JOURNAL OF BACTERIOLOGY,
`0021-9193/00/$04.00⫹0
`Copyright © 2000, American Society for Microbiology. All Rights Reserved.
`
`May 2000, p. 2492–2497
`
`Vol. 182, No. 9
`
`An n-Alkane-Responsive Promoter Element Found in the Gene
`Encoding the Peroxisomal Protein of Candida tropicalis Does
`Not Contain a C6 Zinc Cluster DNA-Binding Motif
`TAMOTSU KANAI, AKIHIRO HARA, NAOKI KANAYAMA, MITSUYOSHI UEDA,
`AND ATSUO TANAKA*
`Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry,
`Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan
`
`Received 22 October 1999/Accepted 9 February 2000
`
`When an asporogenic diploid yeast, Candida tropicalis, is cultivated on n-alkane, the expression of the genes
`encoding enzymes of the peroxisomal ␤-oxidation pathway is highly induced. An upstream activation sequence
`(UAS) which can induce transcription in response to n-alkane (UASALK) was identified on the promoter region
`of the peroxisomal 3-ketoacyl coenzyme A (CoA) thiolase gene of C. tropicalis (CT-T3A). The 29-bp region (from
`ⴚ289 to ⴚ261) present upstream of the TATA sequence was sufficient to induce n-alkane-dependent expression
`of a reporter gene. Besides n-alkane, UASALK-dependent gene expression also occurred in the cells grown on
`oleic acid. Several kinds of mutant UASALK were constructed and tested for their UAS activity. It was clarified
`that the important nucleotides for UASALK activity were located within 10-bp region from ⴚ273 to ⴚ264
`(5ⴕ-TCCTGCACAC-3ⴕ). This region did not contain a CGG triplet and therefore differed from the sequence of
`the oleate-response element (ORE), which is a UAS found on the promoter region of 3-ketoacyl-CoA thiolase
`gene of Saccharomyces cerevisiae. Similar sequences to UASALK were also found on several peroxisomal
`enzyme-encoding genes of C. tropicalis.
`
`Candida tropicalis (strain pK233) is an asporogenic diploid
`yeast, which can utilize n-alkanes as the sole carbon and energy
`source. During utilization of n-alkanes or fatty acids, a pro-
`found development of peroxisomes occurs in the cells, which is
`a major characteristic of this yeast (26). Enzymes localized in
`peroxisomes, such as the enzymes of the fatty acid ␤-oxidation
`pathway and of the glyoxylate pathway, are also induced along
`with the peroxisome proliferation (14, 37).
`Thiolase is an enzyme which catalyzes the final step of the
`␤-oxidation pathway. There are three thiolase isozymes in n-
`alkane-grown C. tropicalis: two acetoacetyl coenzyme A (CoA)
`thiolases (thiolase I), one of which is localized in cytosol (Cs-
`thiolase I) and one of which is localized in the peroxisome
`(Ps-thiolase I), and one peroxisomal 3-ketoacyl-CoA thiolase
`(thiolase III) (17–19). Only Cs-thiolase I is found in the cells
`grown on glucose. Cs-thiolase I and Ps-thiolase I are encoded
`by the same pair of alleles (CT-T1A and CT-T1B) (9, 16), and
`expression of the genes is highly induced on n-alkane, whereas
`low but finite expression occurs in cells grown on glucose (10).
`Thiolase III is encoded by another pair of alleles (CT-T3A and
`CT-T3B) (10), and their expression is highly induced on n-
`alkane but completely repressed on glucose.
`In Saccharomyces cerevisiae, induction of peroxisomal 3-ke-
`toacyl-CoA thiolase (encoded by FOX3/POT1) is mediated via
`an upstream activation sequence (UAS) called the oleate re-
`sponse element (ORE) (3, 12, 30, 31). ORE also exists on the
`upstream regions of genes encoding enzymes relating to the
`␤-oxidation pathway (FOX1 and FOX2) and fatty acid metab-
`olism (SPS19 and ECI1) and proteins relating to peroxisomal
`biogenesis (PEX1 and PEX11) (13). The transcriptional acti-
`
`* Corresponding author. Mailing address: Laboratory of Applied
`Biological Chemistry, Department of Synthetic Chemistry and Biolog-
`ical Chemistry, Graduate School of Engineering, Kyoto University,
`Yoshida, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-5524.
`Fax: 81-75-753-5534. E-mail: atsuo@sbchem.kyoto-u.ac.jp.
`
`vation through ORE occurs by the binding of a heterodimeric
`protein complex consisting of Oaf1p and Oaf2p/Pip2p (12, 13,
`22, 30, 31). Interestingly, OAF2/PIP2 itself is also regulated by
`ORE whereas OAF1 is not (12, 13, 31). However, the effect of
`this difference in the regulation mechanisms is not clear.
`The molecular mechanism underlying the induction of per-
`oxisomal enzymes or peroxisome itself is unclear for C. tropi-
`calis, because no appropriate host-vector system has been
`available. Recently, using ura3 derivatives of C. tropicalis, we
`have developed a transformation system for introducing exog-
`enous DNA into the genomic DNA of C. tropicalis (9). We
`have also cloned an autonomously replicating sequence (ARS)
`from C. tropicalis, which enabled us to introduce exogenous
`DNA into C. tropicalis with a form of episomal vector (6).
`In this study, using the transformation procedure and the
`episomal vector system developed for C. tropicalis, we have
`identified a UAS, which can induce transcription in response to
`n-alkane (designated UASALK), on the promoter region of
`CT-T3A. In comparing its sequence with that of ORE, the
`possibility was suggested that the molecular mechanism induc-
`ing peroxisomal 3-ketoacyl-CoA thiolase in C. tropicalis was
`essentially different from the ORE-mediated induction mech-
`anism in S. cerevisiae.
`
`MATERIALS AND METHODS
`
`Strains and media. C. tropicalis SU-2 (ATCC 20913) (ura3a/ura3b) (5), de-
`rived from C. tropicalis pK233 (ATCC 20336), was used as a host strain for
`transformation. Escherichia coli strain DH5␣ (29) was used for gene manipula-
`tion.
`C. tropicalis was cultivated aerobically at 30°C in a medium containing glucose
`(16.5 g/liter), n-alkane mixture (C10 to C13; 10 ml/liter), oleic acid (5 ml/liter),
`glycerol (20 g/liter), sodium acetate (13.6 g/liter), sodium propionate (10 g/liter),
`or sodium butyrate (11 g/liter) as the sole carbon source (15, 39). The pH was
`adjusted to 5.2 for glucose, n-alkane, oleic acid, and glycerol media or to 6.0 for
`acetate, propionate and butyrate media. Tween 80 (0.5 ml/liter) was added to the
`n-alkane and oleic acid media. The basic composition of the medium was as
`follows: 5.0 g of NH4H2PO4, 2.5 g of KH2PO4, 1.0 g of MgSO4 䡠 7H2O, 0.02 g of
`FeCl3 䡠 6H2O, and 1.0 ml of corn steep liquor per liter of tap water (39).
`
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`VOL. 182, 2000
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`Name
`
`C. TROPICALIS n-ALKANE-RESPONSIVE UAS
`
`2493
`
`TABLE 1. Oligonucleotides used in this study
`
`Sequence
`
`PRT3AJ-1.......................................................................................................5⬘-CATGCTCGAGGTGTTGAATATGTGC-3⬘
`
`T3(⫺550S)......................................................................................................5⬘-TAGGTCGACCCGCGGTATCAACCATCGTCC-3⬘
`T3(⫺473S)......................................................................................................5⬘-TCTGTCGACATTTGGTGGGTCTGCCCCCC-3⬘
`T3(⫺407S)......................................................................................................5⬘-CACGTCGACACATCCCGCTTAGTTGCGGG-3⬘
`T3(⫺382S)......................................................................................................5⬘-CGGGTCGACGAACGTGTATTCCCGTAG-3⬘
`T3(⫺343S)......................................................................................................5⬘-GTCGTCGACTAGTCACCCGCTTCTGCC-3⬘
`T3(⫺310S)......................................................................................................5⬘-GGTGTCGACTCAAAGCTGGCATAAATG-3⬘
`T3(⫺289S)......................................................................................................5⬘-ATAAGTCGACAAAAAAAAGCACAGCATCCTGCACACAAC-3⬘
`T3(⫺281S)......................................................................................................5⬘-CGAAGTCGACGCACAGCATCCTGCACACAAC-3⬘
`T3(⫺270S)......................................................................................................5⬘-ACAGTCGACTGCACACAACCCTGCTCAG-3⬘
`T3(⫺230S)......................................................................................................5⬘-TGGGTCGACAGAAAACCTCGGCTTAAAACC-3⬘
`T3(⫺310X) .....................................................................................................5⬘-CCAGCTCGAGAGGCAAACCCAGAATGGCAG-3⬘
`T3(⫺289X) .....................................................................................................5⬘-CTTCTCGAGCGTCATTTATGCCAGCTTTGA-3⬘
`T3(⫺260X) .....................................................................................................5⬘-CTGCTCGAGGTTGTGTGCAGGATGCTGTGC-3⬘
`
`T3UAS(M1-2)................................................................................................5⬘-GGGCTCGAGGTTGTGTGCAGGATGCTATGCTTTTTTTTG-3⬘
`T3UAS(M2-2)................................................................................................5⬘-GGGCTCGAGGTTGTGTGCAGGATAATGTGCTTTTTTTTG-3⬘
`T3UAS(M3-2)................................................................................................5⬘-GGGCTCGAGGTTGTGTGCAAAATGCTGTGCTTTTTTTTG-3⬘
`T3UAS(M4-2)................................................................................................5⬘-GGGCTCGAGGTTGTGTAAAGGATGCTGTGCTTTTTTTTG-3⬘
`T3UAS(M5-2)................................................................................................5⬘-GGGCTCGAGGTTGTATGCAGGATGCTGTGCTTTTTTTTG-3⬘
`T3UAS(M6-2)................................................................................................5⬘-GGGCTCGAGGTTATGTGCAGGATGCTGTGCTTTTTTTTG-3⬘
`T3UAS(M1-3)................................................................................................5⬘-AAAAGTCGACAAAAAAAAGCATAGCATCCTGCACACAAC-3⬘
`T3UAS(M2-3)................................................................................................5⬘-AAAAGTCGACAAAAAAAAGCACATTATCCTGCACACAAC-3⬘
`T3UAS(M3-3)................................................................................................5⬘-AAAAGTCGACAAAAAAAAGCACAGCATTTTGCACACAAC-3⬘
`T3UAS(M4-3)................................................................................................5⬘-AAAAGTCGACAAAAAAAAGCACAGCATCCTTTACACAAC-3⬘
`T3UAS(M5-3)................................................................................................5⬘-AAAAGTCGACAAAAAAAAGCACAGCATCCTGCATACAAC-3⬘
`T3UAS(M6-3)................................................................................................5⬘-AAAAGTCGACAAAAAAAAGCACAGCATCCTGCACATAAC-3⬘
`T3UAS(WTB-1) ............................................................................................5⬘-AAAAGTCGACAAAAAAAAACACGGCGTCCTGCACACGAC-3⬘
`T3UAS(WTB-2) ............................................................................................5⬘-AGGGCTCGAGGTCGTGTGCAGGACGCCGTGTTTTTTTTTG-3⬘
`
`Plasmid construction. Lac4 encoding Kluyveromyces lactis ␤-galactosidase was
`amplified using primers 5⬘-AACTGTCGACTATGTCTTGCCTTATTCCTGA
`G-3⬘ and 5⬘-CTGTCTCGAGCTTAACGGTCTAATCGTTAATCAG-3⬘. The
`genomic DNA of K. lactis IFO1267 (ATCC8585) was used as a template DNA.
`The amplified Lac4 fragment cut with SalI and XhoI was inserted into the SalI
`site of pUC-URA3, in which the 1.7-kbp C. tropicalis URA3 was inserted into
`pUC19 (11), and the subclone was named pUL4. The ARS of C. tropicalis 1098
`was amplified using primers 5⬘-AAAAGTCGACCACATTTCCCCGAAAAGT
`GCCACC-3⬘ and 5⬘-AAAAGTCGACGGTTAATGTCATGATAATAATGGT
`TTC-3⬘, with pUCNUA1 (6) as a template DNA. Bluescript II(SK⫹) cut with
`SspI was filled in using T4 DNA polymerase and joined with an XhoI linker
`(named Bluescript-Xh), and the amplified ARS fragment cut by SalI was inserted
`into the XhoI site of Bluescript-Xh (named Bluescript-ARS). Bluescript-ARS
`was cut with KpnI, treated with T4 DNA polymerase (blunting), digested with
`SalI, and a 1.4-kbp fragment containing ARS was eluted. This fragment was
`ligated with the SalI-SmaI fragment of pUL4 containing C. tropicalis URA3 and
`LAC4, to make pUAL4.
`All deletion fragments were prepared either by PCR using pT37Bg (11) as a
`template or by annealing of two oligonucleotides. All the oligonucleotides used
`in this study are listed in Table 1. PCR was performed using primer PRT3AJ-1
`the following primers: T3(⫺550S), T3(⫺473S), T3(⫺407S),
`and one of
`T3(⫺382S), T3(⫺343S), T3(⫺310S), T3(⫺289S), T3(⫺270S), or T3(⫺230S).
`Each amplified fragment was cut with SalI and XhoI and inserted into the SalI
`site of pUAL4 to construct plasmid pUTA550, pUTA473, pUTA407, pUTA382,
`pUTA343, pUTA310, pUTA289, pUTA270, and pUTA230, respectively. To
`construct plasmids pUTA311R, pUTA290R, and pUTA261R, PCR was per-
`formed with primer T3(⫺550S) and plus primer T3(⫺310X), T3(⫺289X), and
`T3(⫺260X), respectively. The amplified fragment cut with SalI and XhoI was
`inserted into the SalI site of pUTA230. Plasmids pUTA03F, pUTA03R,
`pUTA04F, and pUTA05F were constructed by the same method as above using
`the following set of primers: T3(⫺310S) and T3(⫺260X) for pUTA03F and
`pUTA03R, T3(⫺310S) and T3(⫺289X) for pUTA04F, and T3(⫺289S) and
`T3(⫺260X) for pUTA05F.
`To construct pUTA11, pUTA12, pUTA13, pUTA14, pUTA15, pUTA16,
`pUTA17, and pUTA18, two complementary oligonucleotides [T3UAS(M1-2)
`and T3UAS(M1-3)
`for pUTA11, T3UAS(M2-2) and T3UAS(M2-3)
`for
`pUTA12, T3UAS(M3-2) and T3UAS(M3-3) for pUTA13, T3UAS(M4-2) and
`T3UAS(M4-3) for pUTA14, T3UAS(M5-2) and T3UAS(M5-3) for pUTA15,
`T3UAS(M6-2) and T3UAS(M6-3) for pUTA16, T3(⫺281S) and T3(⫺260X) for
`pUTA17, and T3UAS(WTB-1) and T3UAS(WTB-2) for pUTA18] were an-
`
`nealed. The annealed fragments were filled in with the Klenow fragment, cut
`with SalI and XhoI, and introduced into the SalI site of pUTA230.
`The nucleotide sequences of all the deletion fragments were checked using
`ABI DNA sequencer model 373.
`␤-Galactosidase assay. ␤-Galactosidase activity was determined by measuring
`the hydrolysis of 4-methylumbelliferyl-␤-D-galactopyranoside (MUG; Molecular
`Probes) (2, 42). Enzyme solution (50 ␮l) in Z buffer (940 ␮l) (24) was incubated
`at 30°C for 1 min, 10 mM MUG solution (10 ␮l) was added, and the increase in
`fluorescence was measured with a Hitachi fluorophotometer model 650-10S
`(excitation, 360 nm; emission, 449 nm). 7-Hydroxy-4-methylcoumarin (Molecu-
`lar Probes) dissolved in 100 mM sodium phosphate buffer (pH 7.0) was used as
`the reference standard. All activities are the mean values of at least two exper-
`iments.
`Other methods. Transformation of C. tropicalis was carried out by electropo-
`ration (1,000 V, 25 ␮F, and 201⍀) (11). The protein concentration was assayed
`by the Bradford method using bovine serum albumin as the standard (1).
`Nucleotide sequence accession number. Nucleotide sequence data of the pro-
`moter region of CT-T3A and CT-T3B will appear in the DDBJ/EMBL/GenBank
`nucleotide sequence databases with the accession numbers AB025647 and
`AB025648, respectively.
`
`RESULTS
`
`To evaluate the activity of promoter elements to induce
`transcription in C. tropicalis, pUAL4, a shuttle vector which
`can replicate in both E. coli and C. tropicalis, was first con-
`structed by the method described in Materials and Methods.
`pUAL4 contains an ARS from C. tropicalis (6), URA3 of C.
`tropicalis (11), and LAC4 encoding ␤-galactosidase of K. lactis
`(27). In Candida yeasts, LAC4 instead of LacZ has usually
`been used as the source of the ␤-galactosidase gene (20, 21,
`23), because several Candida yeasts translate the CUG codon
`as Ser instead of Leu, and LAC4 contains fewer CUG codons
`than LacZ does (3 for LAC4 and 53 for LacZ). A multicloning
`site was introduced before the translation initiation codon of
`
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`2494
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`KANAI ET AL.
`
`J. BACTERIOL.
`
`FIG. 1. Deletion fragments of the CT-T3A promoter and ␤-galactosidase activity in cells grown on n-alkane. The ␤-galactosidase activity after 24 h on n-alkane is
`shown (initial optical density at 570 nm ⫽ 0.2). Arrowheads indicate the direction of inserted fragments in panel B.
`
`LAC4 so that the promoter sequence to be tested could be
`inserted.
`The nucleotide sequences of the upstream regions of CT-
`T3A and CT-T3B (about 1.5 kbp) were determined. A 550-bp
`upstream region of CT-T3A (from ⫺1 to⫺550; UPR-T3A) was
`introduced into pUAL4, and the resulting plasmid (pUTA550)
`was transformed into C. tropicalis SU-2. The transformant was
`then grown on either glucose or n-alkane as the sole carbon
`source, and the intracellular ␤-galactosidase activity was mea-
`sured. ␤-Galactosidase activity in the glucose-grown cells was
`almost negligible (less than 0.1 pmol min⫺1 mg⫺1), while over
`1,000 times more ␤-galactosidase activity was detected for the
`n-alkane-grown cells (Fig. 1A). This result indicated that a
`
`550-bp UPR-T3A contained a sufficient region(s) to induce
`transcription in response to n-alkane.
`A series of deletion fragments of UPR-T3A were constructed
`(pUTA550 to pUTA230), and their abilities to induce tran-
`scription by n-alkane were compared (Fig. 1A). When grown
`on glucose, all deletion mutants showed no detectable ␤-ga-
`lactosidase activity (less than 0.1 pmol min⫺1 mg⫺1). In the
`cells grown on n-alkane, significant levels of ␤-galactosidase
`activity were detected from pUTA550 to pUTA289. The ␤-Ga-
`lactosidase activity dropped sharply between pUTA289 and
`pUTA270, suggesting the existence of a UAS around ⫺270
`to ⫺289. Three internal deletion mutants
`(pUTA261R,
`pUTA290R, and pUTA311R) were also constructed in which
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`VOL. 182, 2000
`
`C. TROPICALIS n-ALKANE-RESPONSIVE UAS
`
`2495
`
`FIG. 2. UAS activity of UASALK and its mutants on n-alkane. The nucleotides different from those of wild-type (WT) UASALK are double underlined. The
`nucleotides in pUTA18 different from those of wild-type UASALK are underlined. The number in parentheses indicates the UAS-dependent transcription inducing
`activity, where the activity of wild-type UASALK was set as 100.
`
`the region between ⫺231 to ⫺260, between ⫺231 to ⫺289, or
`between ⫺231 to ⫺310 was deleted, respectively (Fig. 1A). The
`relatively higher activities detected for pUTA261R than for
`pUTA290R and pUTA311R might be explained by the exis-
`tence of upstream repression sequence (URS) in the region
`between ⫺231 and ⫺260. The putative URS between ⫺231
`and ⫺260 and the UAS between ⫺270 and ⫺289 would be
`present. A noticeable sequence for URS could not be detected
`in the sequence between ⫺231 and ⫺260: 5⬘-CCTGCTCAGT
`GTGACAGGTGGTGGTGTAAT-3⬘.
`To determine the region functioning as the UAS, the fol-
`lowing plasmids were constructed in which the sequence be-
`tween ⫺311 and ⫺261 was inserted into the SalI site of
`pUTA230 (pUTA03F) (Fig. 1B). pUTA230, which contained
`the TATA sequence of UPR-T3A, did not have UAS activity by
`itself (Fig. 1A). ␤-Galactosidase activity in pUTA03F-trans-
`formed cells grown on n-alkane was significantly higher than
`that
`in pUTA230-transformed cells (Fig. 1B). Moreover,
`pUTA03R, in which the same sequence was inserted in the
`opposite direction to pUTA03F, also showed a significant in-
`crease in ␤-galactosidase activity. On the other hand, when
`grown on glucose, neither pUTA03F-transformed nor
`pUTA03R-transformed cells, together with pUTA230-trans-
`formed cells, showed ␤-galactosidase activity (data not shown).
`These results demonstrated the presence of an n-alkane-re-
`sponsive UAS (designated UASALK) in the region between
`⫺311 and ⫺261. Further deletion analysis indicated that the
`29-bp region between ⫺289 and ⫺261 contained sufficient
`sequences for UASALK (pUTA05F in Fig. 1B).
`To find the important nucleotide sequences inside this 29-bp
`region, a series of point mutations were introduced. First, six
`kinds of mutants (M1 to M6) were made in which one or two
`adjacent guanine and/or cytosine nucleotides were changed
`into thymine nucleotides, and their UAS activities were com-
`pared (Fig. 2). Mutants M1, M2, and M6 had almost compa-
`
`rable (over 80%) UAS activity to the wild-type UASALK. On
`the other hand, mutants M3, M4, and M5 had lower UAS
`activity, showing 30, 20, and 59% of the wild-type activity,
`respectively. Moreover, a mutant, in which the adenine stretch
`located between ⫺289 and ⫺282 was deleted (⌬A) had a
`UASALK activity comparable to the wild-type activity. These
`results indicate that nucleotide positions changed in mutant
`M4 (positions ⫺268 and ⫺269) are particularly important for
`UASALK activity. Furthermore, the upstream sequence of CT-
`T3B corresponding to the region of UASALK was tested for its
`UAS activity, and the result indicated that this region also had
`sufficient UAS activity (75% of that of CT-T3A).
`Expression of thiolase III is induced not only by n-alkane but
`also by other carbon sources, such as butyrate (10, 17). There-
`fore, it is of interest to examine whether UASALK induces gene
`expression by other carbon sources. Cells transformed with
`pUTA05F were cultivated on glucose, glycerol, n-alkane, ace-
`tate, propionate, or butyrate as the sole carbon source, and
`intracellular ␤-galactosidase activities were compared (Table
`2). Cells transformed with pUTA230 were used as a control for
`estimating UASALK-independent
`transcriptional activation.
`When the cells were cultivated on glucose, glycerol, or acetate,
`no UASALK-dependent increase of ␤-galactosidase activity was
`observed. On the other hand, in cells grown on propionate or
`butyrate as well as on n-alkane, a UASALK-dependent increase
`of ␤-galactosidase activity was observed. These results demon-
`strate that induction of the expression of the thiolase III gene
`in the propionate- or butyrate-grown cells occurs, at least in
`part, by a common mechanism that acts through UASALK.
`In S. cerevisiae, the transcription of 3-ketoacyl-CoA thiolase
`encoded by FOX3/POT1 is induced by oleic acid (4, 8). Ac-
`cordingly, UASALK was tested to find whether it can induce
`transcription by oleic acid. ␤-Galactosidase activity was in-
`creased in the oleic acid-grown cells harboring pUTA05F (with
`UASALK) or pUTA17 (with ⌬A derivative of UASALK) (activ-
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`J. BACTERIOL.
`
`TABLE 2. Effect of different carbon sources on UASALK-mediated
`transcriptional activation
`
`Carbon source
`
`Glucose
`Glycerol
`n-Alkane
`Acetate
`Propionate
`Butyrate
`
`␤-Galactosidase activity
`(pmol min⫺1 mg⫺1)a
`
`Control
`
`⬍1.0
`18.2
`3.5
`11.1
`24.7
`381
`
`⫹UASALK
`⬍1.0
`15.8
`71.7
`10.9
`127
`1,260
`
`Activation
`(fold)
`
`0.87
`20.5
`0.98
`5.15
`3.30
`
`indicates pUTA230-transformed cells, and ⫹UASALK indicates
`a Control
`pUTA05F-transformed cells. The initial cell density (optical density at 570 nm)
`for each carbon source was as follows: 0.05 for glucose, 0.1 for glycerol, 0.2 for
`n-alkane and acetate, and 0.4 for propionate and butyrate. The cultivation time
`for each condition was as follows; 24 h for glucose- and glycerol-grown cells, 36 h
`for n-alkane- and acetate-grown cells, and 88 h for propionate- and butyrate-
`grown cells.
`
`ity of 22.4 and 26.0 pmol/min/mg, respectively) compared with
`the activity in those harboring pUTA230 (without UASALK)
`(2.51 pmol/min/mg), indicating that UASALK is also active in
`the oleic acid-grown cells. However,
`the cells harboring
`pUTA550 (with total UPR-T3A) had twice the ␤-galactosidase
`activity (45.0 pmol/min/mg) as that of the pUTA05F-harboring
`cells, demonstrating the possible existence of another UAS(s)
`in addition to the UASALK in response to oleic acid on UPR-
`T3A.
`
`DISCUSSION
`
`We have identified the UAS sequence that responds to n-
`alkane (UASALK) in then-alkane-assimilating yeast C. tropi-
`calis. Deletion analysis delimited the sequence of UASALK
`within 29 bp (from positions ⫺289 to ⫺261 of UPR-T3A).
`Further mutation analysis showed that the nucleotides that
`were changed in the M4 mutant (positions ⫺268 and ⫺269)
`were the most critical for the UASALK activity. The 12-bp
`
`FIG. 3. Nucleotide sequences similar to UASALK found on promoters of C.
`tropicalis peroxisomal enzyme genes. POX2 and POX4 encode acyl-CoA oxidase
`(accession numbers for POX2 and POX4 are M18259 and M12160, respectively);
`BFE encodes the bifunctional enzyme (X57854); CAT encodes carnitine acetyl-
`transferase (D84549) (unpublished data); KAT encodes catalase (X13978,
`E01922) (unpublished data); POX18 encodes nonspecific lipid transfer protein
`(X53633 and M24440). The score indicates the number of the nucleotides that
`are the same as those of CT-T3A. The positions of changed nucleotides in the
`UASALK mutants (M3, M4, M5, and M6) are indicated above the sequences.
`Numbers on both sides of the sequences indicate the distance relative to the
`translational start codon.
`
`sequence including these positions was selected, and similar
`motifs were searched for promoters of genes encoding several
`C. tropicalis peroxisomal enzymes (Fig. 3). In this 12-bp se-
`quence,
`the marginal positions were not crucial
`for the
`UASALK activity, because, as for CT-T3B, the marginal posi-
`tions of the corresponding 12-bp sequence were different from
`those of CT-T3A but the region still had the UASALK activity
`(Fig. 2). In POX18 and KAT, regions were found in which
`internal 10 bp of UASALK (5⬘-TCCTGCACAC-3⬘) was com-
`pletely conserved. KAT encodes catalase, a marker enzyme of
`peroxisome, which is highly induced by n-alkane (28, 34, 40,
`41). Therefore, it is reasonable to consider that this region
`functions as a UASALK. POX18 of C. tropicalis (POX18) en-
`codes a nonspecific lipid transfer protein which is induced by
`oleic acid (35, 36). Although it is not clear at present whether
`the expression of C. tropicalis POX18 is induced by n-alkane,
`the expression of Candida maltosa POX18 is inducible by n-
`alkane (7). The UASALK can also induce transcription by oleic
`acid; therefore, it seems probable that the expression of C.
`tropicalis POX18 is induced by n-alkane by the common mech-
`anism through UASALK as in the oleic acid-grown cells. How-
`ever, whether the sequences shown in Fig. 3 actually have the
`UASALK activity should be determined by experiments.
`In C. maltosa, NADPH–cytochrome P-450 reductase which
`is localized in the endoplasmic reticulum, is highly induced by
`n-alkane. By a reporter gene assay, the 0.47-kbp 5⬘ noncoding
`region of the gene was shown to be sufficient for the induction
`on n-tetradecane (25). We compared this region with UASALK.
`Although no region closely homologous to UASALK was de-
`tected, there were two CACAT motifs, the pentanucleotide
`often found in the 5⬘-noncoding regions of P-450alk genes,
`encoding cytochrome P-450, of C. maltosa (25). UASALK of C.
`tropicalis contains a CACACA sequence at its 3⬘ end. Physio-
`logical and genetic evidence suggests that C. tropicalis and C.
`maltosa are closely related strains. Therefore, it is likely that
`the similar activation mechanisms are present in these yeasts,
`in which the CACA motif sequence might play an important
`role.
`Besides n-alkane and oleic acid, UASALK-dependent tran-
`scription also occurred with butyrate and propionate. These
`carbon sources can also induce the proliferation of peroxi-
`somes in C. tropicalis (17, 39). n-Alkane or long-chain fatty
`acids incorporated in C. tropicalis are degraded through the
`fatty acid ␤-oxidation system localized in peroxisomes and are
`ultimately converted into butyryl- or propionyl-CoA. There-
`fore, the results of this study show that these short-chain fatty
`acids and/or their derivatives might be a true inducer(s) that
`causes UASALK-dependent transcriptional activation.
`In S. cerevisiae, the consensus sequence of ORE is suggested
`as inverted repeats of the CGG triplet with a spacing of 15 to
`18 nucleotides (CGGN15–18CCG) (13, 30). The CGG triplet
`repeat is the common consensus sequence for the C6 zinc
`cluster family of fungal transcriptional regulators, such as
`Gal4p (32, 38). In fact, a heterodimeric protein complex con-
`sisting of Oaf1p and Oaf2p/Pip2p, both of which have a C6 zinc
`cluster motif, was identified as the factor that binds to ORE
`(12, 22, 30, 31). On the other hand, UASALK does not contain
`the CGG triplet repeat. This fact strongly suggests that the
`ORE-like regulation mechanism does not exist in C. tropicalis.
`Sloots et al. (33) investigated the regulation mechanism of the
`gene (HDE) encoding the peroxisomal bifunctional enzyme of
`C. tropicalis by introducing its upstream region into S. cerevi-
`siae. By deletion analysis, they identified an oleic acid-respon-
`sive region located between positions ⫺393 and ⫺341. When
`this region was compared with UASALK, no homologous se-
`quence was observed, which supports our notion that these two
`
`LCY Biotechnology Holding, Inc.
`Ex. 1014
`Page 5 of 6
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`VOL. 182, 2000
`
`C. TROPICALIS n-ALKANE-RESPONSIVE UAS
`
`2497
`
`yeasts have differences in the regulation mechanism for the
`induction of peroxisomal enzymes. Further investigation in-
`volving the isolation of the factor(s) binding to UASALK and its
`characterization by comparison with Pip2p will help to clarify
`the activation mechanism of the peroxisomal enzyme genes in
`C. tropicalis.
`
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