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Eukaryotic Cell, February 2005, p. 356-364, Vol. 4, No. 2
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{dagger}

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


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ABSTRACT
 
We have cloned and characterized the gene PYC1, encoding the unique pyruvate carboxylase in the dimorphic 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 medium, as is the case in other eukaryotic microorganisms. This unusual growth phenotype was due to an incomplete 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 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.


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INTRODUCTION
 
Yarrowia lipolytica is a nonconventional yeast that has attracted 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 between 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, catabolism 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 metabolism; 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 several 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-carboxykinase also serves during growth in some nonsugar substrates to provide phosphoenolpyruvate since the pyruvate kinase reaction is irreversible under physiological conditions. In addition to these roles, pyruvate carboxylase has been shown to be an essential factor in the assembly of the peroxisomal oligomeric alcohol oxidase in the methylotrophic yeasts Hansenula polymorpha (now Pichia angusta) and Pichia pastoris (45).



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  		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 tricarboxylic acid cycle. Withdrawal of intermediates of the cycle for biosynthetic 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.

Mutations that abolish pyruvate carboxylase activity make eukaryotic microorganisms unable to grow in glucose-ammonium 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 pyruvate 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 (hereafter 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 demonstrate 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.


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MATERIALS AND METHODS
 
Organisms and growth conditions. Y. lipolytica strain PO1a, MatA leu2-270 ura 3-302 xpr2-322, provided by A. Domínguez (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 µg/ml. Growth was followed by measuring the optical density of the cultures. Transformation of yeasts was done with lithium acetate and heat shock (4, 29).

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'-TGGGGHGGHGCYACYTTYGA-3' and 5'-CATGAAYTGGGCCAGRRTCRCCA-3'. The standard abbreviations 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'-CGTCGGCCTTGAATCGGTGCATA-3' as the inner primer and 6582 5'-CCGTGAGCCTTTGCGATCTCGAT-3' as the outer primer.

To obtain a DNA fragment comprising the YlPYC1 gene, oligonucleotides 6870 5'-TCGCACACCATGTCCAACGTTCC-3' and 6871 5'-TAATTAAGCCCGCACAATCTTGC-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 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 constructed as follows. A platform plasmid, pINA444H-E, to receive the expression 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'-GATATCGCGGCCGCAATTAACAGATAGTTTGCCGGTG-3' and 6665 5'-GGATCCAGATCTGAGCGTGAATTATACG-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 underlined). 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 promoter. 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 (Invitrogen), 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 vortexing. Pyruvate carboxylase activity was assayed spectrophotometrically basically 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 measured was dependent on ATP addition and completely inhibited by incubation with avidin, showing that it was a bona fide pyruvate carboxylase activity. Isocitrate 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.


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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 allowed the design of degenerate oligonucleotides corresponding 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 Materials 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 motif 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 carboxylase 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. lipolytica. The chromosomal copy of YlPYC1 was disrupted by insertion 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 medium glucose-ammonium plates. Unexpectedly, all colonies appeared to grow on this last medium; however, a careful inspection of the plates showed some colonies of slightly smaller size.

We selected them and performed a PCR check of the disruption. One of those giving a pattern compatible with a correct YlPYC1 disruption was subjected to Southern analysis. As can be seen in Fig. 2a, it carries a correct disruption of the YlPYC1 gene. One possibility to explain the unusual growth of the disruptants would be the existence of two genes encoding pyruvate carboxylase activity, as is the case in S. cerevisiae (57, 59); however, measurements of pyruvate carboxylase activity in the disruptant showed that there was not a detectable pyruvate carboxylase activity in the extracts (Table 1). This result indicates that there is only one gene encoding pyruvate carboxylase in Y. lipolytica.



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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.


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  		TABLE 1. Specific activities of pyruvate carboxylase and isocitrate lyase in different strains of Y. lipolyticaa

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 homology to PYC genes different from AY142711 [GenBank] is found. Remarkably, the absence of pyruvate carboxylase activity did not significantly interfere with growth in glucose-ammonium minimal 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 unusual growth phenotype of the Ylpyc1::LEU2 disruptant in such conditions.



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  		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 µl of suspensions of about 3 x 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.

Disruption of YlICL1 in a Ylpyc1::LEU2 strain abolishes growth in glucose-ammonium minimal medium. According to present knowledge, the only anaplerotic route in yeasts besides pyruvate carboxylase is the glyoxylate bypass (23, 35). This bypass appears to be nonfunctional during growth in glucose in the cases studied up to now due to glucose repression (24, 31). However, we observed that glucose-grown cultures of Y. lipolytica always exhibited a significant Icl activity (Table 1). This activity is 8 to 10 times lower than that measured in ethanol or acetate cultures (R. Jardón, personal communication) but could be high enough to support a functional anaplerotic pathway and could explain the growth on glucose-ammonium minimal medium of the Ylpyc1::LEU2 strain. If this hypothesis were correct, disruption of the YlICL1 gene in a Ylpyc1::LEU2 strain should abolish that growth. We isolated this gene and disrupted it in a wild-type and in a Ylpyc1::LEU2 strain as described in Materials and Methods. The correctness of the disruption was ascertained by PCR (Fig. 2b) and measurements of the enzymatic activity (Table 1). It should be noticed that in the cultures in which pyruvate carboxylase activity was absent, a small but consistent increase of Icl activity was measured (Table 1).

Growth assays showed that while single disruptant Ylpyc1::LEU2 and Ylicl1::URA3 strains grew on minimal medium glucose-ammonium plates, the double disruptant Ylpyc1::LEU2 Ylicl1::URA3 was unable to grow in this medium unless Casamino 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 operation 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 results (5), growth on ethanol was not possible whenever Icl was absent.



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  		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 plasmids carry the dominant hygB resistance marker (see Materials and Methods). The picture was taken 4 days after inoculation and incubation at 30°C.

The double Ylpyc1 Ylicl1 mutant lost viability when placed in the nonpermissive glucose-ammonium medium. Twenty-four 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 suspended 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 assess the effect of carbon and nitrogen sources in the medium on the expression of YlPYC1. Comparison of glucose-glutamate and glucose-ammonium medium (Fig. 5) showed that during the exponential phase of growth the YlPYC1 mRNA levels were decreased in the first case, a finding consistent with the lower 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 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).



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  		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.

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 medium 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 indicating 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).



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  		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).


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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 encodes this activity. More conclusive evidence for the gene being YlPYC1 is provided by the lack of pyruvate carboxylase activity when the gene is disrupted, and by the complementation 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 reported (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 difference 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 existence 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 prokaryotic organisms possess two anaplerotic enzymes providing oxaloacetate: pyruvate carboxylase and phosphoenolpyruvate-carboxylase (20, 38, 42, 46), and only disruption of both abolishes growth in glucose-ammonium minimal medium (46). The physiological advantage of the existence of two different reactions 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 activity 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 information from observed phenotypes from one yeast species to another.

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 limited 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 inactivation 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 mutants 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 degree 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 avoidance 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 medium of the double pyc1 icl1 mutant also shows that in Y. lipolytica 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 gluconeogenic carbon sources but also appeared to reduce the rate of growth in glucose. The effect was interpreted as the consequence 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 indicated a decrease in growth rate in glucose of an icl1 mutant, 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 approach.

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 Brewster 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 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. cerevisiae 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 usage between the two yeasts (19) may explain the lack of functional 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 functionality of heterologous proteins (30).

Several differences in behavior or properties of some Y. lipolytica enzymes from central metabolism like hexokinase (47), phosphofructokinase (C.-L. Flores, O. Martínez-Costa, V. Sánchez, C. Gancedo, and J. J. Aragón, unpublished data), 3-phosphoglycerate 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 biotechnological applications.


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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. Domínguez (IMBQ, Salamanca, Spain) and C. Gaillardin (INRA, Grignon, France) for providing yeast strains, plasmids, or technical tips.

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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FOOTNOTES
 
* Corresponding author. Mailing address: Instituto de Investigaciones 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{at}iib.uam.es. Back

{dagger} 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 microbiological community. Back


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Eukaryotic Cell, February 2005, p. 356-364, Vol. 4, No. 2
1535-9778/05/$08.00+0     doi:10.1128/EC.4.2.356-364.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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