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Saccharomyces cerevisiae is one of the best characterized yeasts for the production of heterologous proteins in eukaryotic organisms (Gellissen et al., 1992). The constitutive promoters most commonly used in this yeast are derived from the phosphoglycerate kinase (PGK), alcohol dehydrogenase (ADH) and glyceraldehyde-3-phosphate dehydrogenase (GAP) genes. The inducible promoters commonly used are derived from the galactose-regulated genes GAL1, GAL7 and GAL10 or from the phosphate-regulated gene PHO5, which is induced in response to phosphate starvation (Mendoza-Vega et al., 1994; Schneider and Guarente, 1991).
In recent years, alternative yeast species such as Hansenula polymorpha, Yarrowia lipolytica, Candida tropicalis and Pichia pastoris have gained popularity for the expression of heterologous proteins. P. pastoris has become one of the yeast species most commonly used for academic and commercial purposes (Romanos et al., 1992). Although classical and molecular genetics techniques are well developed for P. pastoris (Higgins and Cregg, 1998), only a few regulated promoters are available for the expression of heterologous proteins.
Most P. pastoris expression vectors use the alcohol oxidase promoter region (pAOX1). One advantage of using this promoter is its cheaper carbon source, such as methanol, to induce the expression of the recombinant proteins. Also, this expression system is highly regulated, favouring its use in the expression of products that could be toxic for the producer host.
In spite of the great number of proteins that have been successfully expressed in P. pastoris using the AOX1 promoter (Cereghino and Cregg, 2000), it is of great interest to have alternative promoter regions regulated by carbon sources other than methanol. This has particular significance for scaled-up fermentations where large volumes of methanol are used, since this product may be a cause of fire hazard. Therefore, several genes which are not induced by methanol (GAP, FLD1, PEX8 and YPT1) have been isolated, and their promoters have been used for the expression of heterologous proteins in P. pastoris (Cereghino and Cregg, 2000).
In this paper, we report the isolation and sequencing of the P. pastoris isocitrate lyase gene (ICL1). We show that the expression of ICL1 gene in P. pastoris is repressed in the presence of glucose and induced in its absence, or in presence of ethanol. Due to this characteristic, the use of this promoter may be an attractive alternative to the conventional pAOX1 promoter for the expression of foreign proteins in P. pastoris.
Materials and methods
Strains and culture conditions
The Escherichia coli strains used in this work were MC1061 (F−araD139 Δ(ara-leu)7696galE15 galK16 Δ(lac)X74 rpsL (Strr) hsdR2 (rk− mk+) mcrA mcrB1) as genomic library host and XL1-Blue (F′::Tn10proA+B+lacIq(lacZ)M15/recA1 endA1 gyrA96 (NaIr) thi hsdR17 (rk− mk+) supE44 relA1 lac) for routine recombinant DNA methods. Growth of bacterial cultures was performed on Luria–Bertani (LB) medium (Sambrook et al., 1989) at 37 °C, supplemented with ampicillin (100 µg/ml) when required.
The yeast strains used in this study were P. pastoris strains BKMY-90 (CIGB, Havana, Cuba) and MP36 his3− (Yong et al., 1992). S. cerevisiae FMY402 (MATaicl1::LEU2 leu2-3, 112 ura3-(fs) his3-11, 15) (Ordiz et al., 1995), kindly provided by Dr Fernando Moreno (Universidad de Oviedo, Spain), was used in the ICL1 complementation assay.
Rich media were based on 1% yeast extract and 2% peptone, and 2% glucose or 3% ethanol were added as carbon sources. Synthetic media (YNB), consisting of 0.67% yeast nitrogen base without amino acids, was supplemented with amino acids as required and with 2% glucose or 3% ethanol as carbon source. Glucose-repressed cultures were harvested at the middle of the exponential phase of growth, when the glucose concentration in the medium was 95 mM. Derepressed cultures were harvested after 24 h of growth, when no glucose was detected in the cultures.
General DNA methods
Routine recombinant DNA methodology was performed according to Sambrook et al. (1989). High specific-activity labelling of DNA hybridization probes was carried out by random hexamer priming (Feinberg and Vogelstein, 1983), using [α32P]dATP (>3000 Ci/mmol, Amersham). For Southern blot analyses, DNA from P. pastoris was digested and transferred to Hybond-N membranes (Amersham) and hybridized overnight at 42 °C in 6× SSC buffer containing 5× Denhardt's solution, 0.5% SDS, 50% formamide and 200 µg/ml denatured, fragmented salmon sperm DNA (Sambrook et al., 1989). Unbound probe was removed by two washes with 2× SSC, 0.1% SDS at room temperature and one with 0.2× SSC, 0.1% SDS at 65 °C. Plasmid DNA isolated from the positive colonies was sequenced by the dideoxy-chain termination method (Sanger et al., 1977), using Sequenase (Version 2.0) Sequencing Kit (Amersham, USB), according to the manufacturer's instructions. Genomic DNA from yeast cells was prepared as described by Sherman et al. (1986).
S. cerevisiae was transformed according to the lithium sulphate method (Ito et al., 1983). Transformation of the MP36 strain of P. pastoris was carried out by electroporation (Becker and Guarente, 1991).
Expression of P. pastoris ICL1 gene in S. cerevisiae
The following construction was made to express the P. pastoris ICL1 gene under its 5′ non-coding region in S. cerevisiae. The 2.8 Kb EcoRI–DraI fragment from the ICL1 gene, which includes 0.68 Kb of its own promoter, and its 3′ terminator region was subcloned into the pRS316 plasmid (Sikorski and Hieter, 1989), previously digested with EcoRI and SmaI, to obtain the plasmid pRS316–ICL1.
The disruption of the ICL1 gene from P. pastoris was done using the HIS3 gene of S. cerevisiae as follows: plasmid pIVICLPp containing the ICL1 gene was digested with EcoRI and DraI and the 2.6 Kb fragment was inserted into pOV10 plasmid (Vincent and Gancedo, 1995), previously digested with SalI, blunt-ended, and then digested with EcoRI. The resulting plasmid was named pOVICL. The pOVICL plasmid was digested with SmaI and BamHI and the 2 Kb XbaI/Klenow–SmaI fragment from plasmid YDp-H (Berben et al., 1991) containing the S. cerevisiae HIS3 gene was ligated into it. The resulting plasmid, pOVICL::HIS3, was digested with XhoI and NotI and the 4 Kb fragment was used to disrupt the chromosomal copy of the ICL1 gene from P. pastoris.
Construction of the dextranase expression vector using the ICL1 promoter of P. pastoris
To express the dexA gene under PICL1, a 1.8 Kb fragment containing the SUC2 signal peptide from S. cerevisiae fused to dextranase coding region was obtained by PCR, using plasmid pPDEX1 (Roca et al., 1996) as template and the primers: (upstream) 5′-atgctagcgcaagctttccttttc-3′; (downstream) 5′-agctcgcgatcagctaatctgcca-3′. The PCR product was inserted into pGEM-T (Promega, USA) and the resulting plasmid named pTVDEX. A 0.68 Kb fragment of the ICL1 promoter was obtained by PCR using plasmid pIVICLPp as template and the primers: (upstream) 5′-cctcgagcttgtaggaattcgca-3′; (downstream) 5′-aaggtgctagcattcttgatatac-3′. The PCR product was inserted into pGEM-T (Promega, USA) to obtain plasmid pTvpICL. The resulting plasmid was digested with XhoI and NheI and the 0.68 Kb fragment ligated into TVDEX previously digested with SalI and NheI to obtain the pIV-2 plasmid.
Finally, the pIV-2 plasmid was cut with EcoRI and EcoRV and the 2.4 Kb fragment was ligated into pPDEX1 vector (Roca et al., 1996), previously digested with the same enzymes. The resulting plasmid, named pPICLDEX, was used in the dextranase expression experiments.
Transformation of P. pastoris and selection of dextranase-producing clones
Plasmid pPICLDEX DNA (5 µg), previously digested with SmaI, was used to transform P. pastoris strain MP36 by electroporation (Becker and Guarente, 1991). His+ transformants were recovered on minimal-agar (G medium) plates (Galzy and Slonimski, 1975). Single colonies were transferred to a minimal-agar plate supplemented with 0.4% blue dextran (Pharmacia) and 3% ethanol to induce the expression of dextranase. The plate was incubated overnight at 30 °C and the formation of clear halos of dextran hydrolysis around the colonies was observed.
Three independent transformants which showed dextranase activity in blue dextran plates were selected for dextranase expression studies in shake-flasks. The transformants were inoculated in 50 ml YNB cultures with 2% glucose or 3% ethanol as carbon source. The activity was measured during the exponential (OD530 nm = 1) and stationary (OD530 nm = 6) phases of growth. Samples of culture supernatant were used for determination of the enzymatic activity.
Cell extracts were prepared according to Blázquez et al. (1993). Isocitrate lyase activity was assayed as described by Dixon and Kornberg (1959). Specific activities were expressed as nmol substrate consumed/min/mg protein in crude extracts.
Dextranase activity was determined in the culture supernatant according to Kosaric et al. (1973). The reducing sugars formed were determined colorimetrically by the dinitrosalicylic acid reagent method (Miller, 1959). One unit (1U) is defined as the amount of enzyme that releases 1 µmol glucose equivalents in 1 min from dextran T-2000 (Pharmacia, Sweden) at 40 °C and pH 5.5.
The initial database searches were performed using Blast (Altschul et al., 1997) with BLOSUM62 substitution matrix and profiles search program, both using the default parameters. The database searches were parsed using GeneQuiz (Scharf et al., 1994), a system for large-scale sequence analysis that allows semi-automatic interactive evaluation. We also used the FASTA algorithm (Pearson and Lipman, 1988) for similarity searches in databases. A consensus multiple alignment was done using the ClustalW program (Thompson et al., 1994).
Protein in extract was determined using the commercial Pierce reagent. Glucose in the medium was determined with glucose oxidase.
Results and discussion
Cloning and sequencing of the ICL1 gene from P. pastoris
The P. pastoris ICL1 gene was isolated from a P. pastoris EcoRI genomic library. Alignment of the amino acid sequences available for the proteins from Saccharomyces cerevisiae, Yarrowia lipolytica, and Candida tropicalis allowed us to design degenerate oligonucleotides corresponding to two conserved regions of the proteins (Figure 1A). A 1.2 Kb fragment isolated by PCR using these degenerated oligonucleotides was cloned into pMOSBlue plasmid (Amersham) and the resulting plasmid was named TvICL. Sequences from both ends showed a high homology (70%) with known ICL genes.
Total genomic DNA was digested with several restriction enzymes and hybridized with the labelled DNA 1.2 kb fragment (Figure 1B). The picture obtained in the Southern blot indicated that P. pastoris contains only one copy of ICL gene, in a 6 Kb DNA band after digestion with EcoRI (lane 3, Figure 1B). The EcoRI-digested DNA was fractionated on an agarose gel, and fragments around 6 Kb were recovered and ligated into the pBluescript plasmid (Amersham). Screening of the library with the 1.2 Kb fragment allowed the isolation of the pIVICLPp-1 plasmid (Figure 1C).
The DNA sequence of a 2.7 Kb genomic fragment was determined (EMBL database Accession No. AJ272040). A 1653 bp open reading frame corresponding to a 551 amino acid polypeptide with a calculated molecular weight of 60.6 kDa was predicted from the DNA sequence (Figure 2). The predicted primary sequence was compared to Iclps of Candida albicans, C. tropicalis, Y. lipolitica, S. cerevisiae, and Kluyveromyces lactis, revealing 73%, 71%, 64%, 64% and 61% identity, respectively. The hexapeptide KKCGHM, conserved in all known Icl proteins and used as a recognition pattern for isocitrate lyases, was found within the P. pastoris protein (boxed in Figure 3).
The 5′ and 3′ non-coding regions contain several characteristics of yeast genes. A putative TATA box (Hernández, 1993) is located at position −96 bp and a possible polyadenylation site is 112 bp downstream from the translation stop codon.
The intracellular localization of isocitrate lyase in S. cerevisiae remained controversial until Taylor et al. (1996) showed by immunocytochemical studies a cytosolic localization of the enzyme. Peroxisomal Iclps are described for C. tropicalis (Oda et al., 1996), Y. lipolitica (Barth and Scheuber, 1993) and A. gossypii (Maeting et al., 1999), which present a PTS1 carboxy-terminal sequence. The carboxy-terminal sequence of isocitrate lyase from P. pastoris does not coincide with the eukaryotic signal S(A,C)-K(H,R)-L for import of the protein into the peroxisome. However, peroxisomal Icl from Aspergillus nidulans and Neurospora crassa lack the PTS1-like carboxy-terminal amino acids S(A,C)–K(H,R)–L (Gainey et al., 1992). Therefore, conclusive evidence for the localization of the enzyme in P. pastoris can only be obtained from further analysis.
Regulation of ICL1 gene in P. pastoris
The expression of Isocitrate lyase in P. pastoris strain BKM90 was measured by the determination of the enzymatic activity in different culture conditions, specifically in repressed (exponential phase) and derepressed (stationary phase) conditions.
In cells grown in glucose as the only carbon source, Icl activity in the exponential growth phase was almost undetectable. In this condition, the expression of the enzyme is not required. In contrast, high levels of Icl activity were detected in stationary phase, consistent with its requirement to metabolize the ethanol produced during the exponential phase. In cells grown in YP medium or YP medium supplemented with ethanol (YPE), the enzyme was detected in both exponential and stationary phases (Figure 4A).
Similar data have been reported for the expression of the lacZ gene, which encodes β-galactosidase, under the control of ICL1 promoter in S. cerevisiae. Using an ICL–lacZ fusion integrated at the ICL1 locus, more than 200-fold induction of β-galactosidase activity was observed after growth on ethanol when compared with glucose-repressed conditions (Schöler and Schüller, 1993).
To determine whether the expression of this enzyme is regulated at the transcriptional level, the steady-state levels of Isocitrate lyase mRNA in cells grown under the conditions described above were analysed by Northern blot (Figure 4B). In comparison with the constitutively expressed actin RNA, ICL1 mRNA was synthesized at a considerable level under derepressed conditions (when glucose is absence in the medium). In addition, glucose not only repressed the transcription of the ICL1 mRNA but also caused a reduction of ICL1 mRNA levels after the shift of ethanol-grown cells to a medium with glucose (Figure 4C). A similar effect of glucose over ICL1 mRNA in S. cerevisiae was also reported by Fernandez et al. (1993). They found that, 30 min after shifting the ethanol grown cells to glucose medium, no specific ICL1 mRNA was detected. In our study the ICL1 mRNA was undetectable just 10 min after addition of glucose to ethanol-grown cells (Figure 4C).
Functional analysis of the ICL1 gene from P. pastoris
The functionality of the ICL gene of P. pastoris was tested by complementation analysis in a strain of S. cerevisiae deficient in the ICL1 gene encoding isocitrate lyase and by disruption of the gene in P. pastoris.
In the first experiment the plasmid pRS316–ICL was introduced into the S. cerevisiae FMY402 mutant strain and transformants were selected on YNB (URA−) minimal medium supplemented with the appropriate amino acids. After initial selection for Ura+ colonies, randomly selected transformants were able to grow in minimal medium plates with ethanol as the only carbon source, showing that the cloned gene was functional in a heterologous host. The same host strain transformed with the pRS316 vector was not able to grow in this medium (data not shown).
Enzymatic activity of isocitrate lyase was also measured in extracts obtained from transformants grown in a medium containing an easily fermentable carbon source, such as glucose. It is interesting to note that Icl activity was detected under both repressed (6 mU/mg) and derepressed conditions (7 mU/mg), suggesting that the heterologous expression of P. pastoris Icl under the control of its own promoter in S. cerevisiae is not repressed by glucose.
This is in contrast to the results reported by Kanai et al. (1996) for the heterologous expression of β-galactosidase in S. cerevisiae using the isocitrate lyase promoter from C. tropicalis, where expression of the LacZ gene was repressed by glucose and enhanced over 300-fold by acetate. In our case, apparently, low values (7 mU/mg) of Icl are sufficient to sustain normal growth of this yeast in ethanol.
Further evidence for the functionality of the cloned gene was obtained by constructing a deletion–substitution mutant. The ICL1 gene of P. pastoris was disrupted by insertion of the S. cerevisiae HIS3 gene. After transformation, one histidine prototrophic transformant was used for further experimentation. A PCR experiment (Figure 5B) confirmed the correct pattern of integration. The disruption resulted in the inability of this strain (MP36/icl1::HIS3) to grow in minimal medium with ethanol as the only carbon source (data not shown) and did not show any detectable isocitrate lyase activity (Figure 5C). This result confirms the essential role of this enzyme for the utilization of ethanol.
Expression of the dextranase structural gene using the promoter of the ICL1 gene
The P. pastoris MP36 strain was transformed with the plasmid pPICLDEX, which contains the dextranase-encoding gene fom P. minioluteum under the control of the ICL1 promoter. His+ transformants were screened to find the clones producing active dextranase, using minimal agar plates containing blue dextran and 3% ethanol as the only carbon source to induce the dextranase expression. All the clones tested (n = 10) formed halos of dextran hydrolysis (Figure 6A).
The chromosomal DNA was isolated from each clone and analysed by Southern blot (data not shown). Three independent transformants with a single copy of the vector properly inserted at the ICL1 locus were grown for further analysis in a 50 ml flask in YNB medium supplemented with 2% glucose or 3% ethanol as the only carbon sources. The colonies contained the pPICLDEX plasmid produced high levels of the dextranase enzyme in the cultures in which the ethanol was used as carbon source or in conditions of absence of glucose (Figure 6B). This result showed that the 5′ non-coding region of the ICL1 gene of the yeast P. pastoris was able to direct the expression of the dexA gene of P. minioluteum. The expression of the dexA gene was regulated in response to the carbon source, being the expression of the protein controlled by the culture conditions used. It is therefore concluded that this fragment could be used as an alternative promoter in the expression system of P. pastoris.
We are grateful to Olivie Vincent, Ahskan Golshani, Bianca Garcia and Jose Carlos Garcia for critical reading of the manuscript. This work was supported by Research Grant No. 3082-327 from The Center for Genetic Engineering and Biotechnology.