Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK1
Roche Products Ltd, Delves Road, Heanor Gate, Heanor, Derbyshire DE75 7SG, UK2
School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK3
Author for correspondence: Donald A. MacKenzie. Tel: +44 1603 255255. Fax: +44 1603 507723. e-mail: donald.mackenzie{at}bbsrc.ac.uk
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ABSTRACT |
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Keywords: stearoyl-CoA desaturase, hexacosenoic acid, fatty acid n-9 desaturation, fatty acid elongation, yeast complementation
Abbreviations: 16:0, palmitic acid; 16:1, palmitoleic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3, -linolenic acid;
-18:3,
-linolenic acid; 20:0, arachidic acid; 20:1, eicosenoic acid; 20:3, dihomo-
-linolenic acid; 20:4, arachidonic acid; 22:0, behenic acid; 22:1, erucic acid; 24:0, lignoceric acid; 24:1, nervonic acid; 26:0, hexacosanoic acid; 26:1, hexacosenoic acid;
9-3, gene encoding fatty acid
9-desaturase homologue; DMOX, 4,4-dimethyloxazoline; ELO2 and ELO3, genes encoding fatty acid elongases; ER, endoplasmic reticulum; FAME, fatty acid methyl ester; LCPUFA, long-chain polyunsaturated fatty acid; ole1 and ole2, genes encoding fatty acid
9-desaturases ole1p and ole2p, respectively; RACE, rapid amplification of cDNA ends; UTR, untranslated region of mRNA
The EMBL accession number for the sequence reported in this paper is AJ278339.
a Present address: Department of Biotechnology, Faculty of Science and Technology, Thammasat University, Rangsit Center, Patumthanee 12121, Thailand.
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INTRODUCTION |
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Multiple fatty acid 9-desaturases, encoded by distinct genes, have been found in a number of other organisms, including rat (Thiede et al., 1986
; EMBL accession no. AB032243), mouse (Tabor et al., 1998
; Zheng et al., 2001
), carp (Tiku et al., 1996
; EMBL accession no. AJ249259), Drosophila melanogaster (Wicker-Thomas et al., 1997
), sesame (Yukawa et al., 1996
), Arabidopsis thaliana (Fukuchi-Mizutani et al., 1998
) and Caenorhabditis elegans (Watts & Browse, 2000
). Although differential expression of these genes has been observed in many cases, the significance of this is not always clear. The three fatty acid
9-desaturase homologues identified in Caenorhabditis elegans displayed different substrate specificities when expressed in yeast but their roles in Caenorhabditis elegans have yet to be elucidated (Watts & Browse, 2000
). In carp, CDS1 and CDS2, encoding two closely related fatty acid
9-desaturases, responded differently to low temperature (Tiku et al., 1996
) and nutritional (S. Polley, M. X. Caddick & A. R. Cossins, personal communication) stimuli and exhibited tissue-specific expression. The differences in expression were thought to be part of co-ordinated, biosynthetic responses to body cooling or dietary changes. In the present paper, we describe the isolation of a gene (
9-3) encoding a third fatty acid
9-desaturase from M. alpina and its functional characterization in yeast.
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METHODS |
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Nucleic acid manipulations.
Degenerate primers with homology to conserved histidine boxes 2 and 3 of fatty acid 9-desaturase genes from S. cerevisiae (Stukey et al., 1990
), Histoplasma capsulatum (Gargano et al., 1995
) and Cryptococcus curvatus (Meesters & Eggink, 1996
) were synthesized on an ABI 394 DNA/RNA synthesizer. Primer combination P5 (5'-CAYMGVTAYACNGAYAC-3'; 192-fold degeneracy) and P2 (5'-RTGRTGRAARTTRTGRT-3'; 64-fold degeneracy) was used to amplify a fragment (P5P2) from M. alpina CBS 528.72 genomic DNA. PCR conditions were 94 °C hot start for 5 min, 30 cycles of 94 °C for 0·5 min, 52 °C for 1·5 min, 72 °C for 1 min, and a final extension at 72 °C for 10 min. The P5P2 fragment was subsequently used to screen a BamHI genomic DNA library from M. alpina CBS 528.72 (Wongwathanarat et al., 1999
) and positive pBK-CMV phagemid clones (Stratagene) were excised in vivo and sequenced. The following primer pairs specific to the M. alpina
9-3 gene were used to analyse intron removal from mRNA by RT-PCR (Wongwathanarat et al., 1999
) under standard PCR conditions at a primer annealing temperature of 52 °C: M9 (5'-TCCTCGGGCAAGAGCTCCAG-3') and M1 (5'-GCGATGAGACCACAGTCGATG-3'), and P5F (5'-ACCATCGTTACACTGACACG-3') and P2R (5'-ACTCGTGGTGGAAGTTGTGG-3'). RT-PCR also identified the
9-3 3' untranslated region (UTR) using the degenerate oligo(dT)18 primer AD99 [5'-AAGCGGCCGC(T)18VN-3'] in the cDNA first-strand synthesis step. Subsequent PCR with gene-specific primer D4 (5'-GAGAAGGGGCAGGCTGTGACT-3') was carried out under standard conditions at a primer annealing temperature of 50 °C. RNA-ligase-mediated rapid amplification of cDNA 5' ends (RLM-RACE), using a First-Choice RLM-RACE kit (Ambion), identified the
9-3 5'-UTR. Gene-specific primers were D9R1 (5'-GCAGTGATGCCCAGTCCGGTG-3') and D9R2 (5'-GGATCGCTTTGATGTGCTTGG-3'). PCRs were carried out as per the kit instructions, using a 60 °C primer annealing temperature.
The ELO3 gene in S. cerevisiae L8-14C was deleted using a PCR-generated kanMX4 cassette (Wach et al., 1994 ; Winzeler et al., 1999
) with 40 bp flanking regions homologous to the ELO3 gene. Forward primer ELO35K (5'- ATTCGGCTTTTTTCCGTTTGTTTACGAAACATAAACAGTCAGCTGAAGCTTCGTACGCTG-3') and reverse primer ELO33K (5'-ATTCTCGCTTCCTATTTAAGCTTTCCTGGAAG-AAGACCTTGATAGGCCACTAGTGGATCTG-3') were used at an annealing temperature of 54 °C to amplify the 1·6 kb ELO3-kanMX4 cassette with vector pFA6a-kanMX4 as template (Wach et al., 1994
). Approximately 1 µg of ELO3 deletion cassette DNA was then transformed into PEG/LiAc-treated whole L8-14C cells, following the method of Gietz et al. (1995)
, and transformants were selected on 16:1/18:1-supplemented medium containing G418 (200 µg ml-1). ELO3-deleted transformants were screened by whole-cell PCR using gene-specific forward and reverse primers ELO3cF (5'-TACCCCAGTTAGGTACTGTG-3') and ELO3cR (5'-CTTTGGGCAGCTAAGGAAATG-3') at an annealing temperature of 58 °C. ELO3-deleted strain L8e1 generated a 1·9 kb PCR fragment, while non-deleted strains gave a 1·5 kb fragment. Deletion of the ELO3 gene in L8e1 was confirmed by analysing this strains fatty acid composition following growth in YNB medium supplemented with 16:1/18:1.
Construction of yeast expression vectors.
Before the transcriptional start point (tsp) of the 9-3 gene was identified by 5'-RACE, a 5'-truncated version of the
9-3 ORF (5'-
9-3), lacking the first 21 codons, was initially cloned into the yeast expression vector pVT100-U (Vernet et al., 1987
; Wongwathanarat et al., 1999
). A synthetic, intronless version of this truncated gene with BamHI and XhoI sites at the 5' end and a BamHI site at the 3' end was created in three steps by overlap extension PCR using a genomic clone as template. First, the following primer combinations were used to amplify three PCR products, B1B2 (413 bp), B3B4 (195 bp) and B5B6 (979 bp) in which the two introns were removed: B1(5'-AAGGATCCCTCGAGAAAAAAATGAAAACGTCTCTCTCCGCT-3') and B2 (5'-AGACCACAGTCGATGGTATC=CAGCAGTGATGCCCAGTCCGGT-3'), B3 (5'-ACCGGACTGGGCATCACTGCTG=GATACCATCGA-CTGTGGTCT-3') and B4 (5'-CTTTTGGGCACCATAGGGGTC=CTTTTCCGTGTCAGTGTAAC-3'), and B5 (5'-GTTACACTGACACGGAAAAG=GACCCCTATGGTG-CCCAAAAG-3') and B6 (5'-AAGGATCCTTATGCGGTGCGACTCACAACAGGTATAG-3'), where the start and stop codons are single-underlined, the relevant restriction sites are double-underlined and the intron deletion sites are indicated by a double hyphen. In the second step, fragments B1B2 and B3B4 were fused together using primers B1 and B4 to generate a 566 bp PCR product (B1B4). Finally, the intronless 1504 bp 5'-
9-3 ORF was created by fusing fragments B1B4 and B5B6 in a PCR with primers B1 and B6. All PCRs were carried out as follows: 5 min hot start at 94 °C, 25 cycles of 94 °C for 0·5 min, 56 °C for 1·5 min, 72 °C for 1 min and a final 10 min extension at 72 °C. The final PCR product (B1B6) was digested with XhoI and BamHI and directionally cloned into pVT100-U. The sequence of the insert was checked using primers ADH1 and ADH2 (Wongwathanarat et al., 1999
) from the yeast ADH1 promoter and terminator regions.
pVT100-U containing the full-length 9-3 ORF was subsequently produced by replacing a 70 bp PstI fragment at the 5' end of the 5'-
9-3 version with a 133 bp PstI fragment containing the extra 21 N-terminal codons of the complete gene. This fragment had been amplified from a
9-3 genomic clone using primers 935P (5'-GCTGCTGCAGGCTCGAGAAAAAAATGGGATCGCTCACTTCG-3') and 933P (5'- CACTGTCGCTGCAGTCACTGTTG-3'), where the
9-3 start codon is single-underlined and the PstI sites are double-underlined. The fragment was generated using TaqPlus Precision (Stratagene) with the following PCR conditions: 1 min hot start at 94 °C, 5 cycles of 1 min at 94 °C, 1 min at 54 °C, 20 s at 72 °C, 20 cycles of 1 min at 94 °C, 1 min at 62 °C, 20 s at 72 °C, followed by 3 min at 72 °C and 10 min at 35 °C. It was then digested with PstI and ligated into purified PstI-cut pVT100-U:5'-
9-3. Clones containing the full-length
9-3 ORF in pVT100-U were identified by PCR and the insert sequenced to verify that the gene was in the correct orientation.
The S. cerevisiae OLE1 ORF was amplified from plasmid pGAL-OLE2.8 (Gonzalez & Martin, 1996 ) which was kindly supplied by Professor C. E. Martin, Rutgers University, Piscataway, New Jersey, USA. An XhoI site was introduced at the 5' end of this gene and a BamHI site at the 3' end using primers 5OLE1 (5'-AACTCGAGAAAAAAATGCCAACTTCTGGAACTAC-3') and 3OLE1 (5'-AAGGATCCTTATACTTAAAAGAACTTACCAGTTTC-3') in a TaqPlus Precision (Stratagene) PCR at an annealing temperature of 52 °C. PCR products were cloned into pCR4-TOPO (Invitrogen) and sequenced. The S. cerevisiae OLE1 ORF was then directionally cloned as an XhoIBamHI fragment into pVT100-U.
Yeast transformation and fatty acid analysis.
pVT100-U containing either the full-length 9-3 ORF or the S. cerevisiae OLE1 ORF was transformed into yeast strains by the PLATE method (Elble, 1992
). Approximately 0·5 µg vector DNA, 100 µg single-stranded herring sperm DNA and one large toothpick of freshly grown yeast cells were used per transformation. Plasmid-containing cells were selected on uracil-minus YNB agar with or without 16:1/18:1 supplementation (Wongwathanarat et al., 1999
). Control transformations were also carried out with pVT100-U lacking any inserts. Transformants were subsequently grown in uracil-minus YNB broth with or without fatty acid supplementation. Fatty acid methyl esters (FAMEs) were prepared as described previously (Wongwathanarat et al., 1999
) and analysed by GC-MS. FAMEs in n-hexane (2 µl) were separated on a 30 m BPX70 0·25 µm column (SGE) in a 5890 Series 2 gas chromatograph (Hewlett Packard) using an injection port temperature of 250 °C and the following column temperatures: 1 min at 110 °C, 110240 °C at a gradient of 5 °C min-1, 10 min at 240 °C. Mass spectra were produced over a scan range of 40500 m/z in a TRIO-1S mass spectrometer (Fisons Instruments VG Organic) that was coupled to the gas chromatograph and operated at an ionization voltage of 70 eV, a source temperature of 200 °C and an interface temperature of 240 °C. To detect FAMEs present at lower concentrations, extracts were concentrated by up to 25-fold before analysing 2 µl as described above. Fatty acid 4,4-dimethyloxazoline (DMOX) derivatives were prepared by reaction of FAMEs with 2-amino-2-methyl-1-propanol at 180 °C for 18 h (Fay & Richli, 1991
). Samples were dissolved in 500 µl iso-hexane and 5 µl analysed by GC-MS on a 100 m CPSil88 column (Chrompack) in a 6890 gas chromatograph (Agilent) with an injection port temperature of 270 °C. The following GC running conditions were used: 35 min at 185 °C; 185190 °C at a gradient of 0·5 °C min-1; 5 min at 190 °C; 190220 °C at a gradient of 0·5 °C min-1; 20 min at 220 °C. The system was pre-calibrated with 68D FAMEs Standard (Nu-Chek Prep.). Mass spectral analysis was performed in electron ionization mode in a 5973 MS (Agilent) over a scan range of 100550 m/z.
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RESULTS AND DISCUSSION |
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The predicted 9-3 protein contained three histidine boxes, four putative transmembrane domains and a C-terminal cytochrome b5 fusion that are typical of a fungal fatty acid desaturase (Fig. 2
) (Napier et al., 1999
; Sperling & Heinz, 2001
). This protein shared 53% aa identity with the M. alpina ole1p and ole2p desaturases and 4050% identity with other fungal fatty acid
9-desaturases. Conversely, it had little or no homology to the other fatty acid desaturases from M. alpina (Michaelson et al., 1998
; Huang et al., 1999
; Sakuradani et al., 1999
), except for about 25% aa identity to the N-terminal cytochrome b5 regions of the
5- and
6-desaturases. A similar low level of amino acid identity was observed with the N-terminal cytochrome b5 regions of a related class of enzymes, the plant sphingolipid desaturases (Sperling et al., 1998
; Libisch et al., 2000
). The
9-3 desaturase was 67 aa residues longer than M. alpina ole1p and ole2p, with the bulk of the difference being a 43 aa extension at the N terminus, but it was similar in size to the fatty acid
9-desaturase from S. cerevisiae (Stukey et al., 1990
). The spacings between histidine boxes and cytochrome b5 domain in
9-3 and the amino acid sequences of these motifs were identical or very similar to those of other fungal
9-desaturases. One interesting observation was that the sequences of histidine box 1 (HRLWSH) and the cytochrome b5 haem-binding pocket [EHPGG(X)10DAT(X)9HS] were identical to those of S. cerevisiae Ole1p and differed from those of M. alpina ole1p and ole2p [HRLWAH and EHPGG(X)10DMT(X)9HS, respectively]. On the other hand, the sequence of histidine box 2 (HRAHH) was the same as that of the other M. alpina
9-desaturases and differed from the yeast sequence (HRIHH). The significance of these sequence differences remains unclear, but may reflect the evolutionary divergence of
9-3 from the M. alpina ole1 and ole2 genes. When displayed on a phylogenetic tree, the
9-3 protein was less related than the Mucor rouxii ole1p to the M. alpina ole1p and ole2p (Fig. 3
). This tree illustrates the clear distinction between animal and fungal fatty acid
9-desaturases and also shows the divergence within species that contain more than one
9-desaturase homologue in the same strain (mouse, rat, carp, Caenorhabditis elegans and M. alpina).
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One major difference seen in yeast 9-3 transformants compared with control strains containing empty vector was the presence of hexacosenoic acid (26:1). Hexacosanoic acid (26:0) is normally found in S. cerevisiae at low concentrations (Welch & Burlingame, 1973
; Oh et al., 1997
), mainly as the hydroxylated fatty acid moiety of sphingolipids (Kohlwein et al., 2001
). Although the amounts of 26:0 and 26:1 in yeast transformants were extremely low (<1% of total fatty acids), the appearance of 26:1 always correlated with the presence of the
9-3 gene (Table 2
). 26:1 was also found in yeast ole1 transformants containing the M. alpina ole1 or ole2 genes (Table 2
). The double bond in 26:1 was determined in all cases to be in the n-9 position by DMOX derivatization (Fig. 5
). In the mass spectrum for 26:1, the molecular ion of 447 m/z units was clearly visible. Radical-induced cleavage occurred uniformly along the alkyl chain, releasing the methyl group at position n-1 (a loss of 15 m/z units) and methylene groups (-CH2-) from positions n-2 onwards (losses of 14 m/z units). The n-9 position of the double bond in 26:1 was evident from the loss of 12 m/z units between positions n-10 and n-11 (320 and 308 m/z units, respectively). Further confirmation that the double bond was indeed at this position came from the characteristic increased abundance of ions resulting from cleavage between positions n-7 and n-8 (348 m/z units) and n-11 and n-12 (294 m/z units). This suggested that the three M. alpina
9-desaturases, ole1p, ole2p and
9-3, also displayed n-9 desaturase activity with very long-chain saturated fatty acid substrates. Conversely, fatty acid elongation of 18:1(n-9) to 26:1(n-9) in these yeast transformants could not be discounted, but this activity would have to be associated in some way with the overproduced
9-desaturase. High levels of an M. alpina
9-desaturase in yeast, as would be expected from the multicopy expression vector used in this study, may have stimulated the activity of endogenous fatty acid elongases (Elo2p/Elo3p), perhaps by interacting with the postulated fatty acid elongase complex in the ER membrane (Kohlwein et al., 2001
).
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ACKNOWLEDGEMENTS |
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Received 8 August 2001;
revised 23 January 2002;
accepted 7 February 2002.