AU720765C - Novel plant enzyme and use thereof - Google Patents

Description

NOVEL PLANT ENZYME AND USE THEREOF

Technical field

The present invention relates to a novel plant enzyme. More specifically, the present invention relates to a method for producing acetylenic compounds in particular acetylenic fatty acids, to a cDNA encoding a plant fatty acid acetylenase, to the use of the said cDNA for transforming oil accumulating organisms for the purpose of producing acetylenic fatty ac¬ ids, and to such oil accumulating organisms per se as well as oils therefrom.

Background of the invention

There is considerable interest, world-wide, in producing chemical feedstocks such as fatty acids for industrial use from renewable plant resources rather than from non-renewable petrochemicals. This concept has broad appeal for both manu¬ facturers and consumers on the basis of resource conservation and in addition provides significant opportunities to develop new industrial crops for agriculture.

There is an enormous diversity of unusual fatty acids in oils from wild plant* species which have been well characterized (see e.g. Badami & Patil, 1981) . Many of these acids are of potential industrial use. This has lead to an interest in do¬ mesticating relevant plant species to enable the agricultural production of particular fatty acids. However the development of genetic engineering combined with a greater understanding of the biosynthesis of unusual fatty acids make it now possi¬ ble to transfer genes coding for key enzymes, involved in the synthesis of a particular fatty acid from a wild species, to a choosen domesticated oilseed crop. In this way specific fatty acids can be produced in high purity and quantities at moderate costs.

One class of fatty acids of particular interest are the acety¬ lenic fatty acids; consisting of an acyl chain having two adjacent carbon atoms linked by an acetylenic or triple bond. Because of their high reactivities they may be ideally suited for the production of coatings, plastics and lubricants. By transferring the genes responsible for the production of a specific acetylenic acid from a wild species to commercial oilseeds, or any other oil accumulating organism that can be easily multiplied, it should be possible to develop a renew¬ able primary source of this oil containing acetylenic fatty acids for industrial uses.

Prior art

The formation of acetylenic bonds in fatty acids in mosses oc¬ curs via the subtraction of hydrogens from a double bond (Kohn et al. , 1994)

Crepis species have seed oils with high contents of acetylenic acids (Badami & Patil, 1981; Hirsinger, 1991) .

Summary of the invention

The present invention provides a new method of producing acetylenic fatty acids from transgenic oil accumulating organisms.

The inventors have characterized an enzyme (acetylenase) that is responsible for the production of 9-octadecen-12-ynoic acid (crepenynic acid) from 9, 12-octadecadienoic acid (linoleic acid) in membrane fractions from developing Crepis alpina seeds. The characterization of the acetylenase from Crepis alpina revealed that the acetylenase had very similar bio¬ chemical properties to the non-heme containing monooxygenases oleate delta 12 and linoleate delta 15 (omega 3) desaturases. Based on the premise that the biochemical similarities observed between the acetylenase and the enzymes producing linoleic and linolenic acid (delta 12 and delta 15 desa¬ turases) would also be associated with similarity in the primary sequence of these proteins a full length cDNA (pCrepl) , encoding a putative acetylenase, was isolated from Crepis alpina .

Initially, two types of cDNA fragments, obtained by using PCR and primers designed by aligning protein sequences of delta 12 desaturases, were characterised from C. alpina . DNA sequence analysis revealed that one was highly homologous to all the other plant endoplasmic reticular (ER) delta 12 desaturases and the castor bean hydroxylase. The other cDNA fragment characterised had a sequence that was homologous to the ER delta 12 desaturase sequences of plants but was divergent not only in a number of non-conserved amino residues but also in a number of amino acid residues that were highly conserved in all delta 12 ER desaturases. Using northern blot analysis the gene encoding this cDNA (pCrepl) was observed to be highly expressed only in a seed specific manner when compared to expression in leaf tissue. Taken together these findings, and a consideration of the unique biochemical nature of an cell in a oilseed, provided strong evidence that the isolated cDNA (pCrepl) from C. alpina encode an enzyme responsible for con¬ verting linoleic acid into crepenynic acid.

Finally, conclusive evidence that the cDNA, pCrepl, from C. alpina encoded a plant acetylenase enzyme was obtained by the expression of this gene in yeast. The expression of this gene together with the addition of linoleic acid when culturing these yeast resulted in the production of a delta 12 acety¬ lenic acid, 9-octadecen-12-ynoic acid (crepenynic acid) , as confirmed by mass spectrometric analysis of extracted yeast fatty acids.

Therefore, in a first aspect, the present invention relates to a method of producing acetylenic compounds, characterized in that a double bond is converted to an acetylenic bond by an acetylenase. In a preferred embodiment of the method, the acetylenic fatty acids are produced by conversion of unsaturated fatty acids to acetylenic fatty acids by a fatty acid acetylenase.

In a second aspect, the invention relates to cDNA coding for acetylenase of the mixed function monoxygenase type containing three conserved histidin motifs (HX₍3 _or 4₎H, HX₍2 _or 3₎HH, and HX ₍2 or ₃)^HH) according to Fig. 1 of the accompanying drawings.

In a further embodiment the invention relates to a cDNA encod¬ ing fatty acid acetylenase, such as Crepis alpina delta 12 acetylenase comprising the sequence according to Fig. 3 of the accompanying drawings or any nucleotide sequences essentially homologous therewith.

A third aspect of the invention concerns use of the above de¬ scribed cDNA for transforming organisms. The organisms may be acetylenic compound accumulating organisms or oil accumulating organisms, respectively.

In a fourth aspect, the invention relates to organisms trans¬ formed with a acetylenase cDNA as described above. The organ¬ isms are acetylenic compound or oil accumulating, examples of the latter being oil crops, oleogeneous yeasts and moulds.

In a fifth aspect, the invention concerns acetylenic componds accumulated in organisms described above.

In a sixth aspect, the invention concerns oils from oil accu¬ mulating organisms described above.

In a preferred embodiment, the present invention relates to transforming oil accumulating organisms with the said isolated cDNA from Crepis alpina seed cDNA library for the purpose of producing acetylenic fatty acids acids and in particular 9-octadecen-12-ynoic acid (crepenynic acid) . Detailed description of the invention

C. alpina seed oil is rich in crepenynic acid [9-octadecen- 12-ynoic acid (Hirsinger, 1989)] . The inventors have studied the biosynthesis of crepenynic acid in C. alpina seeds. The feeding of exogenous l-¹⁴C-labelled free fatty acids to intact developing cotyledons of C. alpina seeds demonstrated that linoleate is a precursor to crepenynic acid. This is contra¬ dictory to previous published results for the biosynthesis of crepenynic acid in Crepis rubra (Haigh & James, 1967) . Although the reaction of acetylenic acid formation in mosses has been shown to be a desatura ion process (Kohn et al.1994), such desaturation processes can be carried out by a variety of different unrelated types of plant enzymes, such as phytoene desaturases (Wieland et al. 1994) or non-heme containing proteins, the latter a class of enzymes of which some show very little amino acid sequence homologies except for three conserved histidin motifs (Shanklin et al. 1994) . It has been suggested that the biosynthesis of acetylenic fatty acids occur by a sequence of intermediates catalyzed by separate en¬ zymatic reactions. For example, acetylenic bonds were thought to be formed as a side pathway of saturated fatty acid syn¬ thesis (Diedrich & Henschel, 1991) ; or via an epoxygenation of a double bond with subsequent conversion to a diol which in its turn is dehydrated in two steps in order to form an acetylenic bond (Van de Loo et al, 1993) . Given these con¬ flicting alternatives the nature of an acetylenase enzyme and its mechanism of action was not known at all nor obvious at the time of the present priority patent application SE 9601236-4.

The enzyme, according to this invention, responsible for the synthesis of crepenynic acid (called the delta 12 acetyle¬ nase) , was shown by the inventors to remain only active in membrane (microsomal) fractions prepared from developing seeds of Crepis alpina, provided that the homogenization buffer contain NADH or NADPH, catalase and free coenzyme A. The char- acterisation of the microsomal acetylenase and its comparison with the delta 12 desaturase (responsible for the desaturation of oleate to linoleate) revealed that these enzymes had very similar properties. Both enzymes required 02 and NADH or NADPH; where both coreductants worked equally well with both enzymes. Cyanide (CN-) and antibodies against cauliflower cy¬ tochrome bs inhibited both these enzymes whereas carbonmonox- ide had no significant effect on either enzyme activity. These data suggested that both enzymes were biochemically similar. The oleate delta 12 hydroxylase from castor bean was also shown to have similar biochemical properties to the delta 12 desaturase despite catalyzing a different reaction (Bafor et al., 1991, Smith et al, 1992) . The castor bean delta 12 hy¬ droxylase gene was later shown to have significant sequence homology to the ER delta 12 desaturase genes (FAD 2 genes) (Van de Loo et al., 1995) . Because the delta 12 acetylenase, like the delta-12 desaturase (FAD2) , catalyzes a dehydrogena- tion between carbons 12 and 13 of an acyl chain, and like the delta 15 desaturase (FAD3) utilized linoleic acid as substrate the inventors considered the possibility that the acetylenase gene should have some sequence homology to the FAD2 and/or the FAD3 genes.

The invention will now be described more closely below in re¬ lation to the accompanying drawings and an Experimental Part.

The drawings sho :

Fig. 1. Restriction map of pCrepl

Fig. 2. Restriction map of pVT-Crepl

Fig. 3. Superimposed single ion chromatograms of ions 333, 365, 367 from FADEA prepared from total fatty acids extracted from yeast strain YN94-1 transformed with pVT-Crepl. The let¬ ters denotes peaks representing the following diethylamide de- rivatives of fatty acids: A, eicosanoic acid; B, eicosaenoic acid; C, 9-octadecen-12-ynoic acid.

Fig. 4. Superimposed single ion chromatograms of ions 333, 365, 367 from FADEA prepared from total fatty acids extracted from yeast strain YN94-1 transformed with empty vector (pVTIOOU; control) . The letters denotes peaks representing the following diethylamide derivatives of fatty acids: A, ei¬ cosanoic acid; B, eicosaenoic acid.

Fig. 5. A total ion chromatogramme of FADEA prepared from fatty acids enriched in the putative 9-octadecen-12 ynoic acid originating from lipid extracts of YN94-1 transformed with pVT-Crepl. The letters denotes peaks representing the follow¬ ing diethylamide derivatives of fatty acids: A, hecadecanoic acid; B, octadecaonoic acid; C, octadeca-9,12-dienoic acid; D. 9-octadecen-12-ynoic acid.

Fig. 6. Mass spectrum of compound corresponding to peak D in Fig.5.

EXPERIMENTAL PART

Cloning of putative acetylenase gene

An alignment of amino acid sequences from different species showed that the membrane bound fatty acid desaturases could be grouped according to the homology of their putative mature protein into three distinct groups (plastid delta 12 desatu¬ rases, ER delta 12 desaturases and delta 15 desaturases,- see Sequence Listing 1) . The castor bean hydroxylase (Van de Loo et al 1995) shared a high homology with the ER delta 12 de¬ saturases to the degree that it was not easily distinguishable from these sequences. Furthermore, the sequences from all three classes of enzymes showed some degree of sequence homo logy with each other. Based on this alignment oligonucleotide primers were designed and synthesised for these three groups of sequences and for a consensus of all of these sequences. The sequence of these primers are given below.

(i) consensus primers (primers designed to a consensus of all three groups of membrane-bound desaturases and the castor bean fatty acid hydroxylase) : sense is GSN CAY GAN TGY GSN CAY antisense is RAN ADR TGR TGN RBN AYR TG.

(ii) plastid delta 12 desaturase primers: sense is TGG MGN TTY AAR CAY GAY MG antisense is GTN S C ATC CAR AAR TGR TA.

(iii) ER delta 12 desaturase primers including the castor bean fatty acid hydroxylase: sense is CAY GAR TGY GGN CAY CAY GC antisense is CCN CKN ARC CAR TCC CAY TC.

(iv) delta 15 desaturase primers: sense is ACN CAY CAY CARAAY CAY GG antisense is CAY TGY TTN CCN CKR TAC CA.

Poly A+ RNA was isolated from developing seeds (100 mg) of C. alpina using a QuickPrep Micro mRNA purifcation kit from Phar¬ macia Biotech. All of the poly A+ RNA from this purification was precipitated and used to synthesise first strand cDNA which was primed with both oligo dT and random hexamers and synthesised with Superscript II reverse transcriptase from Gibco BRL. The polymerase chain reaction (PCR) was then used, with the described primers and this cDNA, to amplify products with the following cycling conditions:1 cycle of 94°C for 2 min, 30 cycles of (94°C, 30 sec; 50°C, 30 sec; 72°C, 30 sec) and finally one cycle of 72°C for 5 min. Products were obtained for all the primers used; particularly noticeable was that the primers against the ER delta 12 de¬ saturases gave significantly more product than from the other primers used. The sizes of the PCR products from the delta 12 and delta 15 primers corresponded to the sizes anticipated.

The PCR products obtained by amplification with the ER delta 12 primers and delta 15 primers were made blunt ended with T4 and klenow polymerases and cloned into the EcoRV site of the plasmid vector Bluescript. DNA sequencing of a number of the clones revealed that at least three distinct sequences had been amplified when using these two sets of primers: (i) a highly conserved delta 15 desaturase sequence (ii) , a highly conserved ER delta 12 sequence and (iii) a sequence (D12V) having homology to the ER delta 12 sequences but showing dis¬ tinct differences even in some amino acid residues that were highly conserved amongst all the other desaturase sequences.

The analysis of fatty acids from C. alpina had indicated that the crepenynic acid was probably present only in seeds. Northern blot analysis at high stringency indicated that the mRNA from the D12V sequence described above was expressed highly in seeds but not in leaves which is consistent with the observation that crepenynic acid was only observed in seeds.

A cDNA library was made from developing seeds from C. alpina using a Uni-ZAP XR cloning kit for cDNA from Stratagene and screened with the random labelled D12V sequence. From this screening it was estimated that cDNAs encoding the D12V sequences were highly abundant; further emphasing the high level of expression of this gene. After the isolation of single hybridising Lambda plaques, pBluescript phagemid was excised using the ExAssist/SOLR system from Stratagene. Phagemids obtained by this were subsequently used to produce double stranded DNA plasmid. From these colonies a full length clone (pCrepl, see Fig. 1) was isolated by using DNA sequenc¬ ing and restriction mapping of isolated plasmid. The insert from pCrepl, a 1.5 kb insert contained in the vector pBluescript SK, was sequenced and from this an open reading frame deduced coding for a 375 aa long protein (Sequence List¬ ing 2) . The analysis of this protein sequence revealed approximately 60% identity and 80% similarity with other plant delta 12 desaturase proteins and had noticeable differences in homology, where, certain residues that were conserved amongst all other desaturases were not in this sequence (see Sequence Listing 1) . Three histidin motifs were present which have been shown to be conserved in a number of non-heme containing mon- oxygenases catalyzing hydroxylation and desaturation reactions (Shanklin et al. 1994).

Expression of the pCrep cDNA and detection of crepenynic acid in transgenic yeast

The pCrepl open reading frame was released from pCrepl on a Smal /Xhol restriction fragment and the 1.5 kb Crepl open reading frame recovered by gelpurification (Langridge et al. , 1980). pVTIOO-U DNA (Vernet et al. , 1987) was digested using PvuII and Xhol. 50 ng PvuII/Xhol-linearized pVTIOO was ligated with 100 ng 1.5 kb Smal/Xhol fragment corresponding to the Crepl open reading frame using T4 DNA ligase (NBL Genen Sci¬ ence Ltd., UK) . Part of the ligation mixture was used to transform competent E. coli DHa cells. One clone (pVT-Crepl) , which contained the expected 1.5 kb insert, was chosen and the contruct checked by digestion with EcoRI, or Hindlll + Xbal. Both digests gave the expected products (approx. 5.3, 2.3 and 0.8 kb for the EcoRI digest, and release of the 1.5 kb open reading frame with the Hindlll + Xbal digest) . pVT-Crepl DNA (see Fig. 2) , or empty vector pVTIOOU, was used to transform Saccharomyces cerevisiae strains YN94-1 and C13-ABYS86, using the PLATE method of Elble (1992) . Overnight yeast transfor¬ mants were spread on SCD minus uracil agar and single colonies were streaked onto fresh selective (minus uracil) plates. The YN94-1 and C13-ABYS86 strains of yeast transformed with pVT-Crepl DNA and with empty vector (pVTIOOU; control) were cultivated in shaking cultures at 28°C for five hours in selective media (without uracil; 400 ml) whereafter 40 ml of cultivation media containing linoleic acid dispersed in Tween 40^® was added to the culture to give a final concentration of 0.03% linoleic acid and 1% Tween 40^® (w/w) . After cultivation for an additional 78h at 28°C the cells were pelleted by cen¬ trifugation and washed by dispersion in 20 ml of 0.1M Tris- HCl buffer pH. 7.8 containing 1% Tween 40^® and repelleted by centrifugation.The cells were further washed by resuspension in 20 ml of 0.1M Tris-HCl buffer pH. 7.8 and pelleted again. The cells were thereafter extracted in a mixture of chloro¬ form/methanol/ 0.15M acetic acid (1:2:0.8 by vol.) in a Braun MSK glass bead cell homogenizer (B. Braun Biotech Interna¬ tional, Melsungen, Germany) at 4000 r.p.m. for 20 s. The yeast lipids were extracted from the mixture into a chloroform phase by adding chloroform and^"0.15M acetic acid to yield final pro¬ portions of 1:1:0.9 (by vol.) of chloroform, methanol and 0.15 M acetic acid . After centrifugation of the mixture the lipid containing chloroform phase was removed and evaporated to dry¬ ness under a stream of nitrogen.

The lipohilic residue were methylated with methanolic HCl (4% w/w) at 85°C for 45 min wherafter the fatty acid methyl esters were extracted into n-hexane. Gas liquid (GC) chromatogrammes of the methyl esters separated on a glass column (2.5m x 3 mm i.d. ) containing 3% SP-2300 on Supelcoport 100/120 mesh (Supelco, Beliefonte, P. USA) revealed a peak with the same retention time as authentic 9-octadecen-12 ynoic acid metyl ester constituting up to 0.3% of total peak areas in samples prepared from yeast transformed with pVT-Crepl but not in samples prepared from yeast transformed with empty vector '(pVTIOOU; control) .

Since acetylenic fatty acid methyl esters can be partially separated from other fatty acid methylesters on silica gel thin layer chromatography, the methylesters prepared from YN94-1 transformed with pVT-Crepl were separated on silica gel 60 thin layer chromatography plates (Merck, Darmstadt, Germany) by developing the plate in hexane/diethyl ether/acetic acid (85:15:1 by vol.) . An area located just be¬ low the main methyl ester area was removed from the plate and the lipids were eluted with methanol/chloroform (2:1) and analyzed by gas liquid chromatography. The fraction were shown to consist of fatty acid methylesters where the peak with the same retention time as 9-octadecen-12 ynoic acid metyl ester made up 12.5% of the total peak area.

The methyl ester fraction enriched in the putative 9- octadecen-12 ynoic acid methyl ester as well as total fatty acid methyl esters prepared from YN94-1 transformed with pVT- Crepl and YN94-1 transformed with empty vector (pVTIOOU; con¬ trol) were hydrolyzed in 2.5M KOH in aqueous methanol (15% methanol, by vol.) at 90°C for 1 h. The free fatty acids were extracted into hexane after acidicifiction with HCl and the hexane phase was evaporated to dryness under a stream of nitrogen.

Fatty acid diethylamides (FADEA) were prepared from the free fatty acids according to Nilsson and Liljenberg (1991) . The FADEA were either injected directly on a gas liquid chromatog¬ raphy coupled to mass spectrometer (GC-MS) or subjected to further purification by silica gel thin layer chromatography by developing the plate in heptane/diethyleter/acteic acid (50:50:1, by vol.) .

The FADEA were analyzed on a Hewlett-Packard 5890 II gas chro- matograph equipped with a DB225 (0.25 mm i.d. x 30 m, J&W, Folsom, USA) in series with a Rtx 2330 (Restek Corp., PA, USA) .fused silica capillary column, coupled with a Hewlett-Packard 5989A mass spectrometer working in electron impact mode at 70 eV. Injection technique was cold splitless at 100°C and then the temperature was raised as quickly as possible to 240°C. Oven temperature was 100°C for 7 min, then 20°C per min to

190°C and then 1°C per min to a final temp, of 225°C where it was kept for another 20 min. The double bond positions were determined according to Nilsson and Liljenberg (1991) .

Single ion chromatogrammes of masses corresponding to the molecular ion of FADEA prepared from total fatty acids from YN94-1 transformed with pVT-Crepl and from YN94-1 transformed with empty vector (pVTIOOU; control) are shown in Fig.5 and Fig.6, respectively. Chromatogram of FADEA from YN94-1 trans¬ formed with pVT-Crepl showed a peak of mass 333 (corresponding to the molecular weigth of 9-octadecen-12 ynoic acid diethyla¬ mide) which was absent in the chromatogram of FADEA from YN94- 1 transformed with empty vector (pVTIOOU; control) . The peak had a retention time of 57.3 min and was located between peaks corresponding to eicosanoic and eicosenoic FADEA derivatives.

A total ion chromatogramme of FADEA prepared from fatty acids enriched in the putative 9-octadecen-12 ynoic acid by thin layer chromatography (as described above) originating from lipid extracts of YN94-1 transformed with pVT-Crepl is shown in Fig. 5. Mass spectrum (Fig.6) of the putative 9- octadecen-12 ynoic acid diethylamide derivative (peak D in Fig.5) showed a gap in mass of 26 amu instead of regular 28 between carbon 7 and 9 indicating a double bond at position 9. Further more there was a gap of 24 amu instead of regular 28 between carbon atom 10 and 12 indicating acetylenic bond at position 12. The peak D produced a mass spectrum identical to that of authentic 9-octadecen-12 ynoic acid diethylamide prepared from oils from Crepis alpina seeds. Thus the peak D in the chromatogram in Fig 5 was unambigously identified as 9- octadecen-12 ynoic acid diethylamide derivative. Since the compound was absent in yeast strains not transformed with the •Crepl cDNA it is clear that the Crepl cDNA codes for a delta- 12 fatty acid acetylenase. REFERENCES

Badami, R.C., and Patil, K,B. (1981) . Strucure and occurrence of unusual fatty acids in minor seed oils. Progress in Lipid Research, 19, 119-53.

Banas, A., Bafor, M., iberg, E., Lenman, M., Stahl, U. and Stymne, S. (1997) Biosynthesis of an acetylenic fatty acid in microsomal preparations from developing seeds of Crepis alpina . In: Williams, J.P., Mobasher, K.U., Lem, N.W. (eds) Physiology, biochemistry and molecular biology of plant lipids. Kluwer Academic Publisher, Dordrecht. In -presε

Birnboim, H.C., and Doly, J. (1979) A rapid alkaline extrac¬ tion procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523

Diedrich, M. & Henschel, K.P. (1991) The natural occurence of unusual fatty acids: Part 3. Acetylenic fatty acids. Nahrung 35, 193-202

Elble, R. (1992) A simple and efficient procedure for trans¬ formation of yeasts. Biotechniques 13, 18-20

Haigh, W.G. & James, A.T. Biochim. Biophys. Acta 137, 391-392

Hirsinger, F. (1989) . New annual oil crops. In Oil crops of the world, (eds. G. Rδbbelen, R.K. Downey and A. Ashri) , pp.518-532. Mc-Graw-Hill Inc.

Kohn, G., Hartmann, E., Stymne, S. & Beutelmann, P. (1994) Biosynthesis of acetylenic acids in the moss Ceratodon pur- pureus. J. Plant Physiol. 144, 265-271 Langridge, J., Langridge, P and Bergquist P.L. (1980) Extrac¬ tion of nucleic acids from agarose gels. Anal. Biochem. 103, 264-271

Nilsson, R. and Liljenberg, C. (1991) The determination of double bond positions in polyunsaturated fatty acids - Gaschromatography/ mass spectrometry of the diethylamide derivative. Phytochemical analysis 2, 253-259

Shanklin, J., Whittle, E. & Fox, B.G. (1994) Eight histidine residues are catalytically essential in membrane -associated iron enzyme, stearoyl-CoA desaturase and are conserved in alkane hydroxylase and xylene monoxygenase. Biochemistry 33, 12787-12794.

Stymne, S. & Lenman, M. (1996) Novel plant enzyme and use thereof. Swedish patent application no. 9601236-4, 96-03-29.

Vernet, T., Dignard, D. and Thomas, D.Y. (1987) A family of yeast expression vectors containing the phafe fl intergenic region. Gene 52, 225-233

Wieland, B., Feil, C, Gloria-Maercker, E., Thumm, G., Lech- ner, M., Bravo, J.M., Poralla, K. & Goetz, F. (1994) Genetic and biochemical analysis of the biosynthesis of the yellow carotenoid 4,4' -diaponeurosporene of Staphylococcus aureus. J. Bacteriol. 176, 7719-7726.

van de Loo, F.J., Broun, P., Turner, S. & Somerville, C. (1995) An oleate D12-hydroxylase from Ricinus communis L. is a fatty acid acyl desaturase homolog. Proc. Natl. Acad. Sci. USA 95, 6743-6747.

van de Loo, F.J., Fox, G. & Somerville, C. (1993) Unusual fatty acids. In: T.S. Moore

Jr (ed.) Lipid Metabolism in Plants. CRC Press, Boca Raton , page 105. INFORMATION ABOUT SEQUENCE LISTING NO 1

Alignment of amino acid sequences from delta 12 ER and plastid desaturases, delta 15 desaturases and from the castor bean hy¬ droxylase. Also included in this alignment is the protein se¬ quence derived from pCrepl (crepis) . Underlined are three hiε- tidin motifs that are conserved in non-heme containing monoxy- genases.

Sequences given in this alignment together with their acces¬ sion numbers are : bnom6des.seq, delta 12 desaturase from Brassica napus (L29214) ; gmom6des.seq, delta 12 desaturase from Glycine max (L29215) ; atom3des.seq, deltal5 desaturase from AraJidopsis thaliana (L22961) ; bnom3des.seq, delta 15 from Brassica napus (L22963) ; rcorrϋdes.seq, delta 15 desaturase from Ricinus communis (L25897) ; siom3des.seq, delta 15 desaturase from oriental sesame (U25817) ; lddlSdes.seq, delta 15 desaturase from Limnanthes douglasii (U17063) ; gsom3des, delta 15 desaturase from Glycine max (L22965) ; atom3bdes.seq, deltalδ desaturase from Arabidopsis thaliana (D17579) ; bnom3Ides.seq, delta 15 from Brassica napus (L22962) ; gsom3bdes.seq, delta 15 desaturase from Glycine max (L22964) ; atdl2des.seq, deltal2 desaturase from Arabidopsis thaliana (L26296) ; gmom6bdes.seq delta 12 desaturase from Glycine max (L43921) ; scoml2des.seq, delta 12 desaturase from S. commersonii (X92847) ; gmom6ades.seq, delta 12 desaturase from Glycine max (L43920) ; rchyd.seq, oleate 12-hydroxylase from Ricinus communis (U22378) ; crepis, Crepis alpina acetylenase from this document. SEQUENCE LISTING 1

1 50 bnomβdes . seq MASRIA DSLFAFTGPQ QCLPRAPK A gmomβdes . seq MACT A DSLLLFKGSY Q.KPVLRRDI atom3des . seq .MANLVLSEC GIRPLPRIYT TPRSNFLSNN N...KFRPSL SSSSYKTSSS bnom3des . seq rcom3des.seq MAAG VLSEC GLRPLPRIYS RPRIGFTSKT TNLLKLRELP DSKSYNLCSΞ sιom3des.seq .MAS VLSEC GLRPLPRVYP KPRTGHPL N SNPTKLRFSR TD GNGSS.. lddlδdes .seq .MASWVLSQY ALNPLPHIFR TPRTSITSHK L TVSHTNNRAT gsom3des .seq .MAT YHQKC GLKPLAPVIP RPRTGAA SS TSRVEF LDTNKWA atom3bdes.seq bnom31des.seq gsom3bdes .seq atdl2des .seq gmomβbdes .seq scoml2des .seq gmomβades .seq rchyd.seq crepis

51 100 bnomβdes.seq SARLSPGVYA VRPIDLLLKG TRRTFLVPAK KRIGCIKAVF VPVAPPSADN gmoπι6des.seq AARYSPGIFS LNSNGLIQKR FRRQRNFVTR NKV VIHAVA IPVQPAPVES atom3des.seq PLSFGLNSRD GFTRNWALNV STPLTTPIFE ESP LEEDNK bnom3des.seq rcom3des.seq FKVSSWSNSK QSNWALNVAV PVNVSTVSGE DDREREEFNG IVN.VDEGKG sιom3des.seq ... FCLSSGI LREKN ALRV SAPLRVLQVE EEEENKEGER VIN.GGEE..

Iddl5des.seq PDLTKLSLIK FRERKLGLRV SAPFQIASTT PE EEDEV gsom3des.seq GPKFQPLRCN LRERNWGLKV SAPLRVASIE EEQKSVDLTN GTNGVEHEKL atom3bdes.seq MW AMDQRTNVNG DPGAGDRKKE bnom3Ides,seq MW AMDQRSNANG D gsom3bdes. seq MV KDTKPLAYAA NNGYQQKGSS atdl2des.seq MGAG GRMPVP.... TSSKKSETDT gmom^βbdes.seq MGAG GRTDVP.... PANRKSEVDP sco l2des.seq MGAG GRMSAP.... NGETEVKRNP gmom^βades .seq MGLAKETTMG GRGRVA.... KVEVQGK.KP rchyd.seq MGGG GRMSTVITSN NSEKKGGSSH crepis MGGG GR GRTSQKPL

101 150 bnomβdes.seq AEDREQLAES YGFKQIGQDL PDNVTLKDIM DTLPKEVFEI DDVKAWKSVL gmomβdes.seq AEYRKQLAED YGFRQVGEPL SDDVTLKDVI NPLPKEVFEI DDVKAWKSVL atom3des.seq QRFDPGAPPP FNLADIRAAI PKHC VKNPW KSLSYWRDV AIVFA bnom3des. seq ..MSYWREL AIVFA rcom3des.seq EFFDAGAPPP FTLADIRAAI PKHCWVKNPW RSMSYVLRDV VWFG siom3des.seq ..FDPGAPPP FKLSDIREAI PKHCWVKDPW RSMGYWRDV AWFG lddlδdes.seq AEFDPGSPPP FKLADIRAAI PKHCWVKNQW RSMSYWRDV VIVLG gsom3des.seq PEFDPGAPPP FNLADIRAAI PKHCWVKDPW RSMSYWRDV IAVFG atom3bdes. seq ERFDPSAQPP FKIGDIRAAI PKHCWVKSPL RSMSYWRDI IAVAA bnom31des.seq ERFDPSAQPP FKIGDIRAAI PKHCWVKSPL RSMSYVARDI FAWA gsom3bdes. seq FDFDPSAPPP FKIAEIRASI PKHCWVKNPW RSLSYVLRDV LVIAA atdl2des.seq TKRVPCEKPP FSVGDLKKAI PPHCFKRSIP RSFSYLISDI IIASC gmomβbdes .seq LKRVPFEKPQ FSLSQIKKAI PPHCFQRSVL RSFSYWYDL TIAFC scoml2des.seq LQKVPTSKPP FTVGDIKKAI PPHCFQRSLI RSFSYWYDL ILVSI gmomβades .seq LSRVPNTKPP FTVGQLKKAI PPHCFQRSLL TSFSYWYDL SFAF rchyd.seq LKRAPHTKPP FTLGDLKRAI PPHCFERSFV RSFSYVAYDV CLSFL crepis MERVSVD.PP FTVSDLKQAI PPHCFKRSVI RSSYYIVHDA IIAYI SEQUENCE LISTING 1 (cont . )

151 200 bnomβdes . seq ISVTSYALGL FMIAKAPWYL LPLAWAWTGT AVTGFFVIGH DCAHKSFSKN gmomβdes . seq ISVTSYALGL FMIΞKAPWYL LPLAWVWTGT AITGFFVIGH DCAHRSFSSN atorα3des . seq LAAGAAYL. .NNWIV WPLYWLAQGT MFWALFVLGH DCGHGSFSND bnom3des . seq LAAGAAYL. .NNWLV WPLYWIAQGT MFWALFVLGH DCGHGSFSND rcom3des . seq LAAVAAYF. .NNWVA WPLYWFCQGT MFWALFVLGH DCGHGSFSNN siom3des . seq LAAVAAYF. .NNWW WPLYWFAQST MFWALFVLGH DCGHGSFSND lddlδdes . seq LAAAAVAA. .NSWAV WPLYWVAQGT MFWALFVLGH DCGHGSFSNN gsom3des . seq LAAAAAYL. .NNWLV WPLYWAAQGT MFWALFVLGH DCGHGSFSNN atom3bdes. seq LAIAAVYV. .DSWFL WPLYWAAQGT LFWAIFVLGH DCGHGSFSDI bnom31des. seq LAVAAVYF. .DSWFF WPLYWAAQGT LFWAIFVLGH DCGHGSFSDI gsom3bdes . seq LVAAAIHF. .DNWLL WLIYCPIQGT MFWALFVLGH DCGHGSFSDS atdl2des . seq FYYVATNYFS LLPQPLSYLA WPLYWACQGC VLTGIWVIAH ECGHHAFSDY gmomβbdes . seq LYYVATHYFH LLPGPLSFRG MAIYWAVQGC ILTGVWVIAH ECGHHAFSDY scoml2des. seq MYYVANTYFH LLPSPYCYIA WPIYWICQGC VCTGIWVNAH ECGHHAFSDY gmomβades.seq IFYIATTYFH LLPQPFSLIA WPIYWVLQGC LLTGVWVIAH ECGHHAFSKY rchyd. seq FYSIATNFFP YISSPLSYVA WLVYWLFQGC ILTGLWVIGH ECGHHAFSEY crepis FYFLADKYIP I PAPLAYLA WPLYWFCQAS ILTGLWVIGH ECGHHAFSDY

201 250 bnomβdes .seq KLVEDIVGTL AFLPLVYPYE PWRFKHDRHH AKTNMLVHDT AWQPVPPEEF gmomβdes .seq KLVEDIVGTL AFMPLIYPYE PWRFKHDRHH AKTNMLREDT AWHPVWKDEF atom3des . seq PKLNSWGHL LHSSILVPYH GWRISHRTHH QNHGHVENDE SWHPMSEKIY bnom3des . seq PRLNSWGHL LHSSILVPYH GWRISHRTHH QNHGHVENDE SWHPMSEKIY rcom3des .seq PKLNSWGHL LHSSILVPYH GWRISHRTHH QNHGHVENDE SWHPLSEKIF siom3des. seq PKLNSWGHI LHSSILVPYH GWRISHRTHH QNHGHVENDE SWHPLSEKIY lddl5des.seq HKLNSWGHL LHSSILVPYH GWRIRHRTHH QNHGHVENDE SWHPMSEKLF gsom3des.seq SKLNSWGHL LHSSILVPYH GWRISHRTHH QHHGHAENDE SWHPLPEKLF atom3bdes .seq PLLNSWGHI LHSFILVPYH GWRISHRTHH QNHGHVENDE SWVPLPERVY bnom31des .seq PLLNTAVGHI LHSFILVPYH GWRISHRTHH QNHGHVENDE SWVPLPEKLY gsom3bdes .seq PLLNSLVGHI LHSSILVPYH GWRISHRTHH QNHGHIEKDE SWVPLTEKIY atdl2des .seq QWLDDTVGLI FHSFLLVPYF SWKYSHRRHH SNTGSLERDE VFVPKQKSAI gmomβbdes . seq QLLDDIVGLI LHSALLVPYF SWKYSHRRHH SNTGSLERDE VFVPKQKSCI scoml2des. seq QWVDDTVGLI LHSALLVPYF SWKYSHRRHH SNTGSLERDE VFVPKPKSQL gmomβades.seq QWVDDWGLT LHSTLLVPYF SWKISHRRHH SNTGSLDRDE VFVPKPKSKV rchyd.seq QLADDIVGLI VHSALLVPYF SWKYSHRRHH SNIGSLERDE VFVPKSKSKI crepis QWVDDTVGFI LHSFLMTPYF SWKYSHRNHH ANTNSLDNDE VYIPKSKAKV

251 300 bnomβdes .seq DS SPVLRKAII FGYGPIRPWL SI AH WVNWHFNLRK gmomβdes .seq ES TPLLRKAII YGYGPFRCWM SI AH WLMWHFDLKK atom3des. seq NTLDK PTRFFRFTLP LVMLAYPFYL WARSPGKK.. ..GSHYHPDS bnom3des. seq KSLDK PTRFFRFTLP LVMLAYPFYL WARSPGKK.. ..GSHYHPDS rcom3des.seq KSLDN VTKTLRFSLP FPMLAYPFYL WSRSPGKK.. ..GSHFHPDS siom3des.seq KNLDT ATKKLRFTLP FPLLAYPIYL WSRSPGKQ.. ..GSHFHPDS lddlδdes .seq RSLDK IALTFRFKAP FPMLAYPFYL WERSPGKT.. ..GSHYHPDS gsom3des .seq RSLDT VTRMLRFTAP FPLLAFPVYL FSRSPGKT.. ..GSHFDPSS atom3bdes . seq KKLPH STRMLRYTVP LPMLAYPLYL CYRSPGKE.. ..GSHFNPYS bnom31des. seq KNLSH STRMLRYTVP LPMLAYPLYL WYRSPGKE.. ..GSHYNPYS gsom3bdes. seq KNLDS MTRLIRFTVP FPLFVYPIYL FSRSPGKE.. ..GSHFNPYS atdl2des.seq KWYGKYLNNP LGRIMMLTVQ F.VLGWPLYL AFNVSGRPYD GFACHFFPNA gmomβbdes .seq KWYSKYLNNP PGRVLTLAVT L.TLGWPLYL ALNVSGRPYD RFACHYDPYG scoml2des .seq GWYSKYLNNP PGRVLSLTIT L.TLGWPLYL AFNVSGRPYD RFACHYDPYG gmomβades . seq AWFSKYLNNP LGRAVSLLVT L.TIGWPMYL AFNVSGRPYD SFASHYHPYA rchyd. seq SWYSKYSNNP PGRVLTLAAT L.LLGWPLYL AFNVSGRPYD RFACHYDPYG crepis ALYYKVLNHP PGRLLIMFIT F.TLGFPLYL FTNISGKKYE RFANHFDPMS SEQUENCE LISTING 1 (cont.)

301 350 bnomβdes . seq .. FRPSEVNR VKISLACVFA FMAVGWPLII YKVGVLGWVK FWLMPWLGYH gmomβdes.seq ..FRPSEVPR VKISLACVFA FIAIGWPLII YKTGIMGWIK FWLMPWLGYH atom3des . seq DLFLPKERKD VLTSTACWTA .MAALLVCLN FTIGPIQMLK LYGIPYWINV bnom3des .seq DLFLPKERND VLTSTACWTA .MAVLLVCLN FVMGPMQMLK LYVIPYWINV rcom3des .seq GLFVPKERKD IITSTACWTA .MAALLVYLN FSMGPVQMLK LYGIPYWIFV siom3des .seq DLFVPNEKKD VITSTVCWTA .MLALLVGLS FVIGPVQLLK LYGIPYLGNV lddl5des .seq DLFVPSEKKD VITSTICWTT .MVGLLIGLS FVMGPIQILK LYWPYWIFV gsom3des.seq DLFVPNERKD VITSTACWAA .MLGLLVGLG FVMGPIQLLK LYGVPYVIFV atom3bdes .seq SLFAPSERKL lATSTTCWSI .MFVSLIALS FVFGPLAVLK VYGVPYIIFV bnom31des .seq SLFAPSERKL lATSTTCWSI .MLATLVYLS FLVGPVTVLK VYGVPYIIFV gsom3bdes. seq NLFPPSERKG lAISTLCWAT .MFSLLIYLS FITSPLLVLK LYGIPYWIFV atdl2des. seq PIYNDRERLQ IYLSDAGILA .VCFGLYRYA AAQGMASMIC LYGVPLLIVN gmomβbdes . seq PIYSDRERLQ IYISDAGVLA .WYGLFRLA MAKGLAWWC VYGVPLLWN scoml2des . seq PIYNNRERLQ IFISDAGVLG .VCYLLYRIA LVKGLAWLVC VYGVPLLWN gmomβades .seq PIYSNRERLL IYVSDVALFS .VTYSLYRVA TLKGLVWLLC VYGVPLLIVN rchyd.seq PIFSERERLQ I IADLGIFA .TTFVLYQAT MAKGLAWVMR IYGVPLLIVN crepis PIFKERERFQ VLLSDLGLLA .VLYGVKLAV AAKGAAWVTC IYGIPVLGVF

351 400 bnomβdes. seq FWMSTFTMVH HTAPH..IPF KPADEWNAAQ AQLNGTVHCD YPSWIEILCH gmomβdes .seq FWMSTFTMVH HTAPY..IPF KYSEEWNRAQ AQLNGTVHCD YPKWIEILCH atom3des.seq MWLDFVTYLH HHGHEDKLPW YRGKEWSYLR GGL.TTLDRD YGLINNIHHD bnom3des .seq MWLDFVTYLH HHGHEDKLPW YRGKEWSYLR GGL.TTLDRD YGLINNIHHD rcom3des.seq MWLDFVTYLH HHGHEDKLPW YRGKAWSYLR GGL.TTLDRD YGWINNIHHD siom3des. seq MWLDLVTYLH HHGHEDKLPW YRGKEWSYLR GGL.TTLDRD YGWINNIHHD lddl5des .seq MWLDFVTYLD HHGHEDKLPW YRGEEWSYLR GGL.TTLDRD YGLINNIHHD gsom3des . seq MWLDLVTYLH HHGHEDKLPW YRGKEWSYLR GGL.TTLDRD YGWINNIHHD atom3bdes.seq MWLDAVTYLH HHGHDEKLPW YRGKEWSYLR GGL.TTIDRD YGIFNNIHHD bnom31des. seq MWLDAVTYLH HHGHDDKLPW YRGKEWSYLR GGL.TTIDRD YGIFNNIHHD gsom3bdes . seq MWLDFVTYLH HHGHHQKLPW YRGKEWSYLR GGL.TTVDRD YGWIYNIHHD atdl2des.seq AFLVLITYLQ H..THPSLPH YDSSEWDWLR GAL.ATVDRD YGILNKVFHN gmomβbdes.seq GFLVLITFLQ H..THPALPH YTSSEWDWLR GAL.ATVDRD YGILNKVFHN scoml2des.seq GFLVLITYLQ H..THPSLPH YDSTEWDWLR GAL.ATCDRD YGVLNKVFHN gmomβades.seq GFLVTITYLQ H..THFALPH YDSSEWDWLK GAL.ATMDRD YGILNKVFHH rchyd. seq CFLVMITYLQ H..THPAIPR YGSSEWDWLR GAM.VTVDRD YGVLNKVFHN crepis I FDIITYLH H..THLSLPH YDSSEWNWLR GAL.STIDRD FGFLNSVLHD

401 450 bnomβdes.seq DINVHIPHHI SPRIPSYNLR AAHQSIQENW GKYTNLATWN WRLMKTIMTV gmomβdes.seq DINVHIPHHI SPRIPSYNLR AAHKSLQENW GQYLNEASWN WRLMKTIMTV atom3des.seq I.GTHVIHHL FPQIPHYHLV EATEAAKPVL GKYYREPDKS .GPLPLHLLE bnom3des.seq I.GTHVIHHL FPQIPHYHLV EATEAAKPVL GKYYREPDKS .GPLPLHLLG rcom3des . seq I.GTHVIHHL FPQIPHYHLV EATEAAKPVM GKYYREPKKS .GPLPLHLLG siom3des.seq I.GTHVIHHL FPQIPHYHLI EATEAAKPVL GKYYREPKKS .APLPFHLLG lddl5des .seq I.GTHVIHHL FPQIPHYHLV EATQAAKPIF GKYYKEPAKS .KPLPFHLID gsom3des . seq I.GTHVIHHL FPQIPHYHLV EATEAAKPVF GKYYREPKKS AAPLPFHLIG atom3bdes .seq I.GTHVIHHL FPQIPHYHLV DATKAAKHVL GRYYREPKTS .GAIPIHLVE bnom31des.seq I.GTHVIHHL FPQIPHYHLV DATKSAKHVL GRYYREPKTS .GAIPIHLVE gsom3bdes . seq I.GTHVIHHL FPQIPHYHLV EATQAAKPVL GDYYREPERS .APLPFHLIK atdl2des.seq ITDTHVAHHL FSTMPHYNAM EATKAIKPIL GDYYQFDGTP WYV gmomβbdes.seq ITDTHVAHHL FSTMPHYHAM EATKAIKPIL GEYYRFDETP FVK scoml2des .seq ITDTHWHHL FSTMPHYNAM EATKAVKPLL GDYYQFDGTP IYK gmomβades.seq ITDTHVAHHL FSTMPHYHAM EATNAIKPIL GEYYQFDDTP FYK rchyd.seq IADTHVAHHL FATVPHYHAM EATKAIKPIM GEYYRYDGTP FYK crepis VTHTHVMHHL FSYIPHYHAK EARDAINTVL GDFYKIDRTP ILK SEQUENCE LISTING 1 (cont . )

451 493 bnomβdes . seq CHVYDKEENY IPFDRLAPEE SQPITFLKKA MPDYAA. gmomβdes . seq CQVYDKEKSL CCLRRTCP.. atom3des . seq ILAKSIKEDH YV SDE GEWYYKADP NLYGEVKVRA D bnom3des . seq ILAKSIKEDH FV SDE GDWYYEADP NLYGEIKVTA E rcom3des . seq SLVRSMKEDH YV SDT GDWYYQKDP KLSGIGGEKT E siom3des .seq DLTRSLKRDH YV SDV GDWYYQTDP QLTGAEKS.. . lddl5des. seq VLLKSLKRDH FV PDT GDIVYYQSDP QISGSLKPE. . gsom3des.seq EIIRSFKTDH FV SDT GDWYYQTDS KINGSSKLE. . atom3bdes.seq SLVASIKKDH YV SDT GDIVFYETDP DLYVYASDKS KIN bnom31des.seq SLVASIKKDH YV SDT GDIVFYETDP DLYVYASDKS KIN gsom3bdes .seq YLIQSMRQDH FV SDT GDWYYQTDS LLLHSQRD atdl2des .seq AMYREAKECI YVEPDREGDK KGVYWYNNKL gmomβbdes .seq AMWREARECI YVEPDQSTES KGVFWYNNKL scoml2des.seq EMWREAKECL YVEKDESSQG KGVFWYKNKL gmomβades.seq ALWREARECL YVEPDEGTSE KGVYWYRNKY rchyd.seq ALWREAKECL FVEPDEGAPT QGVFWYRNKY crepis AMWREAKECI FIEPEKGRES KGVYWY.NKF

SEQUENCE LISTING 2

Nucleotide sequence and derived amino acid sequence of the open reading frame from plasmid pCrepl.

ATGGGTGGCGGTGGCCGTGGTCGGACTTCGCAAAAACCCCTCATGGAACGTGTCTCAGTT M G G G G R G R T S Q K P L M E R V S V

GATCCACCCTTCACCGTGAGTGATCTCAAGCAAGCAATCCCTCCCCATTGCTTCAAGCGA D P P F T V S D L K Q A I P P H C F K R

TCTGTAATCCGTTCCTCTTACTACATAGTCCACGATGCTATTATCGCCTACATCTTCTAC S V I R S S Y Y I V H D A I I A Y I F Y

TTCCTTGCCGACAAATACATTCCGATTCTCCCTGCCCCTCTAGCCTACCTCGCTTGGCCC F L A D K Y I P I L P A P L A Y L A W P

CTTTACTGGTTCTGTCAAGCTAGCATCCTCACCGGCTTATGGGTCATCGGTCACGAATGC L Y W F C Q A S I L T G L W V I G H E C

GGTCACCATGCCTTCAGCGACTACCAGTGGGTTGACGACACTGTGGGCTTCATCCTCCAC G H H A F S D Y Q W V D D T V G F I L H

TCGTTTCTCATGACCCCGTATTTCTCCTGGAAATACAGCCACCGGAACCACCATGCCAAC S F L M T P Y F S W K Y S H R N H H A N

ACAAATTCGCTTGACAACGATGAAGTTTACATCCCCAAAAGCAAGGCCAAAGTCGCGCTT T N S L D N D E V Y I P K S K A K V A L

TACTATAAAGTTCTCAACCACCCACCTGGCCGACTGTTGATTATGTTCATCACCTTCACC Y Y K V L N H P P G R L L I M F I T F T

CTAGGCTTCCCTCTATACCTCTTTACCAATATTTCCGGCAAGAAGTATGAAAGGTTTGCC L G F P L Y L F T N I S G K K Y E R F A

AACCATTTCGACCCCATGAGTCCGATTTTCAAAGAGCGTGAGCGGTTTCAGGTCTTGCTA N H F D P M S P I F K E R E R F Q V L L

TCGGATCTTGGCCTTCTTGCTGTGCTTTACGGAGTTAAACTTGCGGTAGCAGCGAAAGGC S D L G L L A V L Y G V K L A V A A K G

GCCGCCTGGGTGACGTGCATTTACGGAATTCCAGTTTTAGGCGTGTTTATCTTTTTCGAT A A W V T C I Y G I P V L G V F I F F D

ATCATCACCTACTTGCACCACACCCATCTGTCGTTGCCTCATTATGATTCATCTGAATGG I I T Y L H H T H L S L P H Y D S S E W

AACTGGCTCAGAGGGGCTTTGTCAACAATCGATAGGGACTTTGGGTTCCTGAATAGTGTG N W L R G A L S T I D R D F G F L N S V

CTCCATGATGTTACACACACTCACGTTATGCATCATCTGTTTTCATACATTCCACACTAT L H D V T H T H V M H H L F S Y I P H Y

CATGCGAAGGAGGCAAGGGATGCAATCAACACAGTCTTGGGCGACTTTTATAAGATCGAT H A K E A R D A I N T V L G D F Y K I D

AGGACTCCAATTCTGAAAGCAATGTGGAGAGAGGCCAAGGAATGCATCTTCATCGAGCCT R T P I L K A M W R E A K E C I F I E P

GAAAAAGGTAGGGAGTCCAAGGGTGTATATTGGTACAATAAATTCTGA E K G R E S K G V Y W Y N K F *

Claims (1)

1. A method of producing acetylenic compounds, charac¬ terized in that a double bond is converted to an acetylenic bond by an acetylenase.

2. A method according to claim 1, wherein acetylenic fatty acids are produced by conversion of unsaturated fatty acids to acetylenic fatty acids by a fatty acid acetylenase.

3. A method according to claim 2, wherein C18 fatty ac¬ ids with doublebonds at position delta 12 are converted to 12- ynoic acids.

4. A method according to claim 3, wherein linoleic acid is converted to crepenynic acid (9-octadecen-12-ynoic acid) by Crepis alpina delta 12 acetylenase.

5. cDNA coding for acetylenase of the mixed function monoxygenase type containing three conserved histidin motifs

(HX(3 _{or 4})H, HX(₂ or 3) ^H- ^{and HX} (2 or 3)^HH) according to Sequence Listing 1.

6. cDNA according to claim 5 encoding fatty acid acety¬ lenase.

7. cDNA according to claim 6 encoding Crepis alpina delta 12 acetylenase comprising the sequence according to Sequence Listing 2 or any nucleotide sequences essentially homologous therewith.

8. Use of cDNA according to any of the claims 5, 6 or 7 for transforming organisms.

9. Use according to claim 8, wherein the organisms will be capable of accumulating acetylenic compound. 10. Use according to claim 8, wherein the organisms are oil accumulating organisms.

11. Use according to claims 10, wherein the oil accumu¬ lating organisms are selected from the group consisting of oil crops, oleogeneous yeasts and moulds.

12. Organisms transformed with a acetylenase cDNA accord¬ ing to any of the claims 5, 6 or 7.

13. Organisms according to claim 12, which are organisms accumulating acetylenic compounds.

14. Organisms according to claim 12, which are organisms accumulating oil.

15. Organisms according to claim 14, which are selected from the group consisting of oil crops, oleogeneous yeasts and moulds.

16. Acetylenic componds accumulated in organisms accord¬ ing to claim 13.

17. Oils from oil accumulating organisms according to claims 14 or 15.

AMENDED CLAIMS

[received by the International Bureau on 30 July 1997 (30.07.97); original claims 1-17 replaced by amended claims 1-18 (2 pages)]

1. A method of producing acetylenic compounds, charac¬ terized in that C18 fatty acids with doublebonds at position delta 12 are converted to 12-ynoic acids by an acetylenase.

2. A method according to claim 1, wherein linoleic acid is converted to crepenynic acid (9-octadecen-12-ynoic acid) by Crepis alpina delta 12 acetylenase.

3. A DNA sequence coding for acetylenase of the mixed function monoxygenase type containing three conserved histidin motifs ⁽HX(3 _or 4)H, H (2 or 3)^HH' ^an<^ ^HX (2 or 3)^HH) accord^¬ ing to Sequence Listing 1.

4. A DNA sequence according to claim 3 encoding fatty acid acetylenase.

5. A DNA sequence according to claim 4 encoding a delta 12 fatty acid acetylenase.

6. A DNA sequence according to claim 5 encoding Crepis alpina delta 12 acetylenase comprising the sequence according to Sequence Listing 2 or any nucleotide sequences encoding an acetylenase essentially homologous therewith.

7. Use of a DNA sequence according to any of the claims

3. 4, 5 or 6 for transforming organisms.

8. Use according to claim 7, wherein the organisms will be capable of accumulating acetylenic compound.

9. Use according to claim 7, wherein the organisms are oil accumulating organisms. 10. Use according to claim 9, wherein the oil accumulat¬ ing organisms are selected from the group consisting of oil crops, oleogeneous yeasts and moulds.

11. Organisms transformed with an acetylenase DNA accord¬ ing to any of the claims 3, 4, 5 or 6.

12. Organisms according to claim 11, which are organisms accumulating acetylenic compounds.

13. Organisms according to claim 11, which are organisms accumulating oil.

14. Organisms according to claim 13, which are selected from the group consisting of oil crops, oleogeneous yeasts and moulds .

15. A method of obtaining acetylenic componds, comprising accumulation of acetylenic compunds in organisms according to claim 12.

16. A method of obtaining oils, comprising accumulation of oils in organisms according to claims 13 or 14.

17. Acetylenic compounds obtainable by the method accord¬ ing to claim 15.

18. Oils obtainable by the method according to claim 16.