Patent EA-004568-B1 (EA)
Title: Monooxygenase polypeptide with mixed functions, capable to catalize the epoxygenization of carbon bond in molecule of fatty acid, encoding their nucleic acids, methods of use of polypeptides and nucleic acids and transgenic plants
Inventor: GREEN ALLAN
IPC: C12N15/53
Filed: 1998-04-09
Abstract: 1. An isolated nucleic acid molecule which encodes monooxygenase polypeptide Crepis palaestina with mixed functions, is capable of catalysing the epoxygenation of a carbon bond in a fatty acid molecule, wherein said nucleic acid molecule comprises nucleic sequence, selected from group consisting of: (a) nucleic sequence, encoding the monooxygenase polypeptide, with aminoacid sequence SEQ ID No: 2. (b) nucleic sequence SEQ ID No:1 or variants thereof, obtained on the base of genetic code degeneracy. (c) nucleic sequence, which is complimentary to a nucleic indicated in (a) and (b). 2. An isolated nucleic acid molecule which encodes monooxygenase polypeptide Crepis sp., except Crepis palaestina with mixed functions, is capable of catalysing the epoxygenation of a carbon bond in a fatty acid molecule, wherein said nucleic acid molecule comprises nucleic sequence, selected from group consisting of: (a) nucleic sequence, encoding the monooxygenase polypeptide, with aminoacid sequence SEQ ID No: 4. (b) nucleic sequence SEQ ID No:3 or variants thereof, obtained on the base of genetic code degeneracy. (c) nucleic sequence, which is complimentary to a nucleic indicated in (a) and (b). 3. An isolated nucleic acid molecule which encodes monooxygenase polypeptide Vernonia spp with mixed functions, is capable of catalysing the epoxygenation of a carbon bond in a fatty acid molecule, wherein said nucleic acid molecule comprises nucleic sequence, selected from group consisting of: (a) nucleic sequence, encoding the monooxygenase polypeptide, with aminoacid sequence SEQ ID No: 6. (b) nucleic sequence SEQ ID No:5 or variants thereof, obtained on the base of genetic code degeneracy. (c) nucleic sequence, which is complimentary to a nucleic indicated in (a) and (b). 4. The isolated nucleic acid molecule according to claim 1-3, wherein the carbon bond is a double bond in an unsaturated fatty acid molecule. 5. The isolated nucleic acid molecule according to any one of claims 1 to 5 wherein the epoxygenase is a delta6-epoxygenase enzyme, a delta 9-epoxygenase enzyme, a delta12-epoxygenase or a delta15-epoxygenase enzyme. 6. The isolated nucleic acid molecule according to claim 5, wherein the epoxygenase is a delta12-epoxygenase enzyme. 7. The isolated nucleic acid molecule according to claim 2, wherein the plant produces high levels of vernolic acid. 8. The isolated nucleic acid molecule according to claim 2, wherein the plant is a Crepis sp. selected from the list comprising Crepis biennis, Crepis aurea, Crepis conyzaefolia, Crepisintermedia, Crepis occidentalis, Crepis palaestina, Crepis vesicaria and Crepis xacintha. 9. The isolated nucleic acid molecule according to claim 1, having a nucleotide sequence SEQ ID No:1 or a complementary sequence. 10. The isolated nucleic acid molecule according to claim 2, having a nucleotide sequence SEQ ID No:3 or a complementary sequence. 11. The isolated nucleic acid molecule according to claim 3, having a nucleotide sequence SEQ ID No:5 or a complementary sequence. 12. A genetic construct which comprises the isolated nucleic acid molecule according to claim 1, which encodes a monooxygenase polypeptide Crepis palaestina with mixed functions, or part of it, encoding fermentatively active part of said polypeptide, functionally connected with the promoter sequence, wherein said nucleic acid molecule or part of it can be transcripted in the sense or antisense orientation relative to the direction of in vivo transcription of a naturally-occurring monooxygenase gene. 13. A genetic construct which comprises the isolated nucleic acid molecule according to claim 2, which encodes a monooxygenase polypeptide Crepis so., except Crepis palaestina with mixed functions, or part of it, encoding fermentatively active part of said polypeptide, functionally connected with the promoter sequence, wherein said nucleic acid molecule or part of it can be transcripted in the sense or antisense orientation relative to the direction of in vivo transcription of a naturally-occurring monooxygenase gene. 14. A genetic construct which comprises the isolated nucleic acid molecule according to claim 3, which encodes a monooxygenase polypeptide Vernonia galamensis with mixed functions, or part of it, encoding fermentatively active part of said polypeptide, functionally connected with the promoter sequence, wherein said nucleic acid molecule or part of it can be transcripted in the sense or antisense orientation relative to the direction of in vivo transcription of a naturally-occurring monooxygenase gene. 15. A genetic construct which comprises the isolated nucleic acid molecule according to claim 1, which encodes a monooxygenase polypeptide Crepis palaestina with mixed functions, or part of it, capable to modify the expression of said polypeptide, functionally connected with the promoter sequence, wherein said nucleic acid molecule or part of it can be transcripted in the sense or antisense orientation relative to the direction of in vivo transcription of a naturally-occurring monooxygenase gene. 16. A genetic construct which comprises the isolated nucleic acid molecule according to claim 2, which encodes a monooxygenase polypeptide Crepis sp., except Crepis palaestina with mixed functions, or part of it, capable to modify the expression of said polypeptide, functionally connected with the promoter sequence, wherein said nucleic acid molecule or part of it can be transcripted in the sense or antisense orientation relative to the direction of in vivo transcription of a naturally-occurring monooxygenase gene. 17. A genetic construct which comprises the isolated nucleic acid molecule according to claim 23, which encodes a monooxygenase polypeptide Vernonia galamensis with mixed functions, or part of it, capable to modify the expression of said polypeptide, functionally connected with the promoter sequence, wherein said nucleic acid molecule or part of it can be transcripted in the sense or antisense orientation relative to the direction of in vivo transcription of a naturally-occurring monooxygenase gene. 18. Method of reducing the expression of gene, encoding monooxygenase polypeptide with mixed functions, in cell or in microorganism, comprising the administering of genetic construct according to any of claims 12-17, in cell or microorganism and incubation of said cell or in microorganism for a time and under conditions sufficient for expression of nucleic acid molecule, which encodes monooxygenase polypeptide, in said genetic construct. 19. Method according to claim 18, in which the step of genetic construct comprises the stable transformation of the cell or microorganism by genetic construct. 20. Method of preparing of recombinant fermentatively active monooxygenase polypeptide with mixed functions in cell, comprising cell cultivation, comprising genetic construct according to any of claims 12-17, for a time and under conditions sufficient for expression of nucleic acid molecule, which encodes monooxygenase polypeptide, in said genetic construct. 21. Method according to claim 20, comprising the step of administration of genetic construction in a cell before cultivating of the cell. 22. Method of preparing of recombinant fermentatively active monooxygenase polypeptide with mixed functions in transgenic plant, comprising the following steps: (a) administration of genetic construction according to claims 12-17 in cell or tissue of the plant; (b) regeneration of transformed plant from said cell or tissue; and (c) selection of transformed plant with high level of expression in seed of fermentatively active monooxygenase polypeptide, which encodes said genetic construct. 23. The method according to claim 22, wherein the plant is an oilseed species that normally produces high levels of linoleic acid. 24. The method according to claims 22 or 23, wherein the plant is selected from the list comprising Linola flax, oilseed rape, sunflower, safflower, soybean, linseed, sesame, cottonseed, peanut, olive or oil palm, amongst others. 25. Method according to claim 24, wherein flax plant has a low content of linoleic acid. 26. A recombinant monooxygenase polypeptide Crepis palaestina with mixed functions, capable to catalyze epoxygenation of carbon bond in molecule of fatty acid, selected from the group consisting of: (a) polypeptide, comprising amino acid SEQ ID No: 2; (b) polypeptide, comprising the amino acid sequence, encoded by nucleotic sequence SEQ ID No:1 or variants thereof, produced on the base of degeneration of genetic code. 27. A recombinant monooxygenase polypeptide Crepis sp., except Crepis palaestina with mixed functions, capable to catalyze epoxygenation of carbon bond in molecule of fatty acid, selected from the group consisting of: (a) polypeptide, comprising amino acid SEQ ID No: 4; (b) polypeptide, comprising the amino acid sequence, encoded by nucleotic sequence SEQ ID No:3 or variants thereof, produced on the base of degeneration of genetic code. 28. A recombinant monooxygenase polypeptide Vernonia galamensis with mixed functions, capable to catalyze epoxygenation of carbon bond in molecule of fatty acid, selected from the group consisting of: (a) polypeptide, comprising amino acid SEQ ID No: 6; (b) polypeptide, comprising the amino acid sequence, encoded by nucleotic sequence SEQ ID No:5 or variants thereof, produced on the base of degeneration of genetic code. 29. A recombinant polypeptide, according to claim 26, having an amino acid sequence SEQ ID No: 2. 30. A recombinant polypeptide, according to claim 27, having an amino acid sequence SEQ ID No: 4. 31. A recombinant polypeptide, according to claim 28, having an amino acid sequence SEQ ID No: 6. 32. Method of modifying intracellular content of an epoxygenated fatty acid, comprising the cultivation of cell, comprising a genetic construction according to any of claims 12-17, for a time and under conditions sufficient for expression of nucleic acid molecule, which en