Plants have long been a commercially valuable source of oil. Nutritional uses of plant-derived oils have hitherto been dominant, but attention is now turning additionally to plants as a source of industrially useful oils, for example as replacements for or improvements on mineral oils. Oil seeds, such as from rape, have a variety of lipids in them (Hildish & Williams, "Chemical Composition of Natural Lipids", Chapman Hall, London, 1964). There is now considerable interest in altering lipid composition by the use of recombinant DNA technology (Knauf, TIBtech, February 1987, 40-47), but by no means all of the goals have been realised to date for a variety of reasons, in spite of the ever-increasing sophistication of the technology.
Success in tailoring the lipid content of plant-derived oils requires a firm understanding of the biochemistry and genes involved. Broadly, two approaches are available. First, plants may be modified to permit the synthesis of fatty acids which are new (for the plant); so, for example, laurate and/or stearate may be synthesised in rape. Secondly, the pattern and/or extent of incorporation of fatty acids into the glycerol backbone of the lipid may be altered. It is with this latter approach that the present invention is concerned, although the former approach may additionally be used.
Lipids are formed in plants by the addition of fatty acid moieties onto the glycerol backbone by a series of acyl transferase enzymes. There are three positions on the glycerol molecule at which fatty acid (acyl) moieties may be substituted, and the substitution reached at each position is catalysed by a position-specific enzyme: the enzymes are known as 1-, 2- and 3-acyltransferases, respectively.
One, but not the only, current aim of "lipid engineering" in plants is to provide oils including lipids with a high content of erucic (22:1) acid. Erucic acid-containing lipids are commercially desirable for a number of purposes, particularly as replacements to or supplements for mineral oils in certain circumstances, as alluded to above. In the case of oil seed rape (Brassica napus), one of the -most significant oil producing crops in cultivation today, the specificity of the 2-acyltransferase enzyme positively discriminates against the incorporation of erucic acid at position 2. So, even in those cultivars of rape which are able to incorporate erucic acid at positions 1 and 3, where there is no (or at least reduced) discrimination against erucic acid, only a maximum 66% of the fatty acids incorporated into triacyl glycerols can be erucic acid. Such varieties of rape are known as HEAR (high erucic acid rape) varieties.
It would therefore be desirable to increase the erucic acid content of conventional oil seed rape, as well as HEAR varieties; the same can be said of oils of other vegetable oil crops such as maize, sunflower and soya, to name but a few examples. While in principle it may be thought possible to introduce into a desired plant DNA encoding a 2-acyltransferase of different fatty acid specificity, for example from a different plant, in practice there are a number of problems.
First, 2-acyltransferase and 3-acyltransferase are membrane bound, and therefore insoluble, enzymes. They have not been purified. This makes working with them difficult and rules out the use of many conventional DNA cloning procedures. This difficulty does not, paradoxically, lie in the way of cloning the gene (or at least cDNA) encoding the 1-acyltransferase enzyme, which is soluble: in fact, recombinant DNA work has already been undertaken on this enzyme for a completely different purpose, namely the enhancement of chilling resistance in tobacco plant leaves, by Murata et al (Nature 356 710-713 is (1992)).
Secondly, very little is known about the 2- and 3-acyltransferases. There is no idea of their size or how they are targeted to membranes. No nucleotide or amino acid sequence data are available and no antibodies have been raised against them.
Although there has been discussion, therefore, of the desirability of modifying 2-acyltransferase specificity, for example by importing a gene coding for the corresponding enzyme, but of different specificity, from another species, there is a pressing need in the art for the key which enables this work to be done.
USPTO Backgrounds
USPTO Backgrounds