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

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

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

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)

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

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

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

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

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

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.

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

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

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,

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

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.

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

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