Characterization of triglyceride lipase genes of fission yeast Schizosaccharomyces pombe
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- Yazawa, H., Kumagai, H. & Uemura, H. Appl Microbiol Biotechnol (2012) 96: 981. doi:10.1007/s00253-012-4151-8
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Triglycerides (TG) are major storage lipids for eukaryotic cells. In this study, we characterized three genes of fission yeast Schizosaccharomyces pombe, SPCC1450.16c, SPAC1786.01c, and SPAC1A6.05c, that show high homology to Saccharomyces cerevisiae TG lipase genes, TGL3, TGL4, and TGL5. Deletion of each gene increased TG content by approximately 1.7-fold compared to the parental wild-type strain, and their triple deletion mutant further increased TG content to 2.7-fold of the wild-type strain, suggesting that all three genes encode TG lipase and are functioning in S. pombe. The triple deletion mutant showed no growth defect in rich and synthetic medium, but its growth was sensitive to cerulenin, an inhibitor of fatty acid synthesis. This growth defect by cerulenin was restored by adding oleic acid in media, suggesting that these genes were involved in the mobilization of TG in S. pombe. When ricinoleic acid was produced in the triple mutant by introducing CpFAH12 fatty acid hydroxylase gene from Claviceps purpurea, percent composition of ricinoleic acid increased by 1.1-fold compared to the wild-type strain, in addition to a 1.6-fold increase in total fatty acid content per dry cell weight (DCW). In total, the ricinoleic acid production per DCW increased by 1.8-fold in the triple deletion mutant.
KeywordsSchizosaccharomyces pombeYeastTriglyceride lipaseTriglycerideFatty acid
We have been studying the heterologous production of polyunsaturated fatty acids (PUFAs) and fatty acid derivatives in model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. So far, we have produced linoleic (C18:2) and α-linolenic (C18:3n − 3) acids (Kainou et al. 2006), so-called essential fatty acids for human, and dihomo-gamma-linolenic acid (C20:3n − 6), a PUFA with anti-inflammatory effect, in S. cerevisiae (Yazawa et al. 2007). Recently, we are attempting to produce ricinoleic acid (12-hydroxy-octadeca-cis-9-enoic acid, C18:1–OH), an important natural product as a petrochemical replacement in a variety of industry, in S. pombe by introducing oleate Δ12-hydroxylase gene (FAH12). We chose S. pombe instead of S. cerevisiae because the amount of oleic acid (C18:1n − 9), a precursor of ricinoleic acid, is much higher in S. pombe and it reaches to around 75 % of its total fatty acids. Indeed we succeeded to produce more than 50 % ricinoleic acid content to total fatty acids in S. pombe (Holic et al. 2012). In all cases described earlier, the next step to take is to increase their lipid content to apply our knowledge to industrial use because they do not accumulate storage lipids and usually only 5 to 7 % of lipids are produced per dry cell weight (DCW). Since triglycerides (TGs) are the major storage lipids, we are focusing on the study of their metabolism in an effort to increase TG content per cell by removing TG lipase genes and reducing TG degradation in S. pombe.
In S. cerevisiae, a series of recent studies have identified three TG lipase genes (TGL3, TGL4, and TGL5). Whereas enzymes involved in TG synthesis in S. cerevisiae appear both structurally and functionally conserved to mammalian cells, the level of sequence conservation for TG-degrading enzymes is less pronounced. The first characterized S. cerevisiae Tgl1p displays more than 30 % sequence identity to mammalian acid lipases, but it turned out to function as a steryl ester hydrolase rather than a TG lipase in vivo (Jandrositz et al. 2005; Koffel et al. 2005). The first S. cerevisiae TG lipase identified was Tgl3p (Athenstaedt and Daum, 2003), but it lacks any significant structural homology to known lipases in higher eukaryotes. Subsequently, the second and the third TG lipase genes, TGL4 and TGL5, were identified based on the homology to Tgl3p (Athenstaedt and Daum, 2005).
In fission yeast S. pombe, however, studies on metabolism are less advanced than those in S. cerevisiae. S. pombe has been extensively used to study the molecular mechanisms of many aspects of cell physiology, including cell cycle and stress signaling, but little work has been done in the area of lipid cell biology. Its genome sequences predicted candidate genes based on sequence similarity, but no experimental evidence has been obtained so far. In this study, we constructed deletion mutants of candidate genes and examined their impact on TG content to experimentally identify TG lipase genes in S. pombe. We also provided evidence for their biological significance in TG mobilization.
Materials and methods
Strains, media, and growth
Plasmid manipulations were carried out using E. coli DH5α (F-endA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 ∆(argF-lacZ)U169 ϕ80 ∆(lacZ)M15) grown in Luria–Bertani broth, supplemented with ampicillin. The S. pombe strain ARC010-1 (h-leu1-32 ura4-Δ18) (Idiris et al. 2006) was used for this study. S. pombe strains were grown in YES rich media and EMMS synthetic media or EMMS dropout media depending on the selective pressure required to maintain plasmids (Alfa et al.1993). EMMS was used for normal growth, and nitrogen-limited minimal medium (EMM-C/N3) (Holic et al. 2012) was used for the production of ricinoleic acid because our previous experiments indicated that the fatty acid content was higher in the cells grown in EMM-C/N3. EMM medium contained 0.5 % (w/v) ammonium chloride and 2 % (w/v) glucose, but in nitrogen-limited EMM medium (EMM-C/N3), the concentration of ammonium chloride was reduced to 0.1 % and the concentration of glucose was increased to 10 %.
Yeast cells were transformed by using Frozen-EZ Yeast Transformation II kit (Zymo Research, CA, USA) according to the protocol recommended by the manufacturer. For the assay of fatty acids, ARC010-1 strains were pre-cultured overnight at 30 °C in EMMS medium and the resultant cultures were inoculated into fresh EMMS at OD600 = 0.2 unless otherwise specified, and the cultures were shaken at 40 rpm. For the assay of ricinoleic acid production in the CpFAH12 transformant, EMM-C/N3-Leu was used instead of EMMS. Growth in liquid media was monitored by measuring the turbidity of the cells at OD600.
For automatic detection of growth, ARC010-1 disruptants grown in EMMS overnight at 30 °C were inoculated into fresh EMMS at OD630 = 0.05 and the growth curves in liquid media were obtained by monitoring the turbidity of the cells at 630 nm with an automatic detector (Bio-Plotter, Toyo-Sokki, Japan).
The standard techniques of DNA manipulation used in this study are described in Sambrook and Russell (2001). The FAH12 gene from Claviceps purpurea (CpFAH12, Meesapyodsuk and Qiu 2008) was cloned under the nmt1 promoter of S. pombe in pSL10 integration-type plasmid to construct pL2428-9 (Holic et al. 2012). pSL10 is designed to integrate between the leu1-32 and topo2+ loci of chromosome II (Idiris et al. 2010).
Disruption of lipase genes
The open reading frame (ORF) regions of ptl1 (SPCC1450.16c) (+10 bp to +1,658 bp), ptl2 (SPAC1786.01c) (+21 bp to +2,080 bp), and ptl3 (SPAC1A6.05c) (+10 bp to +1534 bp) were removed from the genome of ARC010-1 by the two-step pop-in and pop-out method of ura4 marker, that was named “latency to universal rescue system” or “Latour system” (Hirashima et al. 2006). Their proper disruptions were verified by colony PCR analysis (data not shown).
Fatty acid analysis
Total fatty acid contents were determined by gas chromatographic analysis as described (Kainou et al. 2006). Fatty acid analysis was performed by the application of 0.2-μl aliquots to a gas chromatograph (GC2010, Shimadzu, Japan) equipped with a TC-70 capillary column (30 × 0.25 mm i.d., GL Sciences, Japan) under temperature programming (160–234 °C at 4.5 °C/ min increments). Fatty acid composition was calculated based on the area of each peak, and the amount was determined by comparison with the methylheptadecanoate (C17:0) standard.
Thin-layer chromatography of lipids
Total lipids were extracted from cells by the procedure of Bligh and Dyer (1959). Cells (10 ml of culture each) of S. pombe grown to a stationary phase for 5 days were washed once with water, suspended in 2 ml of methanol, and disrupted by vortexing for 90 s (three times for 30 s each time) together with 0.5 g of glass beads (Brown ϕ = 0.4 to 0.5 mm). Then, 1 ml of chloroform was added to the extract and the tubes were kept shaken for 1 h at room temperature. Liquids were recovered by spinning tubes at 3,000 rpm for 5 min. Glass beads were washed twice with 1.5 ml of chloroform/methanol (1:2) mixture and the liquids were combined into the first solution. Lipids were extracted in chloroform by vortexing the solution with 2 ml each of chloroform and water, and the chloroform phase was separated by spinning them at 1,000 rpm for 5 min. The extracted chloroform was vaporized under nitrogen gas and the dried lipids were suspended in 200 ul of iso-propanol containing 1 % Triton-X100. Samples were kept stored at −80 °C until processed for lipid analysis.
Lipid compositions were analyzed by thin-layer chromatography (TLC). Lipids corresponding to 2.5 OD equivalent cells were separated by TLC (silica gel 60 plate, Merck) using the solvent systems chloroform/acetone/methanol/acetic acid/water (50:20:10:10:5 per volume; developed to one half of the total distance) first for the separation of phospho lipids and then hexane/diethylether/acetic acid (80:40:1 per volume; total distance) for the separation of neutral lipids. Separated lipids were visualized on TLC plates by post-chromatographic staining. Plates were dipped for 10 s into a developing reagent consisting of 4.65 g of MnCl24H2O, 450 ml of methanol, and 30 ml of concentrated sulphuric acid per liter, briefly dried, and heated to 120 °C for 30 min. Subsequently, separated lipids were quantified by densitometric scanning with LAS-1000 image analyzer (Fuji film).
Cellular total lipids were prepared as described earlier. The amount of triglyceride was determined spectrophotometrically by using the L-type WAKO TG-M kit (Wako, Japan) according to the protocol of the manufacturer. The amount of FA in TG was calculated based on the factor derived from triolein (glycerol trioleate) that was assayed with both gas chromatography and this kit. When the same amount of triolein was measured with the L-type WAKO TG-M kit (TG assay) and gas chromatography (FA assay), the value of TG measured by the TG assay kit was 1.18-fold higher than the value of FA measured by gas chromatography.
The S. pombe strains grown to a stationary phase in EMMS at 30 °C were harvested by centrifugation, washed once with PBS buffer (0.137 mM NaCl, 2.68 μM KCl, 10.1 μM Na2HPO4, 1.76 μM KH2PO4), and stained in Nile Red solution (5 μg/ml in PBS) for 10 min in the dark. Nile Red fluorescence images of living cells were detected with an Olympus BX51 fluorescent microscope system in the combination of a 460–495-nm band pass exciter filter (BP460-495 IF) and a 510-nm-long pass barrier filter (BA510 IF) (WIB3 mirror unit). Pictures were taken with a DP30BW CCD camera (Olympus, Japan).
Identification of TG lipase genes in S. pombe
Deletion of ptl1, ptl2, and ptl3 resulted in an increase in TG content
Characterization of double and triple deletion mutants of ptl1, ptl2, and ptl3
To determine whether the functions of ptl1p, ptl2p, and ptl3p are redundant or not, we constructed their double and triple deletions by using a repetitive homologous recombination of Latour system (Hirashima et al. 2006). Like single mutants, all double and triple mutants were viable at 30 °C, and no obvious morphological changes were observed under a light microscope (data not shown), indicating that even their triple deletions did not show synthetic lethality.
Their TG and FA contents are summarized in Fig. 3. As we had expected, the TG contents were further increased in the double deletion of ptl1 plus ptl2 and ptl2 plus ptl3 (2.85 and 2.59-fold), but not in the ptl1 plus ptl3 deletion mutant (1.70-fold, similar to the level of single mutants). The TG content level in the triple mutant (2.68-fold) was similar to that of the double deletion of ptl1 plus ptl2 and ptl2 plus ptl3. In these mutants, the percentage of FA derived from TG increased to around one third (34 to 38 %) of the total FA.
Biological significance of ptl1, ptl2, and ptl3 genes in TG metabolism
To further confirm this point, the triple mutant was grown in the presence of cerulenin but with a supply of exogenous oleic acid at the same time. Under this condition, its growth inhibition was dramatically restored almost to the level of cells grown in the absence of cerulenin (Fig. 7b, see closed triangles). Furthermore, the hampered growth by cerulenin was partially restored by adding oleic acid at t = 15 h in the medium (Fig. 7b, see closed squares). Taken together, these results clearly demonstrated that ptl1, ptl2, and ptl3 genes were functioning for vital TG mobilization in S. pombe.
Expression of CpFAH12 gene in the triple deletion mutant
Fatty acid composition of total fatty acids in the triple mutant expressing CpFAH12 gene
Fatty acid composition in weight %
Total FA (μg)/mg of DCW
[second row: fatty acid contents (μg/mg of dry cell weight)]
0.67 ± 0.06
0.44 ± 0.04
11.07 ± 0.57
3.21 ± 0.40
5.26 ± 0.42
77.94 ± 0.49
46.51 ± 9.17
[0.31 ± 0.03]
[0.20 ± 0.02]
[5.12 ± 0.75]
[1.48 ± 0.11]
[2.47 ± 0.68]
[36.27 ± 7.37]
0.70 ± 0.04
0.47 ± 0.04
10.48 ± 0.25
3.24 ± 0.23
6.85 ± 0.23
76.80 ± 0.15
62.94 ± 3.97
[0.44 ± 0.06]
[0.30 ± 0.05]
[6.37 ± 0.58]
[1.98 ± 0.30]
[4.74 ± 0.16]
[48.16 ± 2.90]
0.55 ± 0.02
0.44 ± 0.02
10.60 ± 0.35
1.07 ± 0.09
0.13 ± 0.00
9.83 ± 0.14
40.67 ± 0.95
6.31 ± 0.34
29.06 ± 0.33
77.37 ± 2.20
[0.43 ± 0.01]
[0.34 ± 0.01]
[8.20 ± 0.04]
[0.82 ± 0.05]
[0.10 ± 0.00]
[7.61 ± 0.33]
[35.48 ± 1.63]
[4.88 ± 0.12]
[22.48 ± 0.39]
0.67 ± 0.01
0.43 ± 0.02
10.87 ± 0.14
1.10 ± 0.08
0.18 ± 0.02
11.40 ± 0.20
32.96 ± 1.14
7.86 ± 0.21
32.93 ± 1.11
124.56 ± 5.50
[0.84 ± 0.03]
[0.54 ± 0.04]
[13.53 ± 0.50]
[1.37 ± 0.15]
[0.23 ± 0.03]
[14.19 ± 0.50]
[41.03 ± 1.50]
[9.80 ± 0.63]
[41.04 ± 2.91]
Studies on metabolism are rare in S. pombe compared to those in S. cerevisiae. To understand the metabolism of TGs, major storage lipids, and apply it to the production of useful FAs in S. pombe, we attempted to identify its TG lipase genes. The predicted lipase genes were selected from the genome database, and their lipase activities were addressed by constructing their deletion mutants. In S. cerevisiae, three genes have been identified to encode TG lipases (TGL3, TGL4, and TGL5) (Athenstaedt and Daum, 2003; Athenstaedt and Daum, 2005), and it was demonstrated that the single deletion of TGL3 or TGL4 increased TG content by 2.39-fold and 1.73-fold, respectively, but deletion of TGL5 did not (0.98-fold) (Athenstaedt and Daum, 2005).
In S. pombe, three genes also have high homology to S. cerevisiae TG lipase genes, and we showed that their disruptions affected the TG and fatty acid contents. Their single deletions showed a similar level of increase in TG content, suggesting that they are functioning equally. Their additive effects were observed in the double deletion mutants of ptl1 plus ptl2 and ptl2 plus ptl3 (2.85- and 2.59-fold increase, respectively), but not in ptl1 plus ptl3 deletion mutant (1.70-fold, similar to the level of single mutants). The TG level in the triple mutant (2.68-fold increase) was similar to that of Δptl1 plus Δptl2 and Δptl2 plus Δptl3, also supporting that the effect of ptl1 plus ptl3 deletions was not additive.
The reason for the non-additive effect of ptl1p plus ptl3p is not clear, but one possibility is the compensation of their expressions. For example, the expression of adh4, the minor alcohol dehydrogenase gene that is not normally produced, is induced when adh1 is disrupted, and it supports fermentative growth in the absence of Adh1p (Sakurai et al. 2004). Thus, the expression levels of ptl1, ptl2, and ptl3 were examined by RT-PCR and normalized based on the act1 expression level to address whether the deletion of one gene affects the expression of other genes to compensate each other (data not shown). The expression levels of ptl1, ptl2, and ptl3 were about 5, 60, and 40 % of act1 in the wild-type strain, and their expression levels were basically not affected by gene disruption of other lipase genes, indicating that their expressions were not compensating each other. Furthermore, in spite of the lower-level expression of ptl1 and a partial conservation of lipolytic motif in ptl1p, ptl1 is not considered to be a minor gene because the single deletion of ptl1 increased the TG level to a similar level as the deletion of another gene.
The other possibility is the formation of a complex between ptl1p and ptl3p. Since the homo-dimer formation of Tgl3p and Tgl4p was predicted based on the genome-wide two-hybrid experiments in S. cerevisiae (Ito et al. 2001), if the products of ptl1p and ptl3p work together by making a complex, the phenomenon could be explained. Furthermore, we should also consider if ptl1p, ptl2p, and ptl3p have additional functions in lipid metabolism because it has been reported recently that Tgl3p and Tgl5p of S. cerevisiae, which are homologs of ptl1p, ptl2p, and ptl3p, had a highly conserved H-(X)4-D aceyltransferase motif, and indeed they had acyltransferase activities (Rajakumari and Daum, 2010). The same motif (169HREQKD174) was found in ptl2p upstream of its lipolytic motif, but in contrast to the deletion of TGL3 or TGL5, no significant decrease in phospholipids was observed in the single deletion mutant of ptl1p, ptl2p, or ptl3p (Fig. 4). However, additional biochemical analyses would be required to examine their additional functions, including their protein level interactions.
To examine whether the TG lipase activity of ptl1p, ptl2p, and ptl3p, predicted from the analysis of the triple deletion mutant, has biological significance, we addressed the mobilization of cellular TG by examining the growth of the triple deletion mutant in the presence of cerulenin. The cerulenin-sensitive phenotype of the triple mutant and its growth recovery by supplementation of oleic acid strongly suggested that they are functioning in TG mobilization in S. pombe cells.
We are studying the possibility of using S. pombe as a host for the production of useful fatty acids and their derivatives because we consider that S. pombe can be a better microbe than S. cerevisiae, owing to its very high C18:1 content. The heterologous expression of CpFAH12 gene in the triple mutant demonstrated that this strain was useful and the production of ricinoleic acid per DCW was increased by 1.8-fold in the triple mutant, suggesting that the prevention of TG degradation by the disruption of TG lipase genes may be one of the strategies to increase lipid content. At the current moment, however, the disadvantage of S. pombe is that its fatty acid content is not sufficiently high to apply transgenic S. pombe strains to industrial applications. S. cerevisiae and S. pombe do not accumulate storage lipids and usually only 5 to 7 % of lipids are produced per DCW, whereas some oleaginous yeasts and fungi have 30 to 50 % lipids. Recently, we have established an S. cerevisiae strain with a total lipid content of 29 % by the DGA1 (diacylglycerol acyltransferase) overexpression in combination with SNF2 disruption and LEU2 overexpression (Kamisaka et al. 2007). Thus, the next step to take is to combine the prevention of TG degradation by TG lipase deletion and the enhancement of TG synthesis by the overexpression of diacylglycerol acyltransferases (Zhang et al. 2003) to assess whether we could further increase TG content in the cells.
A part of this work was supported by an A-STEP (Adaptable and Seamless Technology transfer program through target-driven R&D) research grant from Japan Science and Technology Agency (JST) to HK and HU. We also wish to thank Masakazu Yamaoka, Yasushi Kamisaka, and Kazuyoshi Kimura for continuous encouragement.