EUKARYOTIC CELL, Feb. 2005, p. 356–364 Vol. 4, No.

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1535-9778/05/$08.00⫹0 doi:10.1128/EC.4.2.356–364.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Yarrowia lipolytica Mutants Devoid of Pyruvate Carboxylase Activity

Show an Unusual Growth Phenotype†
Carmen-Lisset Flores* and Carlos Gancedo
Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-Universidad Autónoma de Madrid, Madrid, Spain
Received 26 August 2004/Accepted 2 December 2004

We have cloned and characterized the gene PYC1, encoding the unique pyruvate carboxylase in the dimor-
phic yeast Yarrowia lipolytica. The protein putatively encoded by the cDNA has a length of 1,192 amino acids
and shows around 70% identity with pyruvate carboxylases from other organisms. The corresponding genomic
DNA possesses an intron of 269 bp located 133 bp downstream of the starting ATG. In the branch motif of the
intron, the sequence CCCTAAC, not previously found at this place in spliceosomal introns of Y. lipolytica, was
uncovered. Disruption of the PYC1 gene from Y. lipolytica did not abolish growth in glucose-ammonium me-
dium, as is the case in other eukaryotic microorganisms. This unusual growth phenotype was due to an incom-
plete glucose repression of the function of the glyoxylate cycle, as shown by the lack of growth in that medium
of double pyc1 icl1 mutants lacking both pyruvate carboxylase and isocitrate lyase activity. These mutants grew
when glutamate, aspartate, or Casamino Acids were added to the glucose-ammonium medium. The cDNA from
the Y. lipolytica PYC1 gene complemented the growth defect of a Saccharomyces cerevisiae pyc1 pyc2 mutant, but

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introduction of either the S. cerevisiae PYC1 or PYC2 gene into Y. lipolytica did not result in detectable pyruvate
carboxylase activity or in growth on glucose-ammonium of a Y. lipolytica pyc1 icl1 double mutant.

Yarrowia lipolytica is a nonconventional yeast that has at-
tracted great attention due to its capacity to grow on unusual
carbon sources such as hydrocarbons, its potential as a host for
expression of heterologous proteins and its ability to shift be-
tween a yeast and a hyphal form (for reviews, see references 3
and 4). Surprisingly not much information is available on the
enzymes that participate in the main catabolic routes (22).
Since Y. lipolytica is an obligately respiratory organism, catab-
olism of all carbon sources transits through the tricarboxylic
acid cycle. This is a significant difference with fermentative
yeasts such as Saccharomyces cerevisiae, in which catabolism of
glucose and other sugars proceeds under many conditions,
mainly by fermentation.
The tricarboxylic acid cycle has an amphibolic role in me-
tabolism; it functions not only as an oxidative device coupled to
energy production but also provides building blocks for the
synthesis of important molecules such as porphyrins and sev-
eral amino acids (Fig. 1). Withdrawal of the intermediates of
the cycle for this last purpose would cause a stop of its function
if there were no other reactions to replenish it. In yeasts and
fungi growing in minimal medium, the two known ways to
replenish the tricarboxylic acid cycle are the reaction catalyzed
by pyruvate carboxylase and those catalyzed by isocitrate lyase
and malate synthase, which form the glyoxylate bypass (Fig. 1).
Pyruvate carboxylase coupled with phosphoenolpyruvate-car-
boxykinase also serves during growth in some nonsugar sub-
strates to provide phosphoenolpyruvate since the pyruvate ki-
nase reaction is irreversible under physiological conditions. In
* Corresponding author. Mailing address: Instituto de Investigacio-
nes Biomédicas “Alberto Sols,” CSIC-UAM, C/ Arturo Duperier 4,
E28029 Madrid, Spain. Phone: 34 91 585 44 31. Fax: 34 91 585 4401.
E-mail: clflores@iib.uam.es.
† We dedicate this paper to the memory of Professor Federico
Uruburu, microbiologist and friend who devoted much of his activity in
the Spanish Type Culture Collection to the service of the microbio-
logical community.
addition to these roles, pyruvate carboxylase has been shown to
be an essential factor in the assembly of the peroxisomal oligo-
meric alcohol oxidase in the methylotrophic yeasts Hansenula
polymorpha (now Pichia angusta) and Pichia pastoris (45).
Mutations that abolish pyruvate carboxylase activity make
eukaryotic microorganisms unable to grow in glucose-ammo-
nium medium due to the repressive effect of glucose on the
genes encoding the enzymes of the glyoxylate cycle (37, 45, 53,
57). Two genes, PYC1 and PYC2, encode isoenzymes of pyru-
vate carboxylase in Saccharomyces cerevisiae (57, 59), while
only one has been identified in Pichia pastoris (37) and in
Hansenula polymorpha (45).
Due to the variety of carbon sources used by Y. lipolytica and
to its strictly oxidative metabolism, we are interested in the study
of the anaplerotic reactions that replenish intermediates to the
tricarboxylic acid cycle in this yeast. We report in this work the
cloning and characterization of PYC1 from Y. lipolytica (here-
after YlPYC1). YlPYC1 is a unique gene that encodes pyruvate
carboxylase in this yeast and contains an intron of 269 bp. We
have found that, contrary to the situation in other yeasts, in Y.
lipolytica the absence of pyruvate carboxylase activity does not
preclude growth in glucose-ammonium medium. We demon-
strate that this is due to the incomplete repression by glucose
of the function of the glyoxylate cycle, since a double pyc1 icl1
mutant fails to grow in glucose-ammonium medium.
MATERIALS AND METHODS
Organisms and growth conditions. Y. lipolytica strain PO1a, MatA leu2-270 ura
3–302 xpr2-322, provided by A. Domı́nguez (Salamanca, Spain) was used. An S.
cerevisiae pyc1 pyc2 mutant had been previously constructed in the laboratory
(57). Yeasts were grown at 30°C in 1% yeast extract, 2% peptone, and 2%
glucose or in minimal medium YNB (Difco, Detroit, Mich.) with the carbon and
nitrogen sources indicated in the corresponding experiment at 2% and 40 mM,
respectively. Auxotrophic requirements were added at 0.02 mg/ml and Casamino
Acids (Difco) at 0.5%, when necessary hygromycin B was used at 80 ␮g/ml.
Growth was followed by measuring the optical density of the cultures. Transfor-
mation of yeasts was done with lithium acetate and heat shock (4, 29).

356
VOL. 4, 2005 Y. LIPOLYTICA pyc MUTANTS GROW IN GLUCOSE-AMMONIUM
FIG. 1. Anaplerotic (replenishing) reactions of the tricarboxylic
acid cycle in Y. lipolytica. The strictly respiratory metabolism of Y.
lipolytica directs the catabolism of carbon sources through the tricar-
boxylic acid cycle. Withdrawal of intermediates of the cycle for bio-
synthetic purposes makes necessary the action of anaplerotic enzymes.
In minimal medium with ammonium as the nitrogen source this role is
fulfilled by pyruvate carboxylase, Pyc, or by isocitrate lyase, Icl, and
malate synthase, Mls. Consecutive action of these last two enzymes
constitutes the so-called glyoxylate bypass. The figure does not take
into consideration the possible different subcellular compartmentation
of the reactions.
Isolation of the YlPYC1 gene. We designed degenerate oligonucleotides against
two regions of conserved amino acid sequences in pyruvate carboxylases from
diverse organisms, taking into account the codon bias of Y. lipolytica (http://www
.kazusa.or.jp/codon/). The oligonucleotides were 5⬘-TGGGGHGGHGCYACY
TTYGA-3⬘ and 5⬘-CATGAAYTGGGCCAGRRTCRCCA-3⬘. The standard ab-
breviations to represent ambiguity are used (H ⫽ A ⫹ T ⫹ G; Y ⫽ C ⫹ T; R ⫽
G ⫹ A)(40).
A PCR was performed with these oligonucleotides and Y. lipolytica genomic
DNA as the template. A DNA fragment of ca. 0.9 kb whose sequence had a high
homology to that of other pyruvate carboxylases was isolated. A genomic Y.
lipolytica library (41) donated by C. Gaillardin (INRA, Grignon, France) was
screened (26) with the 0.9-kb fragment as a probe. Three plasmids were isolated,
and one of them, pCL74, containing a 10-kb Y. lipolytica DNA insert was selected
for further work Further sequencing was done by plasmid walking. Since YlPYC1
contains an intron (see Results), the actual initial ATG of the gene was identified
with a 5⬘-rapid amplification of cDNA ends reaction with the RLM-RACE kit
(Ambion 1700, Austin, Tex.) and oligonucleotides 6581 5⬘-CGTCGGCCTTGA
ATCGGTGCATA-3⬘ as the inner primer and 6582 5⬘-CCGTGAGCCTTTGC
GATCTCGAT-3⬘ as the outer primer.
To obtain a DNA fragment comprising the YlPYC1 gene, oligonucleotides
6870 5⬘-TCGCACACCATGTCCAACGTTCC-3⬘ and 6871 5⬘-TAATTAAGCC
CGCACAATCTTGC-3⬘ were used for the 5⬘ and 3⬘ regions, respectively, in a
PCR with Y. lipolytica genomic DNA as the template and the Platinum PCR
357
SuperMix High Fidelity (Invitrogen, Carlsbad, Calif.). The product was cloned
into pGEM-Teasy to produce plasmid pGEM-Teasy-YlPYC.
Y. lipolytica PYC1 cDNA. RNA was obtained from yeasts grown in glucose-
ammonium medium and harvested during the exponential phase of growth. It
was extracted with the Trizol LS reagent (Invitrogen), and cDNA was prepared
with the First-Strand cDNA synthesis kit (Amersham Pharmacia Biotech).
YlPYC1 cDNA was obtained by PCR with oligonucleotides 6870 and 6871 (see
above). The fragment obtained was inserted into plasmid pGEM-Teasy to give
plasmid pGEM-Teasy-YlPYC1cDNA.
Disruption of YlPYC1. To disrupt YlPYC1, plasmid pGEM-Teasy-YlPYC was
digested with StuI, and a fragment of 1.4 kb was eliminated. A 2.1-kb DNA
fragment containing the Y. lipolytica LEU2 gene was obtained from plasmid
pINA62 (4) by digestion with NcoI. It was blunt ended and inserted into the
StuI-digested pGEM-Teasy-YlPYC. The disruption cassette was excised with
NotI and used to transform Y. lipolytica.
Expression of YlPYC1 cDNA in S. cerevisiae. Expression of YlPYC1 cDNA in S.
cerevisiae was from plasmid pCL64, which carries the coding part of YlPYC1
between the S. cerevisiae ADH1 promoter and terminator. To construct pCL64,
plasmid pGEM-Teasy-YlPYC1cDNA (see above) was digested with NotI, and
the 3,587-bp fragment carrying the Y. lipolytica cDNA was inserted into plasmid
pDB20 (6) digested with the same enzyme.
Expression of S. cerevisiae PYC1 or PYC2 in Y. lipolytica. A plasmid to carry S.
cerevisiae PYC1 (ScPYC1) or PYC2 (ScPYC2) situated between the promoter of
the YlTEF1 gene (39) and the terminator of the YlXPR2 gene (16) was con-
structed as follows. A platform plasmid, pINA444H-E, to receive the expression
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cassette was generated by elimination of the HindIII and EcoRI sites from
plasmid pINA444 (8). Plasmid pRWPYlTEF carrying the Y. lipolytica TEF1
promoter flanked by BamHI at the 5⬘ and by EcoRI at the 3⬘ site (a gift of A.
Winkler, Delft, The Netherlands) was digested with EcoRI, blunt ended, and
digested with SacI, and the 465-bp band between the two sites was eliminated.
The XPR2 terminator was obtained by PCR with genomic Y. lipolytica DNA as
the template and oligonucleotides 6664 5⬘-GATATCGCGGCCGCAATTAAC
AGATAGTTTGCCGGTG-3⬘ and 6665 5⬘-GGATCCAGATCTGAGCGTGAA
TTATACG-3⬘. These oligonucleotides create an EcoRV site (italics) and a NotI
site (underlined) at the 5⬘ end and a BamHI site at the 3⬘ site (double under-
lined). The ca. 0.5-kb DNA fragment obtained was inserted into pGEM-Teasy,
recovered by digestion with EcoRV and SacI, and directionally inserted into the
digested pRWPYlTEF. The cassette YlTEF1promoter-YlXPR2 terminator was
extracted from the resulting plasmid by cutting with BamHI and inserted into
pINA444H-E digested with the same enzyme to give plasmid pCL49.
The PYC1 and PYC2 genes from S. cerevisiae (57) were obtained by PCR,
cloned into pGEM-Teasy, excised with NotI, and inserted into pCL49 digested
with the same enzyme to give plasmids pCL53 and pCL77, respectively. These
plasmids were introduced in Y. lipolytica strain CL7B-CJM409, which lacks PYC1
(see Results). A control for the functionality of the inserted PYC1 and PYC2
genes was carried out by testing the complementation of an S. cerevisiae pyc1 pyc2
mutant. To express YlPYC1, ScPYC1 or ScPYC2 in the double mutant Ylpyc1
Ylicl1 plasmid pCL84 was constructed, replacing the YlURA3 marker in plasmid
pCL49 by the hygB resistance marker under the control of the YlHXK1 pro-
moter. Plasmid pCL84 was digested with NotI and YlPYC1, ScPYC1, or ScPYC2
was inserted to generate plasmids pCL85, pCL86, and pCL87, respectively.
Isolation and disruption of the YlICL1 gene. A fragment of 1,467 bp containing
almost the whole YlICL1 gene encoding isocitrate lyase was obtained by PCR
with oligonucleotides 8257 5⬘-CACCTACGCGTCCCTCGACCCCG-3⬘ and
8258 5⬘-GTCCAGCGCCTCTATCAAACTCG-3⬘ and cloned in pGEM-Teasy
to generate plasmid pRJ007 (kindly donated by R. Jardón, this laboratory). To
disrupt YlICL1, this plasmid was digested with EcoRV and a fragment of 1.7 kb
containing the YlURA3 obtained from pINA156 (50) digested with SalI and blunt
ended was ligated into it. The whole disruption cassette was extracted with NotI
and used to transform the corresponding strains of Y. lipolytica.
DNA manipulations. Recombinant DNA techniques were done according to
established protocols. DNA sequencing was performed by the dideoxy chain
termination method (51). All products were sequenced at least twice in each
direction. For northern analysis the yeasts were filtered and flash frozen in liquid
nitrogen as in (7). Total RNA was extracted with the Trizol LS reagent (Invitro-
gen), separated in 1.5% agarose-formaldehyde gels and transferred to Nytran
filters (Schleicher and Schuell, Dassel, Germany). Probes were labeled with 32P
with the Rediprime II Random Prime labeling system (Amersham Biosciences).
As probes in Northern blots a 0.9-kb NcoI-NcoI fragment for YlPYC1 and a
0.7-kb SalI-NcoI fragment for YlICL1 were used.
Preparations of extracts and enzymatic assays. Extracts were made by shaking
yeasts with glass beads in 0.1 M HEPES–1 mM EDTA–1 mM dithiothreitol, pH
7.4, for five periods of 1 min with an interval of 1 min in ice between each
358 FLORES AND GANCEDO EUKARYOT. CELL

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FIG. 2. Disruption of the YlPYC1 and YlICL1 genes in Y. lipolytica. a) Scheme of the disruption of YlPYC1 with YlLEU2, and Southern blot
analysis of the disruption (for details see Materials and Methods). Genomic DNA was digested with BglII or HindIII and probed with the indicated
BamHI-StuI fragment. b) Scheme of the disruption of YlICL1 with YlURA3, and PCR analysis of the disruption with the oligonucleotides shown
by the numbers in italics (for details see Materials and Methods). Numbers indicate distances in kilobases from the left 5⬘ end. Po1a, wild type;
CL7B-CJM 401, Ylpyc1; CJM418, Ylicl1.

vortexing. Pyruvate carboxylase activity was assayed spectrophotometrically ba-
sically as described by van Urk et al. (58). Interference with other NADH-
utilizing systems was avoided by the addition of 2 mM KCN. The activity mea-
sured was dependent on ATP addition and completely inhibited by incubation
with avidin, showing that it was a bona fide pyruvate carboxylase activity. Isocit-
rate lyase was assayed as described in reference 17. Protein was assayed with the
commercial BCA protein assay (Pierce).
Nucleotide sequence accession number. The sequence of the genomic region
of the YlPYC1 gene has been deposited in the GenBank database with accession
number AY142711.
RESULTS
Isolation of the gene encoding pyruvate carboxylase from
Y. lipolytica. Alignment of amino acid sequences from different
eukaryotic pyruvate carboxylases present in the databases al-
lowed the design of degenerate oligonucleotides correspond-
ing to two conserved regions of the protein. A DNA fragment
of 0.9 kb was obtained by PCR with genomic Y. lipolytica DNA
as the template, and this fragment was used as the starting
material to isolate the whole gene as described in detail in Ma-
terials and Methods. The isolated gene, which has been named
YlPYC1, is 3,844 bp long, and as usual in other Y. lipolytica genes
(19), exhibits a high CG content (56%). An examination of the
YlPYC1 sequence revealed a 269-bp-long intron located 133 bp
downstream of the initial ATG. The sequence of the branch mo-
tif of the intron CCCTAAC had not been previously reported
in Y. lipolytica (11) but we have found it in YlPYC1 sequences
from three strains of independent origins, showing that it is a
new, previously unrecognized branch motif in this organism.
The cDNA sequence predicts a protein of 1,192 amino acids
with 70% identity with the pyruvate carboxylases of P. pastoris,
H. polymorpha, and with those encoded by the PYC1 and PYC2
genes from S. cerevisiae.
Disruption of YlPYC1 does not abolish growth in glucose-
ammonium minimal medium. Elimination of pyruvate carbox-
ylase activity abolishes growth in glucose-ammonium minimal
medium in all the eukaryotic microorganisms so far tested (37,
45, 53, 57). We examined if this was also the case in Y. lipoly-
tica. The chromosomal copy of YlPYC1 was disrupted by in-
sertion of the YlLEU2 gene as described in Materials and
Methods. After transformation of the yeast with the disruption
cassette, transformants were selected by leucine prototrophy
on glucose-glutamate plates and replica-plated to minimal me-
dium glucose-ammonium plates. Unexpectedly, all colonies ap-
peared to grow on this last medium; however, a careful inspec-
tion of the plates showed some colonies of slightly smaller size.
We selected them and performed a PCR check of the dis-
ruption. One of those giving a pattern compatible with a cor-
VOL. 4, 2005 Y. LIPOLYTICA pyc MUTANTS GROW IN GLUCOSE-AMMONIUM 359

TABLE 1. Specific activities of pyruvate carboxylase and unusual growth phenotype of the Ylpyc1::LEU2 disruptant in
isocitrate lyase in different strains of Y. lipolyticaa such conditions.
Activity Disruption of YlICL1 in a Ylpyc1::LEU2 strain abolishes
(nmol/min/mg of protein) growth in glucose-ammonium minimal medium. According to
Strain and relevant genotype
Pyruvate Isocitrate present knowledge, the only anaplerotic route in yeasts besides
carboxylase lyase pyruvate carboxylase is the glyoxylate bypass (23, 35). This
Po1a (wild type) 67 ⫾ 9 59 ⫾ 12 bypass appears to be nonfunctional during growth in glucose in
CL7B-CJM 409 (Ylpyc1::LEU2) ⬍1 80 ⫾ 13 the cases studied up to now due to glucose repression (24, 31).
CJM 418 (Ylicl1::URA3) ND ⬍1 However, we observed that glucose-grown cultures of Y. lipo-
a
The strains were grown in minimal glucose-ammonium medium, harvested at lytica always exhibited a significant Icl activity (Table 1). This
an optical density between 0.96 and 1.3, and extracted as described in Materials activity is 8 to 10 times lower than that measured in ethanol
and Methods. Values are means and standard deviations for at least four inde- or acetate cultures (R. Jardón, personal communication) but
pendent cultures. ND, not done.
could be high enough to support a functional anaplerotic path-
way and could explain the growth on glucose-ammonium min-
rect YlPYC1 disruption was subjected to Southern analysis. As imal medium of the Ylpyc1::LEU2 strain. If this hypothesis
can be seen in Fig. 2a, it carries a correct disruption of the were correct, disruption of the YlICL1 gene in a Ylpyc1::LEU2
YlPYC1 gene. One possibility to explain the unusual growth of strain should abolish that growth. We isolated this gene and
the disruptants would be the existence of two genes encoding disrupted it in a wild-type and in a Ylpyc1::LEU2 strain as
pyruvate carboxylase activity, as is the case in S. cerevisiae (57, described in Materials and Methods. The correctness of the
59); however, measurements of pyruvate carboxylase activity in disruption was ascertained by PCR (Fig. 2b) and measure-
the disruptant showed that there was not a detectable pyruvate ments of the enzymatic activity (Table 1). It should be noticed

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carboxylase activity in the extracts (Table 1). This result indi-
cates that there is only one gene encoding pyruvate carboxylase
in Y. lipolytica.
This conclusion has been strengthened after the publication
of the genomic sequence of Y. lipolytica (19) (http://cbi.labri.fr
/Genolevures) in which no other open reading frame with ho-
mology to PYC genes different from AY142711 is found. Re-
markably, the absence of pyruvate carboxylase activity did not
significantly interfere with growth in glucose-ammonium min-
imal medium (Fig. 3) and caused an increase in generation
time of the mutant in that medium of only about 12% (170 min
for the wild type versus 190 min for the pyc1 mutant). These
results open the question about the mechanism that allows the
that in the cultures in which pyruvate carboxylase activity was
absent, a small but consistent increase of Icl activity was mea-
sured (Table 1).
Growth assays showed that while single disruptant Ylpyc1::
LEU2 and Ylicl1::URA3 strains grew on minimal medium glu-
cose-ammonium plates, the double disruptant Ylpyc1::LEU2
Ylicl1::URA3 was unable to grow in this medium unless Cas-
amino Acids, glutamate, and aspartate were added to it (Fig.
3). This result supports our idea that the growth of the Ylpyc1::
LEU2 strain in glucose-ammonium medium is due to the op-
eration of the glyoxylate cycle. Reintroduction of the YlPYC1
cDNA in the double Ylpyc1 Ylicl1 mutant restored growth on
glucose-ammonium (Fig. 4). In agreement with previous re-

FIG. 3. Growth of different strains of Y. lipolytica in medium with glucose or ethanol as the carbon source and ammonium, ammonium plus
Casamino Acids, aspartate, or glutamate as the nitrogen source. The wild-type strain (Wt), the single disruptant strains Ylpyc1::LEU2 (pyc1) and
Ylicl1::URA3 (icl1), and the double disruptant Ylpyc1::LEU2 Ylicl1::URA3 (pyc1 icl1) were grown on plates with the indicated carbon and nitrogen
sources; 5 ␮l of suspensions of about 3 ⫻ 106 cells/ml and successive 20-fold dilutions of each strain were dropped on the plates. The picture was
taken 2 days after inoculation and incubation at 30°C.
360 FLORES AND GANCEDO EUKARYOT. CELL

FIG. 4. Growth of the double Ylpyc1::LEU2 Ylicl1::URA3 mutant on glucose-ammonium is restored by reintroduction of the YlPYC1 gene.
From left to right: growth of the double Ylpyc1 Ylicl1 mutant transformed with plasmids pCL85 carrying the YlPYC1 gene or pCL84, void. These plas-
mids carry the dominant hygB resistance marker (see Materials and Methods). The picture was taken 4 days after inoculation and incubation at 30°C.

sults (5), growth on ethanol was not possible whenever Icl was on the expression of YlPYC1. Comparison of glucose-glutamate
absent. and glucose-ammonium medium (Fig. 5) showed that during
The double Ylpyc1 Ylicl1 mutant lost viability when placed in the exponential phase of growth the YlPYC1 mRNA levels were
the nonpermissive glucose-ammonium medium. Twenty-four decreased in the first case, a finding consistent with the lower

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hours after the transfer to the nonpermissive medium, about
70% of the population remained viable, but after 72 h less than
1% of the population was viable. A wild-type control suspend-
ed for 72 h in a medium without a nitrogen source remained
100% viable. We have no obvious explanation for this behavior.
Analysis of YlPYC1 expression. Due to the anaplerotic role
ascribed to pyruvate carboxylase, it appeared of interest to as-
sess the effect of carbon and nitrogen sources in the medium
requirements of pyruvate carboxylase if replenishment of the
tricarboxylic acid cycle is taken up by glutamate supply. Figure
5a shows that YlICL1 mRNA is clearly detectable in glucose
grown cultures independently of the nitrogen source and also
that in cultures with glutamate its level greatly decreased in the
stationary phase. Mutations in YlPYC1 had influence on the
levels of YlICL1 mRNA and vice versa: the most important
changes were the increase in YlPYC1 mRNA levels during the

FIG. 5. Northern blot analysis of YlPYC1 and YlICL1 transcription. a) mRNA levels of YlPYC1 and YlICL1 in the wild type and in the indicated
mutant strains were compared. The yeast cells were grown in YNB medium with glucose (Glu) as the carbon source and ammonium (NH4⫹) or
glutamate (Glut) as the nitrogen source. b) mRNA levels of YlPYC1 in the wild-type strain grown in YNB medium with ethanol (EtOH) as the
carbon source and glutamate as the nitrogen source. Cells were harvested at the exponential (X) and the stationary (S) phases of growth, and the
extraction and separation of RNA were done as indicated in Material and Methods.
VOL. 4, 2005 Y. LIPOLYTICA pyc MUTANTS GROW IN GLUCOSE-AMMONIUM
FIG. 6. Expression of the heterologous YlPYC1 gene in an S. cerevisiae pyc1 pyc2 mutant and ScPYC1 and ScPYC2 in a Y. lipolytica pyc1 icl1
mutant. a) The Y. lipolytica PYC1 cDNA complements the growth defect of an S. cerevisiae pyc1 pyc2 mutant (Scpyc⫺). b) The S. cerevisiae PYC1
and PYC2 genes do not complement the growth defect of a Y. lipolytica pyc1 icl1 mutant. (For a description of the plasmids used, see Materials
and Methods).
exponential phase of growth in glucose in the Ylicl1 mutant and
the increase of YlICL1 mRNA levels in a Ylpyc1 background
with respect to the wild type (Fig. 5a). A decrease in the level
of YlPYC1 mRNA was observed in ethanol cultures at the
stationary phase (Fig. 5b).
Heterologous cross-complementation of S. cerevisiae pyc1
pyc2, Ylpyc1, and Ylpyc1 Ylicl1 mutants. Expression of the
YlPYC1 cDNA in an S. cerevisiae pyc1 pyc2 mutant restored
wild-type ability to grow in minimal glucose-ammonium me-
dium indicating the functionality of the Y. lipolytica protein in
S. cerevisiae (Fig. 6a). However, when a Ylpyc1::LEU2 strain
was transformed with plasmid pCL53 or pCL77, carrying the S.
cerevisiae PYC1 or PYC2 genes, respectively, no detectable
pyruvate carboxylase activity was found. When these genes were
introduced in a double Yl pyc1 Yl icl1 mutant no growth on
glucose ammonium medium was observed (Fig. 6b) thus indi-
cating that the heterologous proteins were nonfunctional in Y.
lipolytica. This different behavior in the cross-complementation
might be ascribed to the biased codon usage of Y. lipolytica (19).
DISCUSSION
We have identified and cloned the YlPYC1 gene encoding
pyruvate carboxylase in Y. lipolytica. The strong identity in
sequence of the protein deduced with pyruvate carboxylases of
diverse origin (about 70% with those of fungal origin and 40%
with those of other origins) clearly indicate that the gene en-
codes this activity. More conclusive evidence for the gene be-
ing YlPYC1 is provided by the lack of pyruvate carboxylase
activity when the gene is disrupted, and by the complementa-
361
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tion of the S. cerevisiae pyc1 pyc2 growth phenotype when the
corresponding Y. lipolytica cDNA is expressed in such a strain.
The YlPYC1 gene contains an intron of 269 bp situated 133
bp downstream from the starting ATG. In Y. lipolytica introns
in genes encoding glycolytic or related enzymes have been
described for pyruvate kinase and for hexokinase (47, 55, 56)
and others are being detected in the Génolevures project (21).
The most frequently used 5⬘ and 3⬘ splice sites of spliceosomal
introns in Y. lipolytica are different from those more frequently
found in S. cerevisiae (11) and the sites found in YlPYC1 share
those sequences. However, in the branch motif of the YlPYC1
intron, the sequence CCCTAAC exists that has not yet been
described among the series of new branch motifs recently re-
ported (11). The authenticity of this sequence is validated by its
presence in strains from different origins.
As is the case in some yeasts such as Pichia pastoris (37) and
Hansenula polymorpha (45), there is only one gene encoding
pyruvate carboxylase in Y. lipolytica. This is an important dif-
ference with S. cerevisiae, which possesses two genes, PYC1 and
PYC2, encoding two pyruvate carboxylase isoenzymes (57, 59).
These genes appear to have a different regulation of expression
and exhibit some kinetic differences (12, 28), but in spite of
these characteristics, the physiological significance of the exis-
tence of the two genes is not completely understood at present.
In this context it should be borne in mind that S. cerevisiae
appears to have duplicated its genome during evolution and
later lost some of it (25, 32, 48, 60). Interestingly, some pro-
karyotic organisms possess two anaplerotic enzymes providing
oxaloacetate: pyruvate carboxylase and phosphoenolpyruvate-
362 FLORES AND GANCEDO
carboxylase (20, 38, 42, 46), and only disruption of both abol-
ishes growth in glucose-ammonium minimal medium (46). The
physiological advantage of the existence of two different reac-
tions to provide oxaloacetate in these organisms has not yet
been satisfactorily elucidated.
The phenotype produced by the disruption of the YlPYC1
gene shows an important difference with that observed in other
eukaryotic microorganisms lacking this activity. Mutants of
different yeasts and fungi devoid of pyruvate carboxylase ac-
tivity require either aspartate or glutamate to grow in glucose
in a minimal ammonium medium (37, 45, 53, 57), but the pyc1
mutant of Y. lipolytica grew without any of these additions. This
result shows that it is not always possible to transfer informa-
tion from observed phenotypes from one yeast species to an-
other.
We have found that the reason for the lack of obvious growth
phenotype of the pyc1 mutant of Y. lipolytica is the incomplete
catabolite repression of isocitrate lyase in this yeast. This lim-
ited repression contrasts with the situation in other yeasts and
fungi in which the gene encoding Icl is repressed by glucose (9,
36) and the enzyme, in some cases, subjected to catabolite in-
activation by the sugar (1, 43). The explanation proposed for
the behavior of the Ylpyc1 mutant is supported by the lack of
growth in glucose-ammonium minimal medium of a double
Ylpyc1 Ylicl1 mutant.
It has been demonstrated in S. cerevisiae and in P. pastoris
(10, 37) that abolition of catabolite repression restores the
ability to grow in glucose-ammonium minimal medium to mu-
tants devoid of pyruvate carboxylase activity. The proposed
mechanism is that in these conditions, the glyoxylate bypass is
operative. Catabolite repression is very strong in S. cerevisiae
and depends on a strong glycolytic flux (24, 31, 52); reduction
of the flux by different mutations (33, 44, 49) and cultivation
under sugar limitation (15) abolish or at least diminish the
magnitude of the repression.
Yeasts with lower glycolytic capacity may show a lower de-
gree of catabolite repression, and this appears to be the case in
Y lipolytica. The obligate respiratory metabolism of this yeast
and its low rate of glucose utilization (18) could explain why
repression of Icl by glucose is not complete. The physiological
significance of catabolite repression appears to be the avoid-
ance of energy loss due to synthesis and operation of unneeded
enzymes during growth in glucose. The incomplete catabolite
repression in Y. lipolytica might be explained assuming that the
higher ATP yield obtained from glucose by respiration could
allow the energy waste without detrimental effects.
The result of the lack of growth in glucose-ammonium me-
dium of the double pyc1 icl1 mutant also shows that in Y. li-
polytica there is no other physiologically active pathway to
replenish the tricarboxylic acid cycle besides the ones known in
other yeasts and fungi. Barth and Scheuber (5) observed that
deletion of YlICL1 not only affected the use of some gluconeo-
genic carbon sources but also appeared to reduce the rate of
growth in glucose. The effect was interpreted as the conse-
quence of a possible participation of the glyoxylate cycle in the
maintenance of the glycine pool (5).
The effect of the absence of pyruvate carboxylase on growth
in glucose was not much marked as judged from the small
difference in generation time between the wild type and the
mutant. Since the observation of Barth and Scheuber (5) also
EUKARYOT. CELL
indicated a decrease in growth rate in glucose of an icl1 mu-
tant, it seems that neither of the two anaplerotic reactions by
itself is able to cope effectively with the needs of the cell. The
determination of the contribution of each of these reactions to
the needs of a wild-type cell is an interesting question to ap-
proach.
The observed behavior of the YlPYC1 mRNA in glucose-
glutamate versus glucose-ammonium cultures is consistent with
the anaplerotic role ascribed to pyruvate carboxylase. Huet et
al. (28) observed that the transfer of S. cerevisiae cells from a
glutamate medium to an ammonium medium caused a fivefold
increase in the mRNAs corresponding to the PYC1 gene in less
than 30 min, whereas a decrease occurred when the transfer
was done in the opposite direction. The decrease in mRNA
seen in stationary-phase ethanol cultures is reminiscent of the
behavior of the PYC2 gene from S. cerevisiae reported by Brew-
ster et al. (13). These authors observed a fourfold decrease in
the levels of that mRNA after the initial exponential phase of
growth. The mechanisms that produce these changes have not
been clearly elucidated.
YlICL1 mRNA was detected in glucose-grown cultures, a
Downloaded from ec.asm.org by on September 2, 2007
result consistent with the enzymatic activity measurements and
with our proposed mechanism for the growth of the Ylpyc1
mutant. In addition to that, the YlICL1 mRNA showed a small
increase in an Ylpyc1 mutant with respect to the wild type. This
is also in accordance with the increase in Icl activity measured
in this mutant. When the mRNA levels of YlPYC1 during the
exponential phase of growth in glucose were measured in a
Ylicl1 mutant, a small increase was observed. These results
could suggest a coordinated regulation of the transcription of
both genes in Y. lipolytica.
The lack of functional pyruvate carboxylase when the S. cer-
evisiae PYC1 and PYC2 genes are introduced into Y. lipolytica
under the control of a Y. lipolytica gene promoter is noteworthy
if one considers that YlPYC1 complements an S. cerevisiae pyc1
pyc2 mutant. We hypothesize that the difference in codon us-
age between the two yeasts (19) may explain the lack of func-
tional enzyme. The genome from Y. lipolytica is richer in G⫹C
than that of S. cerevisiae and differences in this content have
been reported in some cases to be responsible for lack of func-
tionality of heterologous proteins (30).
Several differences in behavior or properties of some Y. li-
polytica enzymes from central metabolism like hexokinase (47),
phosphofructokinase (C.-L. Flores, O. Martı́nez-Costa, V. Sán-
chez, C. Gancedo, and J. J. Aragón, unpublished data), 3-phos-
phoglycerate kinase (34), pyruvate kinase (27), and pyruvate
carboxylase (this work) with respect to their counterparts in
S. cerevisiae are now well documented. The early separation in
evolution of Y. lipolytica from other yeasts (2, 14, 54) may be at
the basis of the differences observed. All these findings show
that a detailed study of Y. lipolytica metabolism is necessary
before engaging in attempts to use this yeast in biotechnolog-
ical applications.
ACKNOWLEDGMENTS
We thank Juana M. Gancedo (IIB, Madrid, Spain), J. P. van Dijken
and J. Pronk (TU, Delft, The Netherlands), and M. A. Blázquez
(IBMCP, Valencia, Spain) for critical reading and comments on the
manuscript, A. Domı́nguez (IMBQ, Salamanca, Spain) and C. Gail-
lardin (INRA, Grignon, France) for providing yeast strains, plasmids,
or technical tips.
VOL. 4, 2005 Y. LIPOLYTICA pyc MUTANTS GROW IN GLUCOSE-AMMONIUM
This work has been supported by grant BMC-1691-2001-CO2-01
from the Spanish Ministry of Science and Technology to C. Gancedo.
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