&Dgr;9-desaturase gene

Details &Dgr;9-desaturase gene &Dgr;9-desaturase gene

1999-08-27

2002-09-10

Achutamurthy, Ponnathapura (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S254300, C435S252300, C435S134000, C435S071100, C536S023200

Reexamination Certificate

active

06448055

ABSTRACT:

TECHNICAL FIELD
This invention relates to a gene encoding &Dgr;9-desaturase having the activity of desaturating the &Dgr;9-position of a fatty acid. More specifically, the invention relates to a gene encoding &Dgr;9-desaturase of a microorganism belonging to the subgenus Mortierella of the genus Mortierella which is known to accumulate useful, highly unsaturated fatty acids, including arachidonic acid, intracellularly in marked amounts; a process for producing &Dgr;9-desaturase using this gene; an expression vector containing this gene; a transformant transformed with the expression vector; and their utilization.
BACKGROUND ART
Unsaturated fatty acids are synthesized in animals, plants, and microorganisms. Except in higher animals, palmitic acid and stearic acid, which are saturated fatty acids, turn into monounsaturated acids having cis-&Dgr;9 upon desaturation with oxygenases. Then, carbon chain elongation and desaturation are repeated to form unsaturated fatty acids. Such desaturation reactions are each aerobic desaturation relying on a monooxygenation reaction. A saturated fatty acid, such as palmitic acid or stearic acid, is desaturated in the presence of an oxygen atom and NAD(P)H to become a monoenoic acid.
In the present specification, a protein with the activity of desaturating the &Dgr;9-position of a fatty acid is referred to as &Dgr;9-desaturase. &Dgr;9 is a designation complying with the rule that the position of a double bond of a fatty acid is expressed by &Dgr; (delta) combined with the number of the carbon atoms ranging from the carbon atom of its terminal carboxyl group to the carbon atom where the double bond exists. Namely, &Dgr;9 means the presence of a double bond between the 9th and the 10th carbon atom counting from the carbon atom of the terminal carboxyl group. The position of a double bond may be described subsequently to &ohgr; (omega), which represents the number of carbon atoms ranging from the carbon atom of the terminal methyl group of a fatty acid to the carbon atom where the double bond exists.
Of the so biosynthesized unsaturated fatty acids, arachidonic acid (may be designated as “ARA”), dihomo-&ggr;-linolenic acid (may be designated as “DGLA”), and eicosapentaenoic acid (may be designated as “EPA”) are precursors of physiologically active substances having various physiological actions (prostaglandins and thromboxanes). EPA, for example, is commercially available as a health food or a pharmaceutical based on its antithrombotic action or a lipid lowering action. In recent years, ARA and docosahexaenoic acid (may be designated as “DHA”) has been reported to be contained in breast milk, and to be useful for the growth of an infant (“Advances in Polyunsaturated Fatty Acid Research”, Elsevier Science Publishers, 1993, pp. 261-264). Their importance to the height of a fetus and the growth of its brain has also been reported (Proc. Natl. Acad. Sci. USA, 90, 1073-1077 (1993); Lancet, 344, 1319-1322 (1994)).
With this background, moves have been made to add ARA and DHA, the main sources of the difference in fatty acid composition between mother's milk and infant formula, to the infant formula.
In recent years, fish oil has been used for the purpose of adding DHA to infant formula. However, fish oil is an acylglycerol mixture containing many kinds of fatty acids as constituent fatty acids. Since isolation of these components is difficult, fish oil-containing infant formula contains a large amount of EPA as well as DHA. By the action of EPA, conversion from linoleic acid to ARA is suppressed, so that in vivo ARA is decreased. To solve this problem, attention has recently been paid to methods for producing large amounts of desired unsaturated fatty acids, without involving the incorporation of untoward fatty acids, by use of microorganisms, such as Chlorella, algae, molds (filamentous fungi), or bacteria.
DISCLOSURE OF THE INVENTION
The inventors of the present invention have focused on the fact that
Mortierella alpina,
which belongs to the subgenus Mortierella of the genus Mortierella, a genus of filamentous fungi, accumulates marked amounts of fats and oils intracellularly, and its ARA-producing ability is very high. The inventors speculated that various desaturases would be present in these filamentous fungi, and their activity would be very high. Thus, they considered cloning a gene encoding &Dgr;9-desaturase from
Mortierella alpina,
introducing this gene into a microorganism having the ability to produce an unsaturated fatty acid, and increasing the production of an unsaturated fatty acid with the assistance of the microorganism.


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