Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-04-14
2001-12-11
McElwain, Elizabeth F. (Department: 1649)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C800S281000, C435S069100
Reexamination Certificate
active
06329518
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to novel genetic sequences which encode fatty acid epoxygenase enzymes. In particular, the present invention relates to genetic sequences which encode fatty acid &Dgr;12-epoxygenase enzymes as defined herein. More particularly, the present invention provides cDNA and genomic gene sequences which encode plant fatty acid epoxygenases, preferably
Crepeis palaestina
or
Euphorbia lagascae
&Dgr;12-epoxygenases. The genetic sequences of the present invention provide the means by which fatty acid metabolism may be altered or manipulated in organisms such as yeasts, moulds, bacteria, insects, birds, mammals and plants, in particular to convert unsaturated fatty acids to epoxygenated fatty acids therein. The invention extends to genetically modified oil-accumulating organisms transformed with the subject genetic sequences and to the oils derived therefrom. The oils thus produced provide the means for the cost-effective raw materials for use in the efficient production of coatings, resins, glues, plastics, surfactants and lubricants, amongst others.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. Sequence identity numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined after the bibliography.
BACKGROUND TO THE INVENTION
There is considerable interest world-wide in producing chemical feedstock, such as fatty acids, for industrial use from renewable plant sources rather than from non-renewable petrochemicals. This concept has broad appeal to manufacturers and consumers on the basis of resource conservation and provides a significant opportunity to develop new industrial crops for agriculture.
There is a diverse array of unusual fatty acids in nature and these have been well characterised (Badam & Patil, 1981; Smith, 1970). Many of these unusual fatty acids have industrial potential and this has led to interest in domesticating such species to enable agricultural production of particular fatty acids.
One class of fatty acids of particular interest are the epoxy-fatty acids, consisting of an acyl chain in which two adjacent carbon bonds are linked by an epoxy bridge. Due to their high reactivities, they have considerable application in the production of coatings, resins, glues, plastics, surfactants and lubricants. These fatty acids are currently produced by chemical epoxidation of vegetable oils, mainly soybean oil and linseed oil, however this process produces mixtures of multiple and isomeric forms and involves significant processing costs.
Attempts are being made by others to develop some wild plants that contain epoxy fatty acids (eg.
Euphorbia lagascae, Vernonia galamensis
) into commercial sources of these oils. However, problems with agronomic suitability and low yield potential severely limit the commercial utility of traditional plant breeding and cultivation approaches.
The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating the efficiency of commercially-important industrial processes, by the expression of genes isolated from a first organism or species in a second organism or species to confer novel phenotypes thereon. More particularly, conventional industrial processes can be made more efficient or cost-effective, resulting in greater yields per unit cost by the application of recombinant DNA techniques.
Moreover, the appropriate choice of host organism for the expression of a genetic sequence of interest provides for the production of compounds which are not normally produced or synthesized by the host, at a high yield and purity.
However, despite the general effectiveness of recombinant DNA technology, the isolation of genetic sequences which encode important enzymes in fatty acid metabolism, in particular the genes which encode the fatty acid &Dgr;12-epoxygenase enzymes responsible for producing 12,13-epoxy-9-octadecenoic acid (vemolic acid) and 12,13-epoxy-9,15-octadecadienoic acid, amongst others, remains a major obstacle to the development of genetically-engineered organisms which produce these fatty acids.
Until the present invention, there were only limited biochemical data indicating the nature of fatty acid epoxygenase enzymes, in particular &Dgr;12-epoxygenases. However, in
Euphorbia lagascae
, the formation of 12,13-epoxy-9-octadecenoic acid (vernolic acid) from linoleic acid appears to be catalysed by a cytochrome-P450-dependent &Dgr;12 epoxygenase enzyme (Bafor et al., 1993; Blee et al., 1994). Additionally, developing seed of linseed plants have the capability to convert added vernolic acid to 12,13epoxy-9,15-octadecadienoic acid by an endogenous &Dgr;15 desaturase (Engeseth and Stymne, 1996). Epoxy-fatty acids can also be produced by a peroxide-dependent peroxygenase in plant tissues (Blee and Schuber, 1990).
In viork leading up to the present invention, the inventors sought to isolate genetic sequences which encode genes which are important for the production of epoxy-fatty acids, such as 12,13-epoxy-9-octadecenoic acid (vernolic acid) or 12,13-epoxy-9,15-octadecadienoic acid and to transfer these genetic sequences into highly productive commercial oilseed plants and/or other oil accumulating organisms.
SUMMARY OF THE INVENTION
One aspect of the invention provides an isolated nucleic acid molecule which encodes or is complementary to an isolated nucleic acid molecule which encodes a fatty acid epoxygenase.
A second aspect of the invention provides an isolated nucleic acid molecule which hybridizes under at least low stringency conditions to at least 20 contiguous nucleotides of SEQ ID NOs:1 or 3 or 5 or 19 or 20, or a complementary sequence thereto.
A further aspect of the invention provides isolated nucleic acid molecule which comprises a sequence of nucleotides which is at least 65% identical to SEQ ID NO:1 or 3 or 5 or which is at least 75% identical to at least 200 contiguous nucleotides in SEQ ID NOs: 19 or 20, or a complementary sequence thereto.
A further aspect of the invention provides a genetic construct which comprises the isolated nucleic acid molecule supra, in either the sense or antisense orientation, in operable connection with a promoter sequence.
A further aspect of the invention provides a method of altering the level of epoxy fatty acids in a cell, tissue, organ or organism, said method comprising expressing a sense, antisense, ribozyme or co-suppression molecule comprising the isolated nucleic acid molecule supra in said cell for a time and under conditions sufficient for the level of epoxy fatty acids therein to be increased or reduced.
A further aspect of the invention provides a method of producing a recombinant enzymatically active epoxygenase polypeptide in a cell, said method comprising expressing the isolated nucleic acid molecule supra in said cell for a time and under conditions sufficient for the epoxygenase encoded therefor to be produced.
A further aspect of the invention provides a method of producing a recombinant enzymatically active epoxygenase polypeptide in a cell, said method comprising the steps of:
(i) producing a genetic construct which comprises the isolated nucleic acid molecule supra placed operably under the control of a promoter capable of conferring expression on said genetic sequence in said cell, and optionally an expression enhancer element;
(ii) transforming said genetic construct into said cell; and
(iii) selecting transformants which express a functional epoxygenase encoded by the genetic sequence at a high level.
A still further aspect of the invention provides a method of producing a recombinant enzymatically active epoxygenase polypeptide in a transgen
Green Allan
Lenman Marit
Singh Surinder
Stymne Sten
BASF Plant Science GmbH
Greenlee Winner and Sullivan PC
McElwain Elizabeth F.
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