MODIFIED NEUTRAL LIPID ENCAPSULATING PROTEINS AND USES THEREOF
TECHNICAL FIELD
The invention relates to compositions and methods for the production and modification of oil
bodies in various host cell types.
BACKGROUND
In nature, flowering plants efficiently store energy in their seeds through the accumulation of oil,
namely triacylglycerol (TAG) and store it in discreet oil bodies by embedding a phospholipid
protein monolayer around the oil body. These seed crops have been used in a variety of
agricultural applications as feed and more recently also as a feedstock source for biofuels. On a
per weight basis, lipids have approximately double the energy content of either proteins or
carbohydrates and as such, substantial focus has been placed on raising the oil content of various
species, most notably plants. Beyond the energy aspect, the oil bodies themselves also have
unique properties and form the basis for a number of biotechnical applications including but not
limited to the purification of recombinant proteins, formation of multimeric protein complexes,
emulsification and the delivery of bio-actives.
Unfortunately plant seeds represent a very small percentage of total plant biomass and with the
demand for improved agricultural productivity and alternative energies it is recognised that
current oil production from a number of devoted seed crops is insufficient. Research efforts have
focused on not only increasing the productivity of oil production within plant seeds but also oil
production in other cell types and species.
Traditional breeding and mutagenesis have offered incremental successes in this area; however
genetic engineering has made the furthest strides in modifying organisms to produce elevated oil
levels. While certain groups have worked along various parts of the oil synthesis pathway to upregulate
oil production within the seed, others groups have focused on increasing oil in cell types
that represent a larger portion of the biomass.
While genetic engineering has made some progress in increasing oil content in certain targets,
significant challenges still remain. Further productivity increases can still be realized in oil body
production in the seed and the means to produce oil bodies similar to those of a plant seed in
other cell types and species has yet to be achieved.
3
SUMMARY OF THE INVENTION
The present invention provides compositions and methods for producing oil bodies with varying
degrees of stability. The invention involves producing modified oleosins with artificially
introduced cysteine residues. The artificially introduced cysteine residues are preferably
introduced in the N- and C-terminal hydrophilic arms of the modified oleosins.
Expression of the modified oleosins allows for the creation of stable oil bodies beyond the
reproductive tissue of vascular plants into new cell types and even other species. When
combined with a TAG synthesising enzyme, the invention leads to the accumulation and storage
of TAG in eukaryotic cells as stable oil bodies. Compared with an unmodified cell or even one
expressing just a TAG synthesis enzyme, the invention allows for the accumulation of TAG in
excess levels achieved by other means. For example the invention has shown that one can
accumulate higher levels of stable oil bodies beyond the seed, in the vegetative portion of
vascular plants.
Plants with increased levels of TAG in their vegetative tissues provide a valuable energy source
for both animal feedstock and biofuel feedstock applications.
In addition recombinant modified oleosins purified from a host cell (such as E. coli, P. pastoris,
S. ceriviseae, Dunaliella, C. reinhardtii) can be used to generate artificial oil bodies. The
modified oleosins in artificial oil bodies, or those purified form transformed cells, can optionally
be made to cross-link via the cysteine residues in the modified oleosin. The degree of crosslinking
may be controlled manipulating the redox environment. The degree of cross-linking can
also be tailored by altering the number of cysteines in the modified oleosins.
Using combinations of these techniques the oil bodies formed with the modified oleosins can be
tailored for their emulsification properties, to regulate thermal stability, chemical stability, and
peptidase resistance.
The modified oleosins can also be fused to a protein of interest, to form a fusion protein. The
fusion protein (modified oleosin plus protein of interest) can be recombinantly expressed in a
cell or organism. In this way oil bodies containing the expressed fusion proteins can be used to
purify and deliver the protein of interest, for a variety of applications.
4
In addition the oil bodies can protect, or at least delay, degradation and/or biohydrogenation, of
TAG, within the stomach and/or rumen of an animal, allowing the intact individual lipids from
the TAG to be absorbed by the animal in the intestine. Therefore, the invention is also useful in
terms of dietary intake of an animal, particularly through expression of the modified oleosins in
plants.
Polynucleotides encoding modified oleosins with artificially introduced cysteines
In the first aspect the invention provides a polynucleotide encoding a modified oleosin including
at least one artificially introduced cysteine. The term oleosin also includes steroleosin and
caloleosin. The modified oleosin may therefore be selected from a modified oleosin, a modified
caloleosin or a modified steroleosin. In one embodiment the modified oleosin is a modified
oleosin. In another embodiment the modified oleosin is a modified caloleosin. In another
embodiment the modified oleosin is a modified steroleosin. Examples of each type of oleosin
(oleosin, caloleosin and steroleosin) are described herein
In one embodiment, the modified oleosin includes at least two cysteines, at least one of which is
artificially introduced. In a further embodiment, the modified oleosin includes at least two to at
least thirteen (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14 or more) artificially introduced cysteines.
In one embodiment the cysteines are artificially introduced in the N-terminal hydrophilic region
of the oleosin, or in the C-terminal hydrophilic region of the oleosin. In a further embodiment the
modified oleosin incudes at least one cysteine in the N-terminal hydrophilic region, and at least
one cysteine in the C-terminal hydrophilic region. In a further embodiment the cysteines are
distributed substantially evenly over the N-terminal and C-terminal hydrophilic regions of the
oleosin.
In a further embodiment the polynucleotide encodes a fusion protein including the modified
oleosin fused to a protein of interest.
Constructs
In a further aspect the invention provides a genetic construct comprising a polynucleotide of the
invention. In a further aspect the invention provides an expression construct comprising a
polynucleotide of the invention. In one embodiment the polynucleotide in the construct is
operably linked to a promoter sequence. In one embodiment the promoter sequence is capable of
driving expression of the polynucleotide in a vegetative tissue of a plant. In a further
embodiment the promoter sequence is capable of driving expression of the polynucleotide in a
5
seed of a plant. In a further embodiment the promoter sequence is capable of driving expression
of the polynucleotide in the pollen of a plant. In a further embodiment the promoter sequence is
capable of driving expression of the polynucleotide in an E. coli cell. In a further embodiment
the promoter sequence is capable of driving expression of the polynucleotide in a yeast cell. In a
further embodiment the promoter sequence is capable of driving expression of the
polynucleotide in an algal cell.
In another aspect, the invention provides a construct containing a polynucleotide that encodes a
modified neutral lipid protein. In one embodiment, the construct also contains a second
polynucleotide that encodes a triacylglycerol (TAG) synthesizing enzyme. In various
embodiments, the construct can be linked to a promoter sequence capable of driving its
expression in various host cells. As such, the invention also provides use of the constructs to
induce a host cell to express a modified oleosin and/or a TAG synthesizing enzyme. In various
embodiments, the construct expressing a modified oleosin and the construct expressing a TAG
synthesizing enzyme may be driven by the same or by different promoters. In yet another
embodiment the construct is located in an appropriate position and orientation of a suitable
functional endogenous promoter such that the expression of the construct occurs. In various
embodiments, the construct can be expressed in a bacterial, plant, fungal or algal cell. In one
embodiment where the construct is expressed in a plant cell, the cell may be of vegetative, seed,
pollen or fruit tissue.
6
Host cells
In a further aspect the invention provides a host cell comprising a construct of the invention. In
a further aspect the invention provides a host cell genetically modified to comprise a
polynucleotide of the invention. In a further aspect the invention provides a host cell genetically
modified to express a polynucleotide of the invention.
Host cell also expressing a TAG synthesising enzyme
In a further embodiment the host cell is also genetically modified to express a triacylglycerol
(TAG) synthesising enzyme. In a further embodiment the host cell is genetically modified to
comprise a nucleic acid sequence encoding a triacylglycerol (TAG) synthesising enzyme. In a
further embodiment the host cell comprises an expression construct including a nucleic acid
sequence encoding a triacylglycerol (TAG) synthesising enzyme.
In a further embodiment the nucleic acid is operably linked to a promoter sequence. In a further
embodiment the promoter sequence is capable of driving expression of the nucleic acid sequence
in a vegetative tissue of a plant. In one embodiment the promoter sequence is capable of driving
expression of the nucleic acid sequence in a seed of a plant. In one embodiment the promoter
sequence is capable of driving expression of the nucleic acid sequence in the pollen of a plant.
In a further embodiment the promoter sequence is capable of driving expression of the
polynucleotide in an E. coli cell. In a further embodiment the promoter sequence is capable of
driving expression of the polynucleotide in a yeast cell. In a further embodiment the promoter
sequence is capable of driving expression of the polynucleotide in an algal cell.
Host cell types
The host cell may be any type of cell. In on embodiment the host cell is a prokaryotic cell. In a
further embodiment the host cell is a eukaryotic cell. In one embodiment the host cell is selected
from a bacterial cell, a yeast cell, a fungal cell, an insect cell, algal cell, and a plant cell. In one
embodiment the host cell is a bacterial cell. In a further embodiment the host cell is a yeast cell.
In further embodiment the host cell is a fungal cell. In further embodiment the host cell is an
insect cell. In further embodiment the host cell is an algal cell. In a further embodiment the host
cell is a plant cell.
Plants
7
In a further aspect the invention provides a plant comprising a plant cell of the invention. In a
further aspect the invention provides a plant comprising a construct of the invention. In a further
aspect the invention provides a plant genetically modified to comprise a polynucleotide of the
invention. In a further aspect the invention provides a plant genetically modified to express a
polynucleotide of the invention. In a further embodiment the plant expresses a modified oleosin
encoded by the polynucleotide of the invention.
In a further embodiment the modified oleosin is expressed in a vegetative tissue of the plant. In
a further embodiment the modified oleosin is expressed in a seed of the plant. In a further
embodiment the modified oleosin is expressed in the pollen of the plant.
Plant also expresses a TAG enzyme
In a further embodiment the plant is also genetically modified to express a triacylglycerol (TAG)
synthesising enzyme. In a further embodiment the triacylglycerol (TAG) synthesising enzyme is
expressed in the same tissue as the modified oleosin.
In a further embodiment the plant is genetically modified to comprise a nucleic acid sequence
encoding a triacylglycerol (TAG) synthesising enzyme. In a further embodiment the plant
comprises an expression construct including a nucleic acid sequence encoding a triacylglycerol
(TAG) synthesising enzyme.
In a further embodiment the nucleic acid is operably linked to a promoter sequence.
In a further embodiment the promoter sequence is capable of driving expression of the nucleic
acid sequence in a vegetative tissue of a plant. In one embodiment the promoter sequence is
capable of driving expression of the nucleic acid sequence in a seed of a plant. In one
embodiment the promoter sequence is capable of driving expression of the nucleic acid sequence
in the pollen of a plant.
Modified oleosin polypeptides with artificially introduced cysteines
In a further aspect the invention provides a modified oleosin including at least one artificially
introduced cysteine. In a further aspect the invention provides a modified oleosin encode by a
polynucleotide of the invention. In one embodiment, the modified oleosin includes at least two
cysteines, at least one of which is artificially introduced. In a further embodiment, the modified
oleosin includes at least two to at least thirteen (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14 or
more) artificially introduced cysteines.
8
In a further embodiment the modified oleosin includes at least one cysteine in the N-terminal
hydrophilic region, and at least one cysteine in the C-terminal hydrophilic region. In a preferred
embodiment the cysteins are artificially introduced in the N-terminal hydrophilic region of the
oleosin, or in the C-terminal hydrophilic region of the oleosin. Preferablly the cysteins are
distributed substantially evenly between the N-terminal and C-terminal hydrophilic region of the
oleosin.
Fusion proteins with modified oleosins including artificially introduced cysteines
In a further aspect the invention provides a fusion protein comprising a modified oleosin of the
invention and a protein of interest. The fusion protein thus comprises a modified oleosin
portion, and a protein of interest portion.
Oil bodies comprising modified oleosins
In a further aspect the invention provides an oil body comprising a modified oleosin of the
invention. In a further aspect the invention provides an oil body comprising at least two
modified oleosins of the invention. In one embodiment at least two of the modified oleosins are
cross-linked to each other via disulphide bridges between cysteine residues in the modified
oleosins. In a further embodiment the modified oleosins are cross-linked via the artificially
introduced cysteine residues in the modified oleosins.
In a further embodiment the oil body additionally comprises a fusion protein, wherein the fusion
protein includes an oleosin fused to a protein of interest. In this embodiment, the oleosin in the
fusion protein need not include an artificially introduced cysteine. Preferably the oleosin in the
fusion protein does not include an artificially introduced cysteine.
The oil bodies of this embodiment are useful for purifying and delivering the protein of interest,
as discussed in Roberts et al., (2008).
However in this embodiment it is possible to take advantage of the option to vary the
stability/integrity of the oil body provided by presence of the modified oleosins in the oil body,
hence allowing for more stringent purification and delivery procedures.
Oil bodies comprising fusion proteins with modified oleosisn
In a further aspect the invention provides an oil body comprising a fusion protein of the
invention, the fusion protein comprising a modified oleosin of the invention and a protein of
9
interest. The fusion protein thus comprises a modified oleosin portion, and a protein of interest
portion.
In one embodiment the oil body comprises at least two fusion proteins of the invention.
In one embodiment at least two of the fusion proteins are cross-linked to each other via
disulphide bridges between cysteine residues in the modified oleosin portion of the fusion
proteins. In one embodiment the fusion proteins are cross-linked via the artificially introduced
cysteine residues in the modified oleosin portion of the fusion proteins.
In a further embodiment the oil body comprises at least one modified oleosin of the invention.
In a further embodiment at least one fusion protein is cross-linked to at least one modified
oleosin, via a cysteine in the modified oleosin portion of the fusion protein and a cysteine in the
modified oleosin.
Again, the oil bodies of this embodiment are useful for purifying and delivering the protein of
interest, as discussed in Roberts et al., (2008).
However in this embodiment it is possible to take advantage of the option to vary the
stability/integrity of the oil body provided by presence of the modified oleosins in the oil body,
hence allowing for more stringent purification and delivery procedures.
Emulsion
In a further aspect the invention provides an emulsion comprising a modified oleosin of the
invention. In one embodiment the emulsion comprises the modified oleosin and a suitable
carrier. The carrier may be buffered, with the appropriate redox environment to retain the
desired degree of cross-linking of the oleosins.
To resuspend the modified oleosin in the carrier may require sonication or high pressure
homogenising, followed by exposure to the appropriate oxidising conditions.
Compositions
In a further aspect the invention provides a composition comprising a modified oleosin of the
invention. In one embodiment the composition comprises the modified oleosin and a suitable
carrier. The carrier may be buffered, with the appropriate redox environment to attain the
desired degree of cross-linking of the modified oleosins.
10
To resuspend the modified oleosins in the carrier may require sonication or high pressure
homogenising, followed by exposure to the appropriate oxidising conditions.
In a further aspect the invention provides a composition comprising an oil body of the invention.
In one embodiment the composition comprises the oil body and a suitable carrier. The carrier
may be buffered, with the appropriate redox environment to retain the desired degree of crosslinking
of the modified oleosins. In a further embodiment the invention provides a composition
formulated for dermal application comprising an oil body of the invention.
Plants, and parts thereof, comprising oil bodies of the invention
In a further aspect the invention provides a plant, or part thereof, comprising an oil body of the
invention. In a further aspect the invention provides a vegetative tissue of a plant, comprising an
oil body of the invention. In a further aspect the invention provides a seed of a plant, comprising
an oil body of the invention.
Animal feed comprising oil bodies of the invention
In a further aspect the invention provides an animal feed comprising an oil body of the invention.
In a further aspect the invention provides an animal feed comprising a plant, or part thereof, of
the invention.
Methods for producing oil bodies
In a further aspect invention provides a method for producing an oil body, the method
comprising the step of combining:
a) at least two modified oleosins, each including at least one artificially introduced cysteine,
b) triacylglycerol, and
c) phospholipid.
In one embodiment, the modified oleosins each include at least two cysteines, at least one of
which is artificially introduced. In a further embodiment the modified oleosins each include at
least one cysteine in the N-terminal hydrophilic region of the oleosin, and at least one cysteine in
the C-terminal hydrophilic region of the oleosin.
In a further embodiment, the modified oleosin includes at least two to at least thirteen (i.e., 2, 3,
4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14 or more) artificially introduced cysteines.
11
In one embodiment the cysteines are artificially introduced in the N-terminal hydrophilic region
of the oleosins, or in the C-terminal hydrophilic region of the oleosins. In a further embodiment
the cysteines are distributed substantially evenly between the N-terminal and C-terminal
hydrophilic region of the oleosins. In a further embodiment the modified oleosins are crosslinked
via disulphide bridges between cysteine residues in the oleosins. In a further embodiment
embodiment the modified oleosins are cross-linked between the artificially introduced cysteine
residues in the oleosins.
In one embodiment the modified oleosins are part of fusion proteins wherein the fusion proteins
comprise a modified oleosin, and a protein of interest.
In one embodiment the method comprises the additional step of regulating the degree of crosslinking
of modified oleosins in the oil body by controlling the redox environment of the oil body
produced.
All components combined in vivo (in vivo oil bodies)
In one embodiment the components of a), b) and c) are combined within a host cell. In this
embodiment the modified oleosins are preferably expressed in the host cell.
The host cell is preferably genetically modified to express the modified oleosins.
The host cell is preferably comprises a construct of the invention. The host cell is preferably
genetically modified to comprise a polynucleotide of the invention. The host cell is preferably
genetically modified to express a polynucleotide of the invention.
Host cell also expresses a TAG synthesising enzyme
In a further embodiment the host cell is also genetically modified to express a triacylglycerol
(TAG) synthesising enzyme. In a further embodiment the host cell comprises an expression
construct including a nucleic acid sequence encoding a triacylglycerol (TAG) synthesising
enzyme.
In a further embodiment the nucleic acid sequence is operably linked to a promoter sequence. In
one embodiment the promoter sequence is capable of driving expression of the nucleic acid
sequence in a vegetative tissue of a plant. In one embodiment the promoter sequence is capable
of driving expression of the nucleic acid sequence in a seed of a plant. In one embodiment the
promoter sequence is capable of driving expression of the nucleic acid sequence in the pollen of
a plant.
12
In a further embodiment the host cell is also genetically modified to comprise a nucleic acid
sequence encoding a triacylglycerol (TAG) synthesising enzyme. In a further embodiment the
host cell is also genetically modified to express a nucleic acid sequence encoding a
triacylglycerol (TAG) synthesising enzyme.
It will be understood by those skilled in the art that the polynucleotide encoding the modified
oleosin and the nucleic acid sequence encoding a triacylglycerol (TAG) synthesising enzyme can
be placed on the same construct or on separate constructs to be transformed into the host cell.
Expression of each can be driven by the same or different promoters, which may be incuded in
the construct to be transformed. It will also be understood by those skilled in the art that
alternatively the polynucleotide and nucleic acid can be transformed into the cell without a
promoter, but expression of either the polynucleotide and nucleic acid could be driven by a
promoter or promoters endogenous to the cell transformed.
In a further embodiment the host cell forms part of an organism. In a preferred embodiment the
organism is a plant.
In a further embodiment the oil is produced in the vegetative tissues of the plant.
In one embodiment of the method the plant acumulates about 50% to about 400% more lipid
than does a suitable control plant. In a further embodiment of the method the plant acumulates
about 100% to about 300% more lipid than does a suitable control plant. In a further
embodiment of the method the plant acumulates about 150% to about 250% more lipid than does
a suitable control plant. Suitable control plants include non-transformed or wild-type versions of
plant of the same variety and or species as the transformed plant used in the method of the
invention.
In a further embodiment the plant is processed into an animal feed.
In a further embodiment the plant is processed into a biofuel feed stock.
Additional method step to purify the in vivo produced oil bodies
In one embodiment the method includes the additional step of purifying the oil bodies from the
cell or organisim.
Additional method step to vary degree of cross-linking of in vivo produced purified oil bodies
13
In a further embodiment the method comprises the additional step of regulating the degree of
cross-linking of modified oleosins in the in vivo produced purified oil bodies
by controlling the redox environment of the purified oil bodies. In one embodiment the degree
of cross-linking is increased by use of an oxidising environment. In a further embodiment the
degree of cross-linking is decreased by use of a reducing environment.
Components combined in vitro (in vitro / artificial oil bodies)
In certain embodiments the components of a), b) and c) may be combined in vitro.
In one embodiment, the modified oleosin of a) has been recombinantly expressed in, and purified
from a host cell of the invention, before being combined with the components of b) and c).
Additional method step to vary degree of cross-linking of in vitro / artificial oil bodies
In a further embodiment the method comprises the additional step of regulating the degree of
cross-linking by controlling the redox environment in which the components of a), b) and c) are
combined. In one embodiment the degree of cross-linking is increased by combining the
components of a), b) and c) in on oxidising environment. In a further embodiment the degree of
cross-linking is decreased by combining the components of a), b) and c) in a reducing
environment. The degree of cross-linking may also be regulated after the oil body is formed, by
controlling the redox environment in which the oil body is contained.
In a further aspect the invention provides a method of producing a plant that accumulates more
oil than a suitable control plant the method comprising providing a plant transformed with a
polynucleotide of the invention that expresses a modified oleosin encode by the polynucleotide.
In one embodiment the plant is also transformed with a polynucleotide encoding a TAG
synthesising enzyme to express the TAG synthesising enzyme and thus synthesise TAG.
In one embodiment the plant the plant is produced by transforming a single plant, or plant cell,
with both the polynucleotide of any one the invention and the polynucleotide encoding the TAG
synthesising enzyme.
In a further embodiment the plant is produced by crossing a first plant transformed with a
polynucleotide of any one of the invention, with second plant transformed the polynucleotide
encoding the TAG synthesising enzyme, to produce the plant transformed with both a
polynucleotide of the invention, and a polynucleotide encoding the TAG synthesising enzyme.
14
In a further embodiment the oil is TAG. In a further embodiment the oil is produced in the
vegetative tissues of the plant.
In one embodiment of the method the plant acumulates about 50% to about 400% more lipid
than does a suitable control plant. In a further embodiment of the method the plant acumulates
about 100% to about 300% more lipid than does a suitable control plant. In a further
embodiment of the method the plant acumulates about 150% to about 250% more lipid than does
a suitable control plant
In a further embodiment the plant is processed into an animal feed.
In a further embodiment the plant is processed into a biofuel feed stock.
In a further aspect invention provides a method for producing an oil body in a host cell, the
method comprising:
a) introducing into a host cell at least one nucleic acid molecule encoding a modified
oleosin of the invention; and
b) culturing the host cell in order to express the modified oleosin.
In a further aspect invention provides a method for producing an oil body in a host cell, the
method comprising:
a) introducing into a host cell at least one nucleic acid molecule encoding a modified
oleosin of the invention and a nucleic acid molecule encoding a TAG synthesizing
enzyme ; and
b) culturing the host cell in order to express the modified oleosin and the TAG
synthesizing enzyme.
The host cell may be a host cell as herein described.
Oil bodies
In a further aspect invention provides an oil body produced by a method of the invention.
Compositions
In a further aspect the invention provides a composition comprising an oil body of the invention.
In one embodiment the composition comprises the oil body and a suitable carrier. The carrier
may be buffered to provide the appropriate redox environment to retain the desired degree of
15
cross-linking of the modified oleosin. In a further embodiment the invention provides a
composition formulated for dermal application comprising an oil body of the invention.
Plants, and parts thereof, comprising oil bodies of the invention
In a further aspect the invention provides a plant, or part thereof, comprising an oil body of the
invention. In a further aspect the invention provides a vegetative tissue of a plant, comprising an
oil body of the invention. In a further aspect the invention provides a seed of a plant, comprising
an oil body of the invention. In a further aspect the invention provides pollen of a plant,
comprising an oil body of the invention. In a further aspect the invention provides a fruit, or
fruiting body, of a plant, comprising an oil body of the invention.
Animal feed comprising oil bodies of the invention
In a further aspect the invention provides an animal feed comprising an oil body of the invention.
In a further aspect the invention provides an animal feed comprising a plant, or part thereof, of
the invention.
In one embodiment the feed is suitable for a mammalian animal including humans. In a further
embodiment the feed is suitable for non-human mammals. Preferred animals include farm
animals such as but not limited to cows, sheep, horses, goats, pigs, chickens, and the like.
Plants
The modified oleosins may be modified naturally occurring oleosins. The plants from which the
un-modified oleosin sequences are derived may be from any plant species that contains oleosins
and polynucleotide sequences encoding oleosins.
The plant cells, in which the modified oleosins are expressed, may be from any plant species.
The plants, in which the modified oleosins are expressed, may be from any plant species.
In one embodiment the plant cell or plant, is derived from a gymnosperm plant species.
In a further embodiment the plant cell or plant, is derived from an angiosperm plant species.
In a further embodiment the plant cell or plant, is derived from a from dicotyledonous plant
species.
In a further embodiment the plant cell or plant, is derived from a monocotyledonous plant
species.
16
Other preferred plants are forage plant species from a group comprising but not limited to the
following genera: Zea, Lolium, Hordium, Miscanthus, Saccharum, Festuca, Dactylis, Bromus,
Thinopyrum, Trifolium, Medicago, Pheleum, Phalaris, Holcus, Glycine, Lotus, Plantago and
Cichorium.
Other preferred plants are leguminous plants. The leguminous plant or part thereof may
encompass any plant in the plant family Leguminosae or Fabaceae. For example, the plants may
be selected from forage legumes including, alfalfa, clover; leucaena; grain legumes including,
beans, lentils, lupins, peas, peanuts, soy bean; bloom legumes including lupin, pharmaceutical or
industrial legumes; and fallow or green manure legume species.
A particularly preferred genus is Trifolium. Preferred Trifolium species include Trifolium
repens; Trifolium arvense; Trifolium affine; and Trifolium occidentale. A particularly preferred
Trifolium species is Trifolium repens.
Another preferred genus is Medicago. Preferred Medicago species include Medicago sativa and
Medicago truncatula. A particularly preferred Medicago species is Medicago sativa, commonly
known as alfalfa.
Another preferred genus is Glycine. Preferred Glycine species include Glycine max and Glycine
wightii (also known as Neonotonia wightii). A particularly preferred Glycine species is Glycine
max, commonly known as soy bean. A particularly preferred Glycine species is Glycine wightii,
commonly known as perennial soybean.
Another preferred genus is Vigna. A particularly preferred Vigna species is Vigna unguiculata
commonly known as cowpea.
Another preferred genus is Mucana. Preferred Mucana species include Mucana pruniens. A
particularly preferred Mucana species is Mucana pruniens commonly known as velvetbean.
Another preferred genus is Arachis. A particularly preferred Arachis species is Arachis glabrata
commonly known as perennial peanut.
Another preferred genus is Pisum. A preferred Pisum species is Pisum sativum commonly
known as pea.
Another preferred genus is Lotus. Preferred Lotus species include Lotus corniculatus, Lotus
pedunculatus, Lotus glabar, Lotus tenuis and Lotus uliginosus. A preferred Lotus species is
Lotus corniculatus commonly known as Birdsfoot Trefoil. Another preferred Lotus species is
17
Lotus glabar commonly known as Narrow-leaf Birdsfoot Trefoil. Another preferred preferred
Lotus species is Lotus pedunculatus commonly known as Big trefoil. Another preferred Lotus
species is Lotus tenuis commonly known as Slender trefoil.
Another preferred genus is Brassica. A preferred Brassica species is Brassica oleracea,
commonly known as forage kale and cabbage.
Other preferred species are oil seed crops including but not limited to the following genera:
Brassica, Carthumus, Helianthus, Zea and Sesamum.
A preferred oil seed genera is Brassica. A preferred oil seed species is Brassica napus.
A preferred oil seed genera is Brassica. A preferred oil seed species is Brassica oleraceae.
A preferred oil seed genera is Zea. A preferred oil seed species is Zea mays.
A preferred oil seed genera is Carthamus. A preferred oil seed species is Carthamus tinctorius.
A preferred oil seed genera is Helianthus. A preferred oil seed species is Helianthus annuus.
A preferred oil seed genera is Zea. A preferred oil seed species is Zea mays.
A preferred oil seed genera is Sesamum. A preferred oil seed species is Sesamum indicum.
A preferred silage genera is Zea. A preferred silage species is Zea mays.
A preferred grain producing genera is Hordeum. A preferred grain producing species is
Hordeum vulgare.
A preferred grazing genera is Lolium. A preferred grazing species is Lolium perenne.
A preferred grazing genera is Lolium. A preferred grazing species is Lolium arundinaceum.
A preferred grazing genera is Trifolium. A preferred grazing species is Trifolium repens.
A preferred grazing genera is Hordeum. A preferred grazing species is Hordeum vulgare.
Preferred plants also include forage, or animal feedstock plants. Such plants include but are not
limited to the following genera: Miscanthus, Saccharum, Panicum.
A preferred biofuel genera is Miscanthus. A preferred biofuel species is Miscanthus giganteus.
18
A preferred biofuel genera is Saccharum. A preferred biofuel species is Saccharum officinarum.
A preferred biofuel genera is Panicum. A preferred biofuel speices is Panicum virgatum.
DETAILED DESCRIPTION OF THE INVENTION
In this specification where reference has been made to patent specifications, other external
documents, or other sources of information, this is generally for the purpose of providing a
context for discussing the features of the invention. Unless specifically stated otherwise,
reference to such external documents is not to be construed as an admission that such documents,
or such sources of information, in any jurisdiction, are prior art, or form part of the common
general knowledge in the art.
The term “comprising” as used in this specification means “consisting at least in part of”. When
interpreting each statement in this specification that includes the term “comprising”, features
other than that or those prefaced by the term may also be present. Related terms such as
“comprise” and “comprises” are to be interpreted in the same manner.
On a weight for weight basis lipids have approximately double the energy content of either
proteins or carbohydrates. The bulk of the world’s lipids are produced by plants and the densest
form of lipid is as a triacylglycerol (TAG). Dicotyledonous plants can accumulate up to
approximately 60% of their seed weight as TAG which is subsequently used as an energy source
for germination. As such there have been a number of efforts targeted at using seeds rich in oils
to sustainably produce sufficient lipids for both animal and biofuel feed stock.
Given that there is only a limited quantity of TAG able to be produced by seeds alternative
approaches are being made to produce additional lipid (preferentially TAGs) in vegetative
tissues. The majority of these approaches have pursued the up regulation or over expression of
one or several enzymes in the Kennedy pathway in the leaves of plants in order to synthesise
TAG. Typically however, the majority of additional lipids produced by this approach are remobilised
within the plant by a combination of lipases and β-oxidation resulting in a limited
increase in lipid content (usually 2-4% of the DM).
The TAG produced in developing seeds is typically contained within discreet structures called
oil bodies (OBs) which are highly stable and remain as discrete tightly packed organelles
without coalescing even when the cells desiccate or undergo freezing conditions (Siloto et al.,
2006; Shimada et al., 2008). OBs consist of a TAG core surrounded by a phospholipid
monolayer embedded with proteinaceous emulsifiers. The latter make up 0.5-3.5% of the OB; of
19
this, 80-90% is oleosin with the remainder predominantly consisting of the calcium binding
(caloleosin) and sterol binding (steroleosin) proteins (Lin and Tzen, 2004). The emulsification
properties of oleosins derives from their three functional domains which consist of an
amphipathic N-terminal arm, a highly conserved central hydrophobic core (~72 residues) and a
C-terminal amphipathic arm. Similarly, both caloleosin and steroleosin possess hydrophilic N
and C-terminal arms and their own conserved hydrophobic core.
It was previously speculated that the constitutive expression of oleosin or polyoleosin (tandem
head-to-tale fusions of oleosins) with TAG synthesising enzymes in the leaves would result in
the formation of stable oil bodies leading to the accumulation of TAG. We have subsequently
found however, that oleosin and polyoleosins are ineffective and promoting the accumulation of
TAG when co-expressed with DGAT1 in plant leaves (Roberts et al., unpublished data).
The current invention provides modified oleosins which contain one or more artificially
introduced cysteine residues. The encapsulation of the neutral lipids by oleosins containing
engineered cysteines provides an alternative mechanism to accumulate appreciable quantities of
TAG in leaves without the requirement to wait until senescence and without producing extreme
phenotypes. In addition the modified oleosin has a number of other applications involving
modifying OB stability, emulsion properties as well as the generation and purification of
recombinant proteins.
Oil bodies
OBs generally range from 0.5-2.5μm in diameter and consist of a TAG core surrounded by a
phospholipid monolayer embedded with proteinaceous emulsifiers - predominantly oleosins
(Tzen et al, 1993; Tzen, et al 1997). OBs consist of only 0.5-3.5% protein; of this 80-90% is
oleosin with the remainder predominantly consisting of the calcium binding (caleosin) and sterol
binding (steroleosin) proteins (Lin and Tzen, 2004). The ratio of oleosin to TAG within the
plant cell influences the size and number of oil bodies within the cell (Sarmiento et al., 1997;
Siloto et al., 2006).
While OBs are naturally produced predominantly in the seeds and pollen of many plants they are
also found in some other organs (e.g., specific tubers).
Oleosins are comparatively small (15 24 kDa) proteins that are embedded in the surface of OBs.
Oil body stability
20
The suitability of oil bodies, and artificial oil bodies, for the applications discussed above,
among others, is limited at least in part, by their stability. One approach to address oil body
stability was to generate oil bodies comprising so-called polyoleosin. Polyoleosin is the head to
tail fusion of two or more oleosin units (Roberts et al., 2008). Altering the number of oleosin
units enables the properties (thermal stability and degradation rate) of the oil bodies to be
tailored. Expression of polyoleosin in planta leads to incorporation of the polyoleosin units to
the oil bodies as per single oleosin units (Scott et al., 2007). Multiple oleosin units in tandem
head-to-tail arrangements were used to create polyoleosin. Separate constructs (containing from
one to six oleosin repeats) were specifically designed for expression in planta and in E. coli.
The majority of recombinant polyoleosin accumulated in the oil bodies of transgenic plants and
in the inclusion bodies of E. coli. Purified prokaryotically produced polyoleosin was used to
generate artificial oil bodies. Oil body and artificial oil body thermal stability and structural
integrity in proteinase-K were raised by polyoleosin.
However, there are several limiting factors determining the degree of protection/stability that
polyoleosin can provide; these relate to the number of tandem repeats that can be joined before
the process of translation and oil body targeting becomes limiting (Scott et al., 2007); while
another limitation comes from the nature of the oleosin fusion which is achieved by generating a
transcript with a head to tail fusion arrangement. This is essentially a linear protein of
multimeric oleosin repeats that has a number of covalent-links and position of covalent-links per
individual oleosin repeat (i.e., a maximum of one at each end). In addition this arrangement only
affords protection against N-terminal degrading proteins but it does not provide any additional
protection against other proteolytic enzymes that recognise specific internal peptide sequences.
Furthermore, the linking between oleosin units in a polyoleosin molecule formed by tandem
head to tail repeats is not readily altered in situ. While specific protease specific sites could be
engineered into the joining regions in order to break apart fused polyoleosin molecules
embedded into an oil body or artificial oil body they could not be re-fused easily.
Oleosins embedded in oil bodies have previously covalently cross-linked by the addition of
cross-linking agents such as glutaraldehyde or genepin (Peng et al., 2004 & 2006), however, this
random cross-linking requires the addition of cross-linking agents to oil body preparations, and
is not easy to reverse.
Artificial oil bodies
21
Prokaryotically expressed recombinant oleosins can be used to generate artificial oil bodies
(AOBs) who’s properties are very similar to plant derived OBs (Peng et al. 2004; Roux et al.
2004; Chiang et al. 2005; Chiang et al. 2007).
Applications of oil bodies and artificial oil bodies
The unique properties of oil bodies, and their constituent oleosins, form the basis of a number of
biotechnical applications including: purifying recombinant proteins; formation of multimeric
protein complexes; emulsification; delivery of bioactives; generation of multivalent bioactives
and even as a potential flavour enhancer (for reviews see Capuano et al., 2007 and Roberts et al.,
2008).
Emulsions
Emulsions are produced when one or more liquids that are immiscible in another liquid, usually
due to different polarities and thus different hydrophobicities, are uniformly suspended within
that liquid. Examples include oil droplets uniformly dispersed in water, or water droplets
uniformly dispersed in oil. Generation of a relatively stable emulsion requires the use of an
emulsifier, which lowers the interfacial tension between the liquids. The stability of an emulsion
is generally measured in terms of the duration that the uniform dispersion persists under
specified conditions. Emulsifiers are commonly used in the food and cosmetic industry; so need
to have high emulsion stability and be safe for consumption and topical application.
Intact oil bodies containing oleosin naturally form a surfactant-free, oil-in water emulsion. It has
been found that intact oil bodies or oil bodies in which the majority of TAG has been removed
have a broad range of emulsification applications in food, topical personal care (skin creams)
and pharmaceutical formulations (Harada et al., 2002; Deckers et al., 2003; Hou et al., 2003).
Biohydrogenation
It has been demonstrated that the lipid profile of ruminant animal feed in turn influences the lipid
profile of meat and dairy products (Demeyer and Doreau, 1999). Different plants have different
lipid profiles; by selectively feeding animals only plants with the desired lipid profile it is
possible to positively influence the lipid profile of downstream meat and dairy products. In
ruminants the final lipid make up of the meat and milk is not only influenced by the dietary
lipids but is also heavily influenced by biohydrogenation (Jenkins and McGuire 2006; Firkins et
al., 2006; Lock and Bauman, 2004). Biohydrogenation is the hydrogenation of non-reduced
compounds (such as unsaturated fats) by the biota present in the rumen. Biohydrogenation can
22
be prevented/delayed by encapsulating the lipids in a protein or proteins that provide resistance
to microbial degradation (Jenkins and Bridges 2007). The prevention of biohydrogenation by
encapsulating triacylglycerides in polyoleosin or oleosins in planta was reported by Scott et al.,
(2007), Cookson et al., (2009) and Roberts et al., (2008).
Oleosins
Oleosins are comparatively small (15 to 24 kDa) proteins which allow the OBs to become tightly
packed discrete organelles without coalescing as the cells desiccate or undergo freezing
conditions (Leprince et al., 1998; Siloto et al., 2006; Slack et al., 1980; Shimada et al.2008).
Oleosins have three functional domains consisting of an amphipathic N-terminal arm, a highly
conserved central hydrophobic core (~72 residues) and a C-terminal amphipathic arm. The
accepted topological model is one in which the N- and C-terminal amphipathic arms are located
on the outside of the OBs and the central hydrophobic core is located inside the OB (Huang,
1992; Loer and Herman, 1993; Murphy 1993). The negatively charged residues of the N- and Cterminal
amphipathic arms are exposed to the aqueous exterior whereas the positively charged
residues are exposed to the OB interior and face the negatively charged lipids. Thus, the
amphipathic arms with their outward facing negative charge are responsible for maintaining the
OBs as individual entities via steric hinderance and electrostatic repulsion both in vivo and in
isolated preparation (Tzen et al, 1992). The N-terminal amphipathic arm is highly variable and
as such no specific secondary structure can describe all examples. In comparison the C-terminal
arm contains a α-helical domain of 30-40 residues (Tzen et al, 2003). The central core is highly
conserved and thought to be the longest hydrophobic region known to occur in nature; at the
center is a conserved 12 residue proline knot motif which includes three spaced proline residues
(for reviews see Frandsen et al, 2001; Tzen et al, 2003). The secondary, tertiary and quaternary
structure of the central domain is still unclear. Modelling, Fourier Transformation-Infra Red
(FT-IR) and Circular Dichromism (CD) evidence exists for a number of different arrangements
(for review see Roberts et al., 2008).
The properties of the major oleosins is relatively conserved between plants and is characterised
by the following:
 15-25kDa protein corresponding to approximately 140-230 amino acid residues.
 The protein sequence can be divided almost equally along its length into 4 parts which
correspond to a N-terminal hydrophilic region, two centre hydrophobic regions (joined
by a proline knot or knob) and a C-terminal hydrophilic region.
23
 The topology of oleosin is attributed to its physical properties which includes a folded
hydrophobic core flanked by hydrophilic domains. This arrangement confers an
amphipathic nature to oleosin resulting in the hydrophobic domain being embedded in
the phospholipid monolayer (Tzen et al., 1992) while the flanking hydrophilic domains
are exposed to the aqueous environment of the cytoplasm.
 Typically oleosins do not contain cysteines
Preferred oleosins for use in the invention are those which contain a central domain of
approximately 70 non-polar amino acid residues (including a proline knot) uninterrupted by any
charged residues, flanked by two hydrophilic arms.
The term “oleosin” as used herein also includes steroleosin and caloleosin
Steroleosins
Steroleosins comprises an N-terminal anchoring segment comprising two amphipathic α-helices
912 residues in each helix) connected by a hydrophobic anchoring region of 14 residues. The
soluble dehydrogenase domain contains a NADP+- binding subdomain and a sterol-binding
subdomain. The apparent distinction between steroleosins-A and –B occurs in their diverse
sterol-binding subdomains (Lin and Tzen, 2004). Steroleosins have a proline knob in their
hydrophobic domain and contains a sterol-binding dehydrogenase in one of their hydrophilic
arms.
Caloleosins
Caloleosins (Frandsen et al., 2001) have a slightly different proline knot than do the basic
oleosins, and contain a calcium-binding motif and several potential phosphorylation sites in the
hydrophilic arms. Similar to oleosin, caloleosin is proposed to have three structural domains,
where the N- and C-terminal arms are hydrophilic while the central domain is hydrophobic and
acts as the oil body anchor. The N-terminal hydrophilic domain consists of a helix-turn-helix
calcium binding EF-hand motif of 28 residues including an invariable glycine residue as a
structural turning point and five conserved oxygen-containing residues as calcium-binding
ligands (Chen et al., 1999; Frandsen et al., 2001). The C-terminal hydrophilic domain contains
several phosphorylation sites and near the C-terminus is an invariable cysteine that is not
involved in any intra- or inter-disulfide linkages (Peng, 2004). The hydrophilic N- and Ctermini
of caloleosin are approximately 3 times larger than those of oleosin (Lin and Tzen,
24
2004). The hydrophobic domain is thought to consist of an amphipathic α-helix and an
anchoring region (which includes a proline knot).
Examples of oleosin (oleosins, steroleosin and caloleosin) sequences suitable to be modified for
use in the invention, by the addition of at least one artificially introduced cysteine, are shown in
Table 1 below. The sequences (both polynucleotide and polypeptide are provided in the
Sequence Listing)
Table 1
Oleosin Species cDNA
accession no.
SEQ
ID
NO:
Protein
accession no.
SEQ
ID
NO:
Oleosin S. indicum AF302907 34 AAG23840 35
Oleosin S. indicum U97700 36 AAB58402 37
Oleosin A. thaliana X62353 38 CAA44225 39
Oleosin A. thaliana BT023738 40 AAZ23930 41
Oleosin H. annuus X62352.1 42 CAA44224.1 43
Oleosin B. napus X82020.1 44 CAA57545.1 45
Oleosin Z. mays NM_001153560.1 46 NP_001147032.1 47
Oleosin O.sativa AAL40177.1 48 AAL40177.1 49
Oleosin B.oleracea AF117126.1 50 AAD24547.1 51
Oleosin C. arabica AY928084.1 52 AAY14574.1 53
Steroleosin S. indicum AAL13315 54 AAL13315 55
Steroleosin A. napus EU678274 56 ACG69522 57
Steroleosin Z. mays NM_001159142.1 58 NP_001152614.1 59
Steroleosin B. napus EF143915.1 60 ABM30178.1 61
Caloleosin S. indicum AF109921 62 AAF13743 63
Caloleosin G. max AF004809 64 AAB71227 65
25
Caloleosin Z. mays NM_001158434.1 66 NP_001151906 67
Caloleosin B. napus AY966447.1 68 AAY40837 69
Caloleosin C. revoluta FJ455154.1 70 ACJ70083 71
Caloleosin C. sativus EU232173.1 72 ABY56103.1 73
Oleosin, steroleosin and caloleosins are well known to those skilled in the art. Further sequences
from many different species can be readily identified by methods well-known to those skilled in
the art. For example, further sequences can be easily identified by an NCBI Entrez Cross-
Database Search (available at http://www.ncbi.nlm.nih.gov/sites/gquery) using any one of the
terms oleosin, steroleosin and caloleosin.
Plant lipids biosynthesis
All plant cells produce fatty acids from actetyl-CoA by a common pathway localized in plastids.
Although a portion of the newly synthesized acyl chains is then used for lipid biosynthesis
within the plastid (the prokaryotic pathway), a major portion is exported into the cytosol for
glycerolipid assembly at the endoplasmic reticulum (ER) or other sites (the eukaryotic pathway).
In addition, some of the extraplastidial glycerolipids return to the plastid, which results in
considerable intermixing between the plastid and ER lipid pools (Ohlrogge and Jaworski 1997).
The simplest description of the plastidial pathway of fatty acid biosynthesis consists of two
enzyme systems: acetyl-CoA carboxylase (ACCase) and fatty acid synthase (FAS). ACCase
catalyzes the formation of malonyl-CoA from acetyl-CoA, and FAS transfers the malonyl moiety
to acyl carrier protein (ACP) and catalyzes the extension of the growing acyl chain with
malonyl-ACP.
The initial fatty acid synthesis reaction is catalyzed by 3-ketoacyl-ACP III (KAS III) which
results in the condensation of acetyl-CoA and malonyl-ACP. Subsequent condensations are
catalyzed by KAS I and KAS II. Before a subsequent cycle of fatty acid synthesis begins, the 3-
ketoacyl-ACP intermediate is reduced to the saturated acyl-ACP in the remaining FAS reactions,
catalyzed sequentially by the 3-ketoacyl-ACP reductase, 3 hydroxyacyl-ACP dehydrase, and the
enoyl-ACP reductase.
The final products of FAS are usually 16:0 and 18:0-ACP, and the final fatty acid composition of
a plant cell is in large part determined by activities of several enzymes that use these acyl-ACPs
26
at the termination phase of fatty acid synthesis. Stearoyl-ACP desatruase modifies the final
product of FAS by insertion of a cis double bond at the 9 position of the C18:0-ACP. Reactions
of fatty acid synthesis are terminated by hydrolysis or transfer of the acyl chain from the ACP.
Hydrolysis is catalyzed by acyl-ACP thioesterases, of which there are two main types: one
thioesterase relatively specific for 18:1-ACP and a second more specific for saturated acyl-
ACPs. Fatty acids that have been released from ACPs by thioesterases leave the plastid and
enter into the eukaryotic lipid pathway, where they are primarily esterified to glycerolipids on
the ER. Acyl transferases in the plastid, in contrast to thioesterases, terminate fatty acid
synthesis by transesterifying acyl moieties from ACP to glycerol, and they are an essential part
of the prokaryotic lipid pathway leading to plastid glycerolipid assembly.
Triacylglycerol biosynthesis
The only committed step in TAG biosynthesis is the last one, i.e. the addition of a third fatty acid
to an existing diacylglycerol, thus generating TAG. In plants this step is predominantly (but not
exclusively) performed by one of five (predominantly ER localised) TAG synthesising enzymes
including: acyl CoA: diacylglycerol acyltransferase (DGAT1); an unrelated acyl CoA:
diacylglycerol acyl transferase (DGAT2); a soluble DGAT (DGAT3) which has less than 10%
identity with DGAT1 or DGAT2 (Saha et al., 2006); phosphatidylcholine-sterol Oacyltransferase
(PDAT); and a wax synthase (WSD1, Li et al., 2008). The DGAT1 and DGAT2
proteins are eoncoded by two distinct gene families, with DGAT1 containing approximately 500
amino acids and 10 predicted transmembrane domains and DGAT2 has only 320 amino acids
and two transmembrane domains (Shockey et al., 2006).
The term “triacylglycerol synthesising enzyme” or “TAG synthesising enzyme” as used herein
means an enzyme capable of catalysing the addition of a third fatty acid to an existing
diacylglycerol, thus generating TAG. Preferred TAG synthesising enzymes include but are not
limited to: acyl CoA: diacylglycerol acyltransferase1 (DGAT1); diacylglycerol acyl transferase2
(DGAT2); phosphatidylcholine-sterol O-acyltransferase (PDAT) and cytosolic soluble form of
DGAT (soluble DGAT or DGAT3).
Given that endogenous DGAT1 and DGAT2 appear to play roles in mature and senescing leaves
(Kaup et al. 2002; Shockey et al. 2006), it is likely that plants possess a number of feedback
mechanisms to control their activity. Indeed, Zou et al. (2008) recently identified a consensus
sequence (X-Leu-X-Lys-X-X-Ser-X-X-X-Val) within Tropaeolum majus (garden nasturtium)
DGAT1 (TmDGAT1) sequences as a targeting motif typical of members of the SNF1-related
protein kinase-1 (SnRK1) with Ser being the residue for phosphorylation. The SnRK1 proteins
27
are a class of Ser/Thr protein kinases that have been increasingly implicated in the global
regulation of carbon metabolism in plants, e.g. the inactivation of sucrose phosphate synthase by
phosphorylation (Halford & Hardie 1998). Zou et al. (2008) went on to demonstrate that the
obliteration of a potential SnRK1 phosphorylation site in DGAT1 by single point mutation
(Ser197Ala of TmDGAT1) led to the accumulation of significantly higher levels of TAG in the
seed. This mutation increased activity by 38-80%, which led to a 20-50% increase in oil content
on a per seed basis in Arabidopsis.
Phospholipid:DGA acyltransferase (PDAT) forms TAG from a molecule of phospholipid and a
molecule of diacyglycerol. PDAT is quite active when expressed in yeast but does not
appreciably increase TAG yields when expressed in plant seeds. PDAT and a proposed
DAG:DAG transacylase are neutral lipid synthesizing enzymes that produce TAG, but are not
considered part of the Kennedy Pathway.
A combination of wax ester synthase and DGAT enzyme (WS/DGAT) has been found in all
neutral lipid producing prokaryotes studied so far. WS/DAGAT has extraordinary broad activity
on a variety of unusual fatty acids, alcohols and even thiols. This enzyme has a putative
membrane-spanning region but shows no sequence homology to the DGAT1 and DGAT2
families from eukaryotes or the WE synthase from jojoba (Jojoba is the only eukaryote that has
been found to accumulate wax ester).
It should be noted that Lecithin-Cholesterol AcylTransferase (LCAT) and Acylcoenzyme:
Cholesterol AcylTransferase (ACAT) are enzymes that produce sterol esters (a form
of neutral lipid) not TAGs.
In applications requiring the increase of neutral lipids evidence suggests that the higher activity
and broader specificity of DGAT1 relative to DGAT2 is preferential. Where a specific fatty acid
is preferred, such as a long-chain PUFA, DGAT1 is still applicable, provided it accepts the fatty
acid of choice. Plants generally incorporate long chain PUFAs in the sn-2 position. It is not
known whether this is due to high activity of LPAT or low activity of DGAT1 on this substrate.
For the improved specificity for PUFAs, a DGAT2 that prefers these fatty acids may be
preferable, or the properties of DGAT1 could be altered using directed evolution or an
equivalent procedure.
Examples of these TAG synthesising enzymes, suitable for use in the methods and compositions
of the invention, from members of several plant species are provided in Table 2 below. The
sequences (both polynucleotide and polypeptide are provided in the Sequence Listing)
28
Table 2
TAG
synthesising
enzyme
Species cDNA
accession no.
SEQ
ID
NO:
Protein
accession no.
SEQ
ID
NO:
DGAT1 A. thaliana NM_127503 74 NP_179535 75
DGAT1 T. majus AY084052 76 AAM03340 77
DGAT1 Z. mays EU039830 78 ABV91586 79
DGAT2 A. thaliana NM_115011 80 NP_566952 81
DGAT2 B. napus FJ858270 82 AC090187 83
DGAT3
(soluble
DGAT)
A. hypogaea AY875644 84 AAX62735 85
PDAT A. thaliana NM_121367 86 NP_196868 87
PDAT R.
communis
XM_00252130
4
88 XP_002521350 89
The inventions also contemplates use of modified TAG synthesizing enzymes, that are modified
(for example in their sequence by substitutions, insertions or additions an the like) to alter their
specificity and or activity.
TAG accumulation in leaves
A recent field survey of 302 angiosperm species in the north-central USA found that 24% have
conspicuous cytosolic oil droplets in leaves, with usually one large oil droplet per mesophyll cell
(Lersten et al., 2006 [from Slocombe et al 2009]). The role of cytosolic leaf TAG is thought to
be involved in carbon storage and/or membrane lipid re-modelling (for review see Slocombe et
al., 2009). Indeed, in senescing leaves, plastidial fatty acids are partitioned into TAG prior for
further mobilization, and DGAT1 is thought to be instrumental in this process (Kaup et al.,
2002).
29
There have been several attempts to engineer plants to accumulate elevated levels of TAG in
their leaves. The success of these has been somewhat limited by the relatively low level of TAG
that accumulated and in some cases the majority of TAG accumulated in senescing leaves only,
thus limiting the flexibility of harvesting and proportion of crop accumulating TAG at any one
time (Bouvier-Nave et al, 2001; Xu et al., 2005; Winichayakul et al., 2008; Andrianov et al.,
2010; Slocombe et al., 2009 and references therein).
To date the attempts to accumulate TAG in leaves have predominantly focussed on three
particular gene candidates including over expression of DGAT (TAG biosynthesis), mutation of
TGD1 or CTS (resulting in the prevention of lipid remobilisation), and over expression of LEC1,
LEC2 and WRI1 (transcriptional factors involved in storage oil and protein accumulation in
developing seeds). Over expression of TAG and other neutral lipid synthesizing enzymes relies
on the presence of sufficient substrate, in the expanding and or mature leaf this is assumed to be
provided by the plastid (chloroplast in the case of the leaf) which synthesises lipids for
membranes. In photosynthetic leaves of Arabidopsis it has been estimated that the turnover of
membrane lipids is 4% of total fatty acids per day (Bao et al, 2000). In senescing leaves, the
existing plastidal membranes provide the bulk of fatty acids for partitioning into TAG prior to
further mobilization.
Over-expression of the Arabidopsis DGAT1 gene in tobacco leaves results in enhanced TAG
accumulation (Bouvier-Nave et al., 2001), this was later repeated and quantified by Andrianov et
al., (2010). They calculated the TAG level increased 20 fold and lead to a doubling of lipid
content from ~3% to ~6% of dry matter in mature leaves. A further increase to 6.8% was
achieved by the over expression of LEC2 (a master regulator of seed maturation and seed oil
storage) in mature leaves using the inducible Alc promoter (Andrianov et al., 2010). No
estimation of the extractable TAG was given, nor was there any calculation on the accumulation
of TAG in expanding leaves.
Mutations in a permease-like protein TRIGALACTOSYLDIACYLGLYCEROL (TGD1), in
Arabidopsis thaliana caused the accumulation of TAGs, oligogalactolipids and phosphatidate;
this was accompanied by a high incidence of embryo abortion and comparatively poor overall
plant growth (Xu et al., 2005).
Winichayakul et al., (2008) over expressed Arabidopsis thaliana DGAT1 in the leaves of
ryegrass (Lolium perenne) and found this lead to a 50% elevation of total extractable leaf lipid
(from ~4% to 6% of dry matter). Furthermore, the elevated lipid level was present in new leaves
generated by repeated harvests spaced 2-3 weeks apart, indicating that the new emerging leaves
30
were also capable of accumulating additional lipid. However, the elevated lipid level in these
leaves typically began to decline to wild type levels when the leaves were more than 2 weeks old
indicating that the lipids were being re-mobilised via catabolism (release from the glycerol
backbone by lipase followed by β-oxidation).
Slocombe et al., (2009) demonstrated that mutations in the CTS peroxisomal ABC transporter
(cts-2) led to accumulation of up to 1.4% TAG in leaves, particularly during the onset of
senescence. They also ectopically expressed LEC2 during senescence in the cts-2 background;
while this did not elevate the overall accumulation of TAG over the cts-2 mutant it did increase
the accumulation of seed oil type species of TAG in senescing tissue. While cts-2 blocks fatty
acid breakdown it also led to a severe phenotype. Slocombe et al., (2009) concluded that
recycled membrane fatty acids may be able to be re-directed to TAG by expressing the seedprogramme
in senescing tissue or by a block in fatty acid breakdown.
Scott et al., (2007) claimed that the co-expression of a triacylglyceride synthesising enzyme and
polyoleosin (two or more oleosin units fused in a tandem head-to-tail arrangement) would enable
the storage of lipid in a plant cell. Similarly, Cookson et al., (2009) claimed that producing a
single oleosin and a TAG synthesising enzyme within vegetative portions of a plant would lead
to increased number of oil bodies and TAG in the vegetative tissue. Using either of these
techniques leads to a maximum increase in lipid content (not necessarily in the form of TAG) of
up to approximately 50%. Furthermore this level begins to decline as the leaves mature;
typically in leaves greater than 2 weeks old (unpublished data).
Hence, the degree to which TAG can be accumulated in vegetative tissues appears to be limited
to some extent by the fact that the endogenous fixed-carbon recovery machinery catabolises the
TAG.
Leaf senescence – recycling of lipids via TAG intermediates
Leaf senescence is a highly controlled sequence of events leading ultimately to the death of cells,
tissues and finally the whole organ. This entails regulated recruitment of nutrients together with
their translocation from the senescing tissue to other tissues that are still growing and
developing. The chloroplast is the first organelle of mesophyll cells to show symptoms of
senescence and although breakdown of thylakoid membranes is initiated early in the leaf
senescence cascade, the chloroplast envelope remains relatively intact until the very late stages
of senescence. DGAT1 is up-regulated during senescence of Arabidopsis leaves and this is
temporally correlated with increased levels of TAG-containing fatty acids commonly found in
31
chloroplast galactolipids. Recruitment of membrane carbon from senescing leaves, particularly
senescing chloroplasts, to growing parts of the plant is a key feature of leaf senescence, and it
involves de-esterification of thylakoid lipids and conversion of the resultant free fatty acids to
phloem-mobile sucrose. De-esterification of thylakoid lipids appears to be mediated by one or
more senescence induced galactolipases. The formation of TAG appears to be an intermediate
step in the mobilisation of membrane lipid carbon to phloem mobile sucrose during senescence
(Kaup et al., 2002).
Modified oleosins engineered to include artificially introduced cysteines
The modified oleosins of the invention, or for use in the methods of the invention, are modified
to contain at least one artificially introduced cysteine residue. Preferably the engineered oleosins
contain at least two cysteines.
The encapsulation of the neutral lipids by oleosins containing engineered cysteines provides an
alternative mechanism to accumulate appreciable quantities of TAG in leaves without the
requirement to wait until senescence and without producing extreme phenotypes.
Various methods well-known to those skilled in the art may be used in production of the
modified oleosins with artificially introduced cysteines.
Such methods include site directed mutagenesis (US 6,448,048) in which the polynucleotide
encoding an oleosin is modified to introduce a cysteine into the encoded oleosin protein.
Alternatively the polynucleotide encoding the modified oleosins, may be synthesed in its
entirety.
Further methodology for producing modified oleosins of the invention and for use in the
methods of the invention, is provided in the Examples section.
The introduced cysteine may be an additional amino acid (i.e. an insertion) or may replace an
existing amino acid (i.e. a replacement). Preferably the introduced cysteine replaces an existing
amino acid. In a preferred embodiment the replaced amino acid is a charged residue. Preferably
the charged residue is predicted to be in the hydrophilic domains and therefore likely to be
located on the surface of the oil body.
The hydrophilic, and hydrophobic regions/arms of the oleosin can be easily identified by those
skilled in the art using standard methodology (for example: Kyte and Doolitle (1982).
32
The modified oleosins of the invention are preferably range in molecular weight from 5 to 50
kDa, more preferably, 10 to 40kDa, more preferably 15 to 25 kDa.
The modified oleosins of the invention are preferably in the size range 100 to 300 amino acids,
more preferably 110 to 260 amino acids, more preferably 120 to 250 amino acids, more
preferably 130 to 240 amino acids, more preferably 140 to 230 amino acids.
Preferably the modified oleosins comprise an N-terminal hydrophilic region, two centre
hydrophobic regions (joined by a proline knot or knob) and a C-terminal hydrophilic region.
Preferably the modified oleosins can be divided almost equally their length into four parts which
correspond to the N-terminal hydrophilic region (or arm), the two centre hydrophobic regions
(joined by a proline knot or knob) and a C-terminal hydrophilic region (or arm).
Preferably the topology of modified oleosin is attributed to its physical properties which include
a folded hydrophobic core flanked by hydrophilic domains.
Preferably the modified oleosins can be formed into oil bodies when combined with
triacylglycerol (TAG) and phospholipid.
Preferably topology confers an amphipathic nature to modified oleosin resulting in the
hydrophobic domain being embedded in the phospholipid monolayer of the oil body while the
flanking hydrophilic domains are exposed to the aqueous environment outside the oil body, such
as in the cytoplasm.
In one embodiment the modified oleosin of the invention or used in the method of the invention,
comprises a sequence with at least 70% identity the hydrophobic domain of any of the oleosin
protein sequences referred to in Table 1 above.
In one embodiment the modified oleosin of the invention or used in the method of the invention,
comprises a sequence with at least 70% identity to any of the protein sequences referred to in
Table 1 above.
In further embodiment the modified oleosin is essentially the same as any of the oleosins
referred to in Table 1 above, apart from the additional artificially introduced cysteine or
cysteines.
33
In a further embodiment the modified oleosin of the invention or used in the method of the
invention, comprises a sequence with at least 70% identity to the oleosin sequence of SEQ ID
NO: 16.
In further embodiment the modified oleosin has the same amino acid sequence as that of SEQ ID
NO: 16, apart from the additional artificially introduced cysteine or cysteines.
In further embodiment the modified oleosin is has the amino acid sequence of any one of SEQ
ID NO: 16 to 20.
Fusion proteins with modified oleosins
The invention also provides a fusion proteins including a modified oleosin of the invention fused
to a protein of interest.
Preferably the protein of interest is at the N- or C-terminal end of the fusion protein.
Methods for recombinantly expressing fusion proteins are well known to those skilled in the art
(Papapostolou and Howorka, 2009). Production of the fusion protein of the invention may
typically involve fusing the coding sequence of the protein of interest to the coding sequence of
the modified oleosin.
Such fusion proteins may be included in, or expressed in, the oil bodies of the invention and used
to purify and deliver the protein of interest for a variety of applications, as discussed in Roberts
et al, (2008).
However in the invention makes it possible to take advantage of the option to vary the
stability/integrity of the oil body provided by presence of the modified oleosins in the oil body,
hence allowing for more stringent purification and delivery procedures.
Fusion proteins with un-modified oleosins
The invention also involves use of fusion protein including un-modified oleosin fused to a
protein of interest. Production of the fusion protein of the invention may typically involve fusing
the coding sequence of the protein of interest to the coding sequence of the un-modified oleosin.
Preferably the protein of interest is at the N- or C-terminal end of the fusion protein.
34
Such fusion proteins may be included or expressed in the oil bodies of the invention and used to
purify and deliver the protein of interest for a variety of applications, as discussed in Roberts et
al., (2008).
The present invention however, takes advantage of the option to vary the stability/integrity of the
oil body provided by presence of the modified oleosins in the oil body of the invention, hence
allowing for more stringent purification and delivery procedures.
Vegetative tissues
Vegetative tissue include, shoots, leaves, roots, stems. A preferred vegetative tissue is a leaf.
Vegetative tissue specific promoters
An example of a vegetative specific promoter is found in US 6,229,067; and US 7,629,454; and
US 7,153,953; and US 6,228,643.
Pollen specific promoters
An example of a pollen specific promoter is found in US 7,141,424; and US 5,545,546; and US
5,412,085; and US 5,086,169; and US 7,667,097.
Seed specific promoters
An example of a seed specific promoter is found in US 6,342,657; and US 7,081,565; and US
7,405,345; and US 7,642,346; and US 7,371,928.
Fruit specific promoters
An example of a fruit specific promoter is found in US 5,536,653; and US 6,127,179; and US
5,608,150; and US 4,943,674.
Polynucleotides and fragments
The term “polynucleotide(s),” as used herein, means a single or double-stranded
deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15
nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene,
sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA,
mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and
35
purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences,
nucleic acid probes, primers and fragments.
A “fragment” of a polynucleotide sequence provided herein is a subsequence of contiguous
nucleotides that is capable of specific hybridization to a target of interest, e.g., a sequence that is
at least 15 nucleotides in length. The fragments of the invention comprise 15 nucleotides,
preferably at least 16 nucleotides, more preferably at least 17 nucleotides, more preferably at
least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20
nucleotides, more preferably at least 21 nucleotides, more preferably at least 22 nucleotides,
more preferably at least 23 nucleotides, more preferably at least 24 nucleotides, more preferably
at least 25 nucleotides, more preferably at least 26 nucleotides, more preferably at least 27
nucleotides, more preferably at least 28 nucleotides, more preferably at least 29 nucleotides,
more preferably at least 30 nucleotides, more preferably at least 31 nucleotides, more preferably
at least 32 nucleotides, more preferably at least 33 nucleotides, more preferably at least 34
nucleotides, more preferably at least 35 nucleotides, more preferably at least 36 nucleotides,
more preferably at least 37 nucleotides, more preferably at least 38 nucleotides, more preferably
at least 39 nucleotides, more preferably at least 40 nucleotides, more preferably at least 41
nucleotides, more preferably at least 42 nucleotides, more preferably at least 43 nucleotides,
more preferably at least 44 nucleotides, more preferably at least 45 nucleotides, more preferably
at least 46 nucleotides, more preferably at least 47 nucleotides, more preferably at least 48
nucleotides, more preferably at least 49 nucleotides, more preferably at least 50 nucleotides,
more preferably at least 51 nucleotides, more preferably at least 52 nucleotides, more preferably
at least 53 nucleotides, more preferably at least 54 nucleotides, more preferably at least 55
nucleotides, more preferably at least 56 nucleotides, more preferably at least 57 nucleotides,
more preferably at least 58 nucleotides, more preferably at least 59 nucleotides, more preferably
at least 60 nucleotides, more preferably at least 61 nucleotides, more preferably at least 62
nucleotides, more preferably at least 63 nucleotides, more preferably at least 64 nucleotides,
more preferably at least 65 nucleotides, more preferably at least 66 nucleotides, more preferably
at least 67 nucleotides, more preferably at least 68 nucleotides, more preferably at least 69
nucleotides, more preferably at least 70 nucleotides, more preferably at least 71 nucleotides,
more preferably at least 72 nucleotides, more preferably at least 73 nucleotides, more preferably
at least 74 nucleotides, more preferably at least 75 nucleotides, more preferably at least 76
nucleotides, more preferably at least 77 nucleotides, more preferably at least 78 nucleotides,
more preferably at least 79 nucleotides, more preferably at least 80 nucleotides, more preferably
at least 81 nucleotides, more preferably at least 82 nucleotides, more preferably at least 83
36
nucleotides, more preferably at least 84 nucleotides, more preferably at least 85 nucleotides,
more preferably at least 86 nucleotides, more preferably at least 87 nucleotides, more preferably
at least 88 nucleotides, more preferably at least 89 nucleotides, more preferably at least 90
nucleotides, more preferably at least 91 nucleotides, more preferably at least 92 nucleotides,
more preferably at least 93 nucleotides, more preferably at least 94 nucleotides, more preferably
at least 95 nucleotides, more preferably at least 96 nucleotides, more preferably at least 97
nucleotides, more preferably at least 98 nucleotides, more preferably at least 99 nucleotides,
more preferably at least 100 nucleotides, more preferably at least 150 nucleotides, more
preferably at least 200 nucleotides, more preferably at least 250 nucleotides, more preferably at
least 300 nucleotides, more preferably at least 350 nucleotides, more preferably at least 400
nucleotides, more preferably at least 450 nucleotides and most preferably at least 500 nucleotides
of contiguous nucleotides of a polynucleotide disclosed. A fragment of a polynucleotide
sequence can be used in antisense, RNA interference (RNAi), gene silencing, triple helix or
ribozyme technology, or as a primer, a probe, included in a microarray, or used in
polynucleotide-based selection methods of the invention.
The term “primer” refers to a short polynucleotide, usually having a free 3’OH group, that is
hybridized to a template and used for priming polymerization of a polynucleotide
complementary to the target.
The term “probe” refers to a short polynucleotide that is used to detect a polynucleotide
sequence that is complementary to the probe, in a hybridization-based assay. The probe may
consist of a “fragment” of a polynucleotide as defined herein.
Polypeptides and fragments
The term “polypeptide”, as used herein, encompasses amino acid chains of any length but
preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are
linked by covalent peptide bonds. Polypeptides of the present invention, or used in the methods
of the invention, may be purified natural products, or may be produced partially or wholly using
recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a
polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a
polypeptide variant, or derivative thereof.
A “fragment” of a polypeptide is a subsequence of the polypeptide that performs a function that
is required for the biological activity and/or provides three dimensional structure of the
polypeptide. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer
37
or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or
derivative thereof capable of performing the above enzymatic activity.
The term “isolated” as applied to the polynucleotide or polypeptide sequences disclosed herein is
used to refer to sequences that are removed from their natural cellular environment. An isolated
molecule may be obtained by any method or combination of methods including biochemical,
recombinant, and synthetic techniques.
The term “recombinant” refers to a polynucleotide sequence that is removed from sequences that
surround it in its natural context and/or is recombined with sequences that are not present in its
natural context.
A “recombinant” polypeptide sequence is produced by translation from a “recombinant”
polynucleotide sequence.
The term “derived from” with respect to polynucleotides or polypeptides of the invention being
derived from a particular genera or species, means that the polynucleotide or polypeptide has the
same sequence as a polynucleotide or polypeptide found naturally in that genera or species. The
polynucleotide or polypeptide, derived from a particular genera or species, may therefore be
produced synthetically or recombinantly.
Variants
As used herein, the term “variant” refers to polynucleotide or polypeptide sequences different
from the specifically identified sequences, wherein one or more nucleotides or amino acid
residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or
non-naturally occurring variants. Variants may be from the same or from other species and may
encompass homologues, paralogues and orthologues. In certain embodiments, variants of the
inventive polypeptides and polypeptides possess biological activities that are the same or similar
to those of the inventive polypeptides or polypeptides. The term “variant” with reference to
polypeptides and polypeptides encompasses all forms of polypeptides and polypeptides as
defined herein.
Polynucleotide variants
Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%,
more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more
preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more
38
preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more
preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more
preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more
preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more
preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more
preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more
preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more
preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more
preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more
preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more
preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity
to a sequence of the present invention. Identity is found over a comparison window of at least
20 nucleotide positions, preferably at least 50 nucleotide positions, more preferably at least 100
nucleotide positions, and most preferably over the entire length of a polynucleotide of the
invention.
Polynucleotide sequence identity can be determined in the following manner. The subject
polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN
(from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq (Tatiana A. Tatusova,
Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide
sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI
(ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering
of low complexity parts should be turned off.
The identity of polynucleotide sequences may be examined using the following unix command
line parameters:
bl2seq –i nucleotideseq1 –j nucleotideseq2 –F F –p blastn
The parameter –F F turns off filtering of low complexity sections. The parameter –p selects the
appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as
both the number and percentage of identical nucleotides in a line “Identities = “.
39
Polynucleotide sequence identity may also be calculated over the entire length of the overlap
between a candidate and subject polynucleotide sequences using global sequence alignment
programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A full
implementation of the Needleman-Wunsch global alignment algorithm is found in the needle
program in the EMBOSS package (Rice,P. Longden,I. and Bleasby,A. EMBOSS: The European
Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276-
277) which can be obtained from http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The
European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle
global alignments between two sequences on line at http:/www.ebi.ac.uk/emboss/align/.
Alternatively the GAP program may be used which computes an optimal global alignment of
two sequences without penalizing terminal gaps. GAP is described in the following paper:
Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences
10, 227-235.
A preferred method for calculating polynucleotide % sequence identity is based on aligning
sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23,
403-5.)
Polynucleotide variants of the present invention also encompass those which exhibit a similarity
to one or more of the specifically identified sequences that is likely to preserve the functional
equivalence of those sequences and which could not reasonably be expected to have occurred by
random chance. Such sequence similarity with respect to polypeptides may be determined using
the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov
2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
The similarity of polynucleotide sequences may be examined using the following unix command
line parameters:
bl2seq –i nucleotideseq1 –j nucleotideseq2 –F F –p tblastx
The parameter –F F turns off filtering of low complexity sections. The parameter –p selects the
appropriate algorithm for the pair of sequences. This program finds regions of similarity between
the sequences and for each such region reports an “E value” which is the expected number of
times one could expect to see such a match by chance in a database of a fixed reference size
containing random sequences. The size of this database is set by default in the bl2seq program.
40
For small E values, much less than one, the E value is approximately the probability of such a
random match.
Variant polynucleotide sequences preferably exhibit an E value of less than 1 x 10 -6 more
preferably less than 1 x 10 -9, more preferably less than 1 x 10 -12, more preferably less than 1 x
10 -15, more preferably less than 1 x 10 -18, more preferably less than 1 x 10 -21, more
preferably less than 1 x 10 -30, more preferably less than 1 x 10 -40, more preferably less than
1 x 10 -50, more preferably less than 1 x 10 -60, more preferably less than 1 x 10 -70, more
preferably less than 1 x 10 -80, more preferably less than 1 x 10 -90 and most preferably less
than 1 x 10-100 when compared with any one of the specifically identified sequences.
Alternatively, variant polynucleotides of the present invention, or used in the methods of the
invention, hybridize to the specified polynucleotide sequences, or complements thereof under
stringent conditions.
The term "hybridize under stringent conditions", and grammatical equivalents thereof, refers to
the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as
a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or
Northern blot) under defined conditions of temperature and salt concentration. The ability to
hybridize under stringent hybridization conditions can be determined by initially hybridizing
under less stringent conditions then increasing the stringency to the desired stringency.
With respect to polynucleotide molecules greater than about 100 bases in length, typical
stringent hybridization conditions are no more than 25 to 30o C (for example, 10o C) below the
melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987,
Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al.,
1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide
molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G
+ C-log (Na+). (Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed.
Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84:1390). Typical stringent
conditions for polynucleotide of greater than 100 bases in length would be hybridization
conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65oC, 6X
SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1X SSC, 0.1% SDS at
65o C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65oC.
With respect to polynucleotide molecules having a length less than 100 bases, exemplary
stringent hybridization conditions are 5 to 10o C below Tm. On average, the Tm of a
41
polynucleotide molecule of length less than 100 bp is reduced by approximately
(500/oligonucleotide length)o C.
With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., Science.
1991 Dec 6;254(5037):1497-500) Tm values are higher than those for DNA-DNA or DNA-RNA
hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res.
1998 Nov 1;26(21):5004-6. Exemplary stringent hybridization conditions for a DNA-PNA
hybrid having a length less than 100 bases are 5 to 10o C below the Tm.
Variant polynucleotides of the present invention, or used in the methods of the invention, also
encompasses polynucleotides that differ from the sequences of the invention but that, as a
consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity
to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration
that does not change the amino acid sequence of the polypeptide is a “silent variation”. Except
for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be
changed by art recognized techniques, e.g., to optimize codon expression in a particular host
organism.
Polynucleotide sequence alterations resulting in conservative substitutions of one or several
amino acids in the encoded polypeptide sequence without significantly altering its biological
activity are also included in the invention. A skilled artisan will be aware of methods for making
phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).
Variant polynucleotides due to silent variations and conservative substitutions in the encoded
polypeptide sequence may be determined using the publicly available bl2seq program from the
BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/)
via the tblastx algorithm as previously described.
Polypeptide variants
The term “variant” with reference to polypeptides encompasses naturally occurring,
recombinantly and synthetically produced polypeptides. Variant polypeptide sequences
preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%,
more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more
preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more
preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more
preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more
42
preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more
preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more
preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more
preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more
preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more
preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more
preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more
preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more
preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more
preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more
preferably at least 98%, and most preferably at least 99% identity to a sequences of the present
invention. Identity is found over a comparison window of at least 20 amino acid positions,
preferably at least 50 amino acid positions, more preferably at least 100 amino acid positions,
and most preferably over the entire length of a polypeptide of the invention.
Polypeptide sequence identity can be determined in the following manner. The subject
polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the
BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq, which is publicly available from
NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that
filtering of low complexity regions should be turned off.
Polypeptide sequence identity may also be calculated over the entire length of the overlap
between a candidate and subject polynucleotide sequences using global sequence alignment
programs. EMBOSS-needle (available at http:/www.ebi.ac.uk/emboss/align/) and GAP (Huang,
X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-
235.) as discussed above are also suitable global sequence alignment programs for calculating
polypeptide sequence identity.
A preferred method for calculating polypeptide % sequence identity is based on aligning
sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23,
403-5.)
Polypeptide variants of the present invention, or used in the methods of the invention, also
encompass those which exhibit a similarity to one or more of the specifically identified
sequences that is likely to preserve the functional equivalence of those sequences and which
could not reasonably be expected to have occurred by random chance. Such sequence similarity
43
with respect to polypeptides may be determined using the publicly available bl2seq program
from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI
(ftp://ftp.ncbi.nih.gov/blast/). The similarity of polypeptide sequences may be examined using
the following unix command line parameters:
bl2seq –i peptideseq1 –j peptideseq2 -F F –p blastp
Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10 -6 more
preferably less than 1 x 10 -9, more preferably less than 1 x 10 -12, more preferably less than 1 x
10 -15, more preferably less than 1 x 10 -18, more preferably less than 1 x 10 -21, more
preferably less than 1 x 10 -30, more preferably less than 1 x 10 -40, more preferably less than 1
x 10 -50, more preferably less than 1 x 10 -60, more preferably less than 1 x 10 -70, more
preferably less than 1 x 10 -80, more preferably less than 1 x 10 -90 and most preferably 1x10-
100 when compared with any one of the specifically identified sequences.
The parameter –F F turns off filtering of low complexity sections. The parameter –p selects the
appropriate algorithm for the pair of sequences. This program finds regions of similarity between
the sequences and for each such region reports an “E value” which is the expected number of
times one could expect to see such a match by chance in a database of a fixed reference size
containing random sequences. For small E values, much less than one, this is approximately the
probability of such a random match.
Conservative substitutions of one or several amino acids of a described polypeptide sequence
without significantly altering its biological activity are also included in the invention. A skilled
artisan will be aware of methods for making phenotypically silent amino acid substitutions (see,
e.g., Bowie et al., 1990, Science 247, 1306).
Constructs, vectors and components thereof
The term "genetic construct" refers to a polynucleotide molecule, usually double-stranded DNA,
which may have inserted into it another polynucleotide molecule (the insert polynucleotide
molecule) such as, but not limited to, a cDNA molecule. A genetic construct may contain the
necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally,
translating the transcript into a polypeptide. The insert polynucleotide molecule may be derived
from the host cell, or may be derived from a different cell or organism and/or may be a
recombinant polynucleotide. Once inside the host cell the genetic construct may become
integrated in the host chromosomal DNA. The genetic construct may be linked to a vector.
44
The term “vector” refers to a polynucleotide molecule, usually double stranded DNA, which is
used to transport the genetic construct into a host cell. The vector may be capable of replication
in at least one additional host system, such as E. coli.
The term "expression construct" refers to a genetic construct that includes the necessary
elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating
the transcript into a polypeptide. An expression construct typically comprises in a 5’ to 3’
direction:
a) a promoter functional in the host cell into which the construct will be
transformed,
b) the polynucleotide to be expressed, and
c) a terminator functional in the host cell into which the construct will be
transformed.
The term “coding region” or “open reading frame” (ORF) refers to the sense strand of a genomic
DNA sequence or a cDNA sequence that is capable of producing a transcription product and/or a
polypeptide under the control of appropriate regulatory sequences. The coding sequence may, in
some cases, identified by the presence of a 5’ translation start codon and a 3’ translation stop
codon. When inserted into a genetic construct, a “coding sequence” is capable of being
expressed when it is operably linked to promoter and terminator sequences.
“Operably-linked” means that the sequenced to be expressed is placed under the control of
regulatory elements that include promoters, tissue-specific regulatory elements, temporal
regulatory elements, enhancers, repressors and terminators.
The term “noncoding region” refers to untranslated sequences that are upstream of the
translational start site and downstream of the translational stop site. These sequences are also
referred to respectively as the 5’ UTR and the 3’ UTR. These regions include elements required
for transcription initiation and termination, mRNA stability, and for regulation of translation
efficiency.
Terminators are sequences, which terminate transcription, and are found in the 3’ untranslated
ends of genes downstream of the translated sequence. Terminators are important determinants of
mRNA stability and in some cases have been found to have spatial regulatory functions.
The term “promoter” refers to nontranscribed cis-regulatory elements upstream of the coding
region that regulate gene transcription. Promoters comprise cis-initiator elements which specify
45
the transcription initiation site and conserved boxes such as the TATA box, and motifs that are
bound by transcription factors. Introns within coding sequences can also regulate transcription
and influence post-transcriptional processing (including splicing, capping and polyadenylation).
A promoter may be homologous with respect to the polynucleotide to be expressed. This means
that the promoter and polynucleotide are found operably linked in nature.
Alternatively the promoter may be heterologous with respect to the polynucleotide to be
expressed. This means that the promoter and the polynucleotide are not found operably linked in
nature.
A “transgene” is a polynucleotide that is taken from one organism and introduced into a different
organism by transformation. The transgene may be derived from the same species or from a
different species as the species of the organism into which the transgene is introduced.
An “inverted repeat” is a sequence that is repeated, where the second half of the repeat is in the
complementary strand, e.g.,
(5’)GATCTA…….TAGATC(3’)
(3’)CTAGAT…….ATCTAG(5’)
Read-through transcription will produce a transcript that undergoes complementary base-pairing
to form a hairpin structure provided that there is a 3-5 bp spacer between the repeated regions.
Host cells
Host cells may be derived from, for example, bacterial, fungal, yeast, insect, mammalian, algal
or plant organisms. Host cells may also be synthetic cells. Preferred host cells are eukaryotic
cells. A particularly preferred host cell is a plant cell, particularly a plant cell in a vegetative
tissue of a plant.
A “transgenic plant” refers to a plant which contains new genetic material as a result of genetic
manipulation or transformation. The new genetic material may be derived from a plant of the
same species as the resulting transgenic plant or from a different species.
Methods for isolating or producing polynucleotides
The polynucleotide molecules of the invention can be isolated by using a variety of techniques
known to those of ordinary skill in the art. By way of example, such polypeptides can be
46
isolated through use of the polymerase chain reaction (PCR) described in Mullis et al., Eds. 1994
The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference. The
polypeptides of the invention can be amplified using primers, as defined herein, derived from the
polynucleotide sequences of the invention.
Further methods for isolating polynucleotides of the invention include use of all, or portions of,
the polypeptides having the sequence set forth herein as hybridization probes. The technique of
hybridizing labelled polynucleotide probes to polynucleotides immobilized on solid supports
such as nitrocellulose filters or nylon membranes, can be used to screen the genomic or cDNA
libraries. Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65°C
in 5. 0 X SSC, 0. 5% sodium dodecyl sulfate, 1 X Denhardt's solution; washing (three washes of
twenty minutes each at 55°C) in 1. 0 X SSC, 1% (w/v) sodium dodecyl sulfate, and optionally
one wash (for twenty minutes) in 0. 5 X SSC, 1% (w/v) sodium dodecyl sulfate, at 60°C. An
optional further wash (for twenty minutes) can be conducted under conditions of 0.1 X SSC, 1%
(w/v) sodium dodecyl sulfate, at 60°C.
The polynucleotide fragments of the invention may be produced by techniques well-known in
the art such as restriction endonuclease digestion, oligonucleotide synthesis and PCR
amplification.
A partial polynucleotide sequence may be used, in methods well-known in the art to identify the
corresponding full length polynucleotide sequence. Such methods include PCR-based methods,
5’RACE (Frohman MA, 1993, Methods Enzymol. 218: 340-56) and hybridization- based
method, computer/database –based methods. Further, by way of example, inverse PCR permits
acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein,
starting with primers based on a known region (Triglia et al., 1998, Nucleic Acids Res 16, 8186,
incorporated herein by reference). The method uses several restriction enzymes to generate a
suitable fragment in the known region of a gene. The fragment is then circularized by
intramolecular ligation and used as a PCR template. Divergent primers are designed from the
known region. In order to physically assemble full-length clones, standard molecular biology
approaches can be utilized (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.
Cold Spring Harbor Press, 1987).
It may be beneficial, when producing a transgenic plant from a particular species, to transform
such a plant with a sequence or sequences derived from that species. The benefit may be to
alleviate public concerns regarding cross-species transformation in generating transgenic
organisms. Additionally when down-regulation of a gene is the desired result, it may be
47
necessary to utilise a sequence identical (or at least highly similar) to that in the plant, for which
reduced expression is desired. For these reasons among others, it is desirable to be able to
identify and isolate orthologues of a particular gene in several different plant species.
Variants (including orthologues) may be identified by the methods described.
Methods for identifying variants
Physical methods
Variant polypeptides may be identified using PCR-based methods (Mullis et al., Eds. 1994 The
Polymerase Chain Reaction, Birkhauser). Typically, the polynucleotide sequence of a primer,
useful to amplify variants of polynucleotide molecules of the invention by PCR, may be based
on a sequence encoding a conserved region of the corresponding amino acid sequence.
Alternatively library screening methods, well known to those skilled in the art, may be employed
(Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press,
1987). When identifying variants of the probe sequence, hybridization and/or wash stringency
will typically be reduced relatively to when exact sequence matches are sought.
Polypeptide variants may also be identified by physical methods, for example by screening
expression libraries using antibodies raised against polypeptides of the invention (Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) or by
identifying polypeptides from natural sources with the aid of such antibodies.
Computer based methods
The variant sequences of the invention, including both polynucleotide and polypeptide variants,
may also be identified by computer-based methods well-known to those skilled in the art, using
public domain sequence alignment algorithms and sequence similarity search tools to search
sequence databases (public domain databases include Genbank, EMBL, Swiss-Prot, PIR and
others). See, e.g., Nucleic Acids Res. 29: 1-10 and 11-16, 2001 for examples of online resources.
Similarity searches retrieve and align target sequences for comparison with a sequence to be
analyzed (i.e., a query sequence). Sequence comparison algorithms use scoring matrices to
assign an overall score to each of the alignments.
An exemplary family of programs useful for identifying variants in sequence databases is the
BLAST suite of programs (version 2.2.5 [Nov 2002]) including BLASTN, BLASTP, BLASTX,
tBLASTN and tBLASTX, which are publicly available from (ftp://ftp.ncbi.nih.gov/blast/) or
48
from the National Center for Biotechnology Information (NCBI), National Library of Medicine,
Building 38A, Room 8N805, Bethesda, MD 20894 USA. The NCBI server also provides the
facility to use the programs to screen a number of publicly available sequence databases.
BLASTN compares a nucleotide query sequence against a nucleotide sequence database.
BLASTP compares an amino acid query sequence against a protein sequence database.
BLASTX compares a nucleotide query sequence translated in all reading frames against a
protein sequence database. tBLASTN compares a protein query sequence against a nucleotide
sequence database dynamically translated in all reading frames. tBLASTX compares the sixframe
translations of a nucleotide query sequence against the six-frame translations of a
nucleotide sequence database. The BLAST programs may be used with default parameters or the
parameters may be altered as required to refine the screen.
The use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is
described in the publication of Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997.
The “hits” to one or more database sequences by a queried sequence produced by BLASTN,
BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm, align and identify similar
portions of sequences. The hits are arranged in order of the degree of similarity and the length of
sequence overlap. Hits to a database sequence generally represent an overlap over only a
fraction of the sequence length of the queried sequence.
The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce “Expect”
values for alignments. The Expect value (E) indicates the number of hits one can "expect" to see
by chance when searching a database of the same size containing random contiguous sequences.
The Expect value is used as a significance threshold for determining whether the hit to a
database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide
hit is interpreted as meaning that in a database of the size of the database screened, one might
expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by
chance. For sequences having an E value of 0.01 or less over aligned and matched portions, the
probability of finding a match by chance in that database is 1% or less using the BLASTN,
BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.
Multiple sequence alignments of a group of related sequences can be carried out with
CLUSTALW (Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTALW: improving
the sensitivity of progressive multiple sequence alignment through sequence weighting,
positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-
4680, http://www-igbmc.u-strasbg.fr/BioInfo/ClustalW/Top.html) or T-COFFEE (Cedric
49
Notredame, Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel method for fast and accurate
multiple sequence alignment, J. Mol. Biol. (2000) 302: 205-217)) or PILEUP, which uses
progressive, pairwise alignments. (Feng and Doolittle, 1987, J. Mol. Evol. 25, 351).
Pattern recognition software applications are available for finding motifs or signature sequences.
For example, MEME (Multiple Em for Motif Elicitation) finds motifs and signature sequences in
a set of sequences, and MAST (Motif Alignment and Search Tool) uses these motifs to identify
similar or the same motifs in query sequences. The MAST results are provided as a series of
alignments with appropriate statistical data and a visual overview of the motifs found. MEME
and MAST were developed at the University of California, San Diego.
PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmann et al., 1999,
Nucleic Acids Res. 27, 215) is a method of identifying the functions of uncharacterized proteins
translated from genomic or cDNA sequences. The PROSITE database (www.expasy.org/prosite)
contains biologically significant patterns and profiles and is designed so that it can be used with
appropriate computational tools to assign a new sequence to a known family of proteins or to
determine which known domain(s) are present in the sequence (Falquet et al., 2002, Nucleic
Acids Res. 30, 235). Prosearch is a tool that can search SWISS-PROT and EMBL databases
with a given sequence pattern or signature.
Methods for isolating polypeptides
The polypeptides of the invention, or used in the methods of the invention, including variant
polypeptides, may be prepared using peptide synthesis methods well known in the art such as
direct peptide synthesis using solid phase techniques (e.g. Stewart et al., 1969, in Solid-Phase
Peptide Synthesis, WH Freeman Co, San Francisco California, or automated synthesis, for
example using an Applied Biosystems 431A Peptide Synthesizer (Foster City, California).
Mutated forms of the polypeptides may also be produced during such syntheses.
The polypeptides and variant polypeptides of the invention, or used in the methods of the
invention, may also be purified from natural sources using a variety of techniques that are well
known in the art (e.g. Deutscher, 1990, Ed, Methods in Enzymology, Vol. 182, Guide to Protein
Purification,).
Alternatively the polypeptides and variant polypeptides of the invention, or used in the methods
of the invention, may be expressed recombinantly in suitable host cells and separated from the
cells as discussed below.
50
Methods for producing constructs and vectors
The genetic constructs of the present invention comprise one or more polynucleotide sequences
of the invention and/or polynucleotides encoding polypeptides of the invention, and may be
useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms.
The genetic constructs of the invention are intended to include expression constructs as herein
defined.
Methods for producing and using genetic constructs and vectors are well known in the art and
are described generally in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.
Cold Spring Harbor Press, 1987 ; Ausubel et al., Current Protocols in Molecular Biology,
Greene Publishing, 1987).
Methods for producing host cells comprising polynucleotides, constructs or vectors
The invention provides a host cell which comprises a genetic construct or vector of the
invention.
Host cells comprising genetic constructs, such as expression constructs, of the invention are
useful in methods well known in the art (e.g. Sambrook et al., Molecular Cloning : A Laboratory
Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al., Current Protocols in
Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the
invention. Such methods may involve the culture of host cells in an appropriate medium in
conditions suitable for or conducive to expression of a polypeptide of the invention. The
expressed recombinant polypeptide, which may optionally be secreted into the culture, may then
be separated from the medium, host cells or culture medium by methods well known in the art
(e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).
Methods for producing plant cells and plants comprising constructs and vectors
The invention further provides plant cells which comprise a genetic construct of the invention,
and plant cells modified to alter expression of a polynucleotide or polypeptide of the invention,
or used in the methods of the invention. Plants comprising such cells also form an aspect of the
invention.
Methods for transforming plant cells, plants and portions thereof with polypeptides are described
in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory
Manual. Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer to
51
Plants. Springer-Verlag, Berlin.; and Gelvin et al., 1993, Plant Molecular Biol. Manual. Kluwer
Acad. Pub. Dordrecht. A review of transgenic plants, including transformation techniques, is
provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.
Methods for genetic manipulation of plants
A number of plant transformation strategies are available (e.g. Birch, 1997, Ann Rev Plant Phys
Plant Mol Biol, 48, 297, Hellens RP, et al (2000) Plant Mol Biol 42: 819-32, Hellens R et al
Plant Meth 1: 13). For example, strategies may be designed to increase expression of a
polynucleotide/polypeptide in a plant cell, organ and/or at a particular developmental stage
where/when it is normally expressed or to ectopically express a polynucleotide/polypeptide in a
cell, tissue, organ and/or at a particular developmental stage which/when it is not normally
expressed. The expressed polynucleotide/polypeptide may be derived from the plant species to
be transformed or may be derived from a different plant species.
Transformation strategies may be designed to reduce expression of a polynucleotide/polypeptide
in a plant cell, tissue, organ or at a particular developmental stage which/when it is normally
expressed. Such strategies are known as gene silencing strategies.
Genetic constructs for expression of genes in transgenic plants typically include promoters for
driving the expression of one or more cloned polynucleotide, terminators and selectable marker
sequences to detect presence of the genetic construct in the transformed plant.
The promoters suitable for use in the constructs of this invention are functional in a cell, tissue or
organ of a monocot or dicot plant and include cell-, tissue- and organ-specific promoters, cell
cycle specific promoters, temporal promoters, inducible promoters, constitutive promoters that
are active in most plant tissues, and recombinant promoters. Choice of promoter will depend
upon the temporal and spatial expression of the cloned polynucleotide, so desired. The
promoters may be those normally associated with a transgene of interest, or promoters which are
derived from genes of other plants, viruses, and plant pathogenic bacteria and fungi. Those
skilled in the art will, without undue experimentation, be able to select promoters that are
suitable for use in modifying and modulating plant traits using genetic constructs comprising the
polynucleotide sequences of the invention. Examples of constitutive plant promoters include the
CaMV 35S promoter, the nopaline synthase promoter and the octopine synthase promoter, and
the Ubi 1 promoter from maize. Plant promoters which are active in specific tissues, respond to
internal developmental signals or external abiotic or biotic stresses are described in the scientific
52
literature. Exemplary promoters are described, e.g., in WO 02/00894, which is herein
incorporated by reference.
Exemplary terminators that are commonly used in plant transformation genetic construct include,
e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens
nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the
Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solanum tuberosum PI-II
terminator.
Selectable markers commonly used in plant transformation include the neomycin
phophotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which
confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase (bar
gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin
phosphotransferase gene ( hpt) for hygromycin resistance.
Use of genetic constructs comprising reporter genes (coding sequences which express an activity
that is foreign to the host, usually an enzymatic activity and/or a visible signal (e.g., luciferase,
GUS, GFP) which may be used for promoter expression analysis in plants and plant tissues are
also contemplated. The reporter gene literature is reviewed in Herrera-Estrella et al., 1993,
Nature 303, 209, and Schrott, 1995, In: Gene Transfer to Plants (Potrykus, T., Spangenberg. Eds)
Springer Verlag. Berline, pp. 325-336.
The following are representative publications disclosing genetic transformation protocols that
can be used to genetically transform the following plant species: Rice (Alam et al., 1999, Plant
Cell Rep. 18, 572); apple (Yao et al., 1995, Plant Cell Reports 14, 407-412); maize (US Patent
Serial Nos. 5, 177, 010 and 5, 981, 840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996,
877); tomato (US Patent Serial No. 5, 159, 135); potato (Kumar et al., 1996 Plant J. 9, : 821);
cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al., 1987, Plant Cell
Rep. 6, 439); tobacco (Horsch et al., 1985, Science 227, 1229); cotton (US Patent Serial Nos. 5,
846, 797 and 5, 004, 863); grasses (US Patent Nos. 5, 187, 073 and 6. 020, 539); peppermint
(Niu et al., 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al., 1995, Plant Sci.104, 183);
caraway (Krens et al., 1997, Plant Cell Rep, 17, 39); banana (US Patent Serial No. 5, 792, 935);
soybean (US Patent Nos. 5, 416, 011 ; 5, 569, 834 ; 5, 824, 877 ; 5, 563, 04455 and 5, 968, 830);
pineapple (US Patent Serial No. 5, 952, 543); poplar (US Patent No. 4, 795, 855); monocots in
general (US Patent Nos. 5, 591, 616 and 6, 037, 522); brassica (US Patent Nos. 5, 188, 958 ; 5,
463, 174 and 5, 750, 871); cereals (US Patent No. 6, 074, 877); pear (Matsuda et al., 2005, Plant
Cell Rep. 24(1):45-51); Prunus (Ramesh et al., 2006 Plant Cell Rep. 25(8):821-8; Song and Sink
53
2005 Plant Cell Rep. 2006 ;25(2):117-23; Gonzalez Padilla et al., 2003 Plant Cell Rep.22(1):38-
45); strawberry (Oosumi et al., 2006 Planta. 223(6):1219-30; Folta et al., 2006 Planta Apr 14;
PMID: 16614818), rose (Li et al., 2003), Rubus (Graham et al., 1995 Methods Mol Biol.
1995;44:129-33), tomato (Dan et al., 2006, Plant Cell Reports V25:432-441), apple (Yao et al.,
1995, Plant Cell Rep. 14, 407–412), Canola (Brassica napus L.).(Cardoza and Stewart, 2006
Methods Mol Biol. 343:257-66), safflower (Orlikowska et al, 1995, Plant Cell Tissue and Organ
Culture 40:85-91), ryegrass (Altpeter et al, 2004 Developments in Plant Breeding 11(7):255-
250), rice (Christou et al, 1991 Nature Biotech. 9:957-962), maize (Wang et al 2009 In:
Handbook of Maize pp. 609-639) and Actinidia eriantha (Wang et al., 2006, Plant Cell Rep.
25,5: 425-31). Transformation of other species is also contemplated by the invention. Suitable
methods and protocols are available in the scientific literature.
Plants
The term “plant” is intended to include a whole plant, any part of a plant, a seed, a fruit,
propagules and progeny of a plant.
The term ‘propagule’ means any part of a plant that may be used in reproduction or propagation,
either sexual or asexual, including seeds and cuttings.
The plants of the invention may be grown and either self-ed or crossed with a different plant
strain and the resulting hybrids, with the desired phenotypic characteristics, may be identified.
Two or more generations may be grown to ensure that the subject phenotypic characteristics are
stably maintained and inherited. Plants resulting from such standard breeding approaches also
form an aspect of the present invention.
Abbreviations
Oleosin (or Ole)_0-0 means an oleosin without engineered cysteines.
Oleosin (or Ole)_1-1 means an oleosin with one engineered cysteine in each hydrophilic
arm.
Oleosin (or Ole)_1-3 means an oleosin with one engineered cysteine in the N-terminal
hydrophilic arm and three engineered cysteines in the C-terminal hydrophilic arm.
Oleosin (or Ole)_3-1 means an oleosin with three engineered cysteines in the N-terminal
hydrophilic arm and one engineered cysteine in the C-terminal hydrophilic arm.
Oleosin (or Ole)_3-3 means an oleosin with three engineered cysteines in the N-terminal
hydrophilic arm and three engineered cysteines in the C-terminal hydrophilic arm.
54
Oleosin (or Ole)_5-6 means an oleosin with five engineered cysteines in the N-terminal
hydrophilic arm and six engineered cysteines in the C-terminal hydrophilic arm.
Oleosin (or Ole)_6-7 means an oleosin with six engineered cysteines in the N-terminal
hydrophilic arm and seven engineered cysteines in the C-terminal hydrophilic arm.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the sequence of the Oleosin_0-0 and DGAT1 (S205A) construct. CaMV35 is the
Cauliflower Mosais Virus 35S promoter. attB1 is the GATEWAY™ recombination site.
UBQ10 is the intron from the A. thaliana UBQ10 gene. OCS terminator is the octopine synthase
terminator.
Figure 2 shows the Oleosin_1-1 and DGAT1 (S205A) construct arrangement, as transformed
into Arabidopsis thaliana.
Figure 3 shows the sequence of the Oleosin_1-3 and DGAT1 (S205A) construct. CaMV35 is the
Cauliflower Mosais Virus 35S promoter. attB1 is the GATEWAY™ recombination site.
UBQ10 is the intron from the A. thaliana UBQ10 gene. OCS terminator is the octopine synthase
terminator.
Figure 4 shows the Oleosin_3-1 and DGAT1 (S205A) construct. CaMV35 is the Cauliflower
Mosais Virus 35S promoter. attB1 is the GATEWAY™ recombination site. UBQ10 is the
intron from the A. thaliana UBQ10 gene. OCS terminator is the octopine synthase terminator.
Figure 5 shows the Oleosin_3-3 and DGAT1 (S205A) construct. CaMV35 is the Cauliflower
Mosais Virus 35S promoter. attB1 is the GATEWAY™ recombination site. UBQ10 is the
intron from the A. thaliana UBQ10 gene. OCS terminator is the octopine synthase terminator.
Figure 6 shows a map of the construct pRSh1 used for transforming plants. The map shows the
arrangement of the oleosins, with artificially introduced cysteines (in this case Oleo_3-3) under
the control of the CaMV35s promoter as well as Arabidopsis thaliana DGAT1 (S205A) also
under the control of the CaMV35s promoter. Other oleosin sequences and TAG synthesising
enzyme sequences can of course be substituted for Oleo_3-3 and DGAT1 respectively.
Figure 7 shows dot blot comparison of anti-sesame seed oleosin antibodies binding to purified
recombinant sesame seed oleosin with and without engineered cysteine residues.
55
Figure 8 shows immunoblot analysis to detect E. coli expressed oleosin cysteine proteins in
AOBs. Equal volume of AOB (7.5 μL including 2x SDS loading dye without reducing agent)
was loaded per lane. The mM concentration of GSSG is indicated above each lane.
Figure 9 shows SDS and SDS-UREA PAGE/immunoblot analysis of E. coli expressed Ole-0-0,
Ole-1-1 and Ole-3-3. Samples were prepared from inclusion bodies (IB) and artificial oil bodies
(AOBs) in the presence and absence of reducing agents (DTT and -ME) or oxidising agent
(GSSG), where equal amounts of protein were loaded in adjacent lanes.
Figure 10 shows immunoblot analysis of oleosin (Oleo_0-0, Oleo_1-3, Oleo_3-1, and Oleo_3-3,
SEQ ID NOs 11-20) accumulation in the seeds of transgenic Arabidopsis thaliana expressing
both DGAT1 (S205A) and a sesame oleosin under the control of CaMV35S promoters.
Figure 11 shows immunoblot analysis of oleosin (Oleo_0-0, Oleo_1-3, Oleo_3-1, and Oleo_3-3,
SEQ ID NOs 11-20) accumulation in the oil bodies of transgenic Arabidopsis thaliana
expressing both DGAT1 (S205A) and a sesame oleosin under the control of CaMV35S
promoters. The appearance of the oligomeric oleosin bands (dimeric and trimeric) in the
presence of oxidising agent (+) indicates the disulfide bonds are able to form on the outside of
native oil bodies.
Figure 12 shows immunoblot analysis of oleosin (Oleo_0-0, Oleo_1-3, Oleo_3-1, and Oleo_3-3,
SEQ ID NOs 11-20) accumulation in the leaves of transgenic Arabidopsis thaliana expressing
both DGAT1 (S205A) and a sesame oleosin under the control of CaMV35S promoters.
Figure 13 shows immunoblot of recombinant oleosin accumulation (black arrow) in transgenic
Arabidopsis leaves.
Figure 14 shows FAMES GC/MS results demonstratinging accumulation of additional lipids
(black arrows) in Arabidopsis leaves over expressing DGAT1 (S205A) and Ole_3,3.
Figure 15 shows GC/MS results for total leaf lipid profile of wild type and independent lines of
transgenic Arabidopsis containing DGAT1 (S205A) and Ole_3,3. Grey arrow indicates internal
standard. Black arrows indicate additional neutral lipids (wax esters, sterol esters and TAGs.
Open arrows show three lines (41S, 18A and 47C) which accumulate substantial quantities of
neutral lipids in their leaves compared to wild type (and line 50A)
56
Figure 16 shows GC/MS results showing total TAG profile of wild type and transgenic
Arabidopsis (containing DGAT1 (S205A) and Ole_3,3) 2, 3, 4 and 5 weeks after germination.
Black arrows indicate additional TAGs found in transgenic leaves but not wild type.
Figure 17 shows FAMES GC/MS results showing total leaf lipid profiles of wild type and
transgeneic Trifolium repens (containing DGAT1 (S205A) and Ole_3,3).
Figure 18 shows FAMES GC/MS results showing C18:1 and C18:2 leaf lipid profiles of wild
type and transgeneic Trifolium repens (containing DGAT1 (S205A) and Ole_3,3).
EXAMPLES
This invention will now be illustrated with reference to the following non-limiting examples.
EXAMPLE 1: Creating rabbit anti-sesame seed oleosin antibodies
Generating rabbit anti-sesame seed oleosin antibodies
Full length sesame seed oleosin containing a C-terminal His tag (nucleotide sequence is shown
in SEQ ID NO: 1) was expressed in E. coli and inclusion bodies were prepared by standard
techniques. The inclusion bodies were solubilised in Binding Buffer (100 mM phosphate buffer
pH 8.0, 500mM NaCl, 8M urea and 10 mM imidazole) and loaded onto a column containing
equilibrated ion metal affinity chromatography (IMAC) Ni agarose (Invitrogen). Non-bound
proteins were removed from the column by washing with 6 volumes of Wash Buffer
(100 mM phosphate buffer pH 8.0, 500mM NaCl, 6M urea and 50 mM imidazole). Protein was
eluted in 1 vol. aliquots of Elution Buffer (100 mM phosphate buffer pH 8.0, 500 mM NaCl,
6M urea and 250mM imidazole). Eluted fractions were analysed by SDS-PAGE/Coomassie
stain and the protein concentration measured using the Bradford's Assay. 265μg of the
IMAC-purified recombinant oleosin protein was mixed with an equal amount of Freunds
Complete Adjuvant to a final volume of 0.5mL. Following collection of the pre-bleed, the first
injection was administered into multiple sites across the back of the neck and shoulder area of a
rabbit. Booster shots containing 77μg of the purified oleosin were delivered at three and seven
weeks after the primary, and a test bleed of ~3mL was removed for preliminary analysis at nine
weeks. Serum was preserved by the addition of 0.25% v/v phenol and 0.01% v/v merthiolate,
and stored in 200μL aliquots at -20°C.
The sensitivity of the rabbit anti-sesame seed oleosin antibodies was evaluated by immunodotting
which indicated that 0.25ng of sesame seed oleosin could be regularly detected with a
57
1/2,000 dilution of the antibody (Figure 7).
EXAMPLE 2: Design and E. coli expression of modified oleosins containing one or more
artificially introduced cysteine residue
Construct design for expression in E. coli
A number of modified oleosin constructs for expression in E. coli were designed. These
contained either one or three cysteine residues on the N-terminal and C-terminal hydrophilic
arms. The constructs were based on the nucleotide sequence and translated polypeptide
sequence from a sesame seed oleosin, GenBank clone AF091840 which contains no cysteine
residues (SEQ ID NO: 16).
All clones were subcloned into pET29b using engineered NdeI/XhoI sites. In addition, a ProTrp
coding sequence was added to the coding region of the 3' end of the C-terminal hydrophilic arm
to mimic the amino acid residues encoded for by the NcoI site previously engineered by Peng et
al (2006) Stability enhancement of native and artificial oil bodies by genipin crosslink. Taiwan
Patent I 250466.
Oleosin-cysteine proteins mutated to include cysteine residues in both the N- and C- terminal
hydrophilic regions described here are designated Ole-1-1, Ole-1-3, Ole-3-1, and Ole-3-3 (SEQ
ID NO 2, 3, 4, and 5 respectively), where the first and the second numeral digits correspond to
the number of disulfide bonds in the N- and C- terminus, respectively. The standard oleosin
without the cysteine residues was used as a control and was designated as Ole-0-0 (SEQ ID NO
1).
The cysteines were substituted for charged residues predicted to be on the surface of the oil
bodies and are listed below.
N-terminal single cysteine (Ole-1-x) Glu3Cys
N-terminal triple cysteine (Ole-3-x) Glu3Cys Arg12Cys Gln23Cys
C-terminal single cysteine (Ole-x-1) Gln137Cys
C-terminal triple cysteine (Ole-x-3) Gln112Cys Lys123Cys Gln137Cys
The constructs were designed so could be relatively simply sub cloned from the GENEART
provided backbone (pCR4Blunt-TOPO) into pET29b (Novogen) via NcoI/XhoI digestion and
58
ligation. This placed the oleosin coding sequence downstream of the pET29 N-terminal Stag
fusion and upstream of the C-terminal His tag (Figures 1-5 and SEQ ID Nos 1-10). The oleosin
and modified oleosin sequences used are summarised in the Summary of Sequences table.
Expression in E. coli and purification of modified oleosins containing at least one artificially
introduced cysteine
Expression of the recombinant sesame seed oleosins (with and without engineered cysteines) in
the E. coli expression system was evaluated by SDS-PAGE/Coomassie brilliant blue staining
and SDS-PAGE/immunoblot analysis using antibodies raised against the sesame seed oleosin
(described in Example 1).
Expression of recombinant modified oleosin was induced in a freshly inoculated 10mL culture of
E. coli (BL21 Rosetta-Gami) containing an oleosin (with or without engineered cysteine
residues) coding sequence in the pET29 expression vector. The culture was grown at 37°C,
220rpm, until mid log phase (OD6000.5 - 0.7); expression was induced by the addition of IPTG to
1 mM final concentration. The induced culture was incubated at 37°C, 220rpm, for a further
2-3 h. Given the properties of modified oleosin the applicants did not attempt to express it in a
soluble form but rather chose to extract it from inclusion bodies. Aliquots (1mL) of the culture
were transferred to 1.5mL microfuge tubes and the cells pelleted by centrifugation (2655 ×g for
5 min at 4°C).
Pelleted cells were resuspended in BugBuster® Reagent (Merck) at 5 mL/g of wet cell pellet,
with the addition of DNase to 40 μg/mL and mixed gently on a rotator for 30 min followed by
centrifugation at 8000g for 10 min at 4°C. The resultant cell pellet was retreated with
BugBuster® and DNase as above. The remaining soluble protein and suspended cell debris was
separated from the insoluble inclusion bodies by centrifugation at 8000g for 10 min at 4°C.
Recombinant oleosins were further purified from the inclusion bodies using a procedure adapted
from D’Andréa et al. (2007). Briefly: the inclusion body preparation was washed by resuspension
in 200 mM sodium carbonate buffer pH 11 (5 mL per gram of original cell pellet)
and re-pelleted by centrifugation at 8000 ×g for 10 min at 4°C. The washed inclusion body
pellet was again re-suspended in 5 mL 200 mM sodium carbonate buffer per gram of pellet and
added to 9 volumes of freshly prepared chloroform:methanol mix (5:4 v/v) giving a final ratio of
5:4:1 (chloroform:methanol:buffer). The suspension was gently mixed for 5 min which formed a
milky, single phase mixture; this was centrifuged at 10,000 ×g for 10 min at 4°C, and the
supernatant containing the modified oleosin was carefully separated from the pellet and
59
transferred into a new tube. Aliquots of the supernatant were dried down under a stream of
nitrogen and the protein re-solubilised in 8M urea and quantified by Qubit™ (Invitrogen).
EXAMPLE 3: Use of anti-sesame seed oleosin antibodies to bind sesame seed oleosin with
artificially introduced cysteines
A dot-blot was used to compare the ability of the anti-sesame seed oleosin antibodies (Abs)
described in Example 1 to bind to oleosin without cysteines versus the oleosins containing
cysteines (described in Example 2). Dilution series from 12 to 0.25ng of purified Ole-0-0, Ole-
1-3 and Ole-3-1 were spotted onto a pre-equilibrated Hybond-P PVDF Transfer membrane. This
was incubated with the anti-sesame seed oleosin antibodies at 1:2000 as the primary Ab. The
blot was then incubated with the appropriate secondary Ab and developed by
chemiluminescence (Figure 7). The results indicate that on an immunoblot, the anti-sesame seed
oleosin antibodies are up to an order of magnitude more sensitive to the oleosin without cysteine
residues than the oleosins with cysteine residues. As a consequence of the different sensitivities
it was necessary to load different quantities of recombinant protein onto the gels for analysis by
immunoblotting. Despite the non uniform lane loading it is still possible to compare different
oleosins between lanes in terms of their relative distribution between monomeric and oligomeric
forms.
EXAMPLE 4: Creation of artificial oil bodies with E. coli expressed modified oleosins
containing at least one artificially introduced cysteine and altering the degree of cross
linking
Preparation of artificial oil bodies
Artificial oil bodies (AOBs) were then prepared by drying down aliquots of the supernatant
described in Example 3, calculated to contain either 150μg or 1mg of recombinant oleosin.
The process of generating AOBs involved combining PL, TAG, and the recombinant
oleosin/modified oleosin. In the absence of strong chaotropic agents the disruptive force
required to dissociate individual recombinant oleosins from the purified fraction involved several
alternating cycles of sonicating and cooling. This was achieved by solubilising the 150μg and
1mg oleosin/modified oleosin samples in 20μL chloroform containing 150μg PL (Sigma,
Cat#P3644) and mixed with 60μL of purified sesame seed oil (Tzen and Huang 1992) and
940μL of AOB buffer (50mM sodium phosphate buffer pH 8.0, 100mM NaCl). The complete
60
mixture was then sonicated three times for 30sec (Sonics & Materials Vibra~Cell VC600,
600 W, 20 kHz; 1/8" tapered micro-tip probe, power setting #3).
The applicants also found that the purification procedure could be successfully scaled up and
when a 50g cell pellet was used as the starting material it was necessary to substitute the stream
of nitrogen with a rotary vacuum evaporator to remove the chloroform and the majority of the
methanol. At this point the majority of oleosin/modified oleosin precipitated out of the
azeotropic solvent and was separated by centrifugation at 12,000g for 10min.
Inclusion bodies were suspended in 1mL AOB Buffer II (50 mM sodium phosphate, pH 8.0, 100
mM NaCl, 20 mM -mercaptoethanol, 10 mM DTT and 5% [v/v] sesame oil) and then sonicated
4x. AOBs were concentrated by centrifugation at 12,000 rpm for 10min, this resulted in the
formation of a suspension of AOBs overlaying the aqueous fraction. The underlying aqueous
fraction was removed by pipette, and the remaining AOBs were washed (to remove soluble
proteins and reducing agents) by gentle agitation in 1mL AOB Buffer III (50 mM sodium
phosphate, pH 8.0, 100 mM NaCl). After washing, the AOBs were re-concentrated by
centrifugation, and the underlying aqueous fraction removed, then re-suspended by vortexing in
AOB Buffer IV (50 mM sodium phosphate buffer, pH 8.0, 100 mM NaCl, 1 mM GSSG) and the
AOBs stored at 4C for further analyses.
Recombinant Ole-0-0, and all variations of the oleosin-cysteines were successfully expressed
and located in E. coli inclusion bodies (Figure 9). Ole-0-0 was predominantly present as a
monomer (in both inclusion bodies as well as AOBs); this migrated fractionally faster than the
20kDa molecular weight marker (in reducing and non reducing SDS and SDS-UREA PAGE).
Also present were two slower migrating immunoreactive bands of approximately 35 and 36 kDa
which likely correspond to two forms of dimeric oleosin. While Ole-0-0 is not predicted to
contain any cysteine residues the overall intensity and ratio of the two apparent dimers was
influenced by the presence of reducing agents (-ME @ 5% of the sample loading buffer and
10mM DTT).
In the inclusion bodies, the predominant form of Ole-1-1 is monomeric. Only one dimeric form
appeared to be present and this was not influenced by reducing agents or urea. Ole-1-1 from
AOBs (generated in the presence of reducing agent and then in the presence of oxidising agent)
showed a large increase in the ratio of dimer to monomer as well as the formation of trimeric,
tetrameric and likely pentameric oligomers (the electrophoretic focus of these oligomers was
considerably improved in the SDS-UREA gel). Removal of the GSSG and re-introduction of
61
reducing agents to the AOBs resulted in the presence of only monomer and dimer in similar
proportions seen in the inclusion bodies. AOBs generated with Ole-1-1 (in the absence of both
reducing agents and GSSG ) showed the presence of almost equal portions of monomer and
dimer and a small amount of trimer, indicating that the conditions under which the AOBs are
formed have some reducing potential. The subsequent addition of GSSG resulted in an increase
in the oligomeric portions as well as the appearance of a tetrameric form.
While the monomer was the predominant form of Ole-3-3 in the inclusion bodies, a
comparatively high percentage was also present in multiple oligomeric forms. The proportion of
oligomers declined to a small extent with the addition of reducing agent and slightly more by the
addition of both reducing and chaotropic agents. Oligomeric forms of Ole-3-3 that were larger
than a trimer were poorly resolved when the recombinant protein was extracted from AOBs.
The creation of large oligomeric forms was promoted by the addition of GSSG and in the
absence of reducing and chaotropic agents a portion of these oligomeric forms failed to enter the
stacking gel. Combined, these results indicate that on the AOBs, Ole-3-3 was highly crosslinked
and the position of the cross-links was more variable compared to the Ole-3-3 recovered
from the inclusion bodies. This suggests that, despite considerable pre-existing cross-linking
(within the inclusion bodies), on the AOB Ole-3-3 has access to a high number of potential
partners for cross-linking. Similarly for Ole-1-3 and Ole-3-1, the number of cross-linked species
increased when there was more than one cysteine on one or both hydrophilic regions (Figures 8
and 9).
It could be anticipated that in non-reducing SDS-PAGE, oligomers containing the same number
of oleosins but with the disulfide bonds in different positions would migrate differently to each
other. Indeed this can be seen in Figure 8 where the data indicates that the position of the
oleosin arms, relative to one another are at different positions over the oil body. For example the
Ole-1-1 can only form one disulfide bond per arm and this has to form at the same position,
where as the presence of three cysteines enables more than one disulfide bond to form but it also
allows the disulfide bonds to form with different degrees of hydrophilic arm overlap as well as
having multiple oleosins bound to the same arm (Figures 8 and 9).
The addition of SDS and reducing agents (DTT and -ME) decreased the number of oligomeric
complexes (Figure 9). The addition of SDS and urea results in a similar pattern to SDS alone
except that the previously resolved multiple dimeric forms migrated as one and the trimeric and
tetrameric forms appear to be in higher abundance presumably because they are also migrating
as single bands which increases intensity correspondingly (Figure 9). In contrast, the presence
62
of SDS, reducing agent and urea resulted in fewer oligomeric forms of Ole-1-1 and Ole-1-3 but
not Ole-3-1 or Ole-3-3 (Figure 9). In the case of Ole-3-1 and Ole-3-3 it appears that the urea
does not completely denature the disulfides oleosins and may indeed prevent the complete
reduction of the disulfide bonds. It could be that these bonds are formed during the generation of
inclusion bodies (would need to see reduced and non reduced inclusion body preps).
Furthermore, the presence of the dimeric oleosin formed in the absence of engineered cysteine
residues (Figures 8 and 9) indicates that some oligomerisation is due to other types of attraction,
e.g, strong hydrophobic bonding that is not fully disrupted by SDS but can be almost completely
disrupted by the combination of both SDS and urea (Figure 8 and 9).
The effect of increasing the number of potential cross-linking sites in an oleosin peptide on AOB
integrity and emulsion stability can be assessed as follows.
Quantitative Determination of AOB integrity
Assessment of AOB stability and integrity using either absorbance (OD600), direct counting of
AOBs using a hemocytometer, or visual evaluation of coalescence by microscopy proved to be
highly variable and amongst other things was influenced by the: degree of pre-sampling
agitation; quantity of sample removed; time left under the microscope. To avoid this the
applicants devised a simple method to quantify the amount of TAG released from the AOBs into
the surrounding media during a variety of treatments as a means of comparing integrity.
Essentially equal quantities (based on FAMES-GC/MS estimation of TAG and Bradfords
determination of protein) of AOB preparations are made up to a total volume of 200μL using
AOB buffer (containing Proteinase K [PNK] when appropriate at a 1:1 ratio of PNK:total
proteins in OB or AOB samples in a 250μL GC glass insert tubes and covered with a plastic cap.
Following the treatment (elevated temperature or exposure to PNK) 15μL of fish oil (Vitamax®,
Australia) is added to the sample and mixed by vortexing followed by centrifugation at 5,200g
for 1min. The addition of fish oil followed by vortexing enables any TAG that had leaked from
the AOBs to mix with the added fish oil and be floated by brief centrifugation. 4μL of the oil
phase is sampled and subjected to fatty acid methyl esterification (FAME) and then analysed by
GC-MS (Shimadzu model numbers, fitted with a 50mQC2/BPX70-0.25 GC capillary column
(SGE) as described by Browse et al. (1986). In the absence of added fish oil the quantity of
TAG that had leaked from the AOBs was too small to form a samplable visible layer even after
centrifugation, in such a case the maximum volume would have been 6μL. The very different
lipid profiles of fish oil and sesame oil enabled us to easily distinguish the leaked TAG from the
added TAG.
63
Using the internal C15:0 and C17:0 standards the applicants can calculate the absolute amounts
of C18:2 (the major lipid in sesame seed oil) recovered after treatment.
Determination of AOB integrity and emulsion stability at elevated temperature
Oil in water emulsions are less stable at elevated temperatures; hence, it is of interest to
investigate if modified oleosins with varying numbers in introduced cysteines influence AOB
integrity at elevated temperature. To achieve this the applicants determine the integrity (using
the method described above) of OBs and AOBs (containing different oleosins) in a phosphate
buffer (50mM Na-phosphate buffer pH8, 100mM NaCl) at 95C. AOBs are heated for 2h.
Integrity is determined as above.
The effect of higher ratios of crosslinked oleosin:TAG on the stability of AOBs in rumen fluid
can be assessed as follows.
Determination of AOB integrity in rumen fluid
One of the aims of disulfide was to provide some degree of protection from biohydrogenation by
rumen microflora. Assessment of AOB stability with rumen fluid can be assessed as follows.
AOBs are added to an equal volume (25μL) of rumen fluid. Samples are incubated at 39°C for
0, 15, 30, 60, 120 and 240min, at the end of the incubation an equal volume of loading buffer
(Invitrogen) is added, mixed and heated at 70°C for 10min. 15μL of each sample/loading buffer
mix is compared by SDS-PAGE/immunoblot. Integrity is determined as above.
Analysis of AOB integrity in Proteinase K
To investigate the influence of modified oleosin in a controllable and repeatable highly
degradative environment integrity is determined (using the method described above) of AOBs
(containing different modified oleosins) after incubation in an phosphate buffer (50mM Naphosphate
buffer pH8, 100mM NaCl) containing 1:1 (g/g protein) Proteinase K (Invitrogen) at
37°C for 4h. While the maximum activity of Proteinase K is achieved below 65°C the lower
temperature is used in order to reduce the influence of temperature on AOB instability. Integrity
is determined as above.
EXAMPLE 5: Design and in planta expression of modied oleosin containing one or more
artificially introduced cysteines
Construct design for expression in planta
64
The applicants synthesised individual coding sequences for the sesame seed oleosin (based on
GenBank clone AF091840) with different numbers of cysteines in the N- and C-terminal arms.
The coding sequence was flanked by a 5' NotI site and a 3' NdeI site. A separate acceptor
cassette was synthesised containing an attL1 site, a NotI site and NdeI site followed by a nos
termination sequence, a forward facing CaMV35s promoter, the Arabidopsis thaliana DGAT1
(S205A) (SEQ ID NOs 11-20 and Figures 1-5) plus its own UBQ10 intron, an attL2 site. The
sesame seed oleosins with different numbers of cysteines were individually transferred to the
acceptor cassette via the NotI and NdeI sites. Each of these completed cassettes were then
transferred to a plant binary vector pRSh1, Figure 6 (Winichayakul et al., 2008) via the LR
recombination reaction. This placed the oleosin downstream of a CaMV35S promoter (already
contained within pRSh1) and placed a nos terminator (already contained within pRSh1)
downstream of the Arabidopsis DGAT1 (S205A) (Figures 1-5). The nucleotide sequences
encoding the sesame seed oleosins (with cysteines) and DGAT1 were optimised for expression
in Arabidopsis thaliana, this included optimisation of codon frequency, GC content, removal of
cryptic splice sites, removal of mRNA instability sequences, removal of potential
polyadenylation recognition sites, and addition of tetranucleotide stop codon (Brown et al, 1990;
Beelman and Parker, 1995; Rose, 2004; Rose and Beliakoff, 2000; Norris et al., 1993).
It should be noted that the oleosin sequence used is for example only. Any oleosin or steroleosin
or caoleosin sequences could be engineered to contain cross-linking regions. The coding
sequences of the complete ORFs (after splicing) were checked against repeat of the original
oleosin translated sequence and found to be identical over the oleosin coding regions.
Transformation of Arabidopsis thaliana with sesame seed oleosins containing cysteines
Transformation of Arabidopsis thaliana var Columbia (with constructs described above),
analyses of T2 seeds for modified oleosin, immunoblot analysis of Arabidopsis thaliana oil
bodies containing sesame seed oleosin with different numbers of cysteines was performed as
described previously (Scott et al., 2007).
Both the floral-dip (Clough, 1998) and floral-drop methods (Martinez-Trujillo, 2004) were used
in the transformation of Arabidopsis by Agrobacterium tumefaciens GV3101 containing the
binary constructs. T1 seed was collected from the treated plants, germinated and selected by
spraying at 14 d and 21 d post-germination with Basta®. Basta® resistant T1 plants (71, 62 and
23 transformants containing the single sesame seed oleosin, and modified oldeosin constructs
respectively) were transplanted, allowed to self-fertilise, set seed and the T2 seed was collected.
Equal quantities of seed extract from Basta® resistant Arabidopsis plants were analysed by SDS65
PAGE/immunoblot with the anti-sesame seed oleosin antibodies; recombinant sesame seed
oleosin and modified oldeosin of the appropriate size was observed in the majority of samples
(Figure 10). Southern blot analysis was performed on selected T2 lines to determine the number
of insertion sites.
EXAMPLE 6: Extraction and purificiation oil bodies with modified oleosins containing at
least one artificially introduced cysteine from the seeds of Arabidopsis thaliana
Crude Oil Body Preparations from Arabidopsis thaliana seeds
Crude OB preparations were prepared, from seed of plants produced as described in Example 5,
by either grinding 200mg seed with a mortar and pestle containing a spatula tip of sand and
750μL Extraction Buffer (10mM phosphate buffer, pH 7.5 containing 600mM sucrose) or by
homogenising 25mg of seed in 300μL Extraction Buffer using a Wiggenhauser D-130
Homogenizer. A further 750μL Extraction Buffer was added and the slurry in the mortar and
transferred to a 2mL microfuge tube whereas the homogenizer tip was rinsed in 1mL Extraction
Buffer and this volume was added to the homogenised seed. Samples were then centrifuged at
20, 000 ×g for 5min; this left a pellet and aqueous supernatant which was overlaid by an
immiscible oily layer containing both intact and disrupted oil bodies as well as free TAG. The
upper oil layer was gently pushed to the side of the tube, and the aqueous layer and pelleted
material discarded. The oil layer was then re-suspended from the side of the tube by vortexing in
Extraction Buffer and placed in a fresh 2mL microfuge tube. The final volume was made up to
0.5mL with Extraction Buffer.
Purified Oil Body preparations from Arabidopsis thaliana seeds and cross linking cysteine
residues between the engineered oleosins
25 mg of Arabidopsis seed (of plants transformed as described in Example 5) was ground in 300
μl extraction buffer (10 mM Phosphate buffer, pH 7.5 containing 600 mM sucrose) using a
Wiggenhauser D-130 Homogenizer. Seed was ground until crushed and the sample appeared
“creamy” and frothy as starch was released from the seeds. The homogenizer tip was rinsed in 1
ml buffer and this volume was added to the crushed seed. Samples were prepared up to this
point in lots of 4, then centrifuged 14,000rpm for 5 mins. A thin gel loading tip was used to
gently push the oil layer to the side of the tube, and the aqueous layer removed to a fresh tube.
The oil layer was resuspended from the side of the tube using extraction buffer and placed in a
fresh 2 ml tube. The final volume was made up to 0.5 ml (as read on the side of the tube) with
extraction buffer, samples were divided into two and oxidising agent (3mM GSSG) was added to
66
one tube and incubated at room temperature for 10 min. Oil body preparations were then added
to an equal volume of 2 x gel loading buffer and boiled for 5min before loading on to a gel.
Samples were run either on pre-cast NuPAGE Novex 4-12% Bis-Tris Midi Gels(Invitrogen) on a
Criterion gel rig system (Bio-Rad), or NuPAGE® Novex 12% Bis-Tris gradient Gel 1.0 mm, 15
well, cat# NP0343BOX, with NuPAGE® MES SDS Running Buffer (for Bis-Tris Gels only)
(20X), cat# NP0002-02, or on hand-cast Tris-HCl gels. Gels were stained by SafeStain
(Invitrogen) to show total protein loaded or blotted onto Nitrocellulose membrane using the iBlot
system (Invitrogen). In each case, the negative control was a sample extracted from wild type
Columbia seed and the positive control was the same extraction method (although grinding was
by mortar and pestle) performed on wild type sesame seed. 10μl of each sample and the
negative control were loaded onto the gel, and 5μl was used for the positive control.
Following blotting, the membrane was blocked in a solution of 12.5% skim milk powder in
TBST (50 mM Tris pH 7.4, 100 mM NaCl, 0.2 % Tween) for at least 1.5 hours. The membrane
was then washed in TBST 3 x 5 mins before incubating with primary antibody (anti-sesame) at
1/1000 in TBST for 1 hour at room temperature. Following 3 further TBST washes, incubation
with secondary antibody (anti-rabbit) at 1/5000 was carried out for 1 hour at room temperature.
The membrane underwent 3 further washes then the signal was developed using standard
chemiluminesence protocol.
Figure 11 shows the accumulation of sesame seed oleosin units on the oil bodies under the
control of the CaMV35S promoter. It can be seen that recombinant oleosin and polyoleosin was
found to accumulate in the seeds of Arabidopsis thaliana and was correctly targeted to the oil
bodies (Figure 11). In addition, it can be seen that in the presence of oxidising agent for 10
minutes the recombinant oleosins containing cysteines formed cross-links as evidenced by the
appearance of oligomers and corresponding disappearance of the monomeric forms in these
samples and not in the wild type or non oxidised transgenic oil bodies.
The effect of increasing the number of potential cross-linking sites in an oleosin peptide on in
planta OB integrity and emulsion stability can be assessed as follows.
Quantitative Determination of OB integrity
Assessment of OB stability and integrity using either absorbance (OD600), direct counting of
AOBs using a hemocytometer, or visual evaluation of coalescence by microscopy proved to be
highly variable and amongst other things was influenced by the: degree of pre-sampling
67
agitation; quantity of sample removed; time left under the microscope. To avoid this the
applicants devised a simple method to quantify the amount of TAG released from the OBs into
the surrounding media during a variety of treatments as a means of comparing integrity.
Essentially equal quantities (based on FAMES-GC/MS estimation of TAG and Bradfords
determination of protein) of OB preparations are made up to a total volume of 200μL using AOB
buffer (containing Proteinase K [PNK] when appropriate at a 1:1 ratio of PNK:total proteins in
OB samples in a 250μL GC glass insert tubes and covered with a plastic cap. Following the
treatment (elevated temperature or exposure to PNK) 15μL of fish oil (Vitamax®, Australia) is
added to the sample and mixed by vortexing followed by centrifugation at 5,200g for 1min. The
addition of fish oil followed by vortexing enables any TAG that had leaked from the OBs to mix
with the added fish oil and be floated by brief centrifugation. 4μL of the oil phase is sampled
and subjected to fatty acid methyl esterification (FAME) and then analysed by GC-MS
(Shimadzu model numbers, fitted with a 50mQC2/BPX70-0.25 GC capillary column (SGE) as
described by Browse et al. (1986). In the absence of added fish oil the quantity of TAG that had
leaked from the OBs was too small to form a samplable visible layer even after centrifugation, in
such a case the maximum volume would have been 6μL. The very different lipid profiles of fish
oil and sesame oil enabled us to easily distinguish the leaked TAG from the added TAG.
Using the internal C15:0 and C17:0 standards the applicants can calculate the absolute amounts
of C18:2 (the major lipid in sesame seed oil) recovered after treatment.
Determination of OB integrity and emulsion stability at elevated temperature
Oil in water emulsions are less stable at elevated temperatures; hence, it is of interest to
investigate if modified oleosins with varying numbers in introduced cysteines influence OB and
AOB integrity at elevated temperature. To achieve this the applicants determine the integrity
(using the method described above) of OBs (containing different oleosins) in an phosphate
buffer (50mM Na-phosphate buffer pH8, 100mM NaCl) at 95C. AOBs are heated for 2h.
Integrity is determined as above.
The effect of higher ratios of crosslinked oleosin:TAG increase the stability of OBs in rumen
fluid can be assessed as follows:
68
Determination of OB integrity in rumen fluid
One of the aims of disulfide was to provide some degree of protection from biohydrogenation by
rumen microflora. Assessment of OB stability with rumen fluid can be assessed as follows.
OBs are added to an equal volume (25μL) of rumen fluid. Samples are incubated at 39°C for 0,
15, 30, 60, 120 and 240min, at the end of the incubation an equal volume of loading buffer
(Invitrogen) is added, mixed and heated at 70°C for 10min. 15μL of each sample/loading buffer
mix is compared by SDS-PAGE/immunoblot. Integrity is determined as above.
Analysis of OB integrity in Proteinase K
To investigate the influence of modified oleosin in a controllable and repeatable highly
degradative environment integrity is determined (using the method described above) of AOBs
(containing different modified oleosins) after incubation in an phosphate buffer (50mM Naphosphate
buffer pH8, 100mM NaCl) containing 1:1 (g/g protein) Proteinase K (Invitrogen) at
37°C for 4h. While the maximum activity of Proteinase K is achieved below 65°C the lower
temperature is used in order to reduce the influence of temperature on OB instability. Integrity is
determined as above.
EXAMPLE 7: Production of oil bodies in the leaves of Arabidopsis thaliana
In order to produce oil bodies in vegetative tissue, it is necessary to produce triacyclglycerol in

CLAIMS:
1. A polynucleotide encoding a modified oleosin including at least one artificially introduced
cysteine.
2. The polynucleotide of claim 1 in which the modified oleosin includes at least two cysteines,
at least one of which is artificially introduced.
3. The polynucleotide of claim 3 in which, the modified oleosins each include:
i) at least two artificially introduced cysteines,
ii) at least three artificially introduced cysteines,
iii) at least four artificially introduced cysteines,
iv) at least five artificially introduced cysteines,
v) at least six artificially introduced cysteines,
vi) at least seven artificially introduced cysteines,
vii) at least eight artificially introduced cysteines,
viii) at least nine artificially introduced cysteines,
ix) at least ten artificially introduced cysteines,
x) at least eleven artificially introduced cysteines,
xi) at least twelve artificially introduced cysteines,
xii) at least thirteen artificially introduced cysteines, or
xiii) at least fourteen artificially introduced cysteines.
4. The polynucleotide of claim 2 or 3 in which the modified oleosin incudes at least one cysteine
in the N-terminal hydrophilic region, and at least one cysteine in the C-terminal hydrophilic
region.
5. The polynucleotide of claim 4 in which the cysteines are distributed substantially evenly
between the N-terminal and C-terminal hydrophilic regions of the oleosin.
6. The polynucleotide of any one of claims 1 to 5 in which the polynucleotide encodes a fusion
protein including the modified oleosin fused to a protein of interest.
7. A genetic construct, or expression construct, comprising the polynucleotide of any one of
claims 1 to 6.
8. The genetic construct, or expression construct, of claim 7 in which the polynucleotide
construct is operably linked to a promoter sequence.
94
9. The genetic construct, or expression construct, of claim 8 in which the promoter sequence is
capable of driving expression of the polynucleotide in a plant.
10. A host cell comprising a polynucleotide of any one of claims 1 to 6.
11. A host cell genetically modified to express a polynucleotide of any one of claims 1 to 6, or
an expression product of the polynucleotide.
12. A host cell comprising a construct of any one of claims 7 to 9.
13. The host cell of any one of claims 10 to 12 that is also genetically modified to express a
triacylglycerol (TAG) synthesising enzyme.
14. The host cell of claim 13 that comprises an expression construct including a nucleic acid
sequence encoding a triacylglycerol (TAG) synthesising enzyme.
15. The host cell of claim 14 in which the nucleic acid is operably linked to a promoter
sequence.
16. The host cell of claim 15 in which the promoter sequence, linked to the nucleic acid
encoding a triacylglycerol (TAG) synthesising enzyme, is capable of driving expression of the
nucleic acid sequence in a plant.
17. The host cell of any one of claims 10 to 16 that isa host cell selected from a bacterial cell, a
yeast cell, a fungal cell, an insect cell, an algal cell, and a plant cell.
18. The host cell of any one of claims 10 to 16 that is a plant cell.
19. A plant comprising a plant cell of claim 18.
20. The plant of claim 19 that expresses a modified oleosin encoded by the polynucleotide of
any one of claims 1 to 6.
21. The plant of claim 19 that expresses the modified oleosin in a vegetative tissue of the plant.
22. The plant of claim 19 that expresses the modified oleosin in a seed of the plant.
23. The plant of claim 20 that expresses the modified oleosin in the pollen of the plant.
24. The plant of any one of claims 20 to 23 that is also genetically modified to express a
triacylglycerol (TAG) synthesising enzyme.
95
25. The plant of claim 24 in which the triacylglycerol (TAG) synthesising enzyme is expressed
in the same tissue as the modified oleosin.
26. The plant of any one of claims 19 to 25 that expresses about 0.5 to about 4.0 times as much
total lipid as does a suitable control plant.
27. A modified oleosin encoded by the polynucleotide of any one of claims 1 to 6.
28. A modified oleosin including at least one artificially introduced cysteine.
29. The modified oleosin of claim 28 that includes at least two cysteines, at least one of which is
artificially introduced.
30. The modified oleosin of claim 29 including:
i) at least two artificially introduced cysteines,
ii) at least three artificially introduced cysteines,
iii) at least four artificially introduced cysteines,
iv) at least five artificially introduced cysteines,
v) at least six artificially introduced cysteines,
vi) at least seven artificially introduced cysteines.
vii) at least eight artificially introduced cysteines,
viii) at least nine artificially introduced cysteines,
ix) at least ten artificially introduced cysteines,
x) at least eleven artificially introduced cysteines,
xi) at least twelve artificially introduced cysteines,
xii) at least thirteen artificially introduced cysteines, or
xiii) at least fourteen artificially introduced cysteines.
31. The modified oleosin of claim 29 or 30 that includes at least one cysteine in the N-terminal
hydrophilic region, and at least one cysteine in the C-terminal hydrophilic region.
32. The modified oleosin of claim 31 in which the cysteines are distributed substantially evenly
between the N-terminal and C-terminal hydrophilic region of the oleosin.
33. A fusion protein comprising the modified oleosin of any one of claims 27 to 32 and and a
protein of interest.
34. An oil body comprising a modified oleosin of any one of claims 27 to 32.
96
35. An oil body comprising at least two modified oleosins of any one of claims 27 to 32.
36. The oil body of claim 35 in which at least two of the modified oleosins are cross-linked to
each other via at least one disulphide bridges between cysteine residues in the modified oleosins.
37. The oil body of claim 35 in which the modified oleosins are not cross-linked.
38. The oil body in any one of claims 34 to 36 that additionally comprises a fusion protein that
includes an oleosin fused to a protein of interest.
39. The oil body in claim 38 in which the oleosin in the fusion protein does not include an
artificially introduced cysteine.
40. The oil body of claim 38 in which the oleosin in the fusion protein includes an artificially
introduced cysteine in its oleosin portion.
41. The oil body of claim 40 comprising at least two fusion proteins each including an
artificially introduced cysteine.
42. The oil body of claim 41 in which at least two of the fusion proteins are cross-linked to each
other via disulphide bridges between cysteine residues in the modified oleosin portion of the
fusion proteins.
43. An emulsion comprising a modified oleosin of any one of claims 27 to 32.
44. An emulsion comprising an oil body of any one of claims 34 to 42.
45. A composition comprising a modified oleosin of any one of claims 27 to 32.
46. A composition comprising an oil body of any one of claims 34 to 42.
47. The composition of claim 46 comprising the oil body and a suitable carrier.
48. The composition of claim 47 in which the carrier is buffered, with the appropriate redox
environment to attain the desired degree of cross-linking of the modified oleosins.
49. The composition of any one of claims 45 to 48 formulated for dermal application.
50. A plant, or part thereof, comprising an oil body of any one of claims 34 to 42.
51. A vegetative tissue of a plant, comprising an oil body of any one of claims 34 to 42.
97
52. A seed of a plant, comprising an oil body of any one of claims 34 to 42.
53. An animal feed comprising an oil body of any one of claims 34 to 42.
54. An animal feed comprising a plant, or part or tissue thereof, of any one of claims 19 to 26
and 50 to 52.
55. A method for producing an oil body, the method comprising the step of combining:
a) at least two of the modified oleosins of any one of claims 27 to 32, triacylglycerol, and
b) phospholipid.
56. The method of claims 55 comprising the additional step of regulating the degree of crosslinking
of modified oleosins in the oil body by controlling the redox environment of the oil body
produced.
57. The method of claim 55 or 56 in which at least some of the modified oleosins are part of
fusion proteins wherein the fusion proteins comprise a modified oleosin, and a protein of
interest.
58. The method of any one of claims 55 to 57 in which the components of a), b) and c) are
combined within a host cell.
59. The method of claims 58 in which the modified oleosins are expressed in the host cell.
60. The method of claim 58 or 59 in which host cell is genetically modified to express the
modified oleosins.
61. The method of any one of claims 58 to 60 in which the host cell is also genetically modified
to express a triacylglycerol (TAG) synthesising enzyme.
62. The method of any one of claims 58 to 61 in which the host cell forms part of an organism.
63. The method of claim 62 in which the organism is a plant.
64. The method of claim 62 wherein the plant acumulates about 50% to about 400% more lipid
than does a suitable control plant.
65. The method of any one of claims 58 to 64 including the additional step of purifying the oil
bodies from the cell or organism.
98
66. The method of any one of claims 58 to 65 comprising the additional step of regulating the
degree of cross-linking of modified oleosins in the in vivo produced purified oil bodies by
controlling the redox environment of the purified oil bodies.
67. The method of any one of claims 55 to 57 in which the components of a), b) and c) are
combined in vitro.
68. The method of claim 67 comprising the additional step of regulating the degree of crosslinking
by controlling the redox environment in which the components of a), b) and c) are
combined.
69. The method of claim 68 in which the degree of cross-linking is regulated after the oil body is
formed, by controlling the redox environment in which the oil body is contained.
70. An oil body produced by a method of any one of claims 65 to 69.
71. A method of producing oil, the method comprising cultivating a host cell of any one of
claims 10 to 18, or plant of any one of claims 19 to 26 in conditions conducive to the production
of oil.
72. A method of producing a plant that accumulates more oil than a suitable control plant the
method comprising providing a plant transformed with a polynucleotide of any one of claims 1
to 6 that expresses a modified oleosin encode by the polynucleotide.
73. The method of claim of claim 72 wherein the plant is also transformed with a polynucleotide
encoding a TAG synthesising enzyme to express the TAG synthesising enzyme and thus
synthesise TAG.
74. The method of claim 73 wherein the plant is produced by transforming a single plant, or
plant cell, with both the polynucleotide of any one of claims 1 to 6 and the polynucleotide
encoding the TAG synthesising enzyme.
75. The method of claim 72 wherein the plant is produced by crossing a first plant transformed
with a polynucleotide of any one of claims 1 to 6, with second plant transformed the
polynucleotide encoding the TAG synthesising enzyme, to produce the plant transformed with
both a polynucleotide of any one of claims 1 to 6, and a polynucleotide encoding the TAG
synthesising enzyme.
99
76. The method of any one of claims 72 to 75 wherein the plant accumulates about 50% to about
400% more lipid than does a suitable control plant.
77. The method of any one of claims 71 to 76 wherein the oil is TAG.
78. The method of any one of claims 71 to 77 wherein oil is produced in the vegetative tissues
of the plant
79. The method of claim any one of claims 71 to 78 wherein the plant is processed into an
animal feed.
80. The method of any one of claims 71 to 78 wherein the plant is processed into a biofuel feed
stock.
81. A method for producing an oil body in a host cell, the method comprising:
a) introducing into a host cell at least one polynucleotide of any one of claims 1 to 6;
and
b) culturing the host cell in order to express the modified oleosin.
82. A method for producing an oil body in a host cell, the method comprising:
a) introducing into a host cell at least one polynucleotide of any one of claims 1 to 6 and
a nucleic acid molecule encoding a TAG synthesizing enzyme ; and
b) culturing the host cell in order to express the modified oleosin and the TAG
synthesizing enzyme.
83. The method of claim 71, 77, 81 or 82 wherein the host cell is processed into an oil fraction.
84. The method of any one of claims 83 wherein the oil is processed into a fuel, oleochemical or
nutritional or cosmetic oil, a polyunsaturated fatty acid (PUFA) or a combination thereof.