Plant expressing 2-acyltransferase

Details Plant expressing 2-acyltransferase Plant expressing 2-acyltransferase

1998-03-05

2001-02-27

Patterson, Jr., Charles L. (Department: 1652)

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

C800S295000, C800S278000

Reexamination Certificate

active

06194640

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to modified plants. In particular, the invention relates to plants modified such that at least part of the plant (for example seeds of the plant) is capable of yielding a commercially useful oil.
BACKGROUND OF THE INVENTION
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.
SUMMARY AND DETAILED DESCRIPTION OF THE INVENTION
The present invention provides such a key, in the form of a DNA sequence (in the specific case, a cDNA sequence) encoding a 2-acyltransferase. The DNA sequence in
FIG. 1
(SEQ ID NO: 1) from nucleotides 130 to 1254 encodes the 2-acyltransferase from maize (
Zea mays
), including the stop codon.
According to a first aspect of the invention, therefore, there is provided a recombinant or isolated DNA sequence, preferably encoding an enzyme having membrane-bound acyltransferase activity, and selected from:
(i) a DNA sequence comprising the DNA sequence of
FIGS. 1A and 1B
(SEQ ID NO: 1) encoding at least from MET
1
to Stop
375
(SEQ ID NO: 2) or its complementary strand,
(ii) nucleic acid sequences hybridising to the DNA sequence of
FIGS. 1A and 1B
(SEQ ID NO: 1), or its complementary strand, under stringent conditions, and
(iii) nucleic acid sequences which would hybridise to the DNA sequence of
FIGS. 1A and 1B
(SEQ ID NO: 1), or its complementary strand, but for the degeneracy of the genetic code.
Fragments of the above DNA sequences, for example of at least 15, 20, 30, 40 or 60 nucleotides in length, are also within the scope of the invention.
Suitable stringent conditions include salt solutions of approximately 0.9 molar at temperatures of from 35° C. to 65° C. More particularly, stringent hybridisation conditions include 6×SSC, 5×Denhardt's solution, 0.5% SDS, 0.5% tetrasodium pyrophosphate and 50 &mgr;g/ml denatured herring sperm DNA; washing may be for 2×30 minutes at 65° C. in 1×SSC, 0.1% SDS and 1×30 minutes in 0.2×SSC, 0.1% SDS at 65° C.
Nucleic acid sequences within the scope of the first aspect of the invention will generally encode a protein having 2-acyltransferase activity, as that is the activity of the enzyme encoded by the
FIGS. 1A and 1B
nucleic acid sequence (SEQ ID NO: 1). Nucleic acid sequences not encoding a protein having enzymic activity (or the relevant enzymic activity) but otherwise conforming to the first aspect of the invention as set out above may be useful for other purposes (and are therefore also encompassed by the invention); for example they may be useful as probes, which is a utility shared by the nucleic acid sequences of the first aspect of the invention, including the
FIG. 1
sequence itself.
The probe utility arises as follows. As there is likely to be a high degree of homology between acyltransferases of different species (and particularly between 2-acyltransferases of different species) the
FIGS. 1A and 1B
sequence (or part of it, or other sequences within the invention) may be used to probe cDNA or genomic libraries of other species in order to clone DNA sequences encoding acyltransferases having desired specificities. For example, if it is desired to produce oil having a high content of erucic acid esterified to glycerol, a DNA library of any species which naturally makes erucic acid may be probed. Suitable plants include meadow foam (Limnanthes spp., especially
L. alba
and, particularly,
L. douglassi)
and Crambe.
Limnanthes douglassi
is the preferred species, as specificity studies show that there is positive discrimination towards incorporation of erucic acid into position 2 of the triacylglyceride. Libraries of organisms other than the higher plants may be probed; for example, certain bacteria may have an acyltransferase of the desired specificity. DNA in accordance with the invention will in general have a higher degree

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