Novel acyltransferase polynucleotides, poypeptides, and methods of use
TECHNICAL FIELD
The invention relates to compositions and methods for the manipulation of cellular lipid
production and/ or cellular lipid profile.
BACKGROUND
Plant oil is an economically important product not only due to its broad utilization in the food
industry and as a component of feed ingredients but it also has a wide range of applications as
biofuels or in the manufacture of various nutraceutical and industrial products. Within the plant
itself, oil is essential to carry out a number of metabolic processes which are vital to growth and
development particularly during seed germination and early plant growth stages. Considering its
value, there is a growing research interest within the biotechnology field to improve plant oil
production and make the supply more sustainable.
The major component of plant oil is triacylglyceride (TAG). It is the main form of storage lipid
in oil seeds and the primary source of energy for seed germination and seedling development.
TAG biosynthesis via the Kennedy pathway involves sequential acylation steps starting from the
precursor x»-glycerol-3-phosphate (G3P). Firstly, G3P is esterified by an acyl-CoA to form
phosphati dic acid (LPA) in a reaction catalyzed by glycerol-3-phosphate acyltransferase
(GPAT, EC 2.3.1.15). This is followed by a second acylation step catalyzed by /jwphosphatidic
acid acyltransferase (LPAT; EC 2.3.1.51) forming phosphatide acid (PA), a key intermediate in
the biosynthesis of glycerolipids. The PA is then dephosphorylated by the enzyme phosphatidic
acid phosphatase (PAP; EC3.1.3.4) to release the immediate precursor for TAG, the sn- 1,2-
diacylglycerol (DAG). Finally, DAG is acylated in the sn-3 position by the enzyme diacylglycerol
acyltransferase (DGAT; EC 2.3.1.20) to form TAG.
Since this last catalytic action is the only unique step in TAG biosynthesis, DGAT is termed as
the committed triacylglycerol-forming enzyme. As DAG is located at the branch point between
TAG and membrane phospholipid biosyntheses, DGAT potentially plays a decisive role in
regulating the formation of TAG in the glycerolipid synthesis pathway (Lung and Weselake,
2006, Lipids. Dec 2006;41(12):1073-88). There are two different families of DGAT proteins.
The first family of DGAT proteins ("DGAT1") is related to the acyl-coenzyme Axholesterol
acyltransferase ("ACAT") and has been desbried in the U.A. at. 6,100,077 and 6,344,548. A
second family of DGAT proteins ("DGAT2") is unrelated to the DGAT1 family and is
described in PCT Patention Publication WO 2004/01 1671 published Feb. 5, 2004. Other
references to DGAT genes and their use in plants include PCT Publication Nos.
WO2004/01 1,671, WO1998/055.631, and WO2000/001.71 3, and US Patent Publication No.
200301 15632.
DGAT1 is typically the major TAG synfhesising enzyme in both the seed and senescing leaf
(Kaup et al, 2002, Plant Physiol. 129(4):1 616-26; for reviews see Lung and Weselake 2006,
Lipids. 4 1(12):1073-88; Cahoon et al., 2007, Current Opinion in Plant Biology. 10:236-244; and Li
et a/., 2010, Lipids. 45:145-1 57).
Raising the yield of oilseed crops (canola, sunflower, safflower, soybean, corn, cotton, linseed,
flax etc) has been a major target for the agricultural industry for decades. Many approaches
(including traditional and mutational breeding as well as genetic engineering) have been tried,
typically with modest success (Xu et al., 2008, Plant Biotechnol J., 6:799-818 and references
therein).
Although liquid biofuels offer considerable promise the reality of utilising biological material is
tempered by competing uses and the quantities available. Consequently, engineering plants and
microorganisms to address this is the focus of multiple research groups; in particular the
accumulation of triacylglcerol (TAG) in vegetative tissues and oleaginous yeasts and bacteria
(Fortman et al., 2008, Trends Biotechnol 26, 375-381 ; Ohlrogge et al, 2009, Science 324, 1019-
1020). TAG is a neutral lipid with twice the energy density of cellulose and can be used to
generate biodiesel a high energy density desirable biofuel with one of the simplest and most
efficient manufacturing processes. Engineering TAG accumulation in leaves has so far resulted
in a 5-20 fold increase over WT utilising a variety of strategies which includes: the overexpression
of seed development transcription factors (LEC1, LEC2 and WRIl); silencing of
APS (a key gene involved in starch biosynthesis); mutation of CGI-58 (a regulator of neutral lipid
accumulation); and upregulation of the TAG synfhesising enzyme DGAT (diacylglycerol O
acyltransferase, EC 2.3.1 .20) in plants and also in yeast (Andrianov et al, 2009, Plant Biotech J 8,
1-1 1;Mu et al, 2008, Plant Physiol 148, 1042-1054; Sanjaya et / 201 1, Plant BiotechJ 9, 874-883;
Santos-Mendoza et al., 2008, Plant J 54, 608-620; James et al, 2010, Proc Natl Acad Sci U SA
107, 17833-17838; Beopoulos et al, 201 1, Appl Microbiol Biotechnol 90, 1193-1206; Bouvier-
Nave et , 2000, Eur J Biochem 267, 85-96; Durrett et al, 2008, Plant J 54, 593-607. However, it
has been acknowledged that to achieve further increases in TAG, preventing its catabolism may
be crucial within non oleaginous tissues and over a range of developmental stages (Yang and
Ohlrogge, 2009, Plant Physiol 150, 1981-1 989.
Positively manipulating the yield and quality of triacylglycderides (TAG) in eukaryotes is difficult
to achieve. The enzyme diacylglycerol-O-acyltransferase (DGAT) has the lowest specific activity
of the Kennedy pathway enzymes and is regarded as a 'bottleneck' in TAG synthesis.
Attempts have been made previously to improve DGATl by biotechnological methods, with
limited success. For example Nykiforuk et a/., (2002, Biochimica et Biophysica Acta 580:95-
109) reported N-terminal truncation of the r ssic napus DGATl but reported approximately
50% lower activity. McFie e a/., (2010, JBC, 285:37377-37387) reported that N-terminal
truncation of the mouse DGATl resulted in increased specific activity of the enzyme, but also
reported a large decline in the level of protein that accumulated.
Xu e a/., (2008, Plant Biotechnology Journal, 6:799-81 8) recently identified a consensus sequence
(X-Leu-X-Lys-X-X-Ser-X-X-X-Val) within Tropaeolum majus (garden nasturtium) DGATl
(TmDGATl) sequences as a targeting motif typical of members of the SNF -related protein
kinase- 1 (SnRKl) with Ser being the residue for phosphorylation. The SnRKl proteins 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, Plant Mol Biol. 37:735-48. Review). Xu et a/., (2008,
Plant Biotechnology Journal, 6:799-81 8) performed site-directed mutagenesis on six putative
functional regions /motifs of the TmDGATl enzyme. Mutagenesis of a serine residue (S197) in a
putative SnRKl target site resulted in a 38%-80% increase in DGATl activity, and overexpression
of the mutated TmDGATl in Arabidopsis resulted in a 20%—50% increase in oil
content on a per seed basis.
It would be beneficial to provide improved forms of DGATl, which overcome one or more of
the deficiencies in the prior art, and which can be used to increase cellular oil production.
It is an object of the invention to provide an improved DGATl protein and methods for its use
to increase cellular lipid production and/ or at least to provide the public with a useful choice.
SUMMARY OF THE INVENTION
The inventors provide a novel DGATl protein with improved properties over known DGATl
proteins, particularly known DGATl proteins from plants. The novel DGATl protein of the
invention can be expressed in cells to increase cellular lipid accumulation. Expression of the
DGATl protein of the invention in cells results in a higher level of lipid than any of several
other plant DGATl proteins tested by the applicants.
Poyl nucleotide encoding a poylpeptide
In the first aspect the invention provides an isolated polynucleotide encoding a DGATl
polypeptide comprising the sequence of SEQ ID NO:39 (ZmDGATl -long) or a variant or
fragment thereof.
In one embodiment the variant has at least 70% identity to SEQ ID NO:39. In a further
embodiment the variant has DGATl activity.
In a further embodiment the DGATl polypeptide has higher DGATl activity than at least one
other DGATl protein.
In one embodiment the DGATl polypeptide has at least 5%, more preferably at least 10%, more
preferably at least 5%, more preferably at least 20%, more preferably at least 25%, more
preferably at least 30%, more preferably at least 35%, more preferably at least 40%, more
preferably at least 45%, more preferably at least 50% higher DGATl activity relative to the at
least one other DGATl protein.
Preferably the at least one other DGATl protein has the amino acid sequence of the polypeptide
of SEQ ID NO:44 ( ZmDGATl -short).
In one embodiment the DGATl polypeptide has the higher DGATl activity when expressed in
a cell.
In a further embodiment the DGATl polypeptide has higher DGATl activity than any
previously known DGATl protein.
In one embodiment the DGATl polypeptide has the higher DGATl activity when expressed in
a cell.
In one embodiment the DGATl polypeptide has at least 5%, more preferably at least 10%, more
preferably at least 5%, more preferably at least 20%, more preferably at least 25%, more
preferably at least 30%, more preferably at least 35%, more preferably at least 40%, more
preferably at least 45%, more preferably at least 50% higher DGATl activity than any previously
known DGATl protein.
In one embodiment the DGATl polypeptide has the higher DGATl activity when expressed in
a cell.
In a further embodiment the polypeptide of the invention has altered substrate specificity relative
to at least one other DGATl protein.
In one embodiment the DGATl polypeptide has the altered substrate specificity when expressed
in a cell.
Preferably the at least one DGATl protein has the amino acid sequence of the polypeptide of
SEQ ID NO:44 ( ZmDGATl -short).
In a further embodiment the polypeptide of the invention, when expressed in the cell, has altered
substrate specificity relative to any previously known plant DGATl protein.
In one embodiment the DGATl polypeptide has the altered substrate specificity when expressed
in a cell.
In a further embodiment the DGATl protein of the invention is not expressed in naturally
occurring plants.
Polypeptidefragment
Preferably the fragment comprises at least 50 contiguous amino acids, more preferably at least
100 contiguous amino acids, more preferably at least 150 contiguous am o acids, more
preferably at least 200 contiguous amino acids, more preferably at least 250 contiguous amino
acids, more preferably at least 300 contiguous amino acids, more preferably at least 350
contiguous amino acids, more preferably at least 400 contiguous amino acids, more preferably at
least 450 contiguous amino acids of the polypeptide of the invention.
In one embodiment the fragment of the DGATl polypeptide of the invention can confer
increased DGATl activity when added to at least part of another DGATl polypeptide.
Polynucleotide
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of
SEQ ID NO:10 (ZmDGATl-long) or a variant or fragment thereof.
In one embodiment the variant has at least 70% identity to SEQ ID NO:10. In a further
embodiment the variant encodes a polypeptide with DGATl activity.
Poyl nucleotidefragment
In a preferred embodiment, the fragment of the polynucleotide of the invention, encodes a
fragment of the polypeptide of the invention.
Poylpeptide
In a further aspect the invention provides a polypeptide with the sequence of SEQ ID NO:39
(ZmDGATl-long) or a variant or fragment thereof.
In one embodiment the variant has at least 70% identity to SEQ ID NO:39. In a further
embodiment the variant has DGATl activity.
In a further embodiment the DGATl polypeptide has higher DGATl activity than at least one
other DGATl protein.
In one embodiment the DGATl polypeptide has at least 5%, more preferably at least 10%, more
preferably at least 5%, more preferably at least 20%, more preferably at least 25%, more
preferably at least 30%, more preferably at least 35%, more preferably at least 40%, more
preferably at least 45%, more preferably at least 50% higher DGATl activity relative to the at
least one other DGATl protein.
Preferably the at least one DGATl protein has the amino acid sequence of the polypeptide of
SEQ ID NO:44 ( ZmDGATl -short).
In a further embodiment the DGATl polypeptide has higher DGATl activity than any
previously known DGATl protein.
In a further embodiment the polypeptide of the invention, when expressed in the cell, has altered
substrate specificity relative to at least one other DGATl protein.
Preferably the at least one DGATl protein has the amino acid sequence of the polypeptide of
SEQ ID NO:44 ( ZmDGATl-short).
In a further embodiment the polypeptide of the invention, when expressed in the cell, has altered
substrate specificity relative to any previously known plant DGATl protein.
Preferably the fragment comprises at least 50 contiguous amino acids, more preferably at least
100 contiguous amino acids, more preferably at least 150 contiguous amino acids, more
preferably at least 200 contiguous amino acids, more preferably at least 250 contiguous amino
acids, more preferably at least 300 contiguous amino acids, more preferably at least 350
contiguous amino acids, more preferably at least 400 contiguous amino acids, more preferably at
least 450 contiguous amino acids of the polypeptide of the invention.
In one embodiment the fragment of the DGATl polypeptide of the invention can confer
increased DGATl activity when added to at least part of an other DGATl polypeptide.
Construct
In a further embodiment the invention provides a genetic construct comprising a polynucleotide
of the invention.
Cells
In a further embodiment the invention provides a cell comprising a polynucleotide of the
invention. Preferably the cell, or its predecessor, is transformed to comprise the polynucleotide
of the invention.
In a further embodiment the invention provides a cell comprising a genetic construct of the
invention.
In a preferred embodiment the cell expresses the polynucleotide of the invention.
In a preferred embodiment the cell expresses the polypeptide of the invention.
In a preferred embodiment the cell, or its predecessor, is transformed or genetically modified to
expresses the polynucleotide or polypeptide of the invention.
In one embodiment the polypeptide of the invention, when expressed in the cell, has increased
DGAT1 activity relative to at least one other DGAT1 protein.
In one embodiment the DGAT1 polypeptide has at least 5%, more preferably at least 10%, more
preferably at least 5%, more preferably at least 20%, more preferably at least 25%, more
preferably at least 30%, more preferably at least 35%, more preferably at least 40%, more
preferably at least 45%, more preferably at least 50% higher DGAT1 activity relative to the at
least one other DGAT1 protein.
Preferably the at least one DGAT1 protein has the amino acid sequence of the polypeptide of
SEQ ID NO:44 ( ZmDGATl -short).
In a further embodiment the polypeptide of the invention, when expressed in the cell, has
increased DGAT1 activity relative to any previously known plant DGAT1 protein.
In a further embodiment the cell produces more lipid than does a control cell.
In one embodiment the cell produces at least 5% more, preferably at least 10% more, preferably
at least 15% more, preferably at least 20% more, preferably at least 25% more, preferably at least
30% more, preferably at least 35% more, preferably at least 40% more, preferably at least 45%
more, preferably at least 50% more, preferably at least 55% more, preferably at least 60% more,
preferably at least 65% more, preferably at least 70% more, preferably at least 75% more,
preferably at least 80% more, preferably at least 85% more, preferably at least 90% more,
preferably at least 95% more preferably at least 100% more, preferably at least 105% more,
preferably at least 110% more, preferably at least 115% more, preferably at least 120% more,
preferably at least 125% more, preferably at least 130% more, preferably at least 135% more,
preferably at least 140% more, preferably at least 145% more, preferably at least 150% more lipid
than does a control cell.
In a further embodiment the polypeptide of the invention, when expressed in the cell, has altered
substrate specificity relative to at least one other DGAT1 protein.
Preferably the at least one DGAT1 protein has the amino acid sequence of the polypeptide of
SEQ ID NO:44 ( ZmDGATl-short).
In a further embodiment the polypeptide of the invention, when expressed in the cell, has altered
substrate specificity relative to any previously known plant DGAT1 protein.
In a further embodiment the cell has an altered lipid profile relative to a control cell.
In one embodiment the proportion of 16:0 in the triacylglycerol is altered relative to that in a
control cell.
In one embodiment the proportion of 16:0 in the triacylglycerol is altered by at least 1%,
preferably at least 2%, more preferably at least 3%, more preferably at least 4%, more preferably
at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least
8%, more preferably at least 9%, more preferably at least 10%, more preferably at least %,
more preferably at least %, more preferably at least 13%, more preferably at least %, more
preferably at least 5%, more preferably at least 6%, more preferably at least 7%, more
preferably at least 8%, more preferably at least 9%, more preferably at least 20%, relative to
that in a control cell.
In a further embodiment the altered lipid profile has a proportion of 16:0 in the triacylglycerol in
the range 6% to 16%. In this embodiment the proportion of 16:0 in the triacylglycerol is altered
within the range 6% to 16%.
In a further embodiment the proportion of 18:0 in the triacylglycerol is altered relative to that in
a control cell.
In one embodiment the proportion of 18:0 in the triacylglycerol is altered by at least 1%,
preferably at least 2%, more preferably at least 3%, more preferably at least 4%, more preferably
at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least
8%, more preferably at least 9%, more preferably at least 10%, more preferably at least 11%,
more preferably at least %, more preferably at least 13%, more preferably at least 14%, more
preferably at least 5%, more preferably at least 6%, more preferably at least 17%, more
preferably at least 18%, more preferably at least 19%, more preferably at least 20%, relative to
that in a control cell.
In a further embodiment the altered lipid profile has a proportion of 18:0 in the triacylglycerol in
the range 7% to 15%. In this embodiment the proportion of 18:0 in the triacylglycerol is altered
within the range 7% to 15%.
In a further embodiment the proportion of 18:1 in the triacylglycerol is altered relative to that in
a control cell.
In one embodiment the proportion of 18:1 in the triacylglycerol is altered by at least 1%,
preferably at least 2%, more preferably at least 3%, more preferably at least 4%, more preferably
at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least
8%, more preferably at least 9%, more preferably at least 10%, more preferably at least 11%,
more preferably at least 12%, more preferably at least 13%, more preferably at least 14%, more
preferably at least 5%, more preferably at least 6%, more preferably at least 17%, more
preferably at least %, more preferably at least %, more preferably at least 20%, relative to
that in a control cell.
In a further embodiment the altered lipid profile has a proportion of 18:1 in the triacylglycerol in
the range 39% to 55%. In this embodiment the proportion of 18:1 in the triacylglycerol is
altered within the range 39% to 55%.
The control cell may be any cell of the same type that is not transformed with the
polynucleotide, or construct, of the invention to express the polypeptide of the invention. The
control cell may also be transformed with an "empty" vector, wherein the empty vector does not
include an insert sequence corresponding to a polynucleotide of the invnetion or expressing a
polypeptide of the invention.
In one embodiment the control cell is an un transformed cell. In a further embodiment the
control cell is transformed cell to express the polypeptide of SEQ ID NO:44 ( ZmDGATlshort).
In a further embodiment the control cell is transformed cell to express any previously
known plant DGAT1 protein.
Cells also transformed to express an oleosin
In one embodiment the cell is also transformed to express at least one of: an oleosin, steroleosin,
caloleosin, polyoleosin, and an oleosin including at least one artificially introduced cysteine
(WO2011/053169).
In one embodiment the cell is a plant cell.
Plant
In a further embodiment the invention provides a plant comprising a polynucleotide of the
invention. Preferably the plant, or its predecessor, is transformed to comprise the polynucleotide
of the invention.
In a further embodiment the invention provides a plant comprising a genetic construct of the
invention.
Ina further embodiment the the invention provides a plant comprising a plant cell of the
invention.
In a preferred embodiment the plant expresses the polynucleotide of the invention.
In a preferred embodiment the plant expresses the polypeptide of the invention.
In a preferred embodiment the plant, or its predecessor, is transformed or genetically modified
to expresses the polynucleotide or polypeptide of the invention.
In one embodiment the polypeptide of the invention, when expressed in the plant, has increased
DGATl activity relative to at least one other DGATl protein.
In one embodiment the DGATl protein of the invention has at least 5%, more preferably at
least 10%, more preferably at least 15%, more preferably at least 20%, more preferably at least
25%, more preferably at least 30%, more preferably at least 35%, more preferably at least 40%,
more preferably at least 45%, more preferably at least 50% higher DGATl activity relative to the
at least one other DGATl protein.
Preferably the at least one DGATl protein has the amino acid sequence of the polypeptide of
SEQ ID NO:44 ( ZmDGATl -short).
In a further embodiment the polypeptide of the invention, when expressed in the plant, has
increased DGATl activity relative to any previously know plant DGATl protein.
In a further embodiment the plant produces more lipid, in at least one of its tissues or parts, than
does the equivalent tissue or part in a control plant.
In one embodiment the plant produces at least 5% more, preferably at least 0% more,
preferably at least 5% more, preferably at least 20% more, preferably at least 25% more,
preferably at least 30% more, preferably at least 35% more, preferably at least 40% more,
preferably at least 45% more, preferably at least 50% more, preferably at least 55% more,
preferably at least 60% more, preferably at least 65% more, preferably at least 70% more,
preferably at least 75% more, preferably at least 80% more, preferably at least 85% more,
preferably at least 90% more, preferably at least 95% more preferably at least 100% more,
preferably at least 105% more, preferably at least 110% more, preferably at least 115% more,
preferably at least 120% more, preferably at least 125% more, preferably at least 130% more,
preferably at least 135% more, preferably at least 140% more, preferably at least 145% more,
preferably at least 150% more lipid than does a control plant.
In one embodiment the tissue is a vegetative tissue. In one embodiment the part is a leaf. In a
further embodiment the part is a root. In a further embodiment the part is a tuber. In a further
embodiment the part is a corm. In a further embodiment the part is a stalk. In a further
embodiment the part is a stalk of a monoct plant. In a further embodiment the part is from
stover (the dried stalks and leaves of a field crop). Stover is often used as animal fodder, for
example, after the grain of the crop has been harvested
In a preferred embodiment the tissue is seed tissue. In a preferred embodiment the part is a
seed. In a preferred embodiment the tissue is endosperm tissue.
In a further embodiment the plant as a whole produces more lipid than does the control plant as
a whole.
In a further embodiment the plant has an altered lipid profile, in at least one of its tissues or
parts, relative to a control plant.
In one embodiment the proportion of 16:0 in the triacylglycerol is altered relative to that in a
control plant.
In one embodiment the proportion of 16:0 in the triacylglycerol is altered by at least 1%,
preferably at least 2%, more preferably at least 3%, more preferably at least 4%, more preferably
at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least
8%, more preferably at least 9%, more preferably at least 0%, more preferably at least 1%,
more preferably at least 2%, more preferably at least 13%, more preferably at least 14%, more
preferably at least 15%, more preferably at least 6%, more preferably at least 7%, more
preferably at least 8%, more preferably at least 9%, more preferably at least 20%, relative to
that in a control plant.
In a further embodiment the altered lipid profile has a proportion of 16:0 in the triacylglycerol in
the range 6% to 16%. In this embodiment the proportion of 16:0 in the triacylglycerol is altered
within the range 6% to 16%.
In a further embodiment the proportion of 8:0 in the triacylglycerol is altered relative to that in
a control plant.
In one embodiment the proportion of 18:0 in the triacylglycerol is altered by at least 1%,
preferably at least 2%, more preferably at least 3%, more preferably at least 4%, more preferably
at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least
8%, more preferably at least 9%, more preferably at least 0%, more preferably at least 1%,
more preferably at least 2%, more preferably at least 13%, more preferably at least %, more
preferably at least 5%, more preferably at least 6%, more preferably at least 7%, more
preferably at least 8%, more preferably at least 9%, more preferably at least 20%, relative to
that in a control plant.
In a further embodiment the altered lipid profile has a proportion of 18:0 in the triacylglycerol in
the range 7% to 15%. In this embodiment the proportion of 18:0 in the triacylglycerol is altered
within the range 7% to 15%.
In a further embodiment the proportion of 18:1 in the triacylglycerol is altered relative to that in
a control plant.
In one embodiment the proportion of 18:1 in the triacylglycerol is altered by at least 1%,
preferably at least 2%, more preferably at least 3%, more preferably at least 4%, more preferably
at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least
8%, more preferably at least 9%, more preferably at least 0%, more preferably at least 1%,
more preferably at least 2%, more preferably at least 13%, more preferably at least 14%, more
preferably at least 5%, more preferably at least 6%, more preferably at least %, more
preferably at least 8%, more preferably at least 9%, more preferably at least 20%, relative to
that in a control plant.
In a further embodiment the altered lipid profile has a proportion of 18:1 in the triacylglycerol in
the range 39% to 55%. In this embodiment the proportion of 18:1 in the triacylglycerol is
altered within the range 39% to 55%.
In one embodiment the tissue is a vegetative tissue. In one embodiment the part is a leaf. In a
further embodiment the part is a root. In a further embodiment the part is a tuber. In a further
embodiment the part is a corm. In a further embodiment the part is a stalk. In a further
embodiment the part is a stalk of a monoct plant. In a further embodiment the part is a stovum
(stalk and leaf blade).
In a preferred embodiment the tissue is seed tissue. In a preferred embodiment the part is a
seed. In a preferred embodiment the tissue is endosperm tissue.
In a further embodiment the plant as a whole has an altered lipid profile relative to the control
plant as a whole.
The control plant may be any plant of the same type that is not transformed with the
polynucleotide, or construct, of the invention to express the polypeptide of the invention. The
control plant may also be transformed with an "empty" vector, wherein the empty vector does
not include an insert sequence corresponding to a polynucleotide of the invnetion or expressing
a polypeptide of the invention.
In one embodiment the control plant is an untransformed plant. In a further embodiment the
control cell is transformed cell to express the polypeptide of SEQ ID NO:44 ( ZmDGATlshort).
Plant also transformed to express an oleosin
In one embodiment the plant is also transformed to express at least one of: an oleosin, a
steroleosin, a caloleosin, a polyoleosin, and an oleosin including at least one artificially introduced
cysteine (WO 2011/053169).
Plantparts
In a further embodiment the invention provides a part, propagule or progeny of a plant of the
invention.
In a preferred embodiment the part, propagule or progeny comprises at least one of a
polynucleotide, construct, or polypeptide of the invention. Preferably the part, propagule or
progeny, or its predecessor plant, is transformed to comprise the polynucleotide of the
invention.
In a preferred embodiment the part, propagule or progeny expresses at least one of: a
polynucleotide and a polypeptide of the invention.
In a preferred embodiment the part, propagule or progeny expresses a polypeptide of the
invention.
In a preferred embodiment the part, propagule or progeny, or its predecessor plant, is
transformed or genetically modified to expresses the polynucleotide or polypeptide of the
invention.
In a further embodiment the part, propagule or progeny produces more lipid than does a control
part, propagule or progeny, or part, propagule or progeny of a control plant.
In one embodiment the part, propagule or progeny produces at least 5% more, preferably at least
10% more, preferably at least 15% more, preferably at least 20% more, preferably at least 25%
more, preferably at least 30% more, preferably at least 35% more, preferably at least 40% more,
preferably at least 45% more, preferably at least 50% more, preferably at least 55% more,
preferably at least 60% more, preferably at least 65% more, preferably at least 70% more,
preferably at least 75% more, preferably at least 80% more, preferably at least 85% more,
preferably at least 90% more, preferably at least 95% more preferably at least 100% more,
preferably at least 105% more, preferably at least 110% more, preferably at least 115% more,
preferably at least 120% more, preferably at least 125% more, preferably at least 130% more,
preferably at least 135% more, preferably at least 140% more, preferably at least 145% more,
preferably at least 150% more lipid than does a control part, propagule or progeny, or part,
propagule or progeny of a control plant.
In a further embodiment the part, propagule or progeny has an altered lipid profile relative to a
control part, propagule or progeny, or part, propagule or progeny of a control plant.
In one embodiment the proportion of 16:0 in the triacylglycerol is altered relative to that in a
control part, propagule or progeny, or part, propagule or progeny of a control plant.
In one embodiment the proportion of 16:0 in the triacylglycerol is altered by at least 1%,
preferably at least 2%, more preferably at least 3%, more preferably at least 4%, more preferably
at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least
8%, more preferably at least 9%, more preferably at least 0%, more preferably at least 1%,
more preferably at least %, more preferably at least 13%, more preferably at least 1 %, more
preferably at least 15%, more preferably at least 16%, more preferably at least 7%, more
preferably at least 8%, more preferably at least 9%, more preferably at least 20%, relative to
that in a control part, propagule or progeny, or part, propagule or progeny of a control plant.
In a further embodiment the altered lipid profile has a proportion of 16:0 in the triacylglycerol in
the range 6% to 16%. In this embodiment the proportion of 16:0 in the triacylglycerol is altered
within the range 6% to 16%.
In a further embodiment the proportion of 8:0 in the triacylglycerol is altered relative to that in
control part, propagule or progeny, or part, propagule or progeny of a control plant.
In one embodiment the proportion of 18:0 in the triacylglycerol is altered by at least 1%,
preferably at least 2%, more preferably at least 3%, more preferably at least 4%, more preferably
at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least
8%, more preferably at least 9%, more preferably at least 0%, more preferably at least %,
more preferably at least %, more preferably at least 13%, more preferably at least 14%, more
preferably at least 5%, more preferably at least 6%, more preferably at least 7%, more
preferably at least 8%, more preferably at least 9%, more preferably at least 20%, relative to
that in control part, propagule or progeny, or part, propagule or progeny of a control plant.
In a further embodiment the altered lipid profile has a proportion of 18:0 in the triacylglycerol in
the range 7% to 15%. In this embodiment the proportion of 18:0 in the triacylglycerol is altered
within the range 7% to 15%.
In a further embodiment the proportion of 18:1 in the triacylglycerol is altered relative to that in
a control part, propagule or progeny, or part, propagule or progeny of a control plant.
In one embodiment the proportion of 18:1 in the triacylglycerol is altered by at least 1%,
preferably at least 2%, more preferably at least 3%, more preferably at least 4%, more preferably
at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least
8%, more preferably at least 9%, more preferably at least 0%, more preferably at least 1%,
more preferably at least %, more preferably at least 13%, more preferably at least 1 %, more
preferably at least 5%, more preferably at least 6%, more preferably at least 17%, more
preferably at least 8%, more preferably at least 9%, more preferably at least 20%, relative to
that in a control part, propagule or progeny, or part, propagule or progeny of a control plant.
In a further embodiment the altered lipid profile has a proportion of 18:1 in the triacylglycerol in
the range 39% to 55%. In this embodiment the proportion of 18:1 in the triacylglycerol is
altered within the range 39% to 55%.
The control plant may be any plant of the same type that is not transformed with the
polynucleotide, or construct, of the invention to express the polypeptide of the invention.
In one embodiment the control plant is an untransformed plant. In a further embodiment the
control plant is transformed plant to express the polypeptide of SEQ ID NO:44 ( ZmDGATlshort).
Preferably the control the part, propagule or progeny is from a control plant as described above.
In one embodiment the part is from a vegetative tissue. In one embodiment the part is a leaf. In
a further embodiment the part is a root. In a further embodiment the part is a tuber. In a
further embodiment the part is a corm. In a further embodiment the part is a stalk. In a further
embodiment the part is a stalk of a monocot plant. In a further embodiment the part is a
stovum (stalk and leaf blade).
In a further embodiment the part is from a reproductive tissue. In a further embodiment the
part is a seed. In a preferred embodiment the part is from or includes endosperm tissue.
In a further aspect the invention provides an animal feedstock comprising at least one of a
polynucleotide, polypeptide,construct, cell, plant cell, plant part, plant, propagule and progeny of
the invention.
biofuelfeedstock
In a further aspect the invention provides a biofuel feedstock comprising at least one of a
polynucleotide, polypeptide, construct, cell, plant cell, plant part, plant, propagule and progeny of
the invention.
In one embodiment the lipid is an oil. In a further embodiment the lipid is triacylglycerol (TAG)
Methodfor producing lipid/ oil
In a further aspect the invention provides a method for producing a lipid, the method
comprising growing a cell, plant cell or plant that is transformed, or genetically modified, to
express and polynucleotide or polypeptide of the invention wherein the plant produces oil
through the activity of the expressed polypeptide.
In one embodiment the cell, plant cell or plant produces the lipid as a result of the DGAT 1
activity of the polypeptide.
In a further aspect the invention provides a method for producing lipid, the method comprising
extracting lipid from at least one of a cell, plant cell, plant, plant part, propagule and progeny of
the invention.
In one embodiment the lipid is triacylglycerol (TAG).
In a further embodiment the lipid is processed into at least one of:
a) a fuel,
b) an oleochemical,
c) a nutritional oil,
d) a cosmetic oil,
e) a polyunsaturated fatty acid (PUFA), and
f ) a combination of any of a) to e).
In a further aspect the invention provides a method for producing lipid, the method comprising
expressing a DGATl protein of the invention in a cell, plant cell or plant.
In a preferred embodiment expressing the DGATl protein of the invention in the plant leads
production of the lipid in the cell, plant cell or plant.
In one embodiment the method includes the step of transforming a cell, plant cell or plant with a
polynucleotide of the invention encoding the DGATl protein.
In a further embodiment the method includes the step of extracting the lipid from the cell, plant
cell, or plant, or from a part, propagule or progeny of the plant.
In one embodiment the lipid is an oil. In a further embodiment the lipid is triacylglycerol (TAG)
In a further embodiment the lipid is processed into at least one of:
a) a fuel,
b)an oleochemical,
c) a nutritional oil,
d) a cosmetic oil,
e) a polyunsaturated fatty acid (PUFA), and
f ) a combination of any of a) to e).
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. In some embodiments,
the term "comprising" (and related terms such as "comprise and "comprises") can be replaced by
"consisting of (and related terms "consist" and "consists").
The term "DGATl" as used herein means acyl CoA: diacylglycerol acyltransferase (EC 2.3.1.20)
DGATl is typically the major TAG synfhesising enzyme in both the seed and senescing leaf
(Kaup et al, 2002, Plant Physiol. 129(4):1 616-26; for reviews see Lung and Weselake 2006,
Lipids. Dec 2006;41 (12):1 073-88; Cahoon et al., 2007, Current Opinion in Plant Biology. 10:236-
244; and Li et al., 2010, Lipids. 45:145-157).
DGATl contains approximately 500 amino acids and has 10 predicted transmembrane domains
whereas DGAT2 has only 320 amino acids and is predicted to contain only two transmembrane
domains; both proteins were also predicted to have their N - and C-termini located in the
cytoplasm (Shockey et al, 2006, Plant Cell 18:2294-2313). Both DGAT1 and DGAT2 have
orfhologues in animals and fungi and are transmembrane proteins located in the ER.
In most dicotyledonous plants DGATl & DGAT2 appear to be single copy genes whereas there
are typically two versions of each in the grasses which presumably arose during the duplication
of the grass genome (Salse et al, 2008, Plant Cell, 20:1 1-24).
The phrase "increased DGATl activity" means increased specific activity relative to that of the
first and/ or DGATl protein.
An art skilled worker would know how to test the "specific activity" of a DGATl protein or
variant thereof of the invnetion. This may typically be done by isolating, enriching and
quantifying the recombinant DGATl then using this material to determine either the rate of
triaclyglyceride formation and/or the disappearance of precursor substrates (including various
forms of acyl-CoA and DAG) as per Xu et al., (2008), Plant Biotechnology Journal. 6:799-818.
Lipid
In one embodiment the lipid is an oil. In a further embodiment the oil is triacylglycerol (TAG)
Lipid production
In certain embodiments the cell, cells, tissues, plants and plant parts of the invention produces
more lipid than control cells, tissues, plants and plant parts.
Those skilled in the art are well aware of methods for measuring lipid production. This may
typically be done by quantitative fatty acid methyl ester gas chromatography mass spectral
analysis (FAMES GC-MS). Suitable methods are also described in the examples section of this
specification.
Substrate specificity
In certain embodiments, the polypeptides of the invention have altered substrate specificity
relative to other DGATl proteins. Plant DGATl proteins are relatively promiscuous in terms
of the fatty acid substrates and DAG species they are capable of utilisting to generate TAG. As
such they can be considered to have relatively low substrate specificity. However, this can be
modified such that certain fatty acids become a preferred substrate over others. This leads to an
increase in the proportions of the preferred fatty acids in the TAG and decreases in the
proportions of the non preferred fatty acid species. Substrate specificity can be determined by
in vitro quantitiative analysis of TAG production following the addition of specific and known
quantities of purified substrates to known quantities of recombinant DGAT, as per Xu et al.,
(2008), Plant Biotechnology Journal. 6:799-818.
Lipid profile
In a further embodiment the cell, cells, tissues, plants and plant parts of the invention have an
altered lipid profile relative to the control cells, tissues, plants and plant parts.
Those skilled in the art are well aware of methods for assessing lipid profile. This may involve
assessing the proportion or percentage of at least one of the 16:0, 16:1, 18:0, 18:lc9 fatty acid
species present in the lipid. This may typically be done by fatty acid methyl ester (FAME)
analysis (Browse et al., 1986, Anal. Biochem. 152, 141-145). Suitable methods are also described
in the examples section of this specification.
Cells
The DGATl polypeptide of the invention, or as used in the methods of the invention, may be
expressed in any cell type.
In one embodiment the cell is a prokaryotic cell. In a further embodiment the cell is a eukaryotic
cell. In one embodiment the 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 cell is a bacterial cell. In a further
embodiment the cell is a yeast cell. In one embodiment the yeast cell is a S . ceriviseae cell. In
further embodiment the cell is a fungal cell. In further embodiment the cell is an insect cell. In
further embodiment the cell is an algal cell. In a further embodiment the cell is a plant cell.
In one embodiment the cell is a non-plant cell. In one embodiment the non-plant is selected
from E . colt, P. pastoris, S . ceriviseae, D . salina and C. reinhardtii. In a further embodiment the nonplant
is selected from P. pastoris, S . ceriviseae, D . Salina and C. reinhardtii.
In one embodiment the cell is a microbial cell. In another embodiment, the microbial cell is an
algal cell of the division of Chlorophyta (green algae), Rhodophyta (red algae), Phaeophyceae
(brown algae), Bacillariophycaeae (diatoms), or Dinoflagellata (dinoflagellates). In another
embodiment, the microbial cell is an algal cell of the species Chlamydomonas, Dunaliella, Botrycoccus,
Chlorella, Crypthecodinium, Gracilaria, Sargassum, Pleurochrysis, Porp h di , Phaeodactylum,
Haematococcus, Isochrysis, Scenedesmus, Monodus, Cyclotella, Nit^schia, or Parietochloris. In another
embodiment, the algal cell is Chlamydomonas mnhardtii. In yet another embodiment, the cell is
from the genus Yarrowia, Candida, Khodotomla, Rhodosporidittm, Cryptococcus, Trichosporon, Upomyces,
Pjthium, Thraustochytrium, or Ulkenia. In yet another embodiment, the cell is a
bacterium of the genus Rhodococms, Escherichia, or a cyanobacterium. In yet another embodiment,
the cell is a yeast cell. In yet another embodiment, the cell is a synthetic cell.
Plants
The variant DGATl sequences of the invention may be naturally-occurring DGATl sequences.
Preferably the variant DGATl sequences are from plants. In certain embodiments the cells into
which the DGATl proteins of the invention are expressed are from plants. In other
embodiments the DGATl proteins of the invention are expressed in plants.
The plant cells, from which the DGATl proteins of the invention are derived, the plants from
which the plant cells are derived, and the plants in which the DGATl proteins of the invention
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.
Other preferred plants are forage plant species from a group comprising but not limited to the
following genera: Zea, oli m, ordiu , Miscanthus, Saccharum, Festuca, Dactylis, ro s, Thinopyrum,
Trifolium, Medicago, Pheleum, Phalaris, Holms, Glycine, Eotus, Plantago and Cichonum.
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 occidentak. 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 Gyl cine species is Gyl cine 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 Visum. A preferred Visum species is Visum sativum commonly known
as pea.
Another preferred genus is Lotus. Preferred Lotus species include Lotus comkulatus, Lotus
pedunculatus, Lotus glabar, Lotus tenuis and Lotus uliginosus. A preferred Lotus species is Lotus
comkulatus commonly known as Birdsfoot Trefoil. Another preferred Lotus species s 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. A preferred Brassica genus is Camelina. A preferred Camelina
species is Camelina sativa.
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 rass a. A preferred oil seed species is Brassica napus.
A preferred oil seed genera is Brasska. A preferred oil seed species is Brasska okraceae.
A preferred oil seed genera is Carthamus. A preferred oil seed species is Carthamus tinctorius.
A preferred oil seed genera is Uelianthus. A preferred oil seed species is Uelianthus annum.
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 Uordeum. A preferred grain producing species is Uordeum
vulgare.
A preferred grazing genera is Lolium. A preferred grazing species is Uoliu perenne.
A preferred grazing genera is Lolium. A preferred grazing species is Lolium arundinaceum.
A preferred grazing genera is Tnfolium. A preferred grazing species is ri olium repens.
A preferred grazing genera is Uordeum. A preferred grazing species is Uordeum vulgare.
Preferred plants also include forage, or animal feedstock plants. Such plants include but are not
limited to the following genera: Miscanthus, Saccharum, Pankum.
A preferred biofuel genera is Miscanthus. A preferred biofuel species is Miscanthus giganteus.
A preferred biofuel genera is Saccharum. A preferred biofuel species is Saccharum ojficinarum.
A preferred biofuel genera is Pankum. A preferred biofuel speices is Pankum virgatum.
Plant parts, prop agues and progeny
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 progeny, comprising the polynucleotides or constructs of the invention,
and/or expressing the DGAT1 sequences of the invention, also form an part of the present
invention.
Preferably the plants, plant parts, propagules and progeny comprise a polynucleotide or
construct of the invention, and/ or express a DGAT1 sequence of the invention.
Polynucleotides andfragments
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
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.
The term "primer" refers to a short polynucleotide, usually having a free 3Ό H 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.
Poylpeptides andfragments
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.
A "fragment" of a polypeptide is a subsequence of the polypeptide that preferably performs a
function/ activity of and/ or influences three dimensional structure of the polypeptide.
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. The isolated
sequence is preferably separated from the sequences that may be found flanking the sequence in
its naturally occurring 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.
Poyl nucleotide 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
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 the
NCBI website on the World Wide Web at 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 nucleotideseql 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 = " .
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. LongdenJ. 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 the World Wide Web at
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 penaKzing 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 the NCBI website on the World Wide Web at ftp://ftp.ncbi.nih.gov/blast/.
The similarity of polynucleotide sequences may be examined using the following unix command
line parameters:
bl2seq —nucleotideseql —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.
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 0 -6 more
preferably less than 0 -9, more preferably less than x 10 -12, more preferably less than 1 x
10 -15, more preferably less than 1 x 10 -18, more preferably less than x 0 -21, more
preferably less than 0 -30, more preferably less than 1 0 -40, more preferably less than
x 0 -50, more preferably less than x 10 -60, more preferably less than x 0 -70, more
preferably less than x 0 -80, more preferably less than 1 0 -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 30° C (for example, 10° C) below the melting
temperature (Tm) of the native duplex (see generally, Sambrook et i , Eds, 1987, Molecular
Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et a/., 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 ί , 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 65°C, 6X SSC, 0.2% SDS
overnight; followed by two washes of 30 minutes each in X SSC, 0.1% SDS at 65° C and two
washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C.
With respect to polynucleotide molecules having a length less than 100 bases, exemplary
stringent hybridization conditions are 5 to 10° C below Tm. On average, the Tm of a
polynucleotide molecule of length less than 100 bp is reduced by approximately
(500/oligonucleotide length) 0 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 DNARNA
hybrids, and can be calculated using the formula described in Giesen et al, Nucleic Acids
Res. 1998 Nov l;26(21):5004-6. Exemplary stringent hybridization conditions for a DNA-PNA
hybrid having a length less than 00 bases are 5 to 10° 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 the NCBI website on the World Wide
Web at 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,
recombinanfiy 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
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 00 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
the NCBI website on the World Wide Web at ftp://ftp.ncbi.nih.gov/blast/. The default
parameters of bl2seq are utili2ed 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
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 the NCBI website on the
World Wide Web at ftp:/ / ftp.ncbi.nih.gov/blast/. The similarity of polypeptide sequences may
be examined using the following unix command line parameters:
bl2seq - i peptideseql - 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 x 0 -9, more preferably less than x 10 -12, more preferably less than 1 x
10 -15, more preferably less than x 0 -18, more preferably less than x 0 -21, more
preferably less than 0 -30, more preferably less than 0 -40, more preferably less than 1
x 10 -50, more preferably less than x 10 -60, more preferably less than x 0 -70, more
preferably less than x 0 -80, more preferably less than x 0 -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 ad., 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.
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
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.
In certain embodiments the DGAT1 polynucleotides/polypeptides of the invention may be
andvantageously expessed under the contol of selected promoter sequences as described below.
Vegetative tissue specificpromoters
An example of a vegetative specific promoter is found in US 6,229,067; and US 7,629,454; and
US 7,1 53,953; and US 6,228,643.
Pollen specificpromoters
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 specificpromoters
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. A preferred seed specific promoter is the napin
promoter of Brassica n p us (Josefsson et a , 1987, J Biol Chem. 262(25):121 96-201 ; Ellerstrom et
al., 1996, Plant Molecular Biology, Volume 32, Issue 6, pp 1019-1 027).
Fruit specificpromoters
An example of a fruit specific promoter is found in US 5,536,653; and US 6,127,1 79; and US
5,608,150; and US 4,943,674.
Nonphotosynthetic tissuepreferredpromoters
Non-photo synthetic tissue preferred promoters include those preferentially expressed in non
photosynthetic tissues/organs of the plant.
Non-photo synthetic tissue preferred promoters may also include light repressed promoters.
Light repressedpromoters
An example of a light repressed promoter is found in US 5,639,952 and in US 5,656,496.
Root specificpromoters
An example of a root specific promoter is found in US 5,837,848; and US 2004/ 0067506 and US
2001 /0047525.
Tuber specificpromoters
An example of a tuber specific promoter is found in US 6,1 84,443.
Bulb specificpromoters
An example of a bulb specific promoter is found in Smeets et al., (1997) Plant Physiol. 113:765-
771 .
Rhizome preferredpromoters
An example of a rhizome preferred promoter is found Seongjang et al., (2006) Plant Physiol.
142:1 148-1 159.
Endosperm specificpromoters
An example of an endosperm specific promoter is found in US 7,745,697.
Corm promoters
An example of a promoter capable of driving expression in a corm is found in Schenk et al.,
(2001) Plant Molecular Biology, 47:399-412.
Photosythetic tissue preferred promoters
Photosythetic tissue preferred promoters include those that are preferrentially expressed in
photosynthetic tissues of the plants. Photosynthetic tissues of the plant include leaves, stems,
shoots and above ground parts of the plant. Photosythetic tissue preferred promoters include
light regulated promoters.
Light regulatedpromoters
Numerous light regulated promoters are known to those skilled in the art and include for
example chlorophyll a/b (Cab) binding protein promoters and Rubisco Small Subunit (SSU)
promoters. An example of a light regulated promoter is found in US 5,750,385. Light regulated
in this context means light inducible or light induced.
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.
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. Subsequent offspring
or generations of the plant that still contain the new genetic material are also transgenic plants
according to the invention.
Methods for isolating orproducing poyl nucleotides
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 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. For these reasons among others, it is desirable to be able to identify and isolate
orfhologues of a particular gene in several different plant species.
Variants (including orthologues) may be identified by the methods described.
Physical methods
Variant polypeptides may be identified using PCR-based methods (Mullis et a/., 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 e a/., 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 a/.,
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 11-1 6, 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
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 six-frame
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 e a/., 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., Higgms, 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) o T-COFFEE (Cedric
Notredame, Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel method for fast and
accurate multiple sequence alignment, J . Mol. Biol. (2000) 302: 205-21 )) 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 a/., 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 a/., 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 isolatingpoylpeptides
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 a , 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 recombinantiy in suitable host cells and separated from the
cells as discussed below.
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, 987) .
Methods for producing host cells comprisingpoyl nucleotides, 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 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, 997, Ann Rev Plant Phys
Plant Mol Biol, 48, 297, Hellens et al., (2000) Plant Mol Biol 42: 819-32, HeUens 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 literature. Exemplary promoters are described, e.g., in WO 02/00894 and
WO2011/053169, 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 ea mays zein gene terminator, the Ory^a
sativa ADP-glucose pyrophosphorylase terminator and the Solanum tuberosum PITI 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 eta/., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore eta/., 1987, Plant Cell Rep. 6,
439); tobacco (Horsch eta/., 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 eta/.,
1998, Plant Cell Rep. 17, 165); citrus plants (Pena eta/., 1995, Plant Sci.104, 183); caraway (Krens
eta/., 1997, Plant Cell Rep, 17, 39); banana (US Patent Serial No. 5, 792, 935); soybean (US
Patent Nos. 5, 416, 01 ; 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 2005 Plant
Cell Rep. 2006 ;25(2):117-23; Gonzalez Padilla et al, 2003 Plant Cell Rep.22(l):38-45); strawberry
(Oosumi eta/., 2006 Planta. 223(6):1219-30; Folta eta/., 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 eta/., 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 ll(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 eHantha (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.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the nucleic acid sequence and three frame translation of the Arabidopsis thaliana
DGATl transcribed region (SEQ ID NO:116). Exon coding sequences are shown in bold face,
underlined, grey blocks.
Figure 2 shows the nucleic acid sequence and three frame translation of the Zea mays short
DGATl transcribed region (SEQ ID NO:117). This genomic sequence has F469 deleted and
Q67 added compared to the cDNA (EU039830) and peptide (ABV91586) sequences actually
used in this patent. Exon coding sequences are shown in bold face, underlined, grey blocks.
Figure 3 shows the nucleic acid sequence and three frame translation of the Zea mays long
DGATl transcribed region (SEQ ID NO:118) derived from CHORI-201 Maize B73 BAC
Library (available from the World Wide Web at
http://www.ncbi.nlm.nih.gov/nuccore/AC204647; http:/ /bacpac. chori.org/maize201. htm).
Exon coding sequences are shown in bold face, underlined, grey blocks.
Figure 4 shows the peptide sequence of the N-terminal cytoplasmic region of a number of plant
DGATls including both long and short versions from the grasses as well as examples from
dicotyledonous species. Left hand box represents acyl-CoA binding site (Nykiforuk et al, 2002,
Biochimica et Biophysica Acta 1580:95-109). Right hand box represents first transmembrane
region (McFie et ai, 2010, JBC, 285:37377-37387). Left hand arrow represents boundary
between exon 1 and exon 2. Right hand arrow represents boundary between exon 2 and exon 3.
The sequences are AtDGATl (SEQ ID NO:119), BjDGATl (SEQ ID NO:120), BnDGATl -
AF (SEQ ID NO:121), BjDGATl (SEQ ID NO:122), TmajusDGATl (SEQ ID NO:123),
EpDGATl (SEQ ID NO:124), VgDGATl (SEQ ID NO:125), NtDGATl (SEQ ID NO:126),
PfDGATl (SEQ ID NO:127), ZmL (SEQ ID NO:128), SbDGATl (SEQ ID NO:129), OsL
(SEQ ID NO:130), OsS (SEQ ID NO:131), SbDGATl (SEQ ID NO:132), ZmS (SEQ ID
NO:133), PpDGATl (SEQ ID NO:132), SmDGATl (SEQ ID NO:135), EaDGATl (SEQ ID
NO:136), VvDGATl (SEQ ID NO:137), GmDGATl (SEQ ID NO:138), GmDGATl (SEQ
ID NO:139), LjDGATl (SEQ ID NO:140), MtDGATl (SEQ ID NO:141), JcDGATl (SEQ
ID NO:142), VfDGATl (SEQ ID NO:143), RcDGATl (SEQ ID NO:144), PtDGATl (SEQ
ID NO:145), Pt DGAT1 (SEQ ID NO:146).
Figure 5 shows the line-bond structures of the amino acid residues lysine (K) and arginine (R).
EXAMPLES
Example 1: Identification of the DGATl sequence of the invention
Several nucleic acid sequences and polypeptide sequences for the plant type 1 DGATs can be
found by accession number in public domain libraries (Table 1). For creating initial alignments
we used ClustalW (Thompson et al., 1994, Nucleic Acids Res., 22, 4673-4680); these were
manually edited and used to create the models to search the DGAT sequences, using the
HMMER2 package (HMMER 2.3.2 (Oct 2003) Copyright ° 1992-2003 HHMI/Washmgton
University School of Medicine, available from the World Wide Web at http:/ /hmmer.org).
Initial matching of protein sequences against genomic DNA with splice prediction was
performed with the GeneWise package (Birney et al, 2004, Genome Res. 14: 988-995). Some of
the sequences retrieved appeared to have errors; in particular incorrecdy predicted splice sites
which would result in internal deletions that would likely result in non -functional proteins.
While both dicotyledonous and monocotyledonous type 1 DGATs have 16 exons there are
some differences in the position of the splicing. Exon 8 in the dicotylendonous DGATl gene
corresponds to exons 8 and 9 in the monocotyledonous DGATl gene, while exon 14 in the
monocotyledonous gene corresponds to exons 13 and 14 in the dicotyledonous gene. We have
found that the most accurate method for determing the likely genuine coding sequence from
genomic data has been to use Vector NTI Advance (TM) 1 .0 (® 2008 Invitrogen Corporation)
to translate the genome in the three forward reading frames and align these with demonstrated
functional DGATl s from dicotyledonous or monocotyledous species as appropriate (for
example thaliana cDNA NM_127503, protein NP_179535 and Z. mays cDNA EU039830,
protein ABV91586). The genomic sequence and corresponding exon/intron boundary positions
for Arabidopsis thaliana encoding NP_179535 and Zea mays encoding ABV91586 that can be used
as a template for determining other plant DGAT coding regions are shown in Figures 1 and
Figures 2, respectivlely.
Using this method, the applicants have assembled/identified a novel DGATl sequence from Z.
mays DGATl (SEQ ID NO: 10 and SEQ ID NO: 39 [Figure 3]). To the best of the applicant's
knowledge, a functional portion of sequence is not present in any public cDNA database, which
may indicate that the functional protein is not expressed in naturally occurring plants.
The applicants designated this sequence Zea mays DGAT-Long (ZmDGATl-L or Zm-L
DGATl) because the encoded polypeptides is longer than the known Zea mays of SEQ ID NO:
44 (referred to as Zea mays DGATl-short or ZmDGATl-S or Zm-S DGATl) as indicated in
Figure 4.
A similar relationship exists between Oy a sativa DGATl-short, or OsDGATl-S, or Os-S
DGATl (SEQ ID NO:41, and Ory^a sativa DGATl-long, or OsDGATl-L, or Os-L DGATl
(SEQ ID NO:42).
Table 1
Example 2: The DGATl sequence of the invention has surprisingly high activity in
increasing cellular lipid content, and fragments of the DGATl sequence of the invention
are useful in conferring increased activity to other DGATl proteins.
Summary
The applicants compared the activity of the DGATl sequence of the invention to other known
DGATl sequences. Surprisingly the DGATl sequence of the invention showed higher activity,
in increasing cellular lipid content, than any of the other DGATl sequences tested.
Furthermore the applicants have shown that fragments of the DGATl protein of the invention
are useful in conferring increase activity on at least parts of other DGAT proteins.
Materials and methods
Nucleic acid constructs encoding the amino acid sequences, SEQ ID NO: 30 A . fhaliana
DGATl), 34 (T. magus DGATl), 39 (Zea mays DGAT1-L), 4 1 (O. sativa DGAT1-S), 42 (O. satwa
DGAT1-L) and 44 (Table 1) were optimised for expression in Saccharomyces cerevisiae by GeneArt
AG (Germany). These were engineered to have an internal Xhol site within exon 1 encoding the
conserved JV-terminal acyl-Co binding region (identified by Lung and Weselake, 2006, Lipids.
Dec 2006;41(12):1073-88) without altering the amino acid sequence leucine-serine-serine (LSS).
Figure 4 shows alignment of a number of DGATl sequences from plants. The left box shows
the position of the Acyl-CoA binding site.
An E co site was engineered upstream of the 5' coding sequence while an Xbal site was placed
downstream of the 3' stop codon. The internal Xhol and flanking EcoBJ and Xbal sites were
used to generate chimeras between the DGATl sequence of the invention and each of the other
DGATl clones; essentially this fused the N-terminal reputed cytoplasmic region (based on Lung
and Weselake, 2006, Lipids. Dec 2006;41(12):1073-88 and McFie eta/., 2010, JBC, 285:37377-
37387) from one DGATl with the C-terminal ER luminal region of a different DGATl. In
some combinations this resulted in one amino acid change in the remaining cytoplasmic region
downstream of the engineered Xhol site. The putative acyl-Co binding region the L . thaliana
DGATl, T.majus DGATl, Z .m ys- DGATl and O. sativa-l DGATl have an identical amino
acid sequence down stream of the Xhol site (LSSDAIFKQSHA). While in the Z.mays-S DGATl
and O. sativa-S DGAT1 the lysine (K residue is replaced by an arginine (R) residue
(LSSDAIFRQSHA). Since the position of this residue is located 3' to the Xho I site encoded by
LLS then chimeras deriving from one parent containing the lysine and one parent containing the
arginine residue will effectively result in a substitution of this residue. This was considered to be
a minimal disruption since both lysine and arginine are large, positively charged, hydrophilic,
basic amino acids containing a free amine or guanidinium group, respectively at the end of an
aliphatic side chain (Figure 5). The JV-terminal region / C-terminal region domain swapping
constructs, and the parent constructs (highlighted in bold) are shown in in Table 2 , with their
corresponding SEQ ID NOs.
Table 2
Sequences were synfhesised either by GENEART AG (Germany) or GeneScript (USA).
Sequences were optimised for expression in Saccharomyces cerevisiae and flanked with appropriate
incorporated appropriate restriction sites to facilitate the cloning into the pYES2.1 vector
(Invitrogen).
Expression of constructs in S . cerevisiae
The parent DGAT1 constructs and chimeric DGAT1 contstructs were placed into the galactoseinducible
yeast expression vector pYES2.1/V5-His TOPO ® (Invitrogen). This resulted in the
addition of an inframe C-terminal V5 epitope and 6x histidine tag. The chimeric constructs and
the number of their corresponding polypeptide sequences are shown in Table 2 above.
The Saccharomyces cerevisiae quadruple mutant (H1246) in which all four neutral lipid biosynthesis
genes have been disrupted (Sandager et al., 2002, The Journal of Biological Chemistry, 277:6478-
6482) was transformed as per Elble (1992, BioTechniques 13, 18-20) and selected by the ability
to grow in the absence of uracil. Routinely, yeast cells were grown aerobically overnight in a
synthetic medium with 0.67% YNB, without uracil (SC-U) and containing 2% glucose. Cells
from overnight culture were used to inoculate 200 mL of induction medium (SC-U containing
2% galactose and 1% raffinose) to an initial OD 0 of 0.4. Cells were allowed to further grow at
30°C, with shaking at 200 rpm until late stationary phase, normally 48 h. Cells were harvested by
centrifugation at 1500 x _ for 5 min, then cell pellets were washed with distilled water and either
used immediately for subsequent analysis or kept in -80°C until required. Cell pellets for neutral
lipid extraction were freeze-dried for 48 h and stored in -20°C freezer until required.
Lipid analysis of S . cerevisiae
Approximately 10 mg of freeze-dried yeast cell material was accurately weighed then disrupted
using glass beads by vortexing for 1 minute. This lysate was extracted in hot methanolic HC1 for
fatty acid methyl ester (FAME) analysis (Browse et al., 1986, Anal. Biochem. 152, 141-145).
For FA profile analysis approximately 50 mg freeze dried yeast was placed in a 3-mm screw cap
tube, and an equal volume of glass beads added before vortexing at high speed in 3x 1 min
bursts. Following addition of 50 mg of 19:0 TAG internal standard, 2.4 mL of 0.17 M NaCl in
MeOH was added and the mixture vortexed for 15 sec followed by the addition of then 4.8 mL
of heptane and the entire contents mixed.
The solution was then incubated in 80°C water bath for 2 h without shaking. After incubation,
the solution was cooled to room temperature. After cooling, the upper phase (lipidic phase) was
transferred to fresh screw-cap tube and evaporated to dryness under stream of nitrogen gas. The
dried residue was then dissolved in 1mL heptane and mixed thoroughly for TAG SPE
separation using Strata Si-1 Silica column (Phenomenwx, 8B-S012-EAK).
After preconditioning with methanol and equilibrating the Silica column with heptane the 1mL
TAG extract (including 50 g 17:0 TAG Internal Standard was passed through the preequilibrated
column, followed by 1.2 mL of heptane and then 2 mL of chloroform:heptane (1:9
v/v/) and the eluate collected.The total eluate collected was evaporated to dryness under the
stream of N gas and the residue used for FAMEs extraction.
FAMEs of extracted TAG
To the TAG residue above 10 L of internal standard 15:0 FA (4 mg/ mL dissolved in heptane)
and 1 mL of methanolic HC1 (IN) reagent containing 5% of 2,2-dimeethoxypropane (as
water scavenger) were added.
The tube was then flushed with N gas, then sealed immediately with Teflon-lined cap, and
heated at 80°C in a water bath for 1 h. After cooling down, 0.6 mL heptane and 1.0 mL of 0.9%
(w/v) NaCl was added, the mixture vortexed then spun at 500 rpm for 1 min.
From the top heptane layer, 100 m was collected and transfered to a flat-bottom glass insert
fitted into a vial for FAMES GC/MS analysis.
Protein extraction and Trypsin digestion
Yeast cell pellets were washed with lysis buffer (50 mM sodium phosphate, pH 7.4, 1 mM
EDTA, 5% glycerol, 1mM PMSF) then resuspended in 500 m lysis buffer, glass beads were
added and cells disrupted by vortexing 2x at medium speed for 30 seconds. Cell debris was
pelleted by centrifugation at 1000 x g for 5 min, the supernatant transferred to fresh tubes and
total cellular membranes pelleted by ultracentrifugation at 100,000 for 1 h. Membrane
proteins were resuspended in lysis buffer with or without detergent (1% Dodecyl maltoside) and
quantified in a Qubit Fluorometer using the Qubit IT Quantitation Kit.
Trypsin was added to give a final concentration of 25 g/mL to 50 m of protein extract and the
mixture incubated at 30°C for 30 min. The reaction was terminated by addition of Trypsin
inhibitor from Gyl cine max (Sigma-Aldrich catalogue # T6414) to a final concentration of 0.4
m / mL. After addition of trypsin inhibitor, 4x SDS loading dye and lOx reducing agent
(Invitrogen) were added, and the protein incubated at 70°C for 10 min prior to SDS-PAGE
followed by immunoblotting. The blot was probed with either Anti V5-HRP antibody (Cat
#R96125, Invitrogen) at 1:2500 dilution, or anti Kar2 (y-115) antibody produced in rabbit (SC-
33630, Santa Cruz Biotechnology) at 1:200 dilution. Anti Kar2 was used to detect the yeast
protein Kar2, an ER luminaly-located protein (Rose et al, 1989, Cell 57,121 1-1221) which serves as
a control to demonstrate the presence of intact microsomes.
Expression of chimeric DGAT1 in Brassica napus
The same strategy, as described above, was used to generate a variety of chimeric DGATl
constructs for expression in the seeds of Brassica napus. This included the parent DGATl s of T.
majus DGATl, Z. ma s- DGATl and Z. ma s-S DGATl (amino acid SEQ ID NO: 34, 39 and
44 respecitively, Table 1) optimised for expression in Brassica napus by GeneArt AG. The T.
majus construct was engineered to contain a single point mutation S A (Xu et al, 2008, Plant
Biotechnology Journal, 6:799-818). All constructs were engineered to have an optimised Kozak,
Arabidopsis thaliana UBQ10 intron, and tetranucleotide stop codon as per Scott et al., (2010, Plant
Biotechnology Journal, 8:912-917) as indicated in Table 3 below.
Table 3
The same digestion pattern used to generate the chimeras for expression in S . cerevisiae was used
on the B. »<¾i«^optimised constructs to generate the chimeras Tm-ZmL and ZmL-Tm(S189A);
resulting in the peptide sequences listed in Table 4.
Table 4
The parent DGATs and their chimeras were transferred into the Gateway®-compatible binary
vector pMD107 (courtesy of Dr Mark Smith, NRC Saskatoon, SK, Canada, S7N 0 9) which
placed them under the control of the seed- specific napin promoter (Ellerstrom et al., 1996, Plant
Molecular Biology, Volume 32, Issue 6, pp 1019-1027).
Plant transformation
B. napus (cv. DH12075) was transformed via Agrobactenum tumefaciens (GV3101) using the
cotyledon co-cultivation method (adapted from that of Maloney et al., 1989, Plant Cell Rep. 8,
238-242). Control lines contained an empty-vector, and when identified, null sibKng lines were
subsequently used as true controls.
Approximately 200 T0 transformed lines were produced and their corresponding T selfed seeds
were analysed for oil content by GC. Approximately 50 individual transgenic lines (including
control lines) were selected for the next generation (10 plants/line) based on their oil content, or
seed weight (8 lines).
A total of approximately T plants were grown and screened by PCR for copy number and
identification of null sibing lines. T2 seeds were analysed in triplicate for oil content by NMR.
Expression of Z.mays-L and T. majus DGAT1 in Camelina sativa
The strategy above can also be used to generate a variety of chimeric DGAT1 constructs for
expression in the seeds of Camelina sativa and other plants.
Sequences with modifications were synthesised either by GENEART AG (Germany) or
GeneScript (USA). Sequences with modifications were synthesised either by GENEART AG
(Germany) or GeneScript (USA). Sequences were optimised for expression in Brasska species and
included an intron (SEQ ID NO:102) from Arabidopsis th li na DGAT1 —intron 3. Each
sequence was flanked with appropriate attL recombination sites sites to enable the cloning
Gateway ® adapted vectors.
Table 5
DGAT1 DGAT1
Residue C-terminal Additional Type of SEQ
N-terminal C-terminal modification mod information sequence parent parent ID NO:
T.majus T . majus S197A V5-His tag + intron NUCLEIC 103
T.majus T . majus S197A V5-His tag ORF only NUCLEIC 104
T.majus T . majus S197A V5-His tag PEPTIDE 105
Z . mays-L Z. mays-L None V5-His tag + intron NUCLEIC 106
Z . mays-L Z. mays-L None V5-His tag ORF only NUCLEIC 107
Z . mays-L Z. mays-L None V5-His tag PEPTIDE 108
T.majus Z. mays-L None V5-His tag + intron NUCLEIC 109
T.majus Z. mays-L None V5-His tag ORF only NUCLEIC 110
T.majus Z. mays-L None V5-His tag PEPTIDE 11
Z . mays-L T . majus SI 89A V5-His tag + intron NUCLEIC 112
Z . mays-L T . majus S 89A V5-His tag ORF only NUCLEIC 113
Z . mays-L T . majus SI 89A V5-His tag PEPTIDE 114
The parent DGATs and their modified forms were transferred into the Gateway -compatible
binary pRShl Gateway adapted binary vector (Winichayakul et al., 2009, Biotechnol. Appl.
Biochem. 53, 111—122) modified by replacement of the CaMV35S promoter replaced with the
Brasska napus Napin promoter (SEQ ID NO: 115).
Camelina sativa transformation
C. sativa (cf. Calena) were transformed via Agrobacterium tumefaciens (GV3101) using the floral
dip method (adapted from that of Clough and Bent, 1998, Plant J . 16(6):735-745). Essentially
seeds were sown in potting mix in 10 cm pots in a controlled environment, approximately 6
weeks after planting the flowers were dipped for 5-14 minutes under vacuum (70-80 inch Hg) in
an overnight culture of appropriated Agrobacterium GV3101 cells re-suspended in a floral dip
buffer. After vacuum-transformation, plants were kept for 24 h under low light conditions by
partiy covering with a black plastic sheet. Vacuum transformations can be repeated three times at
approximately 10-12 days intervals, corresponding to the flowering duration. Plants were grown
in potting mix in a controlled environment (16-h day length, 21-24 °C, 65-70 % relative
humidity).
The seeds produced can be collected and screened for transformants by germinating and
growing seedlings at 22 °C with continuous light on a half-strength MS medium (pH 5.6)
selection plate containing 1 %(w/v) sucrose, 300 mg/L Timentin, and 25 mg/L DLphosphinofhricin
to select for herbicide resistance. T2 selfed seed populations can also be
screened by immuno blot for the presence of the V5 eptiope.
T selfed seeds may be analysed for oil content by GC. Approximately 50 individual transgenic
lines (including control lines) may be selected for the next generation (10 plants/line) based on
their oil content, or seed weight. T2 plants may be grown and screened by PCR for copy number
and identification of null sibing lines. T2 seeds may be analysed in triplicate for oil content by
NMR or GC/MS.
Results
Addition of fragments of the DGAT1 poylpeptide of the invention to other DGAT1 sequences enhance lipid
production in Saccharomyces cerevisiae relative to that with the other DGAT1 sequences alone.
Tables 8-14 show the lipid yields of a variety of chimeric DGATls in which the N-terminal or
C- terminal region has been derived from the DGAT1 sequence of the invention while the
remainder of the protein has been derived from another plant DGAT1. The lipid yields are
presented either as grams of lipid produced per litre (which therefore compensates for any
differences in growth rate) or have been normalised as a percentage of the lipid yield of the
corresponding unmodified parent DGAT1.
A comparison of parent DGATls with each other, and with each of the chimeric DGATls
made using one donor parent for the N -terminal region, and a different donor parent for the Cterminal
region are shown in Table 5. The parent DGAT1 sequences are highlighted in bold.
Surprisingly the DGAT1 sequence of the invention shows higher activity, in lipid yield
production, than any of the other sequences tested.
The lipid yields at 32 hr have been normalised against the highest lipid-producing parent (Z.
ay s-L and are presented in ascending order.
Table 6
The results also show that addition of the Z. ay s-L N-terminal region to the C-terminal region
of the A . thaliana DGAT1 parent results in increased lipid yield over the full-length A . thaliana
DGAT1 sequence (see SEQ ID NO: 84 versus SEQ ID NO: 59).
The results also show that addition of the Z. mays-L N-terminal or C-terminal region to the Cterminal
or N-terminal region respectively, of the T. majus DGAT1 sequence results in increased
lipid yield over the full-length T. majus DGAT1 sequence (see SEQ ID NO: 88 and 94 versus
SEQ ID NO: 89).
The results also show that addition of the Z. mays-L N-terminal or C-terminal region to the Cterminal
or N-terminal region respectively, of the . sativa—S DGAT1 sequence results in
increased lipid yield over the full-length 0 . sativa-S DGAT1 sequence (see SEQ ID NO: 85 and
69 versus SEQ ID NO: 65).
The results also show that addition of the Z. mays- N-terminal or C-terminal region to the Cterminal
or N-terminal region respectively, of the O. s tiv — DGATl sequence results in
increased lipid yield over the full-length O. sativa-L (see SEQ ID NO: 86 and 75 versus SEQ ID
NO: 71).
The results also show that addition of the Z. mays-L N-terminal or C-terminal region to Cterminal
or N-terminal region respectively, of the Z. mays-S sequence results in increased lipid
yield over the full-length Z. mays-S sequence (see SEQ ID NO: 87 and 81 versus SEQ ID NO:
77).
In summary addition of fragments (either the N-terminal region or C-terminal region) of the Z.
ys- polypeptide of the invention to another DGATl sequence can increase the cellular lipid
yield attainable by the combined sequence over that of the other DGATl sequence.
Addition of fragments of the DGAT1 polypeptide of the invention to other DGAT1 sequences enhance lipid
production in Brassica napus relative to that with the other DGAT1 sequences alone.
Fragments (N-terminal region or C-terminal region) of the Z. ys- polypeptide of the
invention can also be combined with fragments of other plant DGATl s to raise the oil content
in Brassica napus seeds. Tables 21-22 show the seed oil contents from a variety of transgenic
plants expressing such chimeric DGATl s. In Table 6 the seed oil contents are presented both as
a % of Dry Matter (DM) and as a normalised percentage of the seed oil content of the
corresponding unmodified DGATl parents.
Table 7
Tm-ZmL 183-60-6 44.47 17.06 13.82 14.14
ZmL-Tm 185-24-5 45.27 19.16 16.20 15.87
ZmL-Tm 185-24-9 45.14 18.82 15.86 15.54
ZmL-Tm 185-22-1 44.23 16.43 13.53 13.21
ZmL-Tm 185-22-4 43.20 13.71 10.88 10.57
ZmL-Tm 185-22-9 43.49 14.48 11.63 11.31
ZmL-Tm 185-14-10 44.77 17.85 14.91 14.59
ZmL-Tm 185-9-9 43.73 15.11 12.24 11.93
ZmL-Tm 185-8-4 44.02 15.87 12.99 12.67
ZmL-Tm 185-8-7 45.11 18.74 15.79 15.46
ZmL-Tm 185-8-8 44.62 17.45 14.53 14.21
ZmL-Tm 185-8-9 43.48 14.45 11.60 1 .29
In Table 7 the oil contents are presented both on a % of DM basis and as a normalised
percentage of the seed oil content of the corresponding segregating null sibling.
Table 8
Together these results show that addition of fragments (N -terminal or C-terminal) of the Z.
DGAT1-L polypeptide of the invention can be added to parts of the T.majus DGATS1
sequence to increase oil yield relative to that produced by the full length T. majus DGATl.
Discussion
The applicants have thus shown that the novel Z . DGATl -L protein of the invention can
be used to manipulate cellular lipid accumulation. The DGATl of the invention also has higher
activity in increasing cellular lipid content than any other DGATl proteins tested by the
applicants. The applicants have also shown that subsequences, or fragments, of the DGATl of
the invention can be combined with parts of other DGATl to increase activity over that shown
over the other DGATl sequences.

CLAIMS:
1. An isolated polynucleotide encoding a DGATl polypeptide comprising the sequence of
SEQ ID NO:39 (ZmDGATl-long) or a variant or fragment thereof.
2. The isolated polynucleotide of claim 1 wherein the variant has at least 70% identity to
SEQ ID NO:39.
3. The isolated polynucleotide of claim 1 or 2 wherein the variant has DGATl activity.
4. The isolated polynucleotide of any one of claims 1 to 3, wherein the DGATl
polypeptide, when expressed in the cel,l has at least one of
a) higher DGATl activity than at least one other DGATl protein, and
b) altered substrate specificity relative to at least one other DGATl protein.
5. The isolated polynucleotide of claim 4 wherein the at least one other DGATl protein has
the amino acid sequence of the polypeptide of SEQ ID NO:44 ( ZmDGATl -short).
6. The isolated polynucleotide of any one of claims 1 to 5, wherein the DGATl
polypeptide, has at least one of
a) higher DGATl activity than any previously known DGATl protein, and
b) altered substrate specificity relative to any previously known DGATl protein.
7. The isolated polynucleotide, variant or fragment of claim 1 wherein the fragment
comprises at least 50 contiguous amino acids of the sequence of SEQ ID NO:39
(ZmDGATl-long)
8. A genetic construct comprising a polynucleotide of any one of claims 1 to 7.
9. A cell comprising a polynucleotide of the invention of any one of claims 1 to 7.
10. The cell of claim 9 wherein the cell, or its predecessor, is transformed to comprise the
polynucleotide.
11. A cell comprising a genetic construct of claim 8.
12. The cell of any one of claims 8 to 11 that expresses the polynucleotide of any one of
claims 1 to 7.
13. The cell of claim 12 that expresses the DGATl polypeptide.
14. The cell of any one of claims 12 to 14 wherein the expressed DGATl polypeptide has
increased DGATl activity than one or both of:
a) at least one other DGATl protein, and
b) any other previously known DGATl protein
15. The cell of any one of claims 12 to 14 wherein the expressed DGATl polypeptide
altered substrate specificity relative to one or both of:
a) at least one other DGATl protein, and
b) any other previously known DGATl protein.
16. The cell of any one of claims 8 to 15 that produces more lipid than does a control cell.
17. The cell of any one of claims 8 to 16 that has an altered lipid profile relative to a control
cell.
18. The cell of any one of claims 9 to 17 that is a plant cell.
19. The cell of any one of claims 9 to 18 that is also transformed to express at least one of:
an oleosin, steroleosin, caloleosin, polyoleosin, and an oleosin including at least one
artificially introduced cysteine.
20. A plant comprising a polynucleotide of any one of claims 1 to 7.
21. The plant of claim 20 wherein the plant, or its predecessor, has been transformed to
comprise the polynucleotide.
22. A plant comprising a genetic construct of claim 8.
23. A plant comprising a plant cell of claim 18.
24. The plant of any one of claims 1 to 23 wherein plant, or its predecessor, has been
transformed or genetically modified to expresses the polynucleotide or endcoded
polypeptide.
25. The plant of any one of claims 19 to 24 wherein the expressed polypeptide, has
increased DGAT1 activity relative to at least one other DGAT1 protein.
26. The plant of any one of claims 1 to 25 wherein the plant produces more lipid, in at least
one of its tissues or parts, or as a whole, than does a control plant.
27. The plant of any one of claims 19 to 26 wherein the plant has an altered lipid profile, in
at least one of its tissues or parts, or as a whole, relative to a control plant.
28. The plant of any one of claims 19 to 26 wherein the plant is also transformed to express
at least one of: an oleosin, a steroleosin, a caloleosin, a polyoleosin, and an oleosin
including at least one artificially introduced cysteine.
29. A polypeptide with the sequence of SEQ ID NO:39 (ZmD GAT 1-long) or a variant or
fragment thereof.
30. The polypeptide variant or fragment of claim 29 wherein the variant has at least 70%
identity to SEQ ID NO:39.
31. The polypeptide variant or fragment of claim 29 or 30 wherein the variant has DGAT1
activity.
32. A part, propagule or progeny of a plant of any one of claims 20 to 28.
33. A part, propagule or progeny of claim 32 wherein the part, propagule or progeny
comprises at least one of a polynucleotide, construct, plant cell, or polypeptide as defined
in any one of claims 1 to 19 and 29 to 31.
34. The part, propagule or progeny of claim 32 or 33 wherein the part, propagule or
progeny, or its predecessor plant, has been transformed to comprise the polynucleotide
or construct as defined in any one of claims 1 to 8.
35. The part, propagule or progeny of any one of claims 32 or 34 wherein the part,
propagule or progeny expresses the polynucleotide or polypeptide as defined in any one
of claims 1 to 7 and 29 to 31.
36. The part, propagule or progeny of any one of claims 32 or 34 wherein the part,
propagule or progeny produces more lipid than does a control part, propagule or
progeny, or part, propagule or progeny of a control plant.
37. The part, propagule or progeny of any one of claims 32 or 34 wherein the part,
propagule or progeny has an altered lipid profile relative to a control part, propagule or
progeny, or part, propagule or progeny of a control plant.
38. An animal feedstock comprising at least one of a polynucleotide, polypeptide, construct,
cell, plant cell, plant, plant part, propagule and progeny as defined in any one of claims 1
to 30 and 37 to 40.
39. A bio fuel feedstock comprising at least one of a polynucleotide, polypeptide, construct,
cell, plant cell, plant, plant part, propagule and progeny as defined in any one of claims 1
to 30 and 37 to 40.
40. A method for producing a lipid, the method comprising growing a cell, plant cell or plant
that is transformed, or genetically modified, to express and polynucleotide or polypeptide
as defined in any one of claims 1 to 7 and 29 to 31, wherein the plant produces oil
through the activity of the expressed polypeptide.
41. The method of claim 40 wherein the cell, plant cell or plant produces the lipid as a result
of the DGAT1 activity of the polypeptide.
42. A method for producing lipid, the method comprising extracting lipid from at least one
of a cell, plant cell, plant, plant part, propagule and progeny as defined in any one of
claims 9 to 28 and 37 to 40.
43. The method of claim 40 or 42 wherein the lipid is triacylglycerol (TAG).
44. The method of claim 40 or 42 wherein the lipid is processed into at least one of:
a) a fuel,
b) an oleochemical,
c) a nutritional oil,
d) a cosmetic oil,
e) a polyunsaturated fatty acid (PUFA), and
f ) a combination of any of a) to e).