Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se
Reexamination Certificate
1998-09-29
2001-12-25
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
C800S281000, C536S023600, C435S069100, C435S254100, C435S255100, C435S419000
Reexamination Certificate
active
06333448
ABSTRACT:
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 acids, 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 manufacturers 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 domesticating 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 possible 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 acetylenic 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 renewable primary source of this oil containing acetylenic fatty acids for industrial uses.
PRIOR ART
The formation of acetylenic bonds in fatty acids in mosses occurs 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 biochemical 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 desaturases) would also be associated with similarity in the primary sequence of these proteins a full length cDNA (pCrep1) (SEQ ID NO:1), encoding a putative acetylenase (SEQ ID NO:2), 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 (pCrep1) 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 (pCrep1) from
C. alpina
encode an enzyme responsible for converting linoleic acid into crepenynic acid.
Finally, conclusive evidence that the cDNA, pCrep1, 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 acetylenic 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 histidine motifs (His Xaa
(3 or 4)
His and His Xaa
(2 or 3)
His His, and His Xaa
(2 or 3)
His His) according to
FIGS. 8A-8D
of the accompanying drawings.
In a further embodiment the invention relates to a cDNA encoding 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 described 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 transformed with a acetylenase CDNA as described above. The organisms are acetylenic compound or oil accumulating, examples of the latter being oil crops, oleaginous 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 accumulating 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 [0-octadecen12-ynoic acid (Hirsinger, 1989)]. The inventors have studied the biosynthesis of crepenynic acid in
C. alpina
seeds. The feeding of exogenous 1-
14
C-labelled free fatty acids to intact developing cotyledons of
C. alpina
seeds demonstrated that linoleate is a precursor to crepenynic acid. This is contradictory 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 desaturation 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 histidine 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 enzymatic reactions. For example, acetylenic bonds were thought to
Bafor Maureen
Banas Antoni
Dahlqvist Anders
Gummeson Per-Olov
Lee Michael
Browdy and Neimark
McElwain Elizabeth F.
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