Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Ester doai
Patent
1993-11-18
1997-06-17
Rollins, John W.
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Ester doai
800205, 800DIG69, A61K 3123, A01H 104
Patent
active
056397909
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND
Members of several plant familes synthesize large amount of predominantly medium-chain (C8-C14) triacylglycerols in specialized storage tissues, some of which are harvested for production of important dietary or industrial medium-chain fatty acids (F. D. Gunstone, The Lipid Handbook (Chapman & Hall, New York, 1986) pp. 55-112). Laurate (C12:0), for example, is currently extracted from seeds of tropical trees at a rate approaching one million tons annually (Battey, et al., Tibtech (1989) 71:122-125).
The mechanism by which the ubiquitous long-chain fatty acid synthesis is switched to specialized medium-chain production has been the subject of speculation for many years (Harwood, Ann. Rev. Plant Physiol. Plant Mol. Biology (1988) 39:101-138). Recently, Pollard, et al., (Arch. of Blochem. and Biophys. (1991) 284:1-7) identified a medium-chain acyl-ACP thioesterase activity in developing oilseeds of California bay, Umbellularia californica. This activity appears only when the developing cotyledons become committed to the near-exclusive production of triglycerides with lauroyl (12:0) and caproyl (10:0) fatty acids. This work presented the first evidence for a mechanism for medium-chain fatty acid synthesis in plants: During elongation the fatty acids remain esterified to acyl-carrier protein (ACP). If the thioester is hydrolized prematurely, elongation is terminated by release of the medium-chain fatty acid. The Bay thioesterase was subsequently purified by Davies et al., (Arch. Blochem. Biophys. (1991) 290:37-45) which allowed the cloning of a corresponding cDNA and described it use to obtain related clones and to modify the triglyceride composition of plants (WO 91/16421).
SUMMARY OF THE INVENTION
By this invention, further properties and uses of plant medium-chain thioesterases are provided.
In a first embodiment, this invention relates to plant seed and oil derived from that seed, which normally do not contain laurate, but now are found to contain laurate. Seed having as little as 1.0 percent mole laurate are significantly different from wild-type plant species which do not naturally store laurate in seed triglyceride oils. Seed having a minimum of about 15 percent mole laurate, 33 percent laurate or 50 percent laurate are contemplated hereunder. Triglyceride oils in seed or derived from seed with at least two lauroyl fatty acyl groups is likewise contemplated. Brassica seed and oil derived from such seed containing greater than 1.0 percent mole laurate is especially preferred.
In yet a different embodiment, this invention relates to a particular medium-chain thioesterase sequence, the Bay medium-chain thioesterase DNA sequence and to DNA constructs for the expression of this enzyme in a host cell. In particular, a start site for the structural gene sequence upstream to the start site previously reported for this sequence is described.
Other aspects of this invention relate to methods for using a plant medium-chain thioesterase. Expression of a plant medium-chain thioesterase in a bacterial cell to produce medium-chain fatty acids is provided. By this method, quantities of such fatty acids may be harvested in crystalline form from bacteria. Exemplified in the application is the use of E. coli and Bay thioesterase; the fad D E. coli mutant is particularly preferred. In addition, temperature ranges for improved laurate production are described.
Methods to produce an unsaturated medium-chain thioesterase by the use of a plant medium-chain thioesterase are also described herein. It is now found that, even in plants which exclusively produce and incorporate quantities of saturated medium-chain acyl-ACP fatty acids into triglycerides, the thioesterase may have activity against unstaturated fatty acids of the same length.
DESCRIPTION OF THE FIGURES
FIG. 1A. The full length nucleic acid sequence of a bay thioesterase gene (pCGN3822) having an ATG codon at nucleotides 145-147 is provided.
FIG. 1B. The translated sequence of the bay thioesterase gene beginning at the ATG codon at nucleotides 145-147
REFERENCES:
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patent: 4614663 (1986-09-01), Rule
patent: 5000975 (1991-03-01), Tomarelli
Safford et al 1993 Transgenic Research 2:191-198.
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Davies Huw Maelor
Voelker Toni Alols
Calgene Inc.
Rollins John W.
Scherer Donna E.
Schwedler Carl J.
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