WO1999033958A2

WO1999033958A2 - Desaturase

Info

Publication number: WO1999033958A2
Application number: PCT/GB1998/003895
Authority: WO
Inventors: Johnathan A. Napier; Louise Michaelson; Keith Stobart
Original assignee: University Of Bristol
Priority date: 1997-12-23
Filing date: 1998-12-23
Publication date: 1999-07-08
Also published as: HUP0101153A2; JP2002508932A; AU1774899A; EP1042485A2; HUP0101153A3; BR9814434A; CA2315297A1; WO1999033958A3; PL344868A1; CN1283230A; EE200000372A; KR20010033517A

Abstract

This invention relates to cDNA sequences encoding Δ5-fatty acid desaturases comprising the sequences shown in SEQ.1 and SEQ.2.

Description

DESATURASE

This invention relates to DNA sequences encoding Δ5-fatty acid desaturases, the encoded Δ5-fatty acid desaturases, and applications for the Δ5-fatty acid desaturases.

Polyunsaturated fatty acids are important neutraceutically due to their specific health

promoting activities, and biomedically in respect of their potential pharmaceutical

applications in the treatment of specific disease conditions.

Polyunsaturated fatty acids are the precursors for two major classes of metabolites:

prostanoids (which include prostaglandins and thromboxanes) and leukotrienes.

Δ5-fatty acid desaturase catalyses the conversion of dihomogamma linolenic acid (DHL) to

arachidonic (A A) acid, and eicosatetraenoate (ETA) to ecosapentaenoate (EPA), by the

introduction of double bonds at the Δ5 carbon of the respective substrates, and exists as an

endoplasmic reticulum membrane-bound protein in its native state.

Arachidonic acid has a 20 carbon chain with 4 double bonds and is of great importance in

human metabolism since it is a precursor for the synthesis of prostaglandins - 20-carbon chain fatty acids that contain a 5 carbon ring. Prostaglandins are modulators of hormone action and the potential effects of prostaglandins include the stimulation of inflammation,

the regulation of blood flow to particular organs, the control of ion transport across some

membranes, and the modulation of synaptic transmission. Prostaglandins are also

potentially useful as contraceptives due to their ability to suppress progesterone secretion.

Therefore, the ability to modulate prostaglandin synthesis by controlled levels of expression of polyunsaturated fatty acid precursor synthesis is very important both

medically and commercially.

The increased importance of polyunsaturated fatty acids in the food and pharmaceutical

industries has led to an increased demand which has exceeded current production levels

and supplementary sources of high quality, low cost polyunsaturated fatty acids are

required.

Current commercial sources of polyunsaturated fatty acids include selected seed plants,

marine fish and selected mammals, and traditional processing techniques for extracting the

polyunsaturated fatty acids from these sources include solvent extraction, winterization,

urea-adduct formation and distillation. However, present sources have the disadvantages of

seasonal and climatic variations in both production levels and quality, a lack of availability

of plant and fish sources, and the high costs of refining low-grade oils. High costs coupled

with insufficient production levels have retarded the development of commercially

exploitable applications of polyunsaturated fatty acids.

Much effort has gone into developing alternative sources of polyunsaturated fatty acids,

and studies have been carried out to characterise the constituent genes and encoded

proteins of their biosynthesis. The engineering of polyunsaturated fatty acid biosynthesis into oilseeds for example has many advantages for the production of large scale quantities

of, for example, γ-linolenate (GLA), dihomo-γ(-linolenate (DHGLA), arachidonic acid

(AA), eicosapentaenoate (EPA) and docosahezaenoate (DHA). The practicality of this has

been illustrated by the expression of a Borage Δ6 desaturase gene in tobacco resulting in the production of GLA and the octadecatetraenoic acid, 18:4 (Soyanova et al (1997), PNAS

94, 9411-9414). As more of the biosynthetic genes for polyunsaturated fatty acid synthesis

become available, this opens up the possibility of producing at least GLA, AA, EPA and

DHA in oil seeds, as well as controlling the type of lipid assembled. Benefits which would

be obtained from such crops include a cheap and sustainable supply of desirable

polyunsaturated fatty acids on a large scale, tailored polyunsaturated fatty acids profiles to

meet specific nutritional requirements, and in the fine chemical industry, the production of

unusual fatty acids with prescribed levels and locations of unsaturation.

A further approach to the production of polyunsaturated fatty acids is to utilise the

biosynthetic capacity of lower organisms e.g. algae, bacteria, fungi (including

phycomycetes) which can synthesise the entire range of polyunsaturated fatty acids and can

be grown on an industrial scale. Genetic transformation of these organisms will enable the

development of overproducing strains and the manipulation of the polyunsaturated profile

by pathway engineering.

Fungal Δ5 and Δ6 fatty acid desaturases have been cloned, and their sequences disclosed in

WO98/46763, WO98/46764 and WO98/46765.

Polyunsaturated fatty acid metabolism is of greatest importance in human metabolism.

These acids, via the eicosanoids, are fundamental to the proper maintenance of homeostasis

and are linked to serious physiological and pathophysiological syndromes. The inventors have surprisingly isolated and characterised a DNA sequence from the

soil-borne filamentous fungus of the zygomycete class Mortierella alpina encoding a

functional Δ5-fatty acid desaturase.

In addition, the inventors have surprisingly isolated and characterised a DNA sequence

from the nematode worm, Caenorhabditis elegans encoding a functional Δ5-fatty acid

desaturase. This DNA sequence, encoding a functional Δ5-fatty acid desaturase is thought

likely to be more closely related to the human Δ5-fatty acid desaturase than any of the

Δ5-fatty acid desaturase gene sequences isolated so far.

As well as the potential human benefits from the polypeptide encoded by the DNA

sequences of this invention, the DNA sequences of this invention may enable the cloning

of the equivalent human gene and thereby facilitate overproduction of the human DNA

sequence and allow its biomedical exploitation in the treatment of certain human diseases.

Plant and fungal desaturases are mainly integral membrane polypeptides which makes them

difficult to purify and subsequently characterise by conventional methods. Hence,

molecular techniques including the use of mutants and transgenic plants have been adopted

in order to better study lipid metabolism.

A first aspect of the invention provides an isolated animal Δ5-fatty acid desaturase and functional portions thereof.

A second aspect of the invention provides an isolated C. elegans Δ5-fatty acid desaturase. A third aspect of the invention provides a DNA sequence according to a first or second aspect of the invention comprises at least a portion of the sequence shown in SEQ.2 and equivalents to that sequence, or to portions of that sequence, which encode a functional Δ5-fatty acid desaturase by virtue of the degeneracy of the genetic code. Preferably, the DNA sequence is derived from a Caenorhabditis elegans DNA sequence.

Preferably, the gene encoding the Δ5-fatty acid desaturase encoded by the cloned gene is 1341 bp long. The protein is 447 amino acids long with an estimated molecular weight of 57 kDa.

Alternatively, the DNA sequence encodes a functional Δ5-fatty acid desaturase and comprises at least a portion of the sequence shown in SEQ.l and equivalents to that sequence, or to portions of that sequence, which encode a functional Δ5-fatty acid desaturase by virtue of the degeneracy of the genetic code. Preferably, the DNA sequence is derived from a Mortierella alpina DNA sequence.

Preferably, the gene encoding the Δ5-fatty acid desaturase encoded by the cloned gene is 1338 bp long. The protein is 446 amino acids long with an estimated molecular weight of 57 kDa.

Preferably, a DNA sequence according to a third aspect of the invention is functional in a mammal.

Preferably, the DNA sequence is expressed in a mammal.

Preferably, the DNA sequence is expressed in a human.

Preferably, the DNA sequence is obtained by modification of a functional natural gene encoding a Δ-5 fatty acid desaturase.

Preferably, the modification includes modification by chemical, physical, or biological means without removing a catalytic activity of the enzyme which it encodes.

Preferably, the modification improves a catalytic activity of the enzyme which it encodes. Preferably, the biological modification includes recombinant DNA methods and forced evolution techniques.

Preferably, the forced evolution technique is DNA shuffling.

A fourth aspect of the invention provides a polypeptide encoded by a DNA sequence according to a third aspect of the invention.

Preferably, at least a portion of the polypeptide has the sequence shown in SEQ.3 or functional equivalents to that sequence or portions of that sequence. Alternatively, at least a portion of the polypeptide has the sequence shown in SEQ.4 or functional equivalents to that sequence or portions of that sequence.

Preferably, the polypeptide catalyses the conversion of dihomogamma linolenic acid to arachidonic acid.

Preferably, the polypeptide has been modified without removing the catalytic activity of the encoded polypeptide.

Preferably, the polypeptide has been modified in such a way as to introduce a specific level of saturation of a substrate at a specific location within the molecular structure of the substrate.

A fifth aspect of the invention provides a vector containing a DNA sequence of any portion of a DNA sequence according to a third aspect of the invention..

A sixth aspect of the invention provides a method of producing polyunsaturated fatty acids comprising contacting a substrate with a Δ5-fatty acid desaturase according to a first or second aspect of the invention, or a polypeptide according to a fourth aspect of the invention.

A seventh aspect of the invention provides a method of converting dihomogamma linoleic acid to arachidonic acid wherein the conversion is catalysed by a Δ5-fatty acid desaturase according to a first or second aspect of the invention, or a polypeptide according to a fourth aspect of the invention. An eighth aspect of the invention provides an organism engineered to produce high levels of a polypeptide according to a fourth aspect of the invention.

A ninth aspect of the invention provides an organism engineered to produce high levels of a product of a reaction catalysed by a Δ5-fatty acid desaturase according to a first or second aspect of the invention, or by a polypeptide according to a fourth aspect of the invention.

Preferably, the organism has been engineered to carry out the method according to a sixth or seventh aspect of the invention.

Preferably, the organism is a microorganism.

Preferably, the microorganism is selected from algae, bacteria and fungi.

Preferably, the fungi includes phycomycetes. Alternatively, the microorganism is a yeast.

Alternatively, the organism is a plant. Preferably, the plant is selected from oil seed plants.

Preferably, the oil seed plants are selected from oil seed rape, sunflower, cereals including maize, tobacco, legumes including peanut and soybean, safflower, oil palm, coconut and other palms, cotton, sesame, mustard, linseed, castor, borage and evening primrose.

A tenth aspect of the invention provides a seed or other reproductive material derived from an organism according to a ninth aspect of the invention.

Preferably, the organism is a mammal.

An eleventh aspect of the invention provides an isolated multienzyme pathway wherein the pathway includes a Δ5-fatty acid desaturase according to a first or second aspect of the invention.

A twelfth aspect of the invention provides a compound produced by a conversion of a substrate, wherein said conversion is catalysed by a Δ5-fatty acid desaturase according to a first or second aspect of the invention. A thirteenth aspect of the invention provides an intermediate compound produced by the reaction catalysed by a Δ5-fatty acid desaturase according to a first or second aspect of the invention.

A fourteenth aspect of the invention provides a foodstuff or dietary supplement containing a polyunsaturated fatty acid produced by a method according to a sixth aspect of the invention.

A fifteenth aspect of the invention provides a pharmaceutical preparation containing a polyunsaturated fatty acid produced by a method according to a sixth aspect of the invention.

A sixteenth aspect of the invention provides prostaglandins synthesised by a biosynthetic pathway including a catalytic activity of a Δ5-fatty acid desaturase according to a first or second aspect of the invention.

A seventeenth aspect of the invention provides a method for the modulation of prostaglandins synthesis by the control of the levels of expression of a DNA sequence according to a third aspect of the invention.

An eighteenth aspect of the invention provides a probe comprising all or part of a DNA sequence according to a third aspect of the invention,or an equivalent RNA sequence.

A nineteenth aspect of the invention provides a probe comprising all or part of a Δ5-fatty acid desaturase polypeptide according to a fourth aspect of the invention.

A twentieth aspect of the invention provides a method of isolating Δ5-fatty acid desaturases using a probe according to a nineteenth aspect of the invention.

It is possible that the gene of the invention may be transformed into human cells and

exploited in gene therapy techniques at a suitable level in vivo to provide a constant supply

of enzyme converting fatty acids to polyunsaturated fatty acids within the patient's body.

This could be an effective preventative treatment for example, in patients suffering high levels of cholesterol or other medical conditions where administration of polyunsaturated fatty acids may have beneficial disease-preventative effects.

In addition, either whole or part of the DNA sequences of the invention, or whole or part of

the polypeptide sequences of the invention could be used as search probes for research or

diagnostic purposes.

The invention will now be described by way of example only, with reference to the

accompanying drawings, SEQ.1 to SEQ.4, and Figs. 1 to 4, in which:

SEQ.l is a cDNA sequence encoding Δ5-fatty acid desaturase from Mortierella alpina and;

SEQ.2 is a cDNA sequence encoding Δ5-fatty acid desaturase from C. elegans; and

SEQ.3 is the peptide sequence obtained by translating the gene sequence of SEQ.l; and

SEQ.4 is the peptide sequence obtained by translating the gene sequence of SEQ2; and

Fig.1 is a line-up of the gene encoding Mortierella alpina Δ5-fatty acid desaturase with

various Δ6 desaturases and a Δ12 desaturase; and

Fig. 2 is a line-up of the gene encoding Δ5-fatty acid desaturase with the C. elegans Δ6

desaturase and the fungal Δ5 desaturase from M. alpina; and

Fig. 3 is a gas chromatography trace of the fatty acid methyl esters of induced yeast cell

transformants transformed with the Mortierella alpina Δ5-fatty acid desaturase gene and

uninduced yeast cell transformants; and Fig. 4 is a gas chromatography trace of the fatty acid methyl esters of induced yeast cell

transformants transformed with the C. elegans Δ5-fatty acid desaturase gene and

uninduced yeast cell transformants.

Cloning and sequencing of the Δ5-fatty acid desaturase gene from Mortierella alpina

The DNA sequences of the invention encode Δ5-fatty acid desaturases and were cloned

using PCR technology in combination with cDNA library templates and specifically

designed primers. The function of the DNA sequences, namely the conversion of

dihanogamma linolenic acid (DHL) to arachidonic acid (AA), and eicostatetraenoate (ETA)

to ecosapentaenoate (EPA), were verified by expressing the corresponding cDNAs in yeast.

The Δ5-fatty acid desaturase gene from Mortierella alpina was cloned by Polymerase

Chain Reaction (PCR) techniques using cDNA from Mortierella alpina as the template and

specifically designed degenerate oligonucleotide primers (DP) as shown below, based on

the first and third histidine bases of plant Δ12 and Δ15 desaturases previously identified by

Shanklin (Shanklin, J, Whittle, E J & Fox, BG. Biochemistry. 33, 12787-12794 (1994)).

Degenerate oligonucleotide primers (DP)

5'-GCGAATTA(A/T)TIGGICA(T/C)GA(T/C)TG(T/C)GICA-3'

5'-GCGAATTCATIT(G/T)IGG(A/G)AAIA(G/A)(A/G)TG(A/G)TG-3'

where I represents inosine, and the Eco RI sites are underlined. The PCR amplifications were run entirely conventionally on a thermal cycler made by

using a program of 2 minutes at 94 °C then 45 seconds at 94 °C, 1 minute at 55 °C and 1

minute at 72 °C for 32 cycles followed by extension at 72 °C for a further 10 minutes. PCR

amplification products were separated on 1 % agarose gels.

The range of PCR products amplified from the Mortierella alpina cDNA template included

a 660 bp product which was gel purified, cloned into pGEM-T (Promega ^RTM) and

transformed into the Escherichia coli expression host, DH5α.

Primers (P) were designed against the 660 bp product sequence and fragment amplification

carried out by PCR using the cloned 660 bp fragment as a template, and sequence-specific

primers (P) based on the 660 bp product sequence.

Delta B for

5'-GATGCGTCTCACTTTTCA-3'

Delta B rev.

5'-GTGGTGCACAGCCTGGTAGTT-3'

The products of this PCR amplification were gel purified and used as probes to screen a

Mortierella alpina cDNA library. The fragment probe hybridised to 25 out of the 3.5 x 10 ⁵ phage clones screened and one clone was shown, by restriction analysis, to have the

expected size of 1.5 kb. This clone, designated LI 1, was selected for further analysis. Sequence analysis of LI 1 revealed an open reading frame of 1 ,338 bp in length encoding a

polypeptide of 446 amino acids. When analyzed on the protein and genomic databases

using the GCG 8 Program (Devereux J. et al. Nucleic Acids. Res.. 12, 387-395 (1984)),

LI 1 showed a low level 20% identity to the Δ6 desaturase gene from Synechocystis sp.

PCC6803 (Fig. 1).

In Fig. 1, the sequences in the line-up have the following Accession numbers:

S54259 Δ12 Spirulina Accession number : X86736

S54809 Δ6 Spirulina Accession number : X87094

S68358 Putative Sphingolipid desaturase Accession number : X87143

S35157 Δ6 Synechocystis Accession number : LI 1421

PBOR6 Δ6 Borage Accession number U79010

FU2 Δ5 desaturase Accession number AF054824

In addition, although all three histidine boxes characteristic of desaturase enzymes are

present in the translated sequence, the third histidine box located at position 1159 bp in the

sequence contains the variant QXXHH. The translated sequence also contains a

cytochrome bs-like heme-binding domain at the N-terminus which includes the EHPGG

motif whereas previously, this feature has only been observed at the C-terminus of other

fungal desaturases.

Southern blotting of genomic DNA

Sequence specific primers (P) designed against the LI 1 sequence between histidine boxes 1

and 3 of LI 1 , were used in a PCR reaction to amplify a 660 bp region of the LI 1 sequence. The 660 bp PCR product was gel purified and Southern blots of restricted Mortierella

alpina and Mucor circinelloides genomic DNA carried out using the 660 bp fragment as a probe. The results suggest that the gene encoding the Δ5-fatty acid desaturase of the

invention is present in single copy in Mortierella alpina and appears to be absent from

Mucor circinelloides. In addition, there is no detectable Δ5-fatty acid desaturase activity in

Mucor circinelloides.

Expression of the cloned Mortierella alpina gene encoding Δ5-fatty acid desaturase

In order to confirm that the LI 1 sequence encoded a Δ5-fatty acid desaturase enzyme, the

cDNA was subcloned into the yeast expression vector, pYES2, supplied by Invitrogen ™

under the control of the GAL4 polymerase promoter to yield plasmid pYES2/Ll 1. The

expression of LI 1 was checked by in vitro transcription-translation of pYES2/Ll 1 using

the Promega™ coupled Transcription and Translation system. ³⁵S methionine-labelled

translation products were generated which were run on SDS PAGE and visualised by

exposure to autoradiograph film. The estimated molecular weight of the product was 55-60

kD and a control plasmid, pYES2 with no insert, failed to yield any labelled translation

product.

Construct, pYES2/Ll 1, was transformed into yeast Saccharomyces cerevisiae and grown

on uracil-deficient YCA medium. Transformants were selected by virtue of the presence of

the URA3 selectable marker carried by pYES2 Ll 1 and expression of LI 1 was induced by

the addition of galactose to a final concentration of 1% mM. The cultures were grown

overnight in the presence of 0.5 mM dihomo gamma linolenate, detergent (1% tergitol NP-40) and 2% raffinose. Aliquots were harvested at t=0, t=4 hours, and t= 16 hours. Yeast total fatty acids were analysed by GC of methyl esters. The lipids from the induced

and uninduced control samples were transmethylated with 1M HCL in methanol at 80°C

for 1 hr. Fatty acid methyl esters (FAMES) were extracted in hexane. GC analysis of

FAMES was conducted using a Hewlett Packard 58804 Series Gas Chromatograph

equipped with a 25M x 0.32 mm RSL-500 BP bonded capillary column and a flame

ionization detector.

When methyl esters of the total fatty acids isolated from yeast carrying the plasmid

pYes2/Ll 1 and grown in the presence of galactose and dihomo gamma linolenic acid were

analysed by GC an additional peak was observed (see Fig. 3). This extra peak had the

same retention time as the authentic arachidonic acid standard (Sigma) indicating that the

transgenic yeast were capable of desaturating Dihomo gamma linolenic acid at the Δ5

position. No such peaks were observed in any of the control samples (transformation with

pYes2) Fig. 3. The identity of the additional peak was confirmed by GCMS (Kratos

MS80RFA operating at an ionization voltage of 70 eV with a scan range of 500-40 daltons)

which positively identified this compound as arachidonic acid.

This demonstrates that the DNA sequence from Mortierella alpina encodes a functional polypeptide involved in the synthesis of arachidonic acid in the presence of galactose and dihomo gamma linolenate.

Cloning and sequencing of the C. elegans Δ5-fatty acid desaturase gene

Previously, the inventors identified fungal Δ5 and Δ6-fatty acid desaturases from both

plant and animal species which were distinct from previously identified microsomal desaturases. This difference was due to the presence of an N-terminal extension which

showed homology to the electron donor protein cytochrome b₅.

During the characterisation of the fungal (Mortierella alpina) Δ5-fatty acid desaturase and

the C.elegans Δ6-fatty acid desaturase (present on cosmid W08D2 (Accession No.

Z70271)), the inventors identified a related sequence on cosmid T13F2.1 (Accession No.

Z81122) also containing C.elegans DNA likely to encode a fatty acid desaturase.

Analysis of the sequences (using Genefinder program (Wilson, R. et al (1994) Nature, 368,

32-38)) revealed that cosmids W08D2 and T13F2 contained overlapping regions. In

addition, it was found that cosmid T13F2 contained an open reading frame (ORF),

designated T13F2.1, which contained an N-terminal cytochrome b₅ domain (defined by the

diagnostic His-Pro-Gly-Gly motif), as well as three 'histidine boxes' characteristic of all

microsomal desaturases. Further, this putative desaturase contained a variant third histidine

box, with a H→Q substitution for the first histidine in the His-X-X-His-His motif. This

glutamate substitution is present in both plant and animal Δ6-fatty acid desaturases and in

the fungal Δ5-fatty acid desaturase from M.alpina.

The overlap between cosmids T13F2 and W08D2 allowed the determination of the

proximity of the putative desaturase ORF, T13F2.1, to the Δ6-fatty acid desaturase,

revealing that the two sequences were arranged in tandem on chromosome IV, separated by

990 bases from the predicted stop codon of T13F2.1 to the initiating methionine triplet of

the Δ6-fatty acid desaturase. Since sequence analysis predicted that the T13F2.1 ORF was interspersed with a number of introns, heterologous functional expression of genomic DNA was unfeasible. Therefore,

the polymerase chain reaction (PCR) was used to amplify a partial cDNA clone

corresponding to a large predicted exon at the 5'end of the T13F2.1 ORF using the

following primers, CEFOR AND CEREV:

CEFOR:

5' - ATGGTATTACGAGAGCAAGA-3'

CEREV:

5 ' -TCTGGGATCTCTGGTTCTTG-3 '

After initial denaturation at 94 °C for 2 minutes, amplification was performed in 32 cycles

of: 45 seconds at 94 °C, 1 minute at 55 °C, and 1 minute at 72 °C followed by a final

extension at 72 °C for a further 10 minutes.

A DNA fragment of the correct predicted size was amplified (as visualised on a 1 %

agarose gel), the gel band was cut out, the DNA purified and ligated directly into pGEM-T

(Promega), and the resulting plasmid transformed into E.coli DH5α cells. Plasmid DNA

was purified for sequencing using the Qiagen QIAprep miniprep kit, and the nucleotide sequence of the insert determined by automated sequencing using an ABI-377 DNA

sequencer. In order to isolate the complete coding region corresponding to ORF T13F2.1, this isolated

233 bp PCR-amplified fragment was used to screen a mixed stage C.elegans cDNA library

that had been constructed in λZapJl by Prof Yuji Kohara - Mishima, Japan. The screening

was carried out using standard techniques (Sambrook et al (1989) Molecular Cloning. A

Laboratory Manual) using the cloned PCR product as a probe. The DNA fragments were

labelled with α [³²P] d CTP using the Ready to Go DNA-Labelling reaction mix

(Pharmacia). Of 1.4 x 10⁵ pfu screened for hybridization to the 233bp fragment, 5 plaques

gave positive signals and were cored out of the agar plates and eluted into SM buffer . The

resultant phage suspensions were screened for the presence of T13F2.1 by PCR

amplification using CEFOR and CEREV. One clone, designated L4, was purified by 2

additional rounds of plating and hybridisation screening at 65°C using the 233bp fragment

isolated by PCR. Plasmid L4 was released from λ clone L4 by excision and the cDNA

insert sequenced on both strands using a Perkin Elmer AB 1-377 DNA sequencer.

The resulting DNA sequence is shown in SEQ.2, and the predicted amino acid sequence is

shown in SEQ.4.

Functional analysis of L4 in yeast

The complete coding region (coding for 447 amino acids) of L4 was amplified by PCR

using the primers YCEDFor and TCEDRev shown below, which also introduced flanking

H dHJ and Barriffl restriction sites:

YCEDFor: 5'-GCGAAGCTTAAAATGGTATTACGAGAGCAAGAGC-3' (annealing to the initiating methionine is indicated by the bold type face and the Hind HI

restriction site is underlined)

YCEDRev:

5 ' -GCGGGATCC A ATCTAGGC AATCTTTTTAGTC AA-3 '

(annealing to the complement of the stop codon, indicated by the bold type face and the

BarnH I restriction is underlined)

The amplified PCR product containing the complete coding region of L4 was ligated into

the yeast expression vector, pYES2 (Invitrogen), downstream of the GAEl promoter using

H dLII and Rαmlll restriction sites (enzymes supplied by Boehringer Mannheim). The

resulting construct, designated pYΕS2/L4, was transformed into E.coli, and the fidelity of

the PCR-generated insert in plasmid pYES2/L4 was confirmed in vitro by coupled

transcription/translation using the TNT system (Promega). The resulting translation

products were labelled with ³⁵S methionine, separated by SDS-PAGE and visualised by

autoradiography .

The translation product obtained from pYES2/L4 had a molecular weight of approximately

M_r57,000, whereas the control vector, pYES2 with no insert, did not yield a translation

product.

For functional analysis of the L4 coding region the recombinant plasmid was transformed

into S. cerevisiae DBY746 by the lithium acetate method (Elble R. (1992) Bio Techniques

13 18-20). Cells were cultured overnight in a medium containing raffinose as a carbon source, and supplemented by the addition of either linoleic acid (18:2 Δ^{9 12}) or

di-homo-γ-linolenic acid (C20:3 Δ^{8 11,14}) in the presence of 1% tergitol (as described by

Napier et al (1998) Biochem. J. 330 611-614). These fatty acids are not present in S.

cerevisiae but serve as the specific substrates for either the Δ⁶ or Δ⁵-desaturase,

respectively. Expression of the L4 coding region from the GAL1 promoter of the vector

was induced by the addition of galactose to 1%. Growth of the cultures was continued for

16 hours before removal of aliquots for the analysis of fatty acids by GC Total fatty acids

extracted from yeast cultures were analysed by gas chromatography (GC) of methyl esters.

Lipids were transmethylated with 1M HC1 in methanol at 80°C for 1 hr, then fatty acid methyl esters (FAMEs) were extracted in hexane. GC analysis of FAMEs were conducted using a Hewlett Packard 5880A Series Gas chromatograph equipped with a 25 M x 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. Figure 4 shows the result of GC analysis of the fatty

acid methyl esters of transformed yeast strains. An additional peak is apparent in the trace

obtained from induced pYES2/L4 grown in the presence of di-homo-γ-linolenic acid

compared to an empty-vector control. This peak was also absent from uninduced cultures

grown on di-homo-γ-linolenic acid and it is also important to note that pYES2 L4 grown in

the presence of linoleic acid failed to accumulate any novel peaks indicating that this fatty acid is not a substrate for the enzyme encoded by the C. elgans cDNA. The retention time

of the additional peak is identical to that of the authentic methyl-arachidonic acid standard.

The fatty acid produced from di-homo-γ-linolenic acid was further characterised by GCMS (Gas Chromatography Mass Spectrometry) and identified as arachidonic acid. The results

show, therefore, that yeast cells transformed with the plasmid pYES2/L4 had acquired

functional Δ⁵-desaturase activity and were now capable of synthesising arachidonic acid

from the substrate di-homo-γ-linolenic acid. The Δ⁵-desaturase in the transformed yeast

appeared to be an efficient catalyst.

This demonstrates that the DNA sequence from C. elegans encodes a functional

polypeptide involved in the synthesis of arachidonic acid in the presence of galactose and

di-homo-γ-linolenate.

S^Q.2-

1 ATGGTATTAC GAOAGCAAOA GCATGAGCCA TCT CATTA AAATTOATGG

51 AAAATGGTGT CAAATTOACQ ATQCTQTCCT GAGATCACAT CCAGGTGGTA

101 GTGCAATTAC TACCTATAAA AATATGGATG CCACTACCGT AT CCACACA

151 TTCCATACTG GTTCTAAAGA AGCGTATCAA TGGCTGACAG AATTGAAAAA

201 AGAGTGCCCT ACACAAGAAC CAGAGATCCC AGATATTAAG GATGACCCAA

251 TCAAAGGAAT TGATGATGTG AACATGGGAA CTTTCAATAT TTCTGAGAAA

301 CGATCTGCCC AAATAAATAA AAGTTTCACT GATCTACGTA TGCGAGTTCG

351 TGCAGAAGGA CTTATGGA G GATCTCCTTT G TCTACATT AQAAAAATTC

401 TTGAAACAAT CTTCACAATT CTTTTTGCAT TCTACCT CA ATACCACACA

451 TATTATCTTC CATCAGCTAT TCTAATGGGA GTTGCGTGGC AACAATTGGG

501 ATGGTTAATC CATGAATTCG CACATCATCA GTTGTTCAAA AACAGATAC

551 ACAATGATTT GGCCAGCTAT TTCGTTGGAA ACTTTTTACA AGGATTC CA

601 TCTGGTGGTT GGAAAGAGCA GCACAATGTG CATCACGCAG CCACAAATGT

651 TGTTGGACGA GACGGAGATC TTGATTTAGT CCCATTCTAT GCTACAGTGG

701 C-AGAACATCT CAACAATTAT TCTCAGGATT CATGGGT AT GACTCTATTC

751 AGATGGCAAC A GTTCATTG GACATTCATG TTACCATTCC TCCGTCTCTC

801 GTGGCTTCTT CAGTCAATCA TTTTTGTTAG TCAGATGCCA ACTCATTATT

851 ATGACTATTA CAGAAATACT GCGATTTATG AACAGGTTGG TC CTCTTTG

901 CACTGGGCTT GGTCATTGGG TCAATΓGTAT TTCCTACCCG ATTGGTCAAC

951 TAGAATAATG TTCTTCCTTG TTTCTCATCT TGT GGAGGT TTCCTGCTCT

1001 CTCATGTAGT TACTTTCAAT CATTATTCAG TGGAGAAGTT TGCATTGAGC

1051 TCGAACATCA TGTCAAATTA CGCTTGTCTT CAAATCATOA CCACAAOAAA

1101 TATGAGACCT GGAAGATTCA TTGACTGGCT TTGGGGAGGT CTTAACTATC

1151 AGATTGAGCA CCATCTTTTC CCAACGATGC CACGACACAA CTTOAACACT

1201 GTTATGCCAC TTGTTAAGGA GTTTGCAGCA GCAAATGGTT TACCATACAT

1251 GGTCGACGAT TATTTCACAG GATTCTGGCT TGAAATTGAG CAATTCCGAA

1301 ATATTGCAAA TGTTGCTGCT AAATTGACTA AAAAGATTGC CTAG MGTDQGKTFT WEEAAHNTK GDLFLAIRGR VYDVTKFLSR HPGGVDTLLL GAGRDVTPVF EMYHAFGAAD AIMKKYYVGT LVSNELPVFP EPTVFHKTIK TRVEGYFTDR DIDPKNRPEI WGRYALIFGS LIASYYAQLF VPFWERTWL QWFAIIMGF ACAQVGLNPL HDASHFSVTH NPTVWKILGA THDFFNGASY LVWMYQHMLG HHPYTNIAGA DPDVSTFEPD VRRIKPNQKW FVNHINQDMF VPFLYGLLAF KVRIQDINIL YFVKTNDAIR VNPISTWHTV MFWGGKAFFV WYRLIVPLQY LPLGKVLLLF TVADMVSSYW LALTFQANHV VEEVQWPLPD ENGIIQKDWA AMQVETTQDY AHDSHLWTSI TGSLNYQAVH HLFPNVSQHH YPDILAIIKN TCSEYKVPYL VKDTFWQAFA SHLEHLRVLG LRPKEE*

SEϋ^H-The predicted amino acid sequence of L4 the gene which encodes a Δ fatty acid desaturase from C. elegans.

1 MVLREQEHEP FFIKIDGKWC QIDDAVLRSH PGGSAITTYK NMDATTVFHT

51 FHTGSKEAYQ WLTELKKECP TQEPEIPDIK DDPIKGIDDV NMGTFNISEK

101 RSAQINKSFT DLRMRVRAEG LMDGSPLFYI RKILETIFTI LFAFYLQYHT

151 YYLPSAILMG VAWQQLGWLI HEFAHHQLFK NRYYNDLASY FVGNFLQGFS

201 SGGWKEQHNV HHAATNWGR DGDLDLVPFY ATVAEHLNNY SQDSWVMTLF

251 RWQHVHWTFM LPFLRLSWLL QSIIFVSQMP THYYDYYRNT AIYEQVGLSL

301 HWAWSLGQLY FLPDWSTRIM FFLVSHLVGG FLLSHWTFN HYSVEKFALS

351 SNIMSNYACL QIMTTRNMRP GRFIDWLWGG LNYQIEHHLF PTMPRHNLNT

401 VMPLVKEFAA ANGLPYMVDD YFTGFWLEIE QFRNIANVAA KLTKKIA*

Claims

1. An isolated animal ╬ö5-fatty acid desaturase and functional portions thereof.

2. Isolated C. elegans ╬ö5-fatty acid desaturase.

3. A DNA sequence encoding a ╬ö5-fatty acid desaturase according to claim 1 or claim 2.

4. A DNA sequence according to claim 3 and comprising at least a portion of the sequence shown in SEQ.2 and equivalents to that sequence, or to portions of that sequence, which encode a functional ╬ö5-fatty acid desaturase by virtue of the degeneracy of the genetic code.

5. A DNA sequence according to claim 4 derived from a Caenorhabditis elegans DNA sequence.

6. A DNA sequence according to claim 3 encoding a functional ╬ö5-fatty acid desaturase and comprising at least a portion of the sequence shown in SEQ.l and equivalents to that sequence, or to portions of that sequence, which encode a functional ╬ö5 -fatty acid desaturase by virtue of the degeneracy of the genetic code.

7. A DNA sequence according to claim 6 derived from a Mortierella alpina DNA sequence.

8. A DNA sequence according to any one of claims 3 to 7 wherein the DNA sequence is functional in a mammal.

9. A DNA sequence according to claim 8 in which the DNA sequence is expressed in a mammal

10. A DNA sequence according to claim 9 wherein the DNA sequence is expressed in a human.

11. A DNA sequence obtained by modification of a functional natural gene encoding a ╬ö-5 fatty acid desaturase according to claim 1 or claim 2.

12. A DNA sequence according to claim 11 wherein the modification includes modification by chemical, physical, or biological means without removing a catalytic activity of the enzyme which it encodes.

13. A DNA sequence according to claim 12 wherein the modification improves a catalytic activity of the enzyme which it encodes.

14. A DNA sequence according to claim 12 or 13 wherein the biological modification includes recombinant DNA methods and forced evolution techniques.

15. A DNA sequence according to claim 14 wherein the forced evolution technique is DNA shuffling.

16. A polypeptide encoded by a DNA sequence according to any of claims 3 to 15.

17. A polypeptide according to claim 16 wherein at least a portion of the polypeptide has the sequence shown in SEQ.3 or functional equivalents to that sequence or to portions of that sequence.

18. A polypeptide according to claim 16 wherein at least a portion of the polypeptide has the sequence shown in SEQ.4 or functional equivalents to that sequence or to portions of that sequence.

19. A polypeptide according to any of claims 16 to 18 wherein the polypeptide catalyses the conversion of dihomogamma linolenic acid to arachidonic acid.

20. A polypeptide according to any of claims 16 to 19 wherein the polypeptide has been modified without removing the catalytic activity of the encoded polypeptide.

21. A polypeptide according to claim 20 wherein the polypeptide has been modified in such a way as to introduce a specific level of saturation of a substrate at a specific location within the molecular structure of the substrate.

22. A vector containing a DNA sequence or any portion of a DNA sequence according to any of claims 3 to 15.

23. A method of producing polyunsaturated fatty acids comprising contacting a suitable substrate with a ╬ö5-fatty acid desaturase according to claim 1 or 2 or a polypeptide according to claim 16 to 21.

24. A method of converting dihomogamma linolenic acid to arachidonic acid wherein said conversion is catalysed by a ╬ö5-fatty acid desaturase according to claim 1 or 2 or a polypeptide or modified polypeptide according to any of claims 16 to 21.

25. An organism engineered to produce high levels of a polypeptide according to any of claims 16 to 21.

26. An organism engineered to produce high levels of a product of a reaction catalysed by a ╬ö5-fatty acid desaturase according to claim 1 or 2 or by a polypeptide according to any one of claims 16 to 21.

27. An organism which has been engineered to carry out the method of claim 23 or claim 24.

28. An organism according to either of claims 26 and 27 wherein the organism is a microorganism.

29. An organism according to claim 28 wherein a microorganism is selected from algae, bacteria and fungi.

30. An organism according to claim 29 wherein a fungi includes phycomycetes.

31. An organism according to claim 28 wherein said microorganism is a yeast.

32. An organism according to any of claims 25 to 27 wherein the organism is a plant.

33. An organism according to claim 32 wherein the plant is selected from oil seed plants and tobacco.

34. An organism according to claim 33 wherein the oil seed plants are selected from oil seed rape, sunflower, cereals including maize, tobacco, legumes including peanut and soybean, safflower, oil palm, coconut and other palms, cotton, sesame, mustard, linseed, castor, borage and evening primrose.

35. A seed or other reproductive material derived from an organism according to claim 33 or claim 34.

36. An organism according to any of claims 25 to 27 wherein the organism is a mammal.

37. An isolated multienzyme pathway wherein the pathway includes a ╬ö5 desaturase according to claim 1 or 2 or a polypeptide according to any of claims 16 to 21

38. A compound produced by a conversion of a substrate, wherein said conversion is catalysed by a ╬ö5 desaturase according to claim 1 or 2 or by a polypeptide according to any of claims 16 to 21.

39. An intermediate compound produced by the reaction catalysed by a ╬ö5 desaturase according to claim 1 or 2 or by a polypeptide according to any of claims 16 to 21.

40. A foodstuff or dietary supplement containing a polyunsaturated fatty acid produced by a method according to claim 23 or 24.

41. A pharmaceutical preparation containing a polyunsaturated fatty acid produced by a method according to claim 23 or 24.

42. Prostaglandins synthesised by a biosynthetic pathway including a catalytic activity of a ╬ö5 desaturase according to claim 1 or 2 or by a polypeptide according to any of claims 16 to 21.

43. A method for modulation of prostaglandin synthesis by the control of the levels of expression of a DNA sequence according to any of claims 3 to 15.

44. A probe comprising all or part of a DNA sequence according to any of claims 3 to 15 or an equivalent RNA sequence.

45. A diagnostic or search probe comprising all or part of a ╬ö5 desaturase according to claim 1 or 2 or of a polypeptide according to any of claims 16 to 21.

46. A method of isolating ╬ö5 desaturases using a probe according to claim 44 or 45.