Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters fat – fatty oil – ester-type wax – or...
Reexamination Certificate
2001-03-22
2004-11-09
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters fat, fatty oil, ester-type wax, or...
C800S298000, C800S306000, C800S322000, C435S419000, C435S440000, C536S023600
Reexamination Certificate
active
06815579
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a condensing enzyme involved in long chain fatty acid production in plants, including related nucleic acid sequences.
BACKGROUND
Living organisms synthesize a vast array of different fatty acids, which are incorporated into complex lipids. These complex lipids represent both major structural membrane components, and are a major storage product in both plants and animals. In plants, very long chain fatty acids (VLCFAs, chain length C20 or longer) are synthesized predominantly in the epidermal cells where they are either directly incorporated into waxes, or serve as precursors for other aliphatic hydrocarbons found in waxes, including alkanes, primary and secondary alcohols, ketones, aldehydes and acyl-esters (for review see Post-Beittenmiller, 1996). VLCFAs also accumulate in the seed oil of some plant species, where they are incorporated into triacylglycerols (TAGs), as in the
Brassicaceae
, or into wax esters, as in Jojoba. These seed VLCFAs include the agronomically important erucic acid (C22:1), that may be used in the production of lubricants, nylon, cosmetics, pharmaceuticals and plasticizers.
VLCFAs are synthesized by a microsomal fatty acid elongation (FAE) system which involves four enzymatic reactions: (1) condensation of malonyl-CoA with a long chain acyl-CoA, (2) reduction to beta-hydroxyacyl-CoA, (3) dehydration to an enoyl-CoA and (4) reduction of the enoyl-CoA, resulting in the acyl-CoA elongated by two carbons. The condensing enzyme catalyzing reaction (1) is a key activity of the FAE system. It is the rate-limiting enzyme of the VLCFA biosynthetic pathway, which controls the amount of VLCFAs produced (Miller and Kunst, 1997). In addition, the condensing enzyme determines the ultimate VLCFA acyl chain length, and thus their uses in seed oil or wax biosynthesis.
Different condensing enzymes acting on, and producing, acyl chains of different length have recently been characterized. Several groups independently identified the first plant fatty acid elongation gene in
Arabidopsis
, FAE1 (James and Dooner, 1990; Kunst et al., 1992; and Lemleux et al., 1990). FAE1 was subsequently cloned and is described in WO9613582 as catalyzing the conversion of C18 fatty acids to C20-C22 fatty acids. The patent WO9613582 suggests that FAE1 will have activity in a very broad host range. In support of this assertion of broad host range activity, it has been shown that FAE1 has the same activity in yeast as in Arabiqopsis (Miller and Kunst, 1997). A Jojoba protein involved in the synthesis of VLCFAs has also been isolated having relatively high homology to FAE1 (52% amino acid identity), and has been shown to have beta-ketoacyl-coenzyme A synthase (KCS) activity (WO9515387). Broad host range activity for genes encoding KCS has been further evidenced by the Jojoba KCS cDNA. The Jojoba KCS cDNA was able to complement a mutation in a Canola variety of rapeseed (
Brassica napus
), to restore in the variety high levels of VLCFAs (Lassner et al., 1996). An
Arabidopsis
gene (CUT1), required for cuticular wax biosynthesis and pollen fertility, has also been described as encoding a VLCFA condensing enzyme that catalyzes the addition of 2C units to pre-existing C24 or longer fatty acids (Millar et al., 1999).
The broad specificities exhibited by FAE activities provide a means of modifying the synthesis of VLCFAs in a given cell. As evidenced by the accumulation of VLCFAs in tobacco seed expressing FAE1 (Millar and Kunst, 1997), heterologous condensing enzymes may be used to produce VLCFAs in plant species that do not otherwise synthesize VLCFAs. For example, targeted expression of heterologous VLCFA condensing enzymes in seeds may allow the production of crop plants capable of synthesizing VLCFAs of desired lengths in seed oil for industrial applications. New condensing enzymes may also be useful for the manipulation of cuticular waxes which have important functions in many physiological processes in plants, including water balance, protection from UV light, plant-insect interactions, and defense against bacterial and fungal pathogens (Post-Beittenmiller, 1996). As a result, there is a need for new condensing enzymes that may be used alone or in combination with other condensing enzymes to confer on plants and plant tissues the ability to synthesize a range of VLCFAs, including VLCFAs up to C30 in length.
There is also a need for tissue-specific promoters capable of mediating the expression of heterologous condensing enzymes in epidermal cells, which may facilitate the alteration of the wax composition and/or accumulation in plants. This may, in turn, result in the production of crops with increased tolerance to environmental stresses, and/or resistance to pathogens and insects. For example, drought resistance in rice is associated with high wax lines rich in C29, C33 and C35 alkanes (O'Toole and Cruz, 1983; Haque et al., 1992). Increased wax deposition may also be accomplished by overexpression of condensing enzymes with desired acyl chain length specificities using an epidermis-specific promoter, such as the CUT1 promoter (Millar et al., 1999).
The cumulative data, as discussed, from a variety of sources in this field has led to the suggestion that the amounts and acyl chain lengths of VLCFAs, in a wide variety of eukaryotic cells, are regulated by the nature of condensing enzyme expression in the cell (Miller and Kunst, 1997). Condensing enzymes would therefore be useful in a range of biotechnical applications.
SUMMARY
In various aspects, the present invention provides nucleic acid sequence encoding all or part of a new plant long chain fatty acid condensing enzyme (fatty acid elongase), designated herein as KCS2 (for beta-ketoacyl-coenzyme A synthase 2). In some embodiments, KCS2 may mediate the biosynthesis of C18, C20, C22 and C24 fatty acids. The activity of the enzyme is typically characterized by two carbon (malonyl-CoA) additions to C16, C18, C20 and C22 moieties (C16-C22 acyl CoA molecules), i.e. condensation of malonyl-CoA with a C16, C18, C20 or C22 acyl-CoA. The fatty acids produced by the enzyme may for example be saturated 18:0, 20:0, 22:0 and 24:0 fatty acids.
The invention includes recombinant nucleic acid molecules comprising a heterologous nucleic acid coding sequence encoding the plant long chain fatty acid condensing enzyme. In alternative embodiments, the nucleic acid coding sequence may be derived from the
Arabidopsis
KCS2 coding sequence disclosed herein. Alternatively, embodiments include nucleic acids that encode the plant very long chain fatty acid condensing enzyme of the invention, wherein the enzyme has an amino acid sequence that is at least 70% identical to an
Arabidopsis
KCS2 amino acid sequence disclosed herein, when optimally aligned. The nucleic acid coding sequences of the invention also include sequences that hybridize under stringent conditions to a complement of the
Arabidopsis
KCS2 coding sequence disclosed herein. The nucleic acid coding sequences of the invention may also be at least 70% identical to the
Arabidopsis
KCS2 coding sequence, when optimally aligned. Embodiments of the invention include isolated nucleic acid molecules comprising the coding sequences of the invention.
In another aspect, the invention provides recombinant nucleic acid molecules comprising a promoter sequence operably linked to a nucleic acid sequence, wherein the promoter sequence is capable of mediating gene expression in anthers and in very young leaves in
Arabidopsis
. The promoter sequences of the invention may be derived from an
Arabidopsis
KCS2 promoter sequence, as disclosed herein. Promoter sequences of the invention may also hybridize under stringent conditions to the
Arabidopsis
KCS2 promoter sequence disclosed herein. Promoter sequences of the invention may also be at least 70% identical to the
Arabidopsis
KCS2 promoter sequence when optimally aligned.
The invention provides nucleic acid probes comprising probe sequences that hybridize under stringent conditions to a portion of an
Arabidopsi
Clemens Sabine
Kunst Ljerka
Klarquist & Sparkman, LLP
McElwain Elizabeth F.
The University of British Columbia
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