Selective modification of plant fatty acids

Details Selective modification of plant fatty acids Selective modification of plant fatty acids

2001-05-25

2004-05-18

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Plant, seedling, plant seed, or plant part, per se

Higher plant, seedling, plant seed, or plant part

C800S278000, C800S281000, C800S298000

Reexamination Certificate

active

06737564

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to modification of plant fatty acid composition by expression of a plant &Dgr;9 acyl-CoA desaturase, particularly selective and preferential increases in the ratio of oleic acid to stearic acid.
BACKGROUND OF THE INVENTION
Lipids are essential in the composition of all plant cells. Although plant lipids cover a wide range of compounds, the majority of lipids are derived from two important metabolic pathways, the fatty acid biosynthetic pathway and the glycerolipid biosynthetic pathway. Plants naturally produce an assortment of fatty acids which they incorporate into a wide assortment of lipids which perform different functions. Polar glycerolipids (phospholipids and glycolipids), for example, contain two fatty acids attached to both sn-1 and sn-2 positions of the glycerol backbone and a polar headgroup attached to the sn-3 position. Polar glycerolipids play an essential role in cell membrane structure and function. Triacylglycerols, on the other hand, have all three positions of the glycerol backbone esterified with fatty acids and are the major storage lipids in oil-producing plant tissues, such as in plant seeds, and are usually known as plant oils.
The specific properties of a plant oil are dependent on the fatty acid composition of the oil, which in turn affects the nutritional quality of the oil. The health value of high levels of monounsaturates, particularly oleic acid, as the major dietary fat constituent has been established by recent studies. For example, canola oil, which typically contains at least 60% oleic acid (c18:1, &Dgr;9), has been proven effective in lowering cholesterol in human blood. It has also been shown, however, that high levels of all monounsaturated fatty acids are not necessarily beneficial. For example, it has been suggested that palmitoleic acid (c16:1, &Dgr;9) may have certain health disadvantages, such as behaving as a saturated fatty acid in its effect on cholesterol (Nestel et al., 1994, J Lipid Res 35(4):656-662) effecting atrioventicular conduction in the heart (Dhein et al, 1999, Br. J. Pharmacol 128(7) 1375-1384) and correlating with high blood pressure in men at high risk of coronary heart disease (Simon et al., Hypertension Feb. 27, 1996 (2):303-7). As a result, because of these medical and nutritional effects, there is an interest in lowering the level of saturated fatty acids in plant oils beyond certain limits (the limit of allowable saturated fatty acid proportions in canola oil, for example, is 7%).
The fatty acid composition of plant oils is determined both by the genotype of the plant and the plant's response to environmental factors such as light, temperature and moisture. Genetic modification by plant breeding or genetic engineering may be used to modify fatty acid metabolic pathways and thereby modify plant oil composition.
In plants, fatty acids are generally synthesized in the plastid or chloroplast by the FAS system in which the elongating chain is generally esterified to acyl-carrier protein (ACP) as palmitic acid (16:0) and stearic acid (18:0) esterified to ACP (i.e., 16:0-ACP and 18:0-ACP, respectively). A known soluble plant stearoyl-ACP &Dgr;9 desaturase enzyme is located in the chloroplast where it is understood to catalyze the conversion of stearoyl-ACP (18:0-ACP) to oleoyl-ACP (18:1-ACP). These acyl-ACPs may either be used for glycerolipid synthesis in the chloroplast or transported out of chloroplast into the cytoplasm as acyl-CoAs. It is generally believed that the stearoyl-ACP &Dgr;9 enzyme is the only soluble plant desaturase, so that palmitic acid and stearic acid exported from the chloroplast will not undergo further desaturation. Therefore, the level of saturation is largely determined by the amount of saturated fatty acids exported out of the chloroplast and into the cytoplasm.
This situation in plants is in contrast to that known for yeast and mammalian acyl-CoA &Dgr;9 desaturases, which use fatty acids esterified to CoA as substrates, and desaturate both the saturated fatty acids palmitic acid and stearic acid. Mammalian and yeast acyl-CoA &Dgr;9 desaturases have been used to modify levels of saturated fatty acids in plant tissues (U.S. Pat. Nos. 5,866,789 and 5,777,201) and have been shown to result in increased levels of monounsaturated fatty acids, including both oleic and palmitoleic fatty acids, and decreased levels of saturated fatty acids in plant oils. Recently, two genes homologous to the mammalian and yeast acyl-CoA desaturases were isolated from Arabidopsis, ADS1 and ADS2 respectively (Fukuchi-Mizutani et al. (1998) Plant Cell Physiol. 39:247-253). ADS1 and ADS2 share 76% amino acid sequence identity and it has been speculated that these two genes are &Dgr;9 fatty acid desaturases. The Genbank database accession for the ADS1 protein and nucleic acid sequences is D88536, which sets out the sequences as follows:
(SEQ ID NO: 9)
MSLSASEKEENNKKMAADKAEMGRKKRAMWERKWKRLDIVKAFASLFVHF

LCLLAPFNFTWPALRVALIVYTVGGLGITVSYHRNLAHRSFKVPKWLEYF

FAYCGLLAIQGDPIDWVSTHRYHHQFTDSDRDPHSPNEGFWFSHLLWLFD

TGYLVEKCGRRTNVEDLKRQWYYKFLQRTVLYHILTFGFLLYYFGGLSFL

TWGMGIGVAMEHHVTCLINSLCHVWGSRTWKTNDTSRNVWWLSVFSFGES

WHNNHHAPESSARQGLEWWQIDISWYIVRFLEIIGLATDVKLPSESQRRR

MAMVR
(SEQ ID NO: 1)
ccacaaagag tctttttttt ttttctcttc gacttagctt

atacatagtt ttattacaag atgtcattgt cagcctcgga

gaaggaggag aataacaaga aaatggcagc ggacaaggct

gagatgggga ggaagaagag ggcaatgtgg gaaagaaagt

ggaagagatt ggacattgtg aaagcttttg catctctctt

tgtccatttc ctctgtctct tggcgccttt caatttcact

tggccggctt taagagtcgc cctcattgtc tatacggtgg

gtgggctcgg tatcaccgtc tcttaccacc gaaatttggc

tcaccggagc ttcaaagtcc ctaaatggct cgagtatttc

ttcgcttatt gcggccttct tgccattcag ggagatccga

ttgattgggt gagcacacat cgataccatc accagtttac

agattcggat agggacccac atagtcctaa cgaaggattt

tggttcagtc acctcctatg gctatttgat accggttatc

ttgtagaaaa gtgtggaaga aggacaaatg tggaggactt

aaagaggcag tggtactata aattcctcca aagaacagtc

ctttaccaca ttctaacatt tggtttcctc ctctattact

ttggtggttt gtcttttctt acttggggaa tgggtattgg

ggtagcaatg gagcatcatg tgacttgcct cataaactct

ctttgccatg tttggggaag ccgaacttgg aagactaatg

acacttcccg taacgtttgg tggctatcag tattctcgtt

tggagagagc tggcacaaca atcaccacgc cttcgaatcc

tcggcgagac aaggcttaga atggtggcaa atcgacattt

cttggtatat tgtccgcttt ctcgagatta tcggtttggc

tactgatgtt aagttgcctt ccgagagtca acgtcgtcgt

atggcaatgg ttcgttgaag atatggaacg acgtctcgtc

tcatttaagc attagttaat taatgtctac gtacgtttta

agtttttggt aaacgtaaca cttgtaatat tgtgcgatgc

ggtgttgttt tgtgacttgt ggtgtgtgtt tgaaccaact

tgcttaatta agataacgtt cgttttgata tgagcgaaaa

aaaaaaaaaa aaaaaaaa
The Genbank database accession for the ADS2 protein and nucleic acid sequences is D88537, which sets out the sequences as follows:
(SEQ ID NO: 10)
MSVTSTVEENHQKNPSTPAAVEEKKKRRWVFWDRRWRRLDYVKFASFTVH

SLALLAPFYFTWSALWVTFLFYTIGGLGITVSYHRNLAHRSFKVPKWLEY

LLAYCALLAIQGDPIDWVSTHRYHHQFTDSERDPHSPKEGFWFSHLLWIY

DSAYLVSKCGRRANVEDLKRQWFYRFLQKTVLFHILGLGFFLFYLGGMSF

VTWGMGVGAALEVHVTCLINSLCHIWGTRTWKTNDTSRNVWWLSVFSFGE

SWHNNHHAFESSARQGLEWWQIDISWYIVRFFEIIGLATDVKVPTEAQRR

RMAIVR
(SEQ ID NO: 2)
gagaagagaa agagagatcc gaaatgtcgg tgacatcaac

ggtggaggag aaccaccaga aaaatccatc aacgccggcg

gcggtggagg agaagaagaa gaggagatgg gtgttttggg

atagaaggtg gaggagatta gattatgtga aattctcagc

ttctttcact gttcattctc ttgctctctt ggctccgttt

tatttcactt ggtcggctct ttgggttacg tttttgtttt

acaccatcgg tggtcttggt atcaccgtct cttatcatcg

caacttggct caccggagtt tcaaagtccc taaatggctt

gagtatctct tagcctattg tgcccttctc gctattcagg

gagatccgat tgattgggtg agtacacatc gttaccatca

ccagttcacg gattcagaac gtgatccaca tagtcctaag

gaaggttttt ggtttagtca tcttctttgg atctatgact

ctgcctatct tgtttcaaag tgtggaagaa gagcaaacgt

ggaggatttg aagaggcaat ggttttatag gtttcttcag

aaaacagtgc tatttcacat tttaggattg ggtttctttc

tcttctacct tggtggcatg tccttcgtta cttggggaat

gggggtagga gcagcattgg aagtgcacgt gacttgcctc

ataaattcac tctgccatat ttggggcact cgaacttgga

agaccaatga cacttctcgt aatgtttggt ggttatcggt

attttcattt ggagagagtt ggcacaacaa tcatcatgcg

ttcgagtcat cggctagaca aggacttgaa tggtggcaaa

tagacatttc gtggta

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