig. 6. Putrescine catabolism provokes induction of the alc system.
(A) Transcript analysis of the effect of putrescine (Putr) addition to
pregrown mycelia of a wildtype strain (wt) and three strains mutant in
ethanol catabolism: aldA67, gpdA::aldA and aldA67 alcR125.ThegabA
gene encodes GABA permease and is inducible by x-amino acids [31].
The aldA67 mutant exhibits pseudo-constitutive expression of the alcA
and alcR genes (NI *) [13]. (B) Putrescine-induced transcription of the
alcA and gabA genes in the leaky putrescine utilization mutant punA11
compared to that in a wild type strain. Putrescine (1,4-diaminobutane)
was added to uninduced cultures to 25 m

M
. Further experimental de-
tails and abbreviations were as described in the legends to Figs 1 and 2.
3560 M. Flipphi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
inducers of the alc system. Figure 7A clearly shows that
both these esters are inducing despite the fact that the
carbonyl function in ethylpropionate resides in the
b-position, as is the case for the inert compounds methyl-
propionate and 3-pentanone. That the induction provoked
by ethylacetate, apparently the most effective ester, is
considerably less in a strain constitutively overexpressing
ALDH, TgpdA::aldA (data not shown), is in accord with the
formation of acetaldehyde from this ester. Furthermore,
induction of the acetyl-CoA synthetase gene (facA) upon
addition of ethylacetate, propylacetate and ethylpropionate
(Fig. 7A), actually suggests that all esters are hydrolyzed
into acetate during the induction period. In this regard it is
worth noting that wild type facA transcription responds to
acetate and its precursor ethanol (see Fig. 1) but not to
propionate or n-propanol (M. Flipphi & B. Felenbok,
unpublished results) explaining why this gene is not induced
in the presence of methylpropionate.
In conclusion, the results obtained are in agreement with
our previous data and show that aliphatic and cyclic ketones
directly induce the alc genes. There is no correlation between
the inductive capacities of ketones and their structurally
related ester counterparts. In addition, small aliphatic
carboxylic esters induce the alc genes indirectly via the
aldehyde formed from their ethanol or n-propanol moieties.
Acetylester breakdown yields inducing compounds

independently of the
alc
system
The induction of the facA gene by all esters tested except
methylpropionate suggests that carboxylic esters are
degraded during the induction period of 2.5 h. Strong
indications that the hydrolysis of esters is a catalyzed
process come from zymogram analyses of cell-free extracts
and culture medium samples, which evidenced the presence
of multiple intra- and extracellular carboxyl-/acetylesterase
activities. As shown for the intracellular activity in Fig. 8A,
these esterase activities are constitutively produced by the
fungus when grown on lactose and urea. Interestingly, the
esterase spectrum did not change upon addition of typical
inducers of the alc gene system, i.e.
L
-threonine and
2-butanone, nor by the supply of the inducing ester
ethylacetate. Only addition of 2-methylbutyraldehyde to a
lactose-grown culture resulted in induction of an intracel-
lular esterase. However, even this inducible activity does not
depend on AlcR for its expression as the esterase spectrum
in the alc500 deletion mutant was identical to that in a wild
type strain (Fig. 8A).
In line with our previous studies [19], it is to be expected
that the alcohol resulting from ester hydrolysis is oxidized to
an inducing aldehyde. Transcript analysis in a strain
completely lacking the ADHI-encoding alcA gene (alc500-
TalcR) showed that the two AlcR-controlled genes present
in this background, alcR and aldA, are induced by

ethylacetate and ethylpropionate (Fig. 8B). These genes
are also induced in the presence of ethanol despite the fact
that this strain cannot use the latter as a growth substrate.
The acetyl-CoA synthetase gene facA is not ethanol-
inducible in the deletion mutant (M. Flipphi & B. Felenbok,
unpublished results), also indicating that not much acetate is
formed from ethanol. These observations suggest that
inducing amounts of the physiological aldehyde inducer are
Fig. 7. Analysis of the induction of the alc genes by carboxylic esters
and lactones. (A) Transcript analysis of the effect of four carboxylic
esters on the expression of the alcA, alcR and facA genes in a wild type
strain. The inducing ketone 2-pentanone, which is structurally related
to ethylacetate, and the inert ketone 3-pentanone, structurally related
to methylpropionate, provided the controls. The structures of these
latter four compounds are shown below. (B) Northern analysis of wild
type mycelia supplemented with three different lactones. The inducing
ketone cyclohexanone served as the control for induction. The struc-
tures of the circular ketone and its lactone counterpart, d-valero-
lactone, are shown below. Experimental details and abbreviations were
asdescribedinthelegendstoFigs1and2.
Ó FEBS 2003 Activation of alc genes in Aspergillus nidulans (Eur. J. Biochem. 270) 3561
produced from ethanol as well as from ethylesters by other
enzymes in alcA loss-of-function mutants. However, the
limited conversion capacity is clearly not sufficient to
support growth on ethanol. It was surprising to find that
ethylacetate can serve as a sole source of carbon for alc loss-
of-function mutants (not shown). Despite being highly
induced in its presence, the alc genes are dispensable for
growth on this acetylester, strongly suggesting that the
acetate moiety from the ester is preferentially consumed.

From the above analysis it can be questioned whether the
limited, AlcR-independent ethanol conversion capacity in
the alcA deletion mutant is sufficient to produce and
maintain an inducing quantity of acetaldehyde in the
presence of ethylacetate. Induction of the alc system in an
alcA-independent manner could be explained presuming
that acetaldehyde is produced directly from the ethylester by
an AlcR-independent mechanism (Fig. 9). Some mamma-
lian cytochrome P450 isozymes produce carbonyl com-
pounds (aldehydes or ketones) from carboxylic esters
in vitro by oxidative cleavage [52,53]. Interestingly, the
ethanol- and acetone-inducible P450 2E1 from mammals is
also capable of catalyzing conversion of ethanol into acetate
via two subsequent oxidation reactions [54]. Although the
main product is acetate, acetaldehyde is also produced by this
enzyme because the intermediate product/second substrate is
only loosely bound to the catalytic site. Preliminary results
indicate that one putative P450-encoding gene is weakly
but constitutively transcribed in A. nidulans (M. Flipphi &
B. Felenbok, unpublished results). We therefore propose
that the fungus constitutively produces at least one (but
possibly more) P450 oxidase able to form the physiological
aldehyde inducer from ethanol and other alcohols as well as
from inducing carboxylic esters. Initial, AlcR-independent
production of acetaldehyde by P450 could play a crucial role
in triggering ethanol catabolism in A. nidulans.
Acknowledgements
We thank John Clutterbuck and Heather Sealy-Lewis for providing us
with mutant A. nidulans strains, Michael Hynes for plasmids harboring
the facA and facB genes, Irene Garcia for the prnD probe and Joan

Tilburn for the gabA cDNA clone. We are grateful to Sabine Fillinger
Fig. 8. Formation of the physiological aldehyde inducer from acetyl
esters does not depend on the alc system. (A) Zymogram analysis of
intracellular carboxyl-/acetylesterase activity in wild type compared to
that in an alc deletion mutant, alc500. Cell-free extracts from mycelia
subjected to noninduced or induced growth conditions for the alc
genes were prepared as decribed in Materials and methods. Induction
was achieved by adding
L
-threonine (
L
-Thr) (to 50 m
M
)or2-methyl-
butyraldehyde (2MB) (to 2 m
M
) to lactose-grown mycelia. Protein was
separated in a native polyacrylamide gel at pH 8.5. Carboxyl-/acetyl-
esterase activity was resolved directly in the gel as described in Mate-
rials and methods. The zymograms are presented as the negatives.
(B) Northern analysis of the induction of aldA and alcR in the pres-
ence of ethanol or acetylesters in alc500 TalcR, an absolute alcA
deletion mutant (D alcA) (see Materials and methods). Further
experimental details and abbreviations were as described in the legends
to Figs 1 and 2.
Fig. 9. A role for cytochrome P450 in the initial formation of physio-
logical aldehyde inducers from alcohols and carboxylic esters in
A. ni dulans. Constitutively expressed cytochrome P450 isozymes could
oxidize ethanol and ethylesters to yield directly acetaldehyde, trigger-
ing a cascade reaction of coupled expression of alcA-encoded ADHI

and accelerated formation of the physiological inducer. Esterases are
responsible for the energetically more favorable hydrolysis of carb-
oxylic esters to alcohols and carboxylic acids.
3562 M. Flipphi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
who selected the initial alcR transformant in the alc500 deletion mutant
background, Andrew MacCabe for correcting the English, Miguel
Pen
˜
alva for helpful suggestions and Herb Arst for critical reading of the
manuscript. This work was supported by the Centre National de la
Recherche Scientifique, UMR 8621, the Universite
´
Paris-Sud XI and
the European Community, grant QLK3-CT99-00729.
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