From the Arachidonic acid (C20:4
The filamentous fungus M. alpina is unusual in that it
can produce a wide range of polyunsaturated fatty acids. It differs from higher plants in its fatty acid unsaturation as it is able to
produce arachidonic acid (C20:4 In fungi, unsaturated fatty acids are formed in the endoplasmic
reticulum by the action of integral membrane-bound fatty-acid desaturase enzymes, which sequentially insert double bonds into the
acyl chain (5, 6). The Biochemical characterization of membrane-bound desaturases has been
limited because they are difficult to purify due to their hydrophobic
nature. Hence, molecular genetic approaches, particularly the use of
Arabidopsis mutants, have provided much information on
desaturation reactions (9, 10). Analysis of the predicted protein
sequences for the higher plant desaturases together with those from
cyanobacteria, yeast, and mammals revealed the presence of eight highly
conserved histidine residues (11). Mutagenesis studies on the
microsomal Cultures--
M. alpina CBS 210.32 was grown in
liquid culture in potato dextrose medium (Life Technologies, Inc.) at
28 °C with shaking at 200 rpm. After 48 h, mycelium was
harvested and used in fatty acid analysis and various DNA and RNA
extractions.
School of Biological Sciences,
Institute of
Arable Crops Research-Long Ashton Research Station,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
5,8,11,14) is a polyunsaturated fatty acid
synthesized by the
5-fatty acid desaturation of
di-homo-
-linolenic acid (C20:3
8,11,14). In mammals,
it is known to be a precursor of the prostaglandins and the
leukotrienes but it is also accumulated by the filamentous fungus
Mortierella alpina. We have isolated a cDNA encoding
the
5-fatty acid desaturase from M. alpina
via a polymerase chain reaction-based strategy using primers designed
to the conserved histidine box regions of microsomal desaturases, and
confirmed its function by expression in the yeast Saccharomyces
cerevisiae. Analysis of the lipids from the transformed yeast
demonstrated the accumulation of arachidonic acid. The M. alpina
5-desaturase is the first example of a
cloned
5-desaturase, and differs from other fungal
desaturases previously characterized by the presence of an N-terminal
domain related to cytochrome b5.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
5,8,11,14) and
eicosapentaenoic acid (C20:5
5,8,11,14,17) (1). In
filamentous fungi, polyunsaturated fatty acids are present both in the
phospholipids and the triacylglycerols, indicating that they have a
role in membrane structure and as storage oils (2). In animals,
arachidonic acid is a precursor of the short-lived regulatory
molecules, the eicosanoids, which comprise the prostaglandins, the
leukotrienes, and the thromboxanes (3). All mammalian cells except
erythrocytes synthesize eicosanoids, and among their many functions
they are involved in inflammatory response, reproductive function, and
regulation of blood pressure (4).
5-desaturase is responsible for
the conversion of di-homo-
-linolenic acid (C20:3
8,11,14) to arachidonic acid. It is thought to function
like the other microsomal desaturases from higher plants and yeast,
catalyzing aerobic reactions requiring cytochrome
b5 as a co-factor. Electrons are transferred
from NADH-dependent cytochrome b5
reductase, via the heme-containing cytochrome b5
molecule to the fatty acid desaturase (7, 8).
9-desaturase from rat and the
12-desaturase from Synechocystis (11, 12)
showed that all of the conserved histidines were catalytically
essential. Variation is present within the amino acid sequences between
the conserved histidine residues, but sequence alignments have
permitted the design of degenerate oligonucleotides capable of
amplifying novel desaturase gene fragments from various sources
(13-15). Although the approach has yielded gene probes for a variety
of microsomal desaturase activities including
6,
9, and
12, no information is available
yet for a
5-desaturase. Here, we describe the successful
application of this approach to the isolation of a
5-fatty acid desaturase from the fungus
Mortierella alpina.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
RNA Isolation and Manipulation-- Total RNA was isolated from 400 mg wet weight of mycelium using the RNeasy plant mini kit (Qiagen) and 5 µg was reverse transcribed with the Ready-To-GoTM T-primed first strand kit (Amersham Pharmacia Biotech) according to the manufacturers' instructions. The cDNA was used as a template for PCR1 amplification with degenerate primers.
Genomic DNA Isolation-- Approximately 4 g wet weight of mycelium was ground to a fine powder under liquid nitrogen using a precooled mortar and pestle. The ground tissue was added to 10 ml of extraction buffer (10 mM Tris-HCl, pH 8, 10 mM EDTA, 0.5% SDS), thawed, and mixed gently by inversion. Ten ml of phenol:chloroform:isoamyl alcohol (16) were added and mixed gently for 15-30 min. The organic and aqueous phases were separated by centrifugation and the aqueous layer removed to a fresh centrifuge tube. This extraction was repeated until the interface between the two phases was clear, after which an extraction with chloroform:isoamyl alcohol (24:1) was carried out. DNA was concentrated by ethanol precipitation and purified by two rounds of equilibrium centrifugation in a CsCl ethidium bromide density gradient. The band containing genomic DNA was removed from the gradient and diluted with TE buffer. DNA was recovered by ethanol precipitation and dissolved in TE buffer.
PCR-based Cloning--
Highly degenerate primers were
synthesized using inosine for 4-base redundancy and extended at the 5'
ends to include EcoRI restriction sites to facilitate
cloning PCR products. The forward primer was,
5'-GCGAATTC(A/G)TIGGICA(T/C)GA(T/C) TG(T/C)GGICA-3', and
the reverse primer was
5'-GCGAATTCATIT(G/T)IGG(A/G)AAIA(G/A)(A/G)TG(A/G)TG-3', where I stands for inosine and the EcoRI sites are
underlined. The primers were used for PCR amplification of cDNA
reverse transcribed from total RNA. After initial denaturation at
94 °C for 2 min, amplification was performed in 32 cycles of 45 s at 94 °C, 1 min at 55 °C, and 1 min at 72 °C, followed by a
final extension at 72 °C for another 10 min. Amplification products
were fractionated on 1% agarose gels from which selected DNA bands
were purified. They were ligated directly into pGEM-T (Promega) and the
plasmids used to transform into Escherichia coli DH5
cells. Plasmid DNA was purified for sequencing using the Qiagen QIAprep
miniprep kit. Nucleotide sequences were determined using a Perkin Elmer ABI-377 DNA sequencer, and analyzed using the University of Wisconsin GCG software package (17).
Library Screening--
End-adapted cDNA was synthesized from
M. alpina poly(A)+ mRNA using a cDNA
synthesis kit (Amersham Pharmacia Biotech) and ligated into the
EcoRI site of MOSSlox (Amersham Pharmacia Biotech). The
resultant DNA was packaged into phage particles to produce a library of
4×10 6 plaque-forming units with an average insert size of
1.4 kilobases. The library was screened by standard techniques (16)
using cloned PCR products as probes. DNA fragments were labeled with
[
-32P]dCTP using the Ready To GoTM DNA
labeling reaction mix (Amersham Pharmacia Biotech). The full-length cDNA clone L11.1 was purified by successive rounds of plating and
hybridization before it was subjected to plasmid excision in
vivo and the insert was sequenced on both strands.
Southern Blot Analysis--
Aliquots (10 µg) of genomic DNA
from M. alpina and M. circinelloides were
digested with restriction enzymes and fractionated by agarose gel
electrophoresis. DNA was transferred to a Zeta-Probe®
nylon membrane (Bio-Rad) by alkaline capillary transfer for 5-8 h
using 0.4 M NaOH as transfer buffer. To improve transfer of the larger DNA fragments (greater than 4 kilobases), the gel was soaked
in 0.25 M HCl for 15 min to partially depurinate the DNA and then briefly rinsed in water prior to blotting. The filters were
probed with the 660-bp fragment amplified between histidine boxes 1 and
3 using the gene-specific primers 5'-CATGATGCGTCTCACTTTTCA-3' (forward)
and 5'-(G/A)GGTGCACAGCCT GGTAGTT-3' (reverse). Overnight hybridization
was performed at 55 °C in 0.25 M sodium phosphate pH
7.2, 7% SDS, after which the filters were washed in 2× SSC, 0.1% SDS
at the hybridization temperature and x-ray film exposed to it at
80 °C using an intensifying screen (18).
Functional Analysis: Yeast Transformation--
PCR with the
primers MYfor, 5'-GCGGGTACCATGGGTACGGACCAAGGA-3'
(annealing to the initiating methionine indicated by the boldface
type), and MYrev,
5'-GCGGAGCTCCTACTCTTCCTTGGGACG-3' (annealing to
the complement of the stop codon, indicated in boldface type) was used
to amplify the pL11 coding region and to flank it with KpnI
and SacI restriction sites. The amplified PCR product was
ligated into the vector pYES2 (Invitrogen) to generate the plasmid
pYES2/L11 and cloned in E. coli. The fidelity of the cloned PCR product was checked by in vitro transcription and
translation using the TNTTM system (Promega). Translation
products labeled with [35S]methionine were generated,
separated by SDS-polyacrylamide gel electrophoresis, and visualized by
autoradiography. The plasmid was transformed into Saccharomyces
cerevisiae DBY746 by the lithium acetate method (19), and
expression of the transgene was induced by the addition of galactose to
1% (w/v). The yeast culture medium was supplemented with 0.5 mM di-homo--linolenic acid or 0.5 mM linoleic acid in the presence of 1% tergitol as described by Napier et al. (20).
Fatty Acid Analysis-- Total fatty acids extracted from yeast cultures were analyzed by gas chromatography (GC) of methyl esters. Lipids were transmethylated with 1 M HCl in methanol at 80 °C for 1 h, then fatty acid methyl esters (FAMEs) were extracted in hexane. GC analysis of FAMEs was conducted using a Hewlett Packard 5880A Series gas chromatograph equipped with a 25 m × 0.32-mm RSL-500 BP bonded capillary column and a flame ionization detector. Fatty acids were identified by comparison with retention times of FAME standards (Sigma). Relative percentages of the fatty acids were estimated from peak areas. Arachidonic acid was identified by GC-MS using a Krats MS80RFA operating at an ionization voltage of 70 eV, with a scan range of 500-40 daltons.
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RESULTS |
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Cloning of a M. alpina Membrane-bound Desaturase--
The strategy
employed to obtain probes for M. alpina desaturase genes
involved reverse transcriptase PCR with the degenerate primer sequences
used to amplify a plant 6-desaturase gene (15). Inosine
was used at all positions of 4-base degeneracy to maximize the relative
concentrations of individual primers within the degenerate mixtures.
The forward primer mixture was designed to encode histidine box 1, and
the reverse primer mixture encoded the complement of histidine box 3. Total RNA extracted from M. alpina was reverse-transcribed,
and the cDNA product was used as a template in a PCR that yielded
products of various sizes. Several PCR products were generated, and
those of the expected length (600-700 bp) were isolated from a gel for
cloning and sequencing. The DNA sequence of one of the clones (L11)
predicted an open reading frame of 224 amino acids. It displayed some
similarity to other membrane-bound desaturases, including the presence
of the second characteristic histidine box (11). Alignment of the deduced amino acid sequence of this PCR product with the corresponding region of known desaturases indicated the greatest similarity to the
6-desaturase from a cyanobacterium (21), although the
actual level of identity was low and less than 25%. Lipid analysis has shown that in cultures of M. alpina harvested after 48 h arachidonic acid comprises up to 25% of the total
lipid.2 It was therefore
considered possible that L11 could encode part of either a
6- or a
5-desaturase from M. alpina.
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L11.1 Encodes a Protein with a Cytochrome b5-like Heme-binding Domain at the N Terminus-- A heme containing electron donor is required for fatty acid desaturation and cytochrome b5 fulfils this function for the membrane-bound desaturases (5, 8, 22). Fig. 1 shows the presence of a heme binding region characterized by the H-P-G-G motif toward the N terminus and hence the cytochrome b5 and desaturase may exist as a fusion protein. Previously identified fungal desaturases also contain cytochrome b5 fusions, but with the heme-binding region located toward the C terminus (5, 14).
Southern Blot Analysis--
M. circinelloides is a
filamentous zygomycete, which accumulates a triacylglycerol oil rich in
-linolenic acid but lacking arachidonic acid. In all other respects,
it is very similar to M. alpina (6). It thus has genes
encoding
9-,
12-, and
6-desaturase activities and is likely to lack a
5-desaturase gene. Genomic DNA was isolated from
M. alpina and M. circinelloides, digested with
three restriction enzymes and fractionated by agarose gel
electrophoresis. A Southern blot of the gel was probed with the 660-bp
fragment amplified by PCR between histidine boxes 1 and 3 of clone L11.
Fig. 2 shows single hybridizing bands in
each of the digests of M. alpina DNA, but no hybridization was observed to M. circinelloides DNA. In a similar
experiment, a putative
9-desaturase cDNA was found
to hybridize strongly to both the M. circinelloides genome
as well as the M. alpina genome.2 These results
indicate that the desaturase isolated from M. alpina is
encoded by a single copy gene that is not present in M. circinelloides; this suggests that the gene is likely to encode a
5-desaturase rather than a
6-desaturase.
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Functional Analysis of L11.1 in Yeast-- The complete coding region (446 amino acids) of L11.1 was amplified by PCR and inserted into the yeast expression vector pYES2 downstream of the GAL1 promoter. This construct was transformed into E. coli. The fidelity of the PCR-generated insert in plasmid pYES2/L11 was confirmed in vitro by coupled transcription/translation under the control of the T7 RNA polymerase promoter in the vector. The translation products were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. A product of Mr ~56,000 was produced from the plasmid pYES2/L11, whereas the empty vector control (pYES2) failed to yield any protein products (data not shown).
For functional analysis of the L11.1 coding region, the recombinant plasmid was transferred to yeast. Cells were cultured overnight in a medium containing raffinose as a carbon source, and supplemented by the addition of either linoleic acid (18:2
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Identification of a Similar Sequence in C. elegans--
Data base
searching with the M. alpina cDNA identified a high
scoring match to one of the genes on C. elegans cosmid T13F2 (GenBank accession number Z81122). The C. elegans ORF of
T13F2.1 predicts a protein of 454 amino acids. It contains the
characteristics of other membrane-bound desaturases including the three
histidine box domains. It also contains an N-terminal cytochrome
b5 region, and the third histidine box contains
the H Q variant. As the nematode performs
5
desaturation (23), it is possible that this homologue encodes a
5-desaturase gene.
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DISCUSSION |
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A cDNA isolated from M. alpina has been
characterized and found to encode a protein with three histidine box
motifs indicative of a microsomal fatty acid desaturase (11). The
deduced protein sequence also contained the diagnostic features of a
heme-binding cytochrome b5 domain located toward
the N terminus. Evidence that the L11.1 clone encodes a
5-desaturase was obtained by Southern blot analysis,
which indicated the absence of homologous sequences in the genome of
M. circinelloides, a related fungus that produces
-linolenic acid but not arachidonic acid. Functional analysis of the
L11.1 clone in transformed yeast confirmed that it encoded a
5-desaturase gene, since cells growing in a medium
supplemented with di-homo-
-linolenic acid produced arachidonic acid
in significant quantities. The fungal
5-desaturase
appears to differ from the other fungal and plant desaturases that have
been characterized (5, 14). The most closely related sequence
identified was that of the cyanobacterial
6-desaturase
involved in GLA formation (21), with which the fungal
5-desaturase showed only some 22% amino acid identity.
Data base searches have also enabled us to identify a putative animal
5-desaturase in the nematode C. elegans.
It is interesting that the 5-desaturase may exist as a
cytochrome b5 fusion protein. Such fusions have
been identified in a hypothetical desaturase-like sunflower protein
(24) and the
6-desaturase in B. officinalis,
both of which contain N-terminal cytochrome b5
domains (15).
9-Desaturases characterized from other
fungi have also been found to contain cytochrome
b5 domains, but in these examples the diagnostic heme binding site was located toward the C terminus (5, 14). The
5-desaturase from M. alpina, therefore,
appears to be the first fungal fatty acid desaturase described with an
N-terminal cytochrome b5 domain and in this
respect is similar to the "front end"
6-desaturase
of B. officinalis (15, 25) and the
6-desaturase of C. elegans (20). In contrast
to other microsomal desaturases, the microsomal desaturases with
N-terminal cytochrome b5 domains use a
polyunsaturated fatty acid as a substrate and insert a double bond
between the methyl end of the fatty acid and existing double bonds, but
it is unclear how significant a role the fused cytochrome
b5 region plays in this reaction. The OLE1 gene isolated from S. cerevisiae, which
encodes a
9-desaturase, contains a C-terminal cytochrome
b5 domain. OLE1 rescued double
mutants deficient in both OLE1 and the separate microsomal
cytochrome b5. However, when the cytochrome
b5 region was deleted from this gene, the yeast
cells remained fatty acid auxotrophs, even in the presence of the
endogenous yeast cytochrome b5 (5). This
suggests that the cytochrome b5 domain plays an essential role in the desaturation reaction. Whether the cytochrome b5-desaturase fusion proteins are more efficient
awaits further assessment.
Recently, another N-terminal cytochrome b5
fusion protein, Fah1p, has been identified in yeast; it is required for
the -hydroxylation of sphingolipid-associated very-long-chain fatty
acids (26). Interestingly, it contains two of the histidine box motifs
characteristic of membrane-bound desaturases. The
Arabidopsis homologue of Fah1p has no cytochrome
b5 domain but is able to functionally complement a fah1 deletion mutant. This indicates that, in the case of
Fah1p, the N-terminal cytochrome b5 may not be
essential for function (26).
Desaturases isolated from fungal sources have much potential for
further exploitation, particularly in the genetic manipulation of oil
crops to produce novel and diverse polyunsaturated fatty acid products
for the pharmaceutical and nutraceutical industries (27). The
5-desaturase is an essential enzyme in the production of
eicosanoids, and the identification of a M. alpina cDNA
encoding this activity is the first example of its type.
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ACKNOWLEDGEMENTS |
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We thank Prof. S. Shimizu (Kyoto University, Kyoto, Japan) and Prof. R. Herbert (Dundee University, Dundee, UK), for providing M. alpina and M. circinelloides, respectively. We thank Mervyn Lewis for the GC-MS analysis. Institute of Arable Crops Research-Long Ashton receives grant-aided support from the Biotechnology and Biological Sciences Research Council. A. K. S. thanks the Royal Society for equipment grants and the Wolfson foundation for laboratory refurbishment. L. M. thanks Dr. Steve Screen for helpful discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF054824.
§ Supported by a Biotechnology and Biological Sciences Research Council studentship and a Cooperative Awards in Science and Engineering award from Horticulture Research International, Wellesbourne. To whom correspondence should be addressed. Tel.: 44-117-928-7574; Fax: 44-117-925-7374; E-mail: louise.michaelson{at}bristol.ac.uk
1 The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); GC, gas chromatography; FAME, fatty acid methyl ester; MS, mass spectroscopy.
2 L. V. Michaelson, C. M. Lazarus, G. Griffiths, J. A. Napier, and A. K. Stobart, unpublished data.
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REFERENCES |
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