Microbiology Laboratory, RIKEN (Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan.
Peroxisome deficiency represses the expression of n-alkane-inducible YlALK1 encoding cytochrome P450ALK1 in Yarrowia lipolytica
Article first published online: 9 JAN 2006
FEMS Microbiology Letters
Volume 214, Issue 1, pages 31–38, August 2002
How to Cite
Sumita, T., Iida, T., Hirata, A., Horiuchi, H., Takagi, M. and Ohta, A. (2002), Peroxisome deficiency represses the expression of n-alkane-inducible YlALK1 encoding cytochrome P450ALK1 in Yarrowia lipolytica. FEMS Microbiology Letters, 214: 31–38. doi: 10.1111/j.1574-6968.2002.tb11321.x
- Issue published online: 9 JAN 2006
- Article first published online: 9 JAN 2006
- Received 13 May 2002, Revised 24 June 2002, Accepted 24 June 2002
- Cytochrome P450;
- Alkane assimilation;
- Yarrowia lipolytica;
Among the eight genes (YlALK1–YlALK8) encoding P450 cytochromes of the CYP52 family of the n-alkane-assimilating yeast Yarrowia lipolytica, Y1ALK1 is most highly induced by n-alkanes with short hydrocarbon chains, such as n-decane, and involved in the initial hydroxylation of n-alkane. To determine the factors regulating YlALK1 expression, we isolated an n-decane assimilation-deficient mutant, B0-6-1, whose YlALK1 expression level was lower than that of the wild-type. By complementation of the mutation of B0-6-1, we cloned a gene having an open reading frame of 1062 bp. The putative gene product is a protein of 354 amino acids and has significant homology to Pex10ps of other organisms. We named this gene YlPEX10. YlPex10p has a C3HC4 ring finger motif common among Pex10ps in its C-terminal region. This motif was also essential for the function of YlPex10p. Both B0-6–1 and a null mutant of YlPEX10 failed to form peroxisome and showed low-level transcription of YlALK1 after the change of carbon source to n-decane. Furthermore, YlPEX5 and YlPEX6 disruptants also showed low-level transcription of YlALK1 like the YlPEX10 disruptant and B0-6–1 mutant. We propose that in this organism peroxisome deficiency represses the expression of n-alkane-inducible YlALK1 encoding cytochrome P450ALK1.
Cytochrome P450 superfamily members have been found in many microorganisms, including bacteria, archaea, yeasts, and fungi. Most eukaryotic P450s are anchored in the membrane of the endoplasmic reticulum. The cytochromes P450ALK (P450ALKs) that are classified into the CYP52 family have been found in several n-alkane-assimilating yeasts, such as Candida maltosa[1–5], Candida tropicalis[6–10], and Yarrowia lipolytica[11,12]. P450ALKs catalyze terminal monooxygenation of n-alkanes and convert them to long-chain fatty alcohols that are subsequently oxidized to fatty acids. P450ALKs also hydroxylate some alkane metabolites in the omega position to form long-chain dicarboxylic acids.
In a previous study, we cloned eight genes encoding P450ALK species (YlALK1–YlALK8) of Y. lipolytica strain CX161-1B. They were induced by n-tetradecane at various levels. Among them, we demonstrated that only YlALK1 and YlALK2 actually played a role in the assimilation of n-alkanes, and YlALK1 was especially important for the assimilation of short-chain n-alkanes such as n-decane, since a null mutation of YlALK1 almost abolished the growth ability of Y. lipolytica on n-decane.
After n-alkanes are converted into long-chain fatty alcohols and fatty acids, they are transferred to the peroxisomes and further metabolized by the catabolic enzymes therein. Peroxisomes consist of many components, some of which are called peroxins. Peroxins are encoded by PEX genes, and more than 20 different PEX genes have been identified so far in various eukaryotic organisms. Many PEX genes have been isolated from the analysis of the mutants that showed abnormal peroxisomal functions or structures. Such mutants are present in a wide variety of organisms. In yeasts, mutants defective in PEX genes are unable to grow in a medium containing fatty acid or n-alkane as the sole carbon source [15–21].
To elucidate how alkane assimilation is regulated in yeasts, we tried to identify factors that regulate YlALK1 expression when induced by n-alkane. We isolated mutants that were defective in n-decane assimilation. One of the mutants, B0-6-1, showed low YlALK1 promoter activity. By functional complementation of the mutation in mutant B0-6-1, we isolated a gene homologous to PEX10, and named it YlPEX10.
Mutant B0-6–1 and the YlPEX10 disruptant showed lower YlALK1 induction than the wild-type strain. Neither strain had peroxisomes under electron microscopic observation. We noticed that the lower YlALK1 induction was not seen only in the YlPEX10 disruptant and strain B0-6–1 but also in YlPEX5 or YlPEX6 disruptants. We hypothesize that when peroxisomes are not functional, n-alkane metabolic intermediates accumulate, which somehow causes reduced YlALK1 expression.
2Materials and methods
2.1Strains, media and growth conditions
We used Y. lipolytica strain CXAU1 (ade1, ura3) (Table 1). Δpex10 was generated as follows: a 3381-bp Kpn I–Kpn I fragment containing a YlPEX10-coding region was cloned into the Kpn I site of pUC19 to create pPEX10. A 1.7-kb Hin cII fragment of YlURA3 was inserted between two Eco RV sites of YlPEX10 on pPEX10. After Kpn I digestion, a Kpn I–Kpn I fragment carrying the disruption construct was introduced into CXAU1 by electroporation. Disruption of YlPEX10 in the resultant strain Δpex10 was confirmed by Southern hybridization. Southern hybridizations were performed as previously described. Δpex5 and Δpex6 were also generated by disruption of YlPEX5 and YlPEX6 using YlURA3 as a marker gene, respectively. A DNA fragment was PCR-amplified using primers YlPEX5-up and YlPEX5-down (Table 2) and inserted into the Bam HI site on pUC119 to generate pPEX5. A Hin cII fragment of YlURA3 was inserted into the Apa I–Pma CI site on pPEX5. The resultant plasmid was digested by Eco RI and Hin dIII, and a 2.3-kb disruption construct was introduced into CXAU1. Another DNA fragment was PCR-amplified using primers YlPEX6-up and YlPEX6-down (Table 2) and inserted into the Bam HI site on pUC119 to generate pPEX6. A Hin cII fragment of YlURA3 was inserted into the Nde I–Stu I site on pPEX6. The resultant plasmid was digested by Eco RI and Hin dIII, and a 2.2-kb disruption construct was introduced into CXAU1. Transformations of Y. lipolytica strains were performed by electroporation or one-step transformation. Disruption of both YlPEX5 and YlPEX6 was also confirmed by Southern hybridization.
|Strain||Description||Source or reference|
|CXAU1||wild-type strain (MatA, ade1, ura3) generated from CX161-1B||[11,12]|
|B0-6–1||peroxisome-deficient mutant generated from CXAU1||this study|
|Δpex10||YlPEX10 disruptant with YlURA3||this study|
|Δpex6||YlPEX6 disruptant with YlURA3||this study|
|Δpex5||YlPEX5 disruptant with YlURA3||this study|
Escherichia coli strains JM109 (recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, Δ(lac-proAB)/F′[traD36, proAB+, lacIq, lacZΔM15]) and HB101 (supE44, hsdS20 (rB−mB−), recA13, ara-14, proA2, lacY1, galK2, rpsL20, xyl-5, mtl-1, leuB6, thi-1) were used for plasmid preparations and subcloning.
All Y. lipolytica strains were grown at 30°C. The ingredients of YPD, minimal SD and SG media, and minimal media containing n-alkane, and the manner in which n-alkane was supplied to Y. lipolytica cells on solid media were previously described. Oxidized intermediates of n-dodecane were supplied in 0.5% Triton X-100 in solid media. All strains used in this study are listed in Table 1.
2.2Isolation of mutants defective in n-decane assimilation
CXAU1 was cultured until late exponential phase, washed, and diluted until OD600= 1.0. This suspension was applied to UV irradiation for 10–25 s, resulting in a 30–40% survival. It was then plated on a YPD plate and incubated overnight at 30°C. The resulting colonies were transferred to SG and n-decane media containing the required nutrients, and incubated at 30°C. Colonies that were viable on SG but not on n-decane medium were selected.
2.3Mutation in C3HC4 ring finger motif region of YlPEX10
The C341S, H343W, and C346S point mutations were created by PCR using pPEX10 as a template. The respective oligonucleotides C341S-Nae I, H343W-Nae I, and C346S-Nae I, together with B0-6-1-down (Table 2), were used to create C341S, H343W, and C346S. Each PCR product was digested with Nae I and Eco RI, and a 529-bp Nae I–Eco RI fragment was substituted for the wild-type Nae I–Eco RI fragment in YlPEX10 on pPEX10. The resultant plasmids were digested by Kpn I, and 3381-bp fragments were cloned into the Sma I site of pSAT4.
Cells were precultured in 2 ml SG medium for 2 days, seeded at 2% on 10 ml SG medium and cultured for 24 h. They were then centrifuged, washed twice, suspended in 3 ml of YNB containing various carbon sources, and shaken for 6 h. Next the cells were centrifuged again, washed twice, and crushed by glass beads of 0.45–0.5 mm diameter for 3 min in 1 ml Z-buffer (0.06 M Na2HPO4, 0.04 M NaH2PO4, 0.01 M KCl, 0.001 M MgSO4, 0.05 M β-mercaptoethanol). They were centrifuged a final time at 15 000 rpm for 10 min. The supernatants were used for β-galactosidase assays. One unit of activity was equal to 1 nmol ONP produced per minute.
2.5Nucleotide sequence of the YlPEX10 gene and identification of the mutation site
The YlPEX10 gene was sequenced with a SequiTherm EXCEL-II Long-Read Premix DNA Sequencing Kit-LC (Epicenter Technology, Madison, WI, USA) and analyzed with a DNA sequencer (LI-COR model 4000) and GENETYX software as previously described. For the identification of the mutated spot, we amplified the YlPEX10 fragment of the B0-6–1 strain by PCR using the primers B0-6-1-up and B0-6-1-down. The amplified DNA fragments were subcloned into pUC119, sequenced using a DNA Sequencing Kit (BigDye™ Terminator Cycle Sequencing Ready Reaction; Applied Biosystems, Foster City, CA, USA) and analyzed using an ABI Prism™ 310 Genetic Analyzer.
2.6Nucleotide sequence accession numbers
The nucleotide sequence data of YlPEX10 were registered in the DDBJ database under accession number AB036770. The YlPEX10 data were registered by Nicaud and coworkers to EMBL under accession number AJ012084.
3.1Isolation of a mutant defective in n-decane assimilation and low YlALK1 inducibility
To identify various factors involved in the expression of YlALK1, we isolated mutants defective in n-decane assimilation by UV mutagenesis. To select mutants whose YlALK1 expression was specifically affected, we introduced a plasmid bearing a YlALK1 promoter-lacZ fusion construct (pSAT4-YlALK1p-lacZ) into the mutants and measured β-galactosidase activity after the shift of carbon source to n-decane. A mutant strain B0-6–1 showed the lowest β-galactosidase activity among the transformed mutants (Fig. 1). YlALK1 expression was repressed by glycerol but not by glucose as previously reported [11,12]. The mutant grew well on SG medium and was viable at least after 6 h induction on n-decane medium (data not shown); therefore, this low β-galactosidase activity was not due to the decrease in living cells after the change of carbon source.
3.2Cloning of a gene that complements the mutation in B0-6-1
To screen for a gene that complements the mutation in B0-6-1, this mutant was transformed with a cosmid library of the Y. lipolytica genome. Among the approximately 1×104 transformants screened, three resumed growth on n-decane medium. By examining restriction profiles of cloned DNA fragments and by subcloning, we found that the three transformants had plasmids with a common region of Y. lipolytica DNA, and that a 3.4-kb Kpn I–Kpn I fragment complemented the B0-6–1 mutation. By sequencing this fragment, we found an open reading frame. Its putative amino acid sequence had 44% identity to Hansenula polymorpha Pex10p and 31% to Saccharomyces cerevisiae Pex10p. Based on these homologies, we named the gene YlPEX10. In other organisms, PEX10 products are localized in the peroxisome membrane and have been shown to be essential for the normal functioning of peroxisome [26–28], but its function has not been clearly elucidated. Pex10p commonly has a C3HC4 ring finger motif in its C-terminal region, which is essential for the function and is also conserved in YlPex10p.
We confirmed that YlPEX10 functionally complemented the mutation in the mutant strain B0-6–1 (data not shown). We also confirmed that YlPEX10 in B0-6–1 genomic DNA had one mutation, a G to A mutation 471 bp downstream of the initiation codon, which means that the codon TGG was changed to a termination codon TGA (data not shown). By this change, the YlPEX10 gene product should be truncated and not functional in the B0-6–1 mutant.
3.3The C3HC4 ring finger motif is essential for the function of YlPex10p
PEX10 gene products have a common C3HC4 ring finger motif, and this motif is also conserved in YlPex10p. In Pichia pastoris, point mutations that alter conserved residues in this motif abolished the function of Pex10p. To address whether the C3HC4 ring finger motif of YlPex10p is also essential for its function, we created point mutations within it. Cysteine residues in the C3HC4 motif were changed to serine (C341S or C346S), and its histidine residue was changed to tryptophan (H343W). YlPEX10 with the H343W mutation hardly complemented YlPEX10 disruptant (Δpex10) (Fig. 2). Though transformants with YlPEX10 with other single mutations, C341S or C346S, were able to grow on n-decane medium as well as the strain with the wild-type YlPEX10, a transformant with mutant YlPEX10 that carries both C341S and C346S mutations (C2S2) failed to grow in n-decane medium (Fig. 2). These results indicated that the C3HC4 ring finger motif is essential for the function of YlPex10p.
3.4YlPex10p is one of the peroxisomal components but its disruption affects YlALK1 expression
The PEX10 product in other eukaryotes is thought to be one of the peroxisomal membrane components. We examined the peroxisomes of YlPEX10 mutants by electron microscopy. Eight to 10 peroxisomes were present in each wild-type cell grown on n-decane, but peroxisomes were not observed in B0-6–1 and Δpex10 cells after 6 h incubation on n-decane as expected (data not shown). To investigate the subcellular localization of YlPex10p, we generated myc-tagged YlPEX10 in its N-terminal (YlPEX10myc). YlPEX10myc was as functional as YlPEX10. We transformed strain CXAU1 by two plasmids: pSAT4-YlPEX10myc and pSUT5-YlALK1p-GFP-AKL. The former carried YlPEX10myc and the latter carried a fusion gene encoding green fluorescent protein (GFP) with tripeptides AKL in its C-terminal, a modified GFP with a peroxisome-targeting signal [29,30]. As the result of the indirect immunofluorescence analysis of this strain grown by n-decane, we confirmed that the localization of the YlPEX10myc product was the same as for GFP-AKL, and was not in the nucleus (data not shown). This result suggests that YlPex10p localizes to peroxisomes and is not likely a nuclear transcription factor.
Next, Δpex10 was transformed with pSAT4-YlALK1p-lacZ to investigate the effect of YlPEX10 disruption on YlALK1 expression. Fig. 1 shows that induction of the β-galactosidase activity of the transformant by n-decane was moderate, 60–70% lower than that of the wild-type, and similar to the level in strain B0-6–1 with the same YlALK1p-lacZ plasmid. This result indicates that YlPEX10 is relevant to YlALK1 expression, though its product is not a transcription factor.
3.5YlALK1 expression is low in mutants defective in peroxisome
To determine whether the low YlALK1 expression in the YlPEX10 mutants was specifically caused by the loss of PEX10 function or caused by defects in peroxisome formation as a result of YlPEX10 mutations, we generated disruption mutations in PEX5 or PEX6. Pex5p is a PTS1 receptor. It carries peroxisomal proteins with a C-terminal PTS1 to peroxisomes. Pex6p is a member of the AAA protein family of ATPases and functions in the fusion of small peroxisomal vesicles in Y. lipolytica[23,31]. These disruptants, Δpex5 and Δpex6, were unable to grow in n-decane medium like Δpex10. They were transformed with pSAT4-YlALK1p-lacZ. Fig. 3 shows that the resultant transformants showed low YlALK1 expression when incubated in the presence of n-decane. Δpex6 showed β-galactosidase activity almost identical to that of Δpex10, whereas Δpex5 showed much less β-galactosidase activity. The wild-type strain and these disruptants carrying pSAT4-YlLEU2p-lacZ showed almost the same level of β-galactosidase activities regardless of carbon source (data not shown). These disruptants were viable even after 6 h incubation on n-decane (data not shown), and hence their low β-galactosidase activities were not caused by a decrease in cell viability. These results indicate that the low inducibility of YlALK1 is not specific to YlPEX10 mutations and was likely caused by the loss of peroxisome function.
3.6Effects of n-alkane oxidation products on YlALK1 expression
n-Alkanes are oxidized to corresponding long-chain alcohols by n-alkane-inducible P450ALKs and then to fatty aldehydes and fatty acids by oxidation enzymes in microsomes. These oxidation products are transported to peroxisomes and subjected to further oxidation. If peroxisomes are not functional, they should be accumulated in the cell. We therefore presumed that these accumulated intermediates might have prevented full expression of YlALK1.
Y. lipolytica can assimilate n-decane but not oxidized n-decane derivatives as a carbon source. Therefore, we first confirmed that n-dodecane derivatives are assimilated by Y. lipolytica and tested the effect of n-dodecane and its oxidation products –n-dodecanol, 1-dodecanal, and n-lauric acid – on the expression of YlALK1. When the wild-type cells were grown on the respective oxidized derivatives of n-dodecane, induction of the YlALK1 promoter was slight (Fig. 4). Although n-dodecane itself is slightly less effective in induction of YlALK1 than n-decane, the level of induction is still 75–80% of that induced by n-decane.
We next examined whether YlALK1 expression was repressed by these oxidized derivatives of n-dodecane. As shown in Fig. 5, β-galactosidase activity became lower as the amount of n-dodecanol or 1-dodecanal was increased. Lauric acid at 0.5% decreased the level of activity to about one-third of that without lauric acid, and at higher concentrations did not further decrease the YlALK1 expression, probably because lauric acid is not very soluble. These results suggest that the accumulation of oxidation intermediates of n-alkane in Y. lipolytica cells somehow prevents YlALK1 expression.
The Y. lipolytica strain B0-6–1 was isolated as a mutant with low YlALK1 promoter activity in the n-decane medium from a pool of mutants that failed to grow on n-decane as the sole carbon source. The gene that complemented its mutation was expected to encode a regulatory protein necessary for the expression of YlALK1, since the product of YlALK1, cytochrome P450ALK1, is specifically required for the initial oxidation of n-decane. The isolated gene, however, had extensive homology to PEX10 genes of other organisms, and we therefore investigated properties of this Y. lipolytica PEX10, YlPEX10.
Mutant B0-6–1 carried a critical mutation in YlPEX10, as described in Section 3. This mutant and the YlPEX10-disrupted mutant did not form peroxisomes in their visible forms, indicating that YlPEX10 is necessary for peroxisome formation as reported for other yeast PEX10 genes [26,28]. The product of YlPEX10 also has a C3HC4 motif that is common among all PEX10 products [26,28,32]. This motif was also proved to be essential for the function of YlPex10p. YlPex10p was localized in peroxisomes as reported for other Pex10ps [26,28,32]. All these results support the notion that YlPex10p functions as a component of peroxisome but not as a regulatory protein that controls YlALK1 expression.
With respect to why the YlALK1 expression was much lower in strain B0-6–1 and the YlPEX10 disruptant, we presume that peroxisome deficiency of these mutants led to the decrease in YlALK1 promoter activity. When Y. lipolytica is cultured in n-alkane medium, it converts n-alkanes to fatty alcohols by the function of cytochrome P450ALKs and subsequently to fatty aldehydes and fatty acids. These oxidation products of n-alkane are transported to peroxisomes and subjected to β-oxidation. Therefore, the absence of peroxisome or its functional deficiency should result in accumulation of these intermediates or fatty acyl-CoA in cells, which could inhibit the transcription from the YlALK1 promoter. The result of a recent publication from our laboratory, which showed that a deficiency of peroxisomal thiolase also resulted in a substantially reduced expression of YlALK1, also supports this notion. This hypothetical repression of YlALK1 seems to actually have been present, based on the finding that, when a wild-type strain was incubated in the presence of both n-dodecane and its oxidized intermediates, the YlALK1 promoter activity decreased with increasing concentration of intermediates. The extent of decrease was almost similar to those observed in mutant B0-6–1 and Δpex10 that were cultivated in the presence of n-decane. Another finding supporting our hypothesis came from the experiments using other YlPEX gene-disrupted mutants. If peroxisome deficiency of YlPEX10 mutants is really a cause of low YlALK1 promoter activity in the n-decane medium, then these mutants should show similar levels of YlALK1 gene expression. Strains Δpex5 and Δpex6, in which YlPEX5 and YlPEX6 were disrupted, respectively, showed much lower YlALK1 promoter activity than the wild-type strain. YlALK1 promoter activity in Δpex6 that was incubated in the presence of n-decane was about one-third of that in the wild-type, and the activity in Δpex5 was much lower and nearly identical to the level of uninduced activity. Thus it is very likely that the YlALK1 promoter is repressed by peroxisome deficiency, which presumably causes feedback inhibition through the accumulation of end-oxidized n-alkane derivatives. To prove this hypothetical notion, it would be worthwhile to measure the cellular concentration of oxidation intermediates of n-alkane.
Why did Δpex5 show stronger repression of the YlALK1 promoter activity than Δpex6 or Δpex10? Pex5p is a PTS1 receptor that shuttles between the cytosol and peroxisomes. The function of Pex6p is controversial. Pex6p is thought to act in the early step of the peroxisome assembly pathway and to function in small peroxisomal vesicle fusion with Pex1p in Y. lipolytica. However, Pex6p in P. pastoris is thought to act late in the peroxisomal matrix protein import with Pex1p (i.e., after the matrix protein import in which Pex10p functions) so the role of Pex6p (including Pex1p) may be quite different in Y. lipolytica and P. pastoris. Pex10p is an intrinsic component of peroxisome and is thought to function in peroxisome matrix protein transport. According to Rachubinski et al., the stability and abundance of Pex5p were not decreased in the pex6 mutant in Y. lipolytica, so their functions might not be correlated. In P. pastoris, reduced expression of Pex5p was not observed in the PEX10 disruptant. Taken together, these results suggest that Pex5p, Pex6p, and Pex10p may act independently of each other. Disruption of YlPEX5 might have resulted in more severe inhibition of β-oxidation and higher accumulation of oxidation intermediates of n-alkane.
Another possibility is that the low YlALK1 promoter activity in Δpex10 was due to the low concentration of inducer of YlALK1 in cells. If n-alkane uptake is somehow repressed in peroxisome-deficient cells, the YlALK1 expression level should also decrease. When we investigated n-[1-14C]hexadecane uptake in the wild-type strain and Δpex10, Δpex10 incorporated n-[1-14C]hexadecane about eight times less efficiently than the wild-type strain (our unpublished data). Although it is not certain whether n-decane or n-dodecane uptake is also lowered by peroxisome deficiency, this result suggests that lowered n-alkane uptake might also have played a role in the decrease of YlALK1 expression.
The initial goal of this study was to isolate a regulatory mutant defective in the induction of the YlALK1 promoter in response to n-decane by screening mutants defective in n-decane assimilation and in YlALK1 promoter inducibility. However, we found that peroxisome deficiency caused repression of n-alkane-induced YlALK1 expression. This indicates that screening of mutants defective in n-dodecane utilization but able to metabolize dodecanol will help to clarify our understanding of the regulatory genes for YlALK1.
We thank Dr. Y. Sakai for provision of the GFP-AKL gene, and Drs. Y. Nagata, R. Fukuda and K. Umebayashi for their helpful input. This work was performed using the facilities of the Biotechnology Research Center at the University of Tokyo.