Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK1
School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK2
Horticulture Research International, Wellesbourne, Warwick CV35 9EF, 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: Mortierella alpina, 9-desaturase genes, yeast complementation, fatty acid desaturation, oleaginous fungus
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; 20:3, dihomo-
-linolenic acid; 20:4, arachidonic acid (ARA); ER, endoplasmic reticulum; LCPUFA, long-chain polyunsaturated fatty acid; RACE, rapid amplification of cDNA ends; UTR, untranslated region
The GenBank/EMBL accession numbers for the sequences reported in this paper are Y18553 and Y18554 (CBS 528.72 ole1 and ole2 genomic sequences, respectively) and AF0085500 (CBS 210.32 ole1 cDNA).
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INTRODUCTION |
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Known fungal fatty acid desaturases are all endoplasmic reticulum (ER) membrane-bound enzymes which have their active site on the ERs cytoplasmic face. The active site comprises three histidine-rich boxes, normally containing eight essential histidine residues, which fold up to form the di-iron binding site in the native protein (Shanklin et al., 1994 ). Cytochrome b5 is used as the electron donor and in the majority of cases the desaturase is a protein fusion with a cytochrome b5 domain attached at either the N or C terminus. The substrate for the initial
9-desaturation of 18:0 to oleic acid (18:1) is stearoyl-CoA but some of the subsequent desaturation steps, including
12- and
6-desaturation, occur using the fatty acyl chains of phospholipid molecules (Jackson et al., 1998
). M. alpina mutants defective in most of the desaturase activities have been isolated and these have been used, combined in some instances with specific desaturase inhibitors, to alter the fatty acid composition of the fungus (Jareonkitmongkol et al., 1994
; Kawashima et al., 1997
). An alternative approach to manipulating the LCPUFA biosynthetic pathway in M. alpina is to isolate and either overexpress or disrupt the genes encoding the desaturases and elongases. Indeed, the gene encoding the
5-desaturase which converts dihomo-
-linolenic acid (20:3) to 20:4 has been isolated from two different strains of M. alpina (Knutzon et al., 1998
; Michaelson et al., 1998
). The M. alpina
5-desaturase, as predicted, contains three histidine boxes, although one of the essential histidine residues has been replaced with a glutamine, a change which is found in some other desaturases. This enzyme also contains a cytochrome b5 domain fused at the N terminus.
The 9-desaturase carries out the first step in the desaturation pathway which leads to the greatest decrease in fatty acid transition temperature compared to subsequent desaturation reactions (Harwood, 1997
). Because of this,
9-desaturase activity is important in maintaining membrane fluidity and its expression is therefore highly regulated. In several organisms, including Saccharomyces cerevisiae, this control is exerted both at the transcriptional and post-transcriptional level (Choi et al., 1996
; Gonzalez & Martin, 1996
). The
9-desaturase gene (OLE1) has been isolated from a number of yeasts and filamentous fungi and all have a similar structure (Stukey et al., 1990
; Gargano et al., 1995
; Meesters & Eggink, 1996
; Anamnart et al., 1997
; GenBank accession no. AF026401). We have therefore undertaken to isolate the gene encoding this enzyme from M. alpina and to study its expression. Recently, a
9-desaturase gene has been isolated from a patented strain of M. alpina whose gene product displays
9-desaturase activity in Aspergillus oryzae and has a high degree of identity to other
9-desaturases (Sakuradani et al., 1999
). In this paper, we describe the isolation and characterization of two distinct
9-desaturase genes, ole1 and ole2, from two strains of M. alpina which are freely available from fungal culture collections.
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METHODS |
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Amplification of 9-desaturase probes and DNA sequencing.
Degenerate primers with homology to conserved histidine-box and cytochrome b5 regions of 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 DNARNA synthesizer. Primer combinations P3 (5'-TAYCAYAAYTTYCAYCA-3') and P4 (5'-TYSCCSCCSGGRTGNTC-3'), and DESfor (5'-CTKGGYATYACWGCWGG-3') and DESrev (5'-CAGAASGTSGCRTGGTG-3') were used to amplify fragments from 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 (primers P3/P4) or 46 °C (primers DESfor/DESrev) for 1·5 min, 72 °C for 1 min and a final extension at 72 °C for 10 min. Degenerate primers His2for (5'-WSICAYMGIAYICAYCA-3') and His3rev (5'-YTCRTGRTGRAARTTRTG-3') were used at an annealing temperature of 55 °C to amplify fragments from cDNA reverse-transcribed from total RNA of CBS 210.32 as described by Michaelson et al. (1998)
. Primers specific to M. alpina gene
9-1, 91for (5'-CATCACAGCAGGCAAGTAAC-3') and 91rev (5'-GGCGCCGAGCAGTGCGAGCA-3'), were used at an annealing temperature of 62 °C to amplify a
9-1-specific probe. Primer BR99 (5'-AAACGTTTATTACAACAGGC-3') was used in combination with primer 91for at an annealing temperature of 52 °C to generate the remaining part of the
9-1 gene. PCR products were cloned into pCRII, pCR2.1-TOPO (both Stratagene) or pGEM-T (Promega) and the plasmids purified using the Plasmid Mini Purification kit (Qiagen). RT-PCR was carried out using a cDNA first-strand synthesis kit (Amersham Pharmacia Biotech) with an anchored oligo(dT)18 primer, CN95 [5'-CTTCTGGATGTGCGTACTCGAGCT(T)18-3'], followed by PCR with gene-specific primers. DNA sequencing was performed using the PRISM dye terminator kit (Perkin Elmer) and automated sequencer models 373A or 377 (Perkin Elmer). DNA sequences were analysed using the University of Wisconsin GCG software package.
Nucleic acid manipulations.
High molecular mass genomic DNA was isolated from 4-d-old, PDB-grown M. alpina mycelium which had been freeze-ground with liquid nitrogen. DNA was extracted either by the Nucleon Phytopure Plant DNA Extraction kit (Amersham Pharmacia Biotech) or by a standard phenol/chloroform procedure (Michaelson et al., 1998 ). In the latter, DNA was purified either by CsCl/ethidium bromide density gradient equilibrium centrifugation or by using a modification to the Plasmid Midi Purification kit (Qiagen) where the DNA, dissolved in TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 7·0) was diluted approximately 10-fold in modified QBT buffer (Qiagen), lacking 2-propanol and Triton X-100, prior to application to the Qiagen column. Total RNA was isolated either by using the RNeasy Plant Mini kit (Qiagen) with freeze-ground mycelium or the TRIzol reagent (Life Technologies) with freeze-dried mycelium. Southern and Northern blotting were performed using standard procedures for either capillary or vacuum transfer of nucleic acids to nylon membranes. In all cases, hybridization was carried out at 65 °C in Puregene HYB-9 DNA hybridization solution (Flowgen) and blots were subsequently washed in 2xSSC (1xSSC is 0·15 M NaCl, 15 mM sodium citrate, pH 7·0), 0·5% (w/v) SDS and in 0·1xSSC, 0·5% (w/v) SDS at 65 °C. Signals were detected with a Fuji BAS 1500 phosphorimager or by autoradiography on X-ray film. On Northern blots, signals were standardized for fluctuations in RNA loading against the histone H4 transcript (P. Wongwathanarat and others, unpublished results).
Library construction and screening.
A genomic library was constructed in ZAP Express (Stratagene) with BamHI-digested DNA prepared from strain CBS 528.72 following the manufacturers instructions. The library was screened with PCR-amplified
9-desaturase probes which had been labelled with [
-32P]dATP using the Megaprime DNA labelling kit (Amersham Pharmacia Biotech). Approximately 5x104 p.f.u. was used in the primary screen and positive plaques subjected to a secondary screen before in vivo excision of the pBK-CMV phagemids with ExAssist helper phage in E. coli XLOLR (Stratagene). A cDNA library was constructed in
MOSSlox (Amersham Pharmacia Biotech) using EcoRI end-adapted cDNA synthesized from strain CBS 210.32 as described previously (Michaelson et al., 1998
). About 1·5x105 p.f.u. from this library was screened with
9-desaturase probes which had been labelled with [
-32P]dCTP and positive clones in vivo excised in E. coli BM25.8 (Amersham Pharmacia Biotech), a P1 cre recombinase host (Michaelson et al., 1998
). The 5' termini of positive cDNA clones were confirmed or their missing sequences completed by 5'-RACE (rapid amplification of cDNA ends) using the terminal transferase RACE method (Boehringer Mannheim) with cDNA as template and the nested primers RA15' (5'-AGAGTCGATGGTAACCTGCTGT-3') and RA25' (5'-GATACCAAGTCCCGTAGC-3'). The 3' termini of cDNA clones were completed by 3'-RACE using first-strand cDNA synthesized from 5 µg total RNA with the Ready-To-Go T-primed First-Strand kit (Amersham Pharmacia Biotech), according to the manufacturers instructions. PCR was performed using a modified ole1 primer RA13' (5'-GCGAATTCTCATCACTGSCTTTGTCA-3') which contains an EcoRI site (underlined) for cloning purposes and primer cDNA3en (5'-AACTGGAAGAATTCGCGGCCGCAGGAAT-3') which is complementary to the NotI anchor region (underlined) at the 3' end of the first-strand cDNA.
Construction of yeast expression vectors.
The LM9 and 9-2 ORFs were cloned into the yeast expression vector pVT100-U which contains the 2µ origin of replication, the URA3 selection marker and the alcohol dehydrogenase (ADH1) promoter and terminator regions (Vernet et al., 1987
). A synthetic, intronless version of
9-2 with BamHI and HindIII sites at the 5' end and a BamHI site at the 3' end was created by overlap extension PCR using genomic clone
9-2 as template and the following primer combinations: primer A (5'-AAGGATCCAAGCTTAAAAAAATGGCCACTCCCCTCCCCCCA-3') and primer B (5'-CCATAACCGGTGGTATCCTGCAGTAATACCAAGGC-3'), and primer C (5'-GCCTTGGTATTACTGCAGGATACCACCGGTTATGG-3') and primer D (5'-AAGGATCCCTACTCTTCCTTGGAATGGTCGCCATATA-3') where the start and stop codons and the overlap regions are single-underlined and the relevant restriction sites are double-underlined. The two PCR products, 310 and 1098 bp respectively, were then fused using primers A and D to generate a 1·3 kb intronless fragment. 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 was digested with HindIII and BamHI and directionally cloned into pVT100-U. The sequence of the insert was checked using primers from the ADH1 promoter and terminator regions: primer ADH1 (5'-GCTATCAAGTATAAATAGAC-3') and primer ADH2 (5'-GAAATTCGCTTATTTAGAAG-3'), respectively.
Since LM9 is a cDNA clone, PCR was carried out with this as template to create a HindIII site at the 5' end of the ORF and an XbaI site at the 3' end using primer E (5'-AATCTAGAAAGCTTAAAAAAATGGCAACTCCTCTTCCCCCCTC-3') and primer F (5'-AATCTAGACTATTCGGCCTTGACGTGGTCAGTGCC-3') at an annealing temperature of 58 °C. The 1366 bp PCR fragment was digested with HindIII and XbaI, directionally cloned into pVT100-U and the sequence of the insert checked using primers ADH1 and ADH2.
Yeast transformation and fatty acid analysis.
pVT100-U containing either the LM9 or 9-2 ORF was transformed into the S. cerevisiae ole1 mutant strain L8-14C by the lithium acetate/single-stranded carrier DNA/PEG whole cell method of Gietz et al. (1995)
. Undigested vector DNA (200 ng) in sterile TE buffer (pH 8·0) and 250 µg single-stranded herring sperm carrier DNA were incubated with 50 µl competent yeast cells for each transformation. Following lithium acetate/PEG treatment and heat shock at 42 °C for 15 min, the cells were resuspended in 1 ml YNB broth and aliquots plated onto fatty-acid-supplemented YNB agar to select for the URA3+ marker. URA3+ colonies were restreaked onto non-supplemented YNB agar to check for complementation of the ole1 mutation.
Two independent LM9 and 9-2 yeast transformants were grown in 10 ml YNB broth at 25 °C for 4 d for fatty acid analysis. Untransformed L8-14C and transformants containing pVT100-U without an insert were grown in fatty-acid-supplemented YNB broth with and without 20 mg uracil l-1.
9-2 transformants were also grown in YNB broth supplemented individually with 0·5 mM linoleic acid (18:2),
-linolenic acid (18:3),
-linolenic acid (
-18:3), dihomo-
-linolenic acid (20:3) or arachidonic acid (20:4) to test for desaturation of other substrates. In all cases, cells were washed three times with distilled water at 45 °C and freeze-dried prior to transmethylation of the fatty acids. Methanolic HCl (2 ml; Supelco) was added to each dried sample and refluxed at 80 °C for 1 h. Fatty acid methyl esters were extracted with 1·5 ml 0·9% (w/v) NaCl/1 ml hexane and finally resuspended in 0·5 ml hexane. GC analysis was carried out on a BPX70 0·25 µm column (SGE) in a Perkin Elmer AutoSystem GC, with a column temperature of 50250 °C and an injection port temperature of 250 °C. The helium carrier gas pressure was 141 kPa.
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RESULTS AND DISCUSSION |
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Clone LM9 was isolated from the CBS 210.32 cDNA library and contained a 1·66 kb EcoRI insert which encoded a protein of 445 aa with 98% identity to the 9-desaturase from M. alpina 1S-4 described by Sakuradani et al. (1999)
and 4060% identity to other fungal
9-desaturases. This protein displayed the three conserved histidine boxes, C-terminal cytochrome b5 fusion and transmembrane domains characteristic of ER membrane-bound
9-desaturases (Fig. 1a
). The poly(A) tail and part of the 3'-untranslated region (3'-UTR) were missing from this clone. Several attempts at 5'-RACE using gene-specific nested primers RA15' and RA25' only extended the 5' end of the cDNA by 32 bp, suggesting that the transcription start site was close to this point. 3'-RACE with primers RA13' and cDNA3en completed the 3'-UTR and included the poly(A) tail. A consensus poly(A) addition signal, AATAAA (Gurr et al., 1987
), was present in this gene 160 bp downstream from the TAG stop codon. On assembling the complete LM9 sequence, the total length of the cDNA was 1·74 kb. On Southern blots of CBS 210.32 genomic DNA, digested with either BamHI or HindIII and probed with fragment His2for/His3rev, at least two strongly hybridizing bands were seen per track, indicating that this strain may also have more than one
9-desaturase gene (data not shown).
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Genomic clone 9-2 was isolated using P3/P4 as probe and contained a 3342 bp BamHI insert which also encoded a protein of 445 aa. This protein showed less identity (86%) to the M. alpina ole1 gene product. The single intron of 115 bp disrupted the ORF in the same position as in
9-1 but had the more common 5' splice site GTATGT, a 3' splice site, TAG, and consensus lariat sequences CATCAAC and TCTCAAC, 42 and 18 bp, respectively, from the 3' end (Gurr et al., 1987
). A less common poly(A) addition signal, AT(A)6TAATAA, was located 23 bp downstream from the TAG stop codon. The organization of this gene, designated ole2, is outlined in Fig. 1(c)
.
Functional characterization of the M. alpina 9-desaturase genes in S. cerevisiae
To confirm the in vivo function of the two putative 9-desaturase genes, the CBS 210.32 ole1 and CBS 528.72 ole2 ORFs were expressed in L8-14C, an ole1 mutant of S. cerevisiae (Stukey et al., 1989
). Each ORF was cloned into the yeast expression vector pVT100-U (Vernet et al., 1987
) with the consensus sequence (A)6 immediately 5' of the ATG start codon of each gene, a sequence which is associated with highly expressed S. cerevisiae genes (Hamilton et al., 1987
). Both ORFs formed transcripts in yeast transformants, as determined by Northern analysis (data not shown), and complemented the ole1 mutation since the transformants grew without 16:1 and 18:1 supplementation. Fatty acid analysis of the transformants showed that both 16:1 and 18:1 were produced but that the ratio of 18:1 to 16:1 was higher than in wild-type S. cerevisiae (Table 1
). M. alpina only produces negligible amounts of 16:1 and this result confirms that the M. alpina
9-desaturases had a substrate preference for 18:0 compared with 16:0, unlike the S. cerevisiae enzyme. There also appeared to be a difference in fatty acid composition between the ole1 (LM9) and ole2 (
9-2) transformants, indicating that the 14% difference in amino acid identity between the two proteins may have some significance for
9-desaturase activity. ole2 (
9-2) yeast transformants fed with a range of unsaturated fatty acids failed to desaturate these further, confirming that the ole2 protein only had
9-desaturase activity (data not shown).
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Supplementation of CBS 210.32 cultures with a variety of unsaturated fatty acids containing a 9-unsaturated bond reduced ole1 transcript levels, with 18:1, 18:2 and
-18:3 having the most pronounced effect (Fig. 3
). This repression has been observed in several fungi (Choi et al., 1996
; Meesters & Eggink, 1996
). In S. cerevisiae the regulatory sequences responsible for transcriptional control, the 111 bp FAR (fatty-acid-regulated) element containing repeated CCCGGG motifs and the sequence GGGGTTAGC, have been identified in the ole1 promoter (Choi et al., 1996
). In addition, an as yet undefined sequence in the 5'-UTR is required for post-transcriptional regulation of the S. cerevisiae ole1 gene (Gonzalez & Martin, 1996
). Similar sequences could not be found in the promoter regions or 5'-UTRs of the M. alpina ole1 and ole2 genes.
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(2) Both ole1 and ole2 ORFs complemented the ole1 mutation in S. cerevisiae and showed a substrate preference for 18:0 compared with 16:0.
(3) The ole1 gene was expressed in all strains of M. alpina which were studied and showed transcriptional regulation in response to supplementation with 9-unsaturated fatty acids.
(4) Transcription of the ole2 gene was only detected in one of the six strains of M. alpina which were examined, suggesting that gene expression may be strain-specific or induced under certain physiological conditions.
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ACKNOWLEDGEMENTS |
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Received 17 March 1999;
revised 25 May 1999;
accepted 18 June 1999.