PLANT DGAT-1 VARIANTS

VARIANTES VEGETALES DE DGAT-1

English Abstract

Modified DGAT-1 polypeptides and variants with enhanced or modified
diacylglycerol
transferase activity, and transgenic microbes and plants cells comprising the
modified
DGAT-1 polypeptides.

Claims

WHAT IS CLAIMED IS:
1. A modified Type 1 diacylglycerol acyltransferase polypeptide resulting in
either or both
increased oil production or altered fatty acid composition of oil, compared to
the unmodified
polypeptide having the sequence of SEQ ID NO: 1 (Figure 2), wherein the
modified
polypeptide has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to
SEQ ID
NO: 1 and comprises at least one of the following amino acid substitutions:
.cndot. A threonine amino acid substitution at a position corresponding to
position 10 of SEQ
ID No. 1 to an asparagine;
.cndot. A methionine acid amino acid substitution at a position
corresponding to position 11
of SEQ ID No. 1 to a threonine.
.cndot. A proline amino acid substitution at a position corresponding to
position 12 of SEQ ID
No. 1 to a serine.
.cndot. A glycine amino acid substitution at a position corresponding to
position 16 of SEQ
ID No. 1 to a serine.
.cndot. A leucine acid amino acid substitution at a position corresponding
to position 20 of
SEQ ID No. 1 to a histidine.
.cndot. An aspartic acid amino acid substitution at a position
corresponding to position 21 of
SEQ ID No. 1 to a glycine.
.cndot. An arginine acid amino acid substitution at a position
corresponding to position 24 of
SEQ ID No. 1 to a serine,
.cndot. A lysine amino acid substitution at a position corresponding to
position 27 of SEQ ID
No. 1 to a glutamic acid.
.cndot. A lysine amino acid substitution at a position corresponding to
position 27 of SEQ ID
No. 1 to an arginine.
.cndot. An arginine amino acid substitution at a position corresponding to
position 29 of SEQ
ID No. 1 to a histidine.
.cndot. A serine amino acid substitution at a position corresponding to
position 32 of SEQ ID
No. 1 to a tyrosine.

.cndot. A serine amino acid substitution at a position corresponding to
position 33 of SEQ ID
No. 1 to a threonine.
.cndot. A glycine amino acid substitution at a position corresponding to
position 35 of SEQ
ID No. 1 to an arginine.
.cndot. A glycine amino acid substitution at a position corresponding to
position 35 of SEQ
ID No. 1 to a glutamic acid.
.cndot. A valine amino acid substitution at a position corresponding to
position 41 of SEQ ID
No. 1 to a leucine.
.cndot. A threonine amino acid substitution at a position corresponding to
position 42 of SEQ
ID No. 1 to an isoleucine.
.cndot. An alanine amino acid substitution at a position corresponding to
position 46 of SEQ
ID No. 1 to a proline.
.cndot. An arginine amino acid substitution at a position corresponding to
position 51 of SEQ
ID No. 1 to a glutamine.
.cndot. A valine amino acid substitution at a position corresponding to
position 52 of SEQ ID
No, 1 to an aspartic acid.
.cndot. A valine amino acid substitution at a position corresponding to
position 52 of SEQ ID
No. 1 to an isoleucine.
.cndot. A serine amino acid substitution at a position corresponding to
position 54 of SEQ ID
No. 1 to a threonine.
.cndot. A valine amino acid substitution at a position corresponding to
position 56 of SEQ ID
No. 1 to an Isoleucine.
.cndot. A glutamine amino acid substitution at a position corresponding to
position 60 of SEQ
ID No. 1 to a glutamic acid.
.cndot. An alanine amino acid substitution at a position corresponding to
position 66 of SEQ
ID No. 1 to a serine.
.cndot. A glutamic acid amino acid substitution at a position corresponding
to position 70 of
SEQ ID No, 1 to a glycine.
.cndot. A glutamic acid amino acid substitution at a position corresponding
to position 70 of
SEQ ID No. 1 to a lysine.
51

.cndot. A glutamic acid amino acid substitution at a position corresponding
to position 70 of
SEQ ID No, 1 to a valine.
.cndot. A serine amino acid substitution at a position corresponding to
position 74 of SEQ ID
No. 1 to a phenylalanine.
.cndot. An asparagine amino acid substitution at a position corresponding
to position 81 of
SEQ ID No. 1 to an aspartic acid.
.cndot. A valine amino acid substitution at a position corresponding to
position 82 of SEQ ID
No. 1 to a glutamic acid.
.cndot. A valine amino acid substitution at a position corresponding to
position 82 of SEQ ID
No. 1 to a methionine.
.cndot. A glutamic acid amino acid substitution at a position corresponding
to position 100 of
SEQ ID No. 1 to an aspartic acid.
.cndot. An alanine amino acid substitution at a position corresponding to
position 107 of SEQ
ID No. 1 to a threonine.
.cndot. An isoleucine amino acid substitution at a position corresponding
to position 108 of
SEQ ID No. 1 to a threonine.
.cndot. A lysine amino acid substitution at a position corresponding to
position 110 of SEQ
ID No. 1 to an asparagine.
.cndot. A serine amino acid substitution at a position corresponding to
position 112 of SEQ
ID No. 1 to an arginine.
.cndot. An alanine amino acid substitution at a position corresponding to
position 114 of SEQ
ID No. 1 to an aspartic acid.
.cndot. An alanine amino acid substitution at a position corresponding to
position 114 of SEQ
ID No. 1 to a proline.
.cndot. A leucine amino acid substitution at a position corresponding to
position 116 of SEQ
ID No. 1 to an isoleucine.
.cndot. A valine amino acid substitution at a position corresponding to
position 125 of SEQ
ID No. 1 to a glycine.
.cndot. A valine amino acid substitution at a position corresponding to
position 125 of SEQ
ID No. 1 to a phenylalanine.
52

.cndot. A leucine amino acid substitution at a position corresponding to
position 136 of SEQ
ID No. 1 to an isoleucine.
.cndot. A leucine amino acid substitution at a position corresponding to
position 136 of SEQ
ID No. 1 to a phenylalanine.
.cndot. An isoleucine amino acid substitution at a position corresponding
to position 143 of
SEQ ID No. 1 to a phenylalanine.
.cndot. An isoleucine amino acid substitution at a position corresponding
to position 143 of
SEQ ID No. 1 to a valine.
.cndot. A methionine amino acid substitution at a position corresponding to
position 161 of
SEQ ID No. 1 to a lysine.
.cndot. A leucine amino acid substitution at a position corresponding to
position 164 of SEQ
ID No. 1 to a phenylalanine.
.cndot. An alanine amino acid substitution at a position corresponding to
position 172 of SEQ
ID No. 1 to a glycine.
.cndot. A lysine amino acid substitution at a position corresponding to
position 183 of SEQ
ID No. 1 to an arginine
.cndot. A cysteine amino acid substitution at a position corresponding to
position 184 of SEQ
ID No. 1 to a tyrosine.
.cndot. A leucine amino acid substitution at a position corresponding to
position 193 of SEQ
ID No. 1 to a phenylalanine.
.cndot. A methionine amino acid substitution at a position corresponding to
position 199 of
SEQ ID No. 1 to a threonine.
.cndot. A threonine amino acid substitution at a position corresponding to
position 200 of
SEQ ID No. 1 to an isoleucine.
.cndot. A glutamic acid amino acid substitution at a position corresponding
to position 201 of
SEQ ID No. 1 to a valine.
.cndot. A valine amino acid substitution at a position corresponding to
position 202 of SEQ
ID No. 1 to an isoleucine.
.cndot. A leucine amino acid substitution at a position corresponding to
position 224 of SEQ
ID No. 1 to a phenylalanine.
53

.cndot. A leucine amino acid substitution at a position corresponding to
position 224 of SEQ
ID No. 1 to a valine.
.cndot. An asparagine amino acid substitution at a position corresponding
to position 248 of
SEQ ID No. 1 to an isoleucine
.cndot. An asparagine amino acid substitution at a position corresponding
to position 248 of
SEQ ID No. 1 to a tyrosine.
.cndot. A serine amino acid substitution at a position corresponding to
position 262 of SEQ
ID No. 1 to a threonine acid.
.cndot. A cysteine amino acid substitution at a position corresponding to
position 286 of SEQ
ID No. 1 to a glycine.
.cndot. A cysteine amino acid substitution at a position corresponding to
position 286 of SEQ
ID No. 1 to a tyrosine.
.cndot. A cysteine amino acid substitution at a position corresponding to
position 286 of SEQ
ID No. 1 to a serine.
.cndot. An isoleucine amino acid substitution at a position corresponding
to position 287 of
SEQ ID No. 1 to a valine.
.cndot. A lysine amino acid substitution at a position corresponding to
position 289 of SEQ
ID No. 1 to an asparagine.
.cndot. A glycine amino acid substitution at a position corresponding to
position 290 of SEQ
ID No. 1 to a serine.
.cndot. A glycine amino acid substitution at a position corresponding to
position 290 of SEQ
ID No. 1 to an alanine
.cndot. A phenylalanine amino acid substitution at a position corresponding
to position 302 of
SEQ ID No. 1 to a cysteine.
.cndot. A phenylalanine amino acid substitution at a position corresponding
to position 302 of
SEQ ID No. 1 to an isoleucine.
.cndot. A phenylalanine amino acid substitution at a position corresponding
to position 302 of
SEQ ID No. 1 to a leucine.
.cndot. A phenylalanine amino acid substitution at a position corresponding
to position 308 of
SEQ ID No. 1 to a leucine.
54

.cndot. An isoleucine amino acid substitution at a position corresponding
to position 314 of
SEQ ID No. 1 to a methionine.
.cndot. A lysine amino acid substitution at a position corresponding to
position 322 of SEQ
ID No. 1 to a glutamic acid.
.cndot. A lysine amino acid substitution at a position corresponding to
position 326 of SEQ
ID No. 1 to an asparagine.
.cndot. A lysine amino acid substitution at a position corresponding to
position 326 of SEQ
ID No. 1 to a glutamine.
.cndot. An aspartic acid amino acid substitution at a position
corresponding to position 328 of
SEQ ID No. 1 to a glutamic acid.
.cndot. A glycine amino acid substitution at a position corresponding to
position 332 of SEQ
ID No. 1 to an alanine.
.cndot. A glycine amino acid substitution at a position corresponding to
position 332 of SEQ
ID No. 1 to a valine.
.cndot. A valine amino acid substitution at a position corresponding to
position 336 of SEQ
ID No. 1 to a methionine.
.cndot. A valine amino acid substitution at a position corresponding to
position 341 of SEQ
ID No. 1 to a leucine.
.cndot. A tyrosine amino acid substitution at a position corresponding to
position 386 of SEQ
ID No. 1 to a phenylalanine.
.cndot. An arginine amino acid substitution at a position corresponding to
position 388 of
SEQ ID No. 1 to a serine.
.cndot. A methionine amino acid substitution at a position corresponding to
position 392 of
SEQ ID No. 1 to a lysine.
.cndot. An arginine amino acid substitution at a position corresponding to
position 409 of
SEQ ID No. 1 to a serine.
.cndot. An asparagine amino acid substitution at a position corresponding
to position 410 of
SEQ ID No. 1 to a lysine.
.cndot. A leucine amino acid substitution at a position corresponding to
position 422 of SEQ
ID No. 1 to a phenylalanine.

.cndot. An arginine amino acid substitution at a position corresponding to
position 437 of
SEQ ID No. 1 to a histidine.
.cndot. A leucine acid amino acid substitution at a position corresponding
to position 438 of
SEQ ID No. 1 to a histidine.
.cndot. A leucine acid amino acid substitution at a position corresponding
to position 438 of
SEQ ID No. 1 to an isoleucine.
.cndot. A leucine amino acid substitution at a position corresponding to
position 441 of SEQ
ID No. 1 to a proline.
.cndot. A leucine amino acid substitution at a position corresponding to
position 441 of SEQ
ID No. 1 to a valine.
.cndot. A leucine amino acid substitution at a position corresponding to
position 445 of SEQ
ID No. 1 to a valine.
.cndot. An isoleucine amino acid substitution at a position corresponding
to position 447 of
SEQ ID No. 1 to a phenylalanine.
.cndot. A phenylalanine amino acid substitution at a position corresponding
to position 449 of
SEQ ID No. 1 to a leucine.
.cndot. A phenylalanine amino acid substitution at a position corresponding
to position 449 of
SEQ ID No. 1 to a cysteine.
.cndot. A glutamine amino acid substitution at a position corresponding to
position 450 of
SEQ ID No. 1 to a histidine.
.cndot. A leucine acid amino acid substitution at a position corresponding
to position 453 of
SEQ ID No. 1 to a methionine.
.cndot. An asparagine amino acid substitution at a position corresponding
to position 458 of
SEQ ID No. 1 to an aspartic acid.
.cndot. A methionine amino acid substitution at a position corresponding to
position 467 of
SEQ ID No. 1 to a lysine.
.cndot. A phenylalanine amino acid substitution at a position corresponding
to position 473 of
SEQ ID No. 1 to an isoleucine.
.cndot. A phenylalanine amino acid substitution at a position corresponding
to position 473 of
SEQ ID No. 1 to a leucine.
56

.cndot. A phenylalanine amino acid substitution at a position corresponding
to position 477 of
SEQ ID No. 1 to a leucine.
.cndot. A methionine amino acid substitution at a position corresponding to
position 484 of
SEQ ID No. 1 to an isoleucine.
.cndot. A methionine amino acid substitution at a position corresponding to
position 484 of
SEQ ID No. 1 to a leucine.
.cndot. A methionine amino acid substitution at a position corresponding to
position 484 of
SEQ ID No. 1 to a valine.
.cndot. A valine amino acid substitution at a position corresponding to
position 486 of SEQ
ID No. 1 to a glutamic acid;
.cndot. An arginine amino acid substitution at a position corresponding to
position 496 of
SEQ ID No. 1 to a proline; or
.cndot. A leucine amino acid at position corresponding to position 493
converted to a stop
codon resulting in the formation of a modified DGAT polypeptide missing the
final
nine carboxy-terminal amino acids.
2. An isolated polynucleotide comprising:
(a) a nucleotide sequence encoding a polypeptide of claim 1;
(b) a nucleotide sequence encoding a polypeptide having enhanced or modified
diacylglycerol acyltransferase activity, wherein the nucleotide sequence has
at least 80%,
85%, 90%, 95%, 98% or 99% sequence identity to a nucleotide sequence of claim
2(a);
(c) a nucleotide sequence encoding a polypeptide having enhanced or modified
diacylglycerol acyltransferase activity, wherein the nucleotide sequence
hybridizes under
stringent conditions to a nucleotide sequence of claim 2(a); or
(d) a complement of the nucleotide sequence of (a), (b) or (c), wherein the
complement and the nucleotide sequence consist of the same number of
nucleotides
57

and are 100% complementary.
3. A recombinant construct which encodes a modified polypeptide as claimed
in claim 1.
4. A transgenic oleaginous microbial or oilseed cell comprising the
recombinant construct
of claim 2.
5. The transgenic cell of claim 4 comprising a soybean, corn, canola,
sunflower, flax,
cotton, or safflower cell.
6. The transgenic cell of claim 4 comprising a yeast cell,
7. A method of making a transgenic cell having enhanced DGAT activity
resulting in
increased oil production and/or modified oil content when compared to a non-
transgenic cell,
the method comprising: (a) transforming at least one cell with a recombinant
construct of
claim 2,
8. The method of claim 7 wherein the transgenic cell is a plant cell and
said cell is
regenerated into a fully functional fertile whole plant that exhibits
increased oil production
and/or a modified oil content.
9. The method of claim 8 wherein the enhanced DGAT activity results in a
modification of
fatty acid profile of the lipid content of said cell.
10. A fully functional fertile whole plant that exhibits increased oil
production and/or a
modified oil content, comprising cells as claimed in claim 4.
11. A fully functional fertile whole -plant that exhibits increased oil
production and/or a
modified fatty acid composition, comprising a modified DGAT-1 polypeptide
variant as
58

claimed in claim 1, which plant is the result of non-transgenic methods of
altering the plant
genome.
12. The plant of claim 11 which is the result of a mutation and selection
technique, such as
TILLING, or a genome editing technique such as the use of zinc finger
nucleases,
transcription activator-like effectors, homing meganucleases, or a CRISPR/Cas
system.
59

Description

CA 2959039 2017-02-27
PLANT DGAT-1 VARIANTS
Field of the Invention
[0001] The present invention relates to modified diacylglycerol
acyltransferase (DGAT)
polypeptides with enhanced or modified oil formation ability.
Background
[0002] Demand for vegetable oils for food and fuel continues to increase.
Total vegetable oil
accumulation per area can be enhanced by maximizing the amount of oil
accumulated in each
seed. Plant breeding and genetics have demonstrated that it may be possible to
manipulate
seed oil content by a variety of means.
[0003] Strategies that have been advanced for increasing seed oil content
include: reduction
of other seed components such as lignin, protein, starch, and soluble
carbohydrates, increasing
oil-formation tissues, increasing availability of key nutrients and various
aspects of metabolic
intervention including manipulation of transcription factors, and biosynthetic
enzyme
capacity,
[0004] Biosynthetic capacity can be influenced by front-end loading whereby
the availability
of the initial building blocks for oil synthesis are increased, and end-
product unloading
whereby the synthesis of the final product, triacylglycerol (TAG) is
increased. One of the rate
limiting steps for oil synthesis in seeds is the final step in TAG
biosynthesis controlled by the
enzyme DGAT (EC 2.3.1.20).
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[0005] DGAT utilizes sn-1,2- or sn-1,3-diacylglycerol (DAG) and acyl-CoA as
substrates in
TAG biosynthesis. DGAT activity resides in at least two distinct membrane
bound
polypeptides, referred to as DGAT type 1 or DGAT-1 and DGAT type 2 or DGAT-2.
The
level of DGAT activity in the developing seed may have a substantial effect on
the flow of
carbon into TAG. This hypothesis is supported by forward and reverse genetics,
revealing
that, in several plant species, mutations in DGAT-1 directly affect oil
content and quality.
Over-expression of plant DGAT-1 has been used to stimulate oil deposition in a
model plant
Arabidopsis thaliana and in an oil crop Brass/ca napus, under both greenhouse
and field
conditions. Similar results have been obtained with the heterologous
expression of a fungal
DGAT-2 in soybean (Glycine max).
[0006] There remains a need in the art for variants of DGAT-1 which may allow
enhanced
production of oil in oleaginous microorganisms and plants.
Summary of the Invention
[0007] To explore the full potential of DGAT in oilseed metabolic engineering,
it is desirable
to better understand the enzyme's mechanism of action and regulation. However,
since
DGAT-1 is an integral membrane protein with multiple transmembrane domains,
the
resolution of its three-dimensional structure and the ensuing experiments that
would shed
light on the structure¨function relationship are currently beyond reach.
[0008] In one aspect, the invention may comprise a modified DGAT-1 polypeptide
variant as
described herein. When compared to the unmodified wildtype Type 1 DGAT
polypeptide of
SEQ ID No 1, the variant may have enhanced or modified diacylglycerol
transferase activity.
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CA 2959039 2017-02-27
[0009] In another aspect, the invention may comprise an isolated
polynucleotide comprising:
(a) a nucleotide sequence encoding a modified DGAT-1 polypeptide having
enhanced or
modified diacylglycerol transferase activity;
(b) a nucleotide sequence encoding a modified DGAT-1 polypeptide having
enhanced or
modified diacylglycerol acyltransferase activity, wherein the nucleotide
sequence has at least
80%, 85%, 90%, 95%, 98% or 99% sequence identity to a nucleotide sequence of
paragraph
(a) above;
(c) a nucleotide sequence encoding a modified DGAT-1 polypeptide having
enhanced or
modified diacylglycerol acyltransferase activity, wherein the nucleotide
sequence hybridizes
under stringent conditions to a nucleotide sequence encoding a modified DGAT-1
polypeptide
having enhanced or modified diacylglycerol transferase activity; or
(d) a complement of the nucleotide sequence of (a), (b) or (c), wherein the
complement and the nucleotide sequence consist of the same number of
nucleotides and are
100% complementary,
[0010] In another aspect, the invention may comprise a recombinant construct
which encodes
a modified DGAT-1 polypeptide having enhanced or modified diacylglycerol
transferase
activity.
[0011] In another aspect, the invention may comprise a trans genie yeast or
oilseed cell
comprising a recombinant construct described herein.
100121 In another aspect, the invention may comprise a method of making a
transgenic cell
having enhanced DGAT activity resulting in increased oil production and/or
modified fatty
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CA 2959039 2017-02-27
acid composition, when compared to a non-transgenic cell, the method
comprising the step of
the transformation of at least one cell with a recombinant construct described
herein.
[0013] In another aspect, the invention may comprise a fully functional
fertile whole plant
that exhibits increased oil production and/or a modified oil content,
comprising a modified
DGAT-1 polypeptide variant as described herein, which plant is the result of
transgenic or
non-transgenic methods of altering the plant genome. Non-transgenic methods of
altering the
plant genome include genome alteration and selection techniques, such as
TILLING, or
genome editing techniques such as the use of zinc finger nucleases,
transcription activator-like
effectors, homing meganucleases, or a CRISPR/Cas system.
Brief Description of the Drawings
[0014] The following drawings form part of the specification and are included
to further
demonstrate certain embodiments or various aspects of the invention. In some
instances,
embodiments of the invention can be best understood by referring to the
accompanying
drawings in combination with the detailed description presented herein. The
description and
accompanying drawings may highlight a certain specific example, or a certain
aspect of the
invention. However, one skilled in the art will understand that portions of
the example or
aspect may be used in combination with other examples or aspects of the
invention.
[0015] Figure 1. Oil content of the yeast strains hosting BnDGAT-1 variants
analyzed by
GC/MS. WT is a control yeast strain 1 hosting the native BnDGAT-1; VEC is a
control yeast
strain 2 hosting an empty yeast expression vector.
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CA 2959039 2017-02-27
[0016] Figure 2. Eighty-two amino acid substitutions were identified from the
50 DGAT-1
mutants, shown in comparison to unmodified Type 1 DGAT polypeptide (SEQ ID NO:
1).
[0017] Figure 3, growth curves of selected single site mutants. Please refer
to Table 2 for the
amino acid substitution of the BnDGAT1 variants, 31, control yeast strain 2
hosting an empty
yeast expression vector (VEC); 32, control yeast strain 1 hosting the native
BnDGAT-1 (WT).
[0018] Figure 4 shows neutral lipid content of selected single site mutants.
Please refer to
Table 2 for the amino acid substitution of the BnDGAT1 variants. 31, control
yeast strain 2
hosting an empty yeast expression vector (VEC); 32, control yeast strain 1
hosting the native
BnDGAT-1 (WT).
[0019] Figure 5 shows total fatty acid content of yeast cells harvested at 52
hours (early
stationary phase). Please refer to Table 2 for the amino acid substitution of
the BnDGAT1
variants. 31, control yeast strain 2 hosting an empty yeast expression vector
(VEC); 32,
control yeast strain 1 hosting the native BnDGAT-1 (WT).
[0020] Figure 6 shows gene expression level of the selected DGAT-1 single site
mutants.
Please refer to Table 2 for the amino acid substitution of the BnDGAT1
variants. 31, control
yeast strain 2 hosting an empty yeast expression vector (VEC); 32, control
yeast strain 1
hosting the native BnDGAT-1 (WT).
[0021] Figure 7 shows protein expression level of the selected mutants. Please
refer to Table
2 for the amino acid substitution of the BnDGAT1 variants. 31, control yeast
strain 2 hosting
an empty yeast expression vector (VEC); 32, control yeast strain 1 hosting the
native
BnDGAT-1 (WT).
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CA 2959039 2017-02-27
[0022] Figure 8 shows enzyme activity of the selected single site mutants.
Please refer to
Table 2 for the amino acid substitution of the BnDGAT1 variants. 31, control
yeast strain 2
hosting an empty yeast expression vector (VEC); 32, control yeast strain 1
hosting the native
BnDGAT-1 (WT).
[0023] Figure 9 shows transient expression of BnDGAT1 mutants in N.
benthamiana
Detailed Description
[0024] The present invention is the result of efforts to enhance the catalytic
efficiency of
neutral lipid synthesis in transgenic cells. Directed evolution is a general
term applied to a
collection of techniques to generate mutated proteins and to select variants
with desirable
properties. Directed evolution may target enzymatic attributes such as:
activity, substrate
preference and specificity, stability, pH optima or solvent tolerance. The
combination of
directed evolution with a high throughput selection system may allow
identification of
improved but rare events.
[0025] Randomly mutagenized libraries of B. napus DGAT-1 were generated in a
yeast
expression vector using error-prone PCR. The mutagenized libraries were used
to transform a
Saccharomyces cerevisiae yeast strain devoid of neutral lipid biosynthetic
capacity.
Transformants that showed increased lipid synthesis were analyzed using a high
throughput
positive selection system. This process eliminates mutated variants of the
gene which have
lost neutral lipid synthase activity.
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CA 2959039 2017-02-27
[0026] As used herein, the recited terms have the following meanings. All
other terms and
phrases used in this specification have their ordinary meanings as one of
skill in the art would
understand. Such ordinary meanings may be obtained by reference to technical
dictionaries
known to and accepted by those skilled in the art.
[0027] In the context of this disclosure, a number of terms may be used. The
following
definitions or abbreviations are provided.
[0028] The term "fatty acids" refers to long chain aliphatic acids (alkanoic
acids) of varying
chain length, typically from about 12 to 22 carbon atoms in length, although
both longer and
shorter chain-length acids are known. Fatty acids are classified as saturated
or unsaturated.
The term "saturated fatty acids" refers to those fatty acids that have no
"double bonds"
between the carbon atoms in the carbon chain. In contrast, "unsaturated fatty
acids" comprise
"double bonds" between the carbon atoms. "Monounsaturated fatty acids" have
only one
"double bond", while "polyunsaturated fatty acids" (or "PUFAs") have at least
two double
bonds.
[0029] "Microbial oils" or "single cell oils" are those oils naturally
produced by
microorganisms (e.g., algae, oleaginous yeasts and filamentous fungi) during
their lifespan.
The term "oil" refers to a lipid substance that is liquid at 25 C. and
usually polyunsaturated.
In contrast, the term "fat" refers to a lipid substance that is solid at 25
C. and usually
saturated.
[0030] "Neutral lipids" refer to those lipids commonly found in cells in lipid
bodies as storage
fats and oils and are so called because at cellular pH, the lipids bear no
charged groups.
Generally, they are completely non-polar with no affinity for water. Neutral
lipids generally
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CA 2959039 2017-02-27
refer to mono-, di-, and/or triesters of glycerol with fatty acids, also
called monoacylglycerol,
diacylglycerol or triacylglycerol, respectively (or collectively,
acylglycerols). A hydrolysis
reaction must occur to release free fatty acids from acylglycerols.
[0031] The term "triacylglycerol(s)" or its abbreviation "TAG(s)", also known
as
"triglyceride(s)" or "TG" or "oil", refer to neutral lipids composed of three
fatty acyl residues
esterified to a glycerol molecule (and such terms will be used interchangeably
throughout the
present disclosure herein). Such oils can contain long chain PUFAs, as well as
shorter
saturated and unsaturated fatty acids and longer chain saturated fatty acids.
Thus, "oil
biosynthesis" generically refers to the synthesis of TAGs in the cell.
[0032] The term "DGAT" refers to a diacylglycerol acyltransferase (also known
as an acyl-
CoA-diacylglycerol acyltransferase or a diacylglycerol 0-acyltransferase) (EC
2.3.1.20). This
enzyme is responsible for the conversion of acyl-CoA and 1,2-diacylglycerol to
TAG and
CoA (thereby involved in the terminal step of TAG biosynthesis). Two families
of DGAT
enzymes exist: DGAT-1 and DGAT-2.
[0033] The term "nucleic acid" means a polynucleotide and includes single or
double-
stranded polymer of deoxyribonucleotide or ribonueleotide bases. Nucleic acids
may also
include fragments and modified nucleotides. Thus, the terms "polynucleotide",
"nucleic acid
sequence", "nucleotide sequence" or "nucleic acid fragment" are used
interchangeably and is a
polymer of RNA or DNA that is single- or double-stranded, optionally
containing synthetic,
non-natural or altered nucleotide bases. Nucleotides (usually found in their
5'-monophosphate
form) are referred to by their single letter designation as follows: "A" for
adenylate or
deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or
deosycytidylate, "G"
8

CA 2959039 2017-02-27
for guanylate or deoxyguanylate, "U" for uridlate, "T" for deosythymidylate,
"R" for purines
(A or G), "Y" for pyrimidiens (C or T), "IC for G or T, "H" for A or C or T,
"I" for inosine,
and "N" for any nucleotide. Unless otherwise indicated, a particular nucleic
acid sequence
also implicitly encompasses conservatively modified variants thereof (e.g.,
degenerate codon
substitutions) and complementary sequences as well as the sequence explicitly
indicated,
Specifically, degenerate codon substitutions may be achieved by generating
sequences in
which the third position of one or more selected (or all) codons is
substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucl. Acids Res., 19:508 (1991);
Ohtsuka et al., J.
Biol. Chem., 260:2605 (1985); Rossolini et al., Mol. Cell, Probes, 8:91
(1994).
100341 A "variant" of a molecule is a sequence that is substantially similar
to the sequence of
the native molecule. For nucleotide sequences, variants include those
sequences that, because
of the degeneracy of the genetic code, encode the identical amino acid
sequence of the native
protein. Naturally occurring allelic variants such as these can be identified
with the use of
well-known molecular biology techniques, as, for example, with polymerase
chain reaction
(PCR) and hybridization techniques. Variant nucleotide sequences also include
synthetically
derived nucleotide sequences, such as those generated, for example, by using
site-directed
mutagenesis that encode the native protein, as well as those that encode a
polypeptide having
amino acid substitutions.
[0035] "Operably-linked" refers to the association of nucleic acid sequences
on single nucleic
acid fragment so that the function of one is affected by the other. For
example, a regulatory
DNA sequence is said to be "operably linked to" or "associated with" a DNA
sequence that
codes for an RNA or a polypeptide if the two sequences are situated such that
the regulatory
9

CA 2959039 2017-02-27
DNA sequence affects expression of the coding DNA sequence (i.e., that the
coding sequence
or functional RNA is under the transcriptional control of the promoter).
Coding sequences can
be operably-linked to regulatory sequences in sense or antisense orientation.
In another
example, the complementary RNA regions of the invention can be operably
linked, either
directly or indirectly, 5' to the target mRNA, or 3' to the target mRNA, or
within the target
mRNA, or a first complementary region is 5' and its complement is 3' to the
target mRNA.
[0036] The term "conserved domain" or "motif' means a set of amino acids
conserved at
specific positions along an aligned sequence of evolutionarily related
proteins. While amino
acids at other positions can vary between homologous proteins, amino acids
that are highly
conserved at specific positions indicate amino acids that are essential in the
structure, the
stability, or the activity of a protein. Because they are identified by their
high degree of
conservation in aligned sequences of a family of protein homologues, they can
be used as
identifiers, or "signatures", to determine if a protein with a newly
determined sequence
belongs to a previously identified protein family.
[0037] The terms "homology", "homologous", "substantially similar" and
"corresponding
substantially" are used interchangeably herein. They refer to nucleic acid
fragments wherein
changes in one or more nucleotide bases do not affect the ability of the
nucleic acid fragment
to mediate gene expression or produce a certain phenotype. These terms also
refer to
modifications of the nucleic acid fragments of the instant invention such as
deletion or
insertion of one or more nucleotides that do not substantially alter the
functional properties of
the resulting nucleic acid fragment relative to the initial, unmodified
fragment. It is therefore

CA 2959039 2017-02-27
understood, as those skilled in the art will appreciate, that the invention
encompasses more
than the specific exemplary sequences.
[0038] Moreover, the skilled artisan recognizes that substantially similar
nucleic acid
sequences encompassed by this invention are also defined by their ability to
hybridize (under
moderately stringent conditions, e.g., 0.5XSSC (Saline Sodium Citrate, 20XSSC
= 3.0 M
NaC1/ 0.3 M trisodium citrate), 0.1% SDS (sodium dodecyl sulphate), 60 deg.
C.) with the
sequences exemplified herein, or to any portion of the nucleotide sequences
disclosed herein
and which are functionally equivalent to any of the nucleic acid sequences
disclosed herein.
Stringency conditions can be adjusted to screen for moderately similar
fragments, such as
homologous sequences from distantly related organisms, to highly similar
fragments, such as
genes that duplicate functional enzymes from closely related organisms. Post-
hybridization
washes determine stringency conditions.
[0039] The term "selectively hybridizes" includes reference to hybridization,
under stringent
hybridization conditions, of a nucleic acid sequence to a specified nucleic
acid target
sequence to a detectably greater degree (e.g., at least 2-fold over
background) than its
hybridization to non-target nucleic acid sequences and to the substantial
exclusion of non-
target nucleic acids. Selectively hybridizing sequences typically have about
at least 80%
sequence identity, or 90% sequence identity, up to and including 100% sequence
identity (i.e.,
fully complementary) with each other.
[0040] The term "stringent conditions" or "stringent hybridization conditions"
includes
reference to conditions under which a probe will selectively hybridize to its
target sequence.
Stringent conditions are sequence-dependent and will be different in different
circumstances.
11

CA 2959039 2017-02-27
By controlling the stringency of the hybridization and/or washing conditions,
target sequences
can be identified which are 100% complementary to the probe (homologous
probing).
Alternatively, stringency conditions can be adjusted to allow some mismatching
in sequences
so that lower degrees of similarity are detected (heterologous probing).
Generally, a probe is
less than about 1000 nucleotides in length, optionally less than 500
nucleotides in length.
[0041] Typically, stringent conditions will be those in which the salt
concentration is less than
about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or
other salts) at pH
7.0 to 8.3 and the temperature is at least about 30 deg. C. for short probes
(e.g., 10 to 50
nucleotides) and at least about 60 deg. C. for long probes (e.g., greater than
50 nucleotides).
Stringent conditions may also be achieved with the addition of destabilizing
agents such as
formamide. Exemplary low stringency conditions include hybridization with a
buffer solution
of 30 to 35% formamide, 1 M NaCl, 1% SDS at 37 deg. C., and a wash in 1X to 2X
SSC at 50
to 55 deg. C. Exemplary moderate stringency conditions include hybridization
in 40 to 45%
formamide, 1 M NaC1, 1% SDS at 37 deg. C., and a wash in 0.5X to 1X SSC at 55
to 60 deg.
C. Exemplary high stringency conditions include hybridization in 50%
formamide, 1 M NaC1,
1% SDS at 37 deg. C., and a wash in 0.1X SSC at 60 to 65 deg. C.
[0042] The "Clustal V method of alignment" corresponds to the alignment method
labeled
Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins,
D. G. et al.
(1992) Comput. Appl. Biosci. 8:189.491) and found in the MegAlign.TM program
of the
LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). For
multiple alignments, the default values correspond to GAP PENALTY=10 and GAP
LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation
of
12

CA 2959039 2017-02-27
percent identity of protein sequences using the Clustal method are KTUPLE=1,
GAP
PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these
parameters are KTUPLE=2, GAP PENALTY=5, WINDOW-4 and DIAGONALS
SAVED=4. After alignment of the sequences using the Clustal V program, it is
possible to
obtain a "percent identity" by viewing the "sequence distances" table in the
same program.
[0043] "BLASTN method of alignment" is an algorithm provided by the National
Center for
Biotechnology Information (NCBI) to compare nucleotide sequences using default

parameters.
[0044] "Gene" refers to a nucleic acid fragment that expresses a specific
protein, including
regulatory sequences preceding (5' non-coding sequences) and following (3' non-
coding
sequences) the coding sequence. "Native gene" refers to a gene as found in
nature with its
own regulatory sequences. "Chimeric gene" refers to any gene that is not a
native gene,
comprising regulatory and coding sequences that are not found together in
nature.
Accordingly, a chimeric gene may comprise regulatory sequences and coding
sequences that
are derived from different sources, or regulatory sequences and coding
sequences derived
from the same source, but arranged in a manner different than that found in
nature. A
"foreign" gene refers to a gene not normally found in the host organism, but
that is introduced
into the host organism by gene transfer. Foreign genes can comprise native
genes inserted into
a non-native organism, or chimeric genes. A "transgene" is a gene that has
been introduced
into the genome by a transformation procedure.
[0045] "Coding sequence" refers to a DNA sequence that codes for a specific
amino acid
sequence. "Regulatory sequences" refer to nucleotide sequences located
upstream (5' non-
13

CA 2959039 2017-02-27
coding sequences), within, or downstream (3' non-coding sequences) of a coding
sequence,
and which influence the transcription, RNA processing or stability, or
translation of the
associated coding sequence. Regulatory sequences may include, but are not
limited to:
promoters, translation leader sequences, introns, polyadenylation recognition
sequences, RNA
processing sites, effector binding sites and stem-loop structures.
"Promoter" refers to a DNA sequence capable of controlling the expression of a
coding
sequence or functional RNA. The promoter sequence consists of proximal and
more distal
upstream elements, the latter elements often referred to as enhancers.
Accordingly, an
"enhancer" is a DNA sequence that can stimulate promoter activity, and may be
an innate
element of the promoter or a heterologous element inserted to enhance the
level or tissue-
specificity of a promoter. Promoters may be derived in their entirety from a
native gene, or be
composed of different elements derived from different promoters found in
nature, or even
comprise synthetic DNA segments. It is understood by those skilled in the art
that different
promoters may direct the expression of a gene in different tissues or cell
types, or at different
stages of development, or in response to different environmental conditions.
It is further
recognized that since in most cases the exact boundaries of regulatory
sequences have not
been completely defined, DNA fragments of some variation may have identical
promoter
activity. Promoters that cause a gene to be expressed in most cell types at
most times are
commonly referred to as "constitutive promoters".
[0046] "PCR" or "polymerase chain reaction" is a technique for the synthesis
of large
quantities of specific DNA segments and consists of a series of repetitive
cycles (Perkin
Elmer Cetus Instruments, Norwalk, Conn.). Typically, the double-stranded DNA
is heat
14

CA 2959039 2017-02-27
denatured, the two primers complementary to the 3' boundaries of the target
segment are
annealed at low temperature and then extended at an intermediate temperature.
One set of
these three consecutive steps is referred to as a "cycle". Error prone PCR or
epPCR is a
variation of a PCR method. Normally the replication of DNA by PCR is extremely
specific. In
error prone PCR, mistakes are intentionally induced in the base pairing during
DNA synthesis
that results in the introduction of errors in the newly synthesized
complementary DNA strand.
[0047] The term "recombinant" refers to an artificial combination of two
otherwise separated
segments of sequence, e.g., by chemical synthesis or by the manipulation of
isolated segments
of nucleic acids by genetic engineering techniques.
[0048] The terms "plasmid", "vector" and "cassette" refer to an extra
chromosomal element
often carrying genes that are not part of the central metabolism of the cell,
and usually in the
form of circular double-stranded DNA fragments. Such elements may be
autonomously
replicating sequences, genome integrating sequences, phage or nucleotide
sequences, linear or
circular, of a single- or double-stranded DNA or RNA, derived from any source,
in which a
number of nucleotide sequences have been joined or recombined into a unique
construction
which is capable of introducing a promoter fragment and DNA sequence for a
selected gene
product along with appropriate 3' untranslated sequence into a cell.
"Transformation cassette"
refers to a specific vector containing a foreign gene and having elements in
addition to the
foreign gene that facilitates transformation of a particular host cell.
"Expression cassette"
refers to a specific vector containing a foreign gene and having elements in
addition to the
foreign gene that allow for enhanced expression of that gene in a foreign host
(i.e., to a
discrete nucleic acid fragment into which a nucleic acid sequence or fragment
can be moved.)

CA 2959039 2017-02-27
[0049] The terms "recombinant construct", "expression construct", "chimeric
construct",
"construct", and "recombinant DNA construct" are used interchangeably herein.
A
recombinant construct comprises an artificial combination of nucleic acid
fragments, e.g.,
regulatory and coding sequences that are not found together in nature. For
example, a
chimeric construct may comprise regulatory sequences and coding sequences that
are derived
from different sources, or regulatory sequences and coding sequences derived
from the same
source, but arranged in a manner different than that found in nature. Such a
construct may be
used by itself or may be used in conjunction with a vector. If a vector is
used, then the choice
of vector is dependent upon the method that will be used to transform host
cells as is well
known to those skilled in the art. For example, a plasmid vector can be used.
The skilled
artisan is well aware of the genetic elements that must be present on the
vector in order to
successfully transform, select and propagate host cells comprising any of the
isolated nucleic
acid fragments of the invention.
[0050] The term "expression", as used herein, refers to the production of a
functional end-
product (e.g., a mRNA or a protein [either precursor or mature]).
[00511 The term "introduced" means providing a nucleic acid (e.g., expression
construct) or
protein into a cell. Introduced includes reference to the incorporation of a
nucleic acid into a
eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into
the genome of
the cell, and includes reference to the transient provision of a nucleic acid
or protein to the
cell. Introduced includes reference to stable or transient transformation
methods, as well as
sexually crossing. Thus, "introduced" in the context of inserting a nucleic
acid fragment (e.g.,
a recombinant DNA construct/expression construct) into a cell, means
"transfection" or
16

CA 2959039 2017-02-27
"transformation" or "transduction" and includes reference to the incorporation
of a nucleic
acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid
fragment may be
incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid
or mitochondrial
DNA), converted into an autonomous replicon, or transiently expressed (e.g.,
transfected
mRNA).
[0052] "Mature" protein refers to a post-translationally processed polypeptide
(i.e., one from
which any pre- or propeptides present in the primary translation product have
been removed).
"Precursor" protein refers to the primary product of translation of mRNA
(i.e., with pre- and
propeptides still present). Pre- and propeptides may be but are not limited to
intracellular
localization signals.
[0053] "Stable transformation" refers to the transfer of a nucleic acid
fragment into a genome
of a host organism, including both nuclear and organellar genomes, resulting
in genetically
stable inheritance. In contrast, "transient transformation" refers to the
transfer of a nucleic
acid fragment into the nucleus, or DNA-containing organelle, of a host
organism resulting in
gene expression without integration or stable inheritance. Host organisms
containing the
transformed nucleic acid fragments are referred to as "transgenic" organisms.
[0054] As used herein, "transgenic" refers to a plant or a cell which
comprises within its
genome a heterologous polynucleotide. Preferably, the heterologous
polynucleotide is stably
integrated within the genome such that the polynucleotide is passed on to
successive
generations. The heterologous polynucleotide may be integrated into the genome
alone or as
part of an expression construct. Transgenic is used herein to include any
cell, cell line, callus,
tissue, plant part or plant, the genotype of which has been altered by the
presence of
17

CA 2959039 2017-02-27
heterologous nucleic acid including those transgenics initially so altered as
well as those
created by sexual crosses or asexual propagation from the initial transgenic.
The term
=
"transgenic" as used herein does not encompass the alteration of the genome
(chromosomal or
extra-chromosomal) by conventional plant breeding methods or by naturally
occurring events
such as random cross-fertilization, non-recombinant viral infection, non-
recombinant bacterial
transformation, non-recombinant transposition, or spontaneous mutation.
[0055] The term "oleaginous" refers to those organisms that tend to make or
store lipids. The
term "oleaginous microorganism" refers to those microorganisms that make or
store oil. It is
not uncommon for oleaginous microorganisms to accumulate in excess of about
25% of their
dry cell weight as oil. Non-limiting examples of oleaginous microorganisms
include yeast
such as the following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium,
Cryptococcus, Trichosporon and Lipomyces.
[0056] The term "plant" refers to whole plants, plant organs, plant tissues,
seeds, plant cells,
seeds and progeny of the same. Plant cells include, without limitation, cells
from seeds,
suspension cultures, embryos, meristematic regions, callus tissue, leaves,
roots, shoots,
gametophytes, sporophytes, pollen and microspores.
[0057] "Progeny" comprises any subsequent generation of a plant.
[0058] To the extent that the following description is of a specific
embodiment or a particular
use of the invention, it is intended to be illustrative only, and not limiting
of the claimed
invention. The following description is intended to cover all alternatives,
modifications and
equivalents that are included in the spirit and scope of the invention, as
defined in the
appended claims. References in the specification to "one embodiment", "an
embodiment",
18
_

CA 2959039 2017-02-27
etc., indicate that the embodiment described may include a particular aspect,
feature,
structure, or characteristic, but not every embodiment necessarily includes
that aspect, feature,
structure, or characteristic. Moreover, such phrases may, but do not
necessarily, refer to the
same embodiment referred to in other portions of the specification. Further,
when a particular
aspect, feature, structure, or characteristic is described in connection with
an embodiment, it is
within the knowledge of one skilled in the art to affect or connect such
aspect, feature,
structure, or characteristic with other embodiments, whether or not explicitly
described.
[0059] Increase in Cellular Lipid Content
[0060] Methods to increase the oil content of oleaginous cells, plants and/or
plant seeds are
desirable. Previous transgenic studies have shown that DGAT has a fundamental
role in
controlling oil production in yeasts and plants. The present invention
comprises novel
modifications made to the primary amino acid sequence of a DGAT polypeptide in
an attempt
to increase DGAT specific activity.
[0061] As used herein, "enhanced or modified diacylglycerol transferase
activity" is activity
resulting in increased oil production, or modified fatty acid composition of
the oil produced,
or both, in a seed, plant or oleaginous microorganism.
[0062] In one embodiment, BnaC.DGAT-1.a (GenBank# 31\1224473) (Greer et al.,
2015) was
selected as the starting point for mutagenesis.
[0063] A variety of different DNA sequence diversity generating procedures can
be used for
generating modified nucleic acid sequences that include but are not limited to
chemical
mutagenesis, radiation and processes such as DNA shuffling. In one embodiment,
the
19

CA 2959039 2017-02-27
mutations may be derived by use of error-prone PCR, which is a method where
random
mutantions are inserted into any DNA sequence. Typically, the replication of
DNA by the
polymerase is extremely reliable, however in error prone PCR the fidelity of
the Taq DNA
polymerase is modulated by alteration of the composition of the reaction
buffer. Under these
conditions, the polymerase makes errors in base pairing during DNA synthesis
that result in
the introduction of nucleotide base changes in the newly synthesized
complementary DNA
strand. By carefully controlling the buffer composition, the frequency of mis-
incorporation of
nucleotide bases, and therefore the number of errors introduced into the
sequence, may be
regulated. In directed evolution experiments, the substitution frequency is
normally
established at around 1 - 3 base pair substitutions per kilobase of DNA.
[0064] For optimal results, a Taq DNA polymerase that does not have proof-
reading ability is
used. The proof-reading, or auto-correction of nucleotide sequence, is a
property that is found
in many commercially available Taq DNA polymerases. However, use of a proof-
reading
DNA polymerase in an error prone PCR reaction will result in the automatic
correction of the
mismatched nucleotides, and any mutations that were introduced during the
reaction will be
lost.
[0065] High Throughput Screening (HTS)
[0066] Regardless of the method used to generate the random modifications,
directed
evolution requires a reliable high throughput system of screening (HTS)
capable of selecting
variants with desired characteristics from a vast number of generated mutants
(Bershtein et al,
2008). One method available to assess DGAT activity is based on the use of
radio-labeled
substrates and involve separation of radio-labeled TAG from other products in
the reaction

CA 2959039 2017-02-27
mixture using thin layer chromatography (Coleman, 1992). The specific activity
of
heterologously expressed DGAT is relatively low, typically measured in pmol of
TAG per mg
of protein. Moreover, the need to prepare microsomal fractions further limits
the throughput
and consequently the potential to study large populations of mutagenized DGAT-
1. Another
method which may circumvent this problem comprises a positive selection system
combined
with a rapid in situ fluorescence assay that correlates with DGAT enzyme
activity (Siloto
2009a,b).
[0067] Yeast (Saccharomyces cerevisiae), lacking TAG synthase activity
(quadruple
knockout DGA1, LR01, ARE1 and ARE2) is viable under normal growth conditions
despite
the lack of neutral lipid production (Sandager et al., 2002), but exhibits
reduced growth rates
compared to wild type on growth medium supplemented with diacylglycerol or
fatty acids.
[0068] This knock-out yeast strain may be used in a positive selection system
for genes
conferring neutral lipid synthase activity. The cells which have neutral lipid
synthase activity
will grow significantly faster, allowing their apparent positive selection.
The growth media
may be supplemented with a fatty acid such as oleic acid, in concentrations
from about 25 t,t1V1
to about 1000 }1M.
[0069] A method to estimate lipid content of oleaginous microorganisms based
on a
fluorescent dye, Nile Red, was reported by Kimura et al., (2004). Nile Red
staining can be
used to quantify neutral lipids such as TAG and Sterol ester (TB), due to the
fact that the
fluorescence intensity is much higher for neutral lipids than for polar
lipids. The maximum
wavelength emission of Nile Red conjugated with neutral lipids is different
from the
maximum of the dye-polar lipid complex (Greenspan et al., 1985). Therefore,
both contents of
21

CA 2959039 2017-02-27
neutral lipids and activity levels of neutral lipid synthases may be
quantified by measuring the
fluorescence of cells stained with Nile Red.
[0070] In vitro evolution of neutral lipid synthases to enhance enzymatic
activity and modify
substrate selectivity can be performed by combining directed evolution with
high throughput
neutral lipid synthesis assays.
[0071] Screening can be performed on populations of mutagenized neutral lipid
synthase
genes in order to select variants with increased activity with the acyl chain
of interest by a
process of molecular evolution. Selection is performed by incorporating the
free fatty acid of
interest in the solid medium or by growing pre-selected yeast cells in the
liquid medium
containing the fatty acid and measuring the accumulation of neutral lipids by
the fluorescent
assay.
[0072] Embodiments of the invention can be combined with other analytical
procedures that
require analysis of a large number of individual samples arrayed in a large
multi-well plate,
such as 96-well or 384-well plates used commonly in the art.
[0073] The fluorescent assay for neutral lipid synthase activity can be
combined with
fluorescent cell sorting (FACS) to increase the efficiency of selection and
the throughput
(approximately one million individual cells per hour). The methods described
herein may be
used either individually or in combination to identify or isolate TAG synthase
enzymes with
enhanced or specialized activity.
[0074] Characterization of the Mutations
22

CA 2959039 2017-02-27
[0075] After yeast strains producing elevated amounts of TAG are detected by
HTS, they
may be cultured in large volume (e.g. 25 ml or 100 ml liquid medium) and the
biomass
harvested for oil analysis by GC/MS and specific DGAT-1 activity assay to
verify the impact
of amino acid substitution on enzyme activity. The plasmids containing DGAT-1
gene
variants maybe extracted for sequencing to identify the mutant sites. The
impact on oil
synthesis is tested in the yeast system described in the current invention,
The amino acid sites
identified as significantly contributing to TAG synthesis can be further
investigated by
saturated sited mutagenesis to find the optimal mutation. This is accomplished
by replacement
of an identified mutant amino acid with other amino acids in order to
determine the optimal
replacement at any one site. The DGAT-1 variants can then be expressed in
Arabidopsis and
oilseed crops to test their impact on TAG synthesis in plants.
[0076] Expression of selected mutations in plants.
[0077] To test their impact on seed oil biosynthesis, BnDGAT-1 variants,
comprising
mutations of interest, can be expressed in Arabidopsis, canola, or other
oilseeds. Homozygous
plants with stable expression of BnDGAT-1 variants can be identified after
selfing and growth
to maturity. Gas chromatographic (GC) analysis is used to determine seed oil
content and
fatty acid composition.
[0078] Many different procedures are well known to researchers in the field of
plant
molecular biology that result in plant transformation and recombinant gene
expression in
transgenic plants. Generally, transformation methods can be grouped into two
basic strategies;
physical methods to introduce foreign DNA into plant cells and Agrobacterium-
mediated
transformation of plant cells, (Borampuram et al., 2011).
23

CA 2959039 2017-02-27
[0079] A widely practiced general method of achieving plant transformation
comprises the
use of Agrobacteria (as reviewed in Gelvin, 2003).
[0080] Generally, methods for plant transformation are well known in the art
and detailed
procedures have been published and patented for transformation of well-known
plant species
such as tobacco, (Conley et al. 2011), canola, (US 5,188,958), Arabidopsis,
(US 6,353,155),
corn, (US 5,981,840), cotton, (US 5,998,207), oil palm, (US 8,017,837), and
soybean, (US
5,024,944).
[0081] In addition to DNA sequences required for transport and integration of
foreign DNA
into the host nuclear or plastid genomes, transformation vectors additionally
comprise DNA
sequences that are needed for gene expression. Such DNA sequences include
promoters,
enhancers, terminators, selectable markers, targeting and intervening
sequences.
[0082] Organ specific promoters, especially those that direct expression to
developing
embryos in seeds are useful for expression of DGAT-1 variants for increased
oil production in
seeds. In a specific embodiment, the Brassica seed-specific napin promoter was
used. (Siloto
et al, 2009a).
[0083] Promoters that are active in plant seeds are well known in the
literature and elements
for the regulation of seed specific expression have been described for
Brassica, (Rask et al,
1998) and many other species as described in patents including: US 6,437,220,
US 5,504,200,
US 6,320,102, US 5,530,184, 6,013,862.
[0084] Gene activity can be increased by a variety of means. Enhancers are
short DNA
regions, (50-100 bp), of DNA that can bind proteins that activate
transcription of a gene or
24

CA 2959039 2017-02-27
genes. Examples include sequences related to physiological induction such as
heat, pathogen
attack, (Mitsuhara et al., 1996), and viral promoter sequences such as double
35S, (Kay et al.,
1987), and AMV RNA4, (Datla et al., 1993). A database of cis-acting regulatory
elements has
been created, (Lescot et al., 2002). Gene expression can also be increased by
intron
sequences, (Parra et al., 2011).
100851 In order that transgenic plants can be selectively recovered from
transformed cells,
transformation vectors often comprise a gene that codes for a product that
allows the
identification and or selection of transformed plants. Examples of gene
products that can be
used as visual reporter genes to recognize transformants include GUS, (Beta-
glucuronidase)
that imparts a blue pigment, or GFP, the green fluorescent protein that can be
detected via
fluorescence.
[0086] Gene products that impart a negative or positive selection can be used
effectively to
select transformed cells, (Miki et al., 2004). Examples of selectable markers
that have been
used extensively include those that impart antibiotic resistance such as to
kanamycin,
hygromycin or streptomycin, or provide tolerance to herbicides such as
phosphinotricin or
glyphosate. A variety of strategies have also been used to remove selectable
marker genes
because of concerns that such genes may have a negative environmental impact,
(Gleave et
al., 1999).
[0087] In one embodiment, the transformation of ilrabidopsis with BnDGAT-1
variants was
undertaken using the binary vector pGreen 0229 under the control of a napin
seed specific
promoter (Jiang et al., 1995) and rubisco terminator. Cloning was conducted
using the
phosphorothioate-based method (Blanusa et al., 2010). The T-DNA of pGreen 0229
vector

CA 2959039 2017-02-27
also contains a phosphinothricin acetyltransferase cassette conferring
resistance to
phosphinotricin. The resulting binary vectors were transformed into
Agrobacterium
tumefaciens (strain GV 3101) through electroporation. Recombinant A.
tumefaciens strains,
each containing one BnDGAT-1 variant, was cultivated individually until
reaching the desired
optical density for plant transformation.
[0088] A. thaliana (ecotype Columbia) transformation was conducted using a
modified floral
dipping method (Clough and Bent, 1998). Briefly, sixty to eighty 4-inch pots,
each containing
five plants, were treated with the A. tumefaciens mix twice with an interval
of two weeks
between each dipping. Plants were cultivated to maturity and harvested. Ti
plants were
selected in medium containing phosphinotricin and seedlings were transferred
to soil. The
seeds were harvested and used for segregation analysis to obtain homozygous
plants. The
seeds were then grown with controls under exactly the same condition to the
next generation.
Subsequently, seeds were harvested for oil analysis by GC.
[00891 The transformation of canola with BnDGAT-1 variants was undertaken
using the
binary vector pGreen 0029 under the control of a napin seed specific promoter
(Jiang et al.,
1995) and rubisco terminator. Cloning was conducted using the phosphorothioate-
based
method (Blanusa et al., 2010). The T-DNA of pGreen 0029 vector has resistance
to
kanamycin. The constructs were transformed to A. tumefaciens strains as
described above, and
the strains were used to transform B. napus L. cv DH12075, a line of canola
presenting
favourable agronomic characteristics which can be used for development of
commercial
cultivars. B. napus was transformed using the established method of Moloney et
al., (1989),
26

CA 2959039 2017-02-27
[0090] Once mutations that result in an increase of oil synthesis in yeast or
plant cells that
were identified using an initial B. napus DGAT 1 DNA sequence and high
throughput
screening in the yeast system, equivalent changes or mutations could be made
to endogenous
DGAT genes of any species by any number of known methods. Therefore, in one
aspect, the
invention may comprise modified plants which have mutations corresponding to
the
mutations of the modified DGAT-1 variants described herein, and which exhibit
enhanced or
modified diacylglycerol transferase activity, which plants have been produced
using DNA
editing methods. The term "DNA editing" refers to a type of genetic
manipulation in which
DNA is inserted, replaced or removed from a genome using engineered nucleases,
The
nucleases create specific double-stranded DNA breaks at specified locations
where after the
induced break is repaired by the endogenous processes of homologous
recombination or non-
homologous end-joining. A number of sequence specific nucleases are known that
can be
used to target specific DNA sequences for deletion, modification or insertion,
(Podcvin et al,
2013). Modification methods include zinc-finger nucleases, (ZFNs, Urnov et
al., 2010, Miller
et al., 2007), TALENs, (Bedell et al., 2012, Joung and Sander, 2013),
Meganucleases, (Puchta
and Fauser, 2013) and CRISPR/Cas9, (Ran et al, 2013, Belhaj et al, 2013, Shan
et al, 2013).
[0091] Once specific sites that confer altered activity of lipid biosynthesis
enzymes are
identified by induced mutation and high through-put screening then these
individual sites can
be modified using genome-editing nucleases.
100921 Zinc-finger nucleases or ZFNs, are artificial restriction enzymes
generated by the
fusion of a zinc-finger DNA-binding domain to a DNA cleavage domain. Zinc
finger nuclease
domains can be designed to target specific desired DNA sequences thus enabling
targeted
27

CA 2959039 2017-02-27
modification of any DNA sequence. Exemplary ZFNs and methods of using them are

described in US Patent Application No. 20120329067.
[0093] Transcription activator-like effector nucleases or TALENs are
artificial restriction
enzymes generated by the fusion of a TAL-effector DNA-binding domain to a DNA
cleavage
domain. Transcription activator-like effectors, (TALEs) can be engineered to
bind to any
desired DNA sequence, The combination of a specifically engineered TALE with a
DNA
cleavage domain results in DNA cleavage at a specific desired DNA sequence.
Exemplary
TALEs, TALENs and methods of using them are described in US Patent No.
8440431.
[0094] A CRISPR.Cas9 system is a prokaryotic defense system that confers
resistance to
foreign genetic elements such as plasmids and bacteriophage. CRISPRs,
(Clustered Regularly
Interspaced Short Palindromic Repeats) are segments of DNA comprising short
repetitions of
DNA sequences interspersed by segments of "spacer DNA" obtained from previous
exposure
to viruses or plasmids. Cas9, (CRISPR associated protein 9) is an RNA-guided
DNA
endonuclease enzyme. CRISPR/Cas9 systems are described in U.S. Patent No.
8697359, and
an exemplary use of a CRISPR/Cas9 system to engineer plant genomes is
described in PCT
Application WO 2014/144155 (US Patent Application No. 20140273235).
[0095] Meganucleases are endodeoxyribonucleases characterized by a large DNA
recognition
site of about 12 ¨ 40 base pairs. Modification of the recognition sequence
through protein
engineering allows the replacement, elimination or modification of DNA
sequences in a
highly targeted way. For example, US Patent No. 8338157 described rationally
engineered
meganucleases and their use in producing engineered maize plants.
28

CA 2959039 2017-02-27
[0096] For example random mutations could be made in endogenous DNA sequences
of a
DGAT gene of any species or any additional gene involved in lipid biosynthesis
using
TILLING, (McCallum et al., 2000). TILLING is an abbreviation of Targeted
Induced Local
Lesions in Genomes and is a method for the directed identification of
mutations in a specific
=
gene. The method combines chemical mutagenesis (with a chemical mutagen such
as Ethyl
Methanesulfonate, (EMS)) with a DNA screening-technique that identifies single
base-pair
mutations in a selected gene. The TILLING method relies on the formation of
DNA
heteroduplexes that are formed when DNA sequences are amplified using PCR and
then
heated and slowly cooled. A "bubble" forms at the mismatch of the two DNA
strands which is
then cleaved by a single-stranded DNA nuclease. Sequencing of the TILLING
induced
mutations would then allow discovery of mutations at sites previously
determined to have an
impact on oil synthesis or composition. TILLING provides a method which
combines high
density of mutations with rapid mutational screening to discover induced
lesions. Mutations
which correspond to the mutations of the modified DGAT-1 variants identified
herein, may
then be selected. TILLING methods are described in US Patent Application No.
20040053236.
[0097] Examples
[0098] The present invention is further defined in the following Examples. It
should be
understood that these Examples, while indicating preferred embodiments of the
invention, are
given by way of illustration only. From the above discussion and these
Examples, one skilled
in the art can ascertain the essential characteristics of this invention, and
without departing
from the spirit and scope thereof, can make various changes and modifications
of the
29

CA 2959039 2017-02-27
invention to adapt it to various usages and conditions. Thus, various
modifications of the
invention in addition to those shown and described herein will be apparent to
those skilled in
the art from the foregoing description. Such modifications are also intended
to fall within the
scope of the appended claims.
Example 1
[0099] Amino Acid Substitutions that resulted in higher oil in yeast.
[00100] As shown in Table 1, 50 DGAT-1 variants which exhibited increased oil
content
were identified using the yeast Nile red selection system. The expression of
these mutants in
both wild type and H1246 yeast strains resulted in higher oil content than
native BnDGAT-1.
All yeast strains were cultured in 20 ml liquid medium and the cells were
harvested for oil
analysis by GC/MS. As shown in Figure 1, GC/MS assay confirmed that all
mutants resulted
in higher oil content in 111246 yeast.
Table 1. BnDGAT-1 variants that resulted in higher oil content in yeast
strains determined by
the Nile red assay method. WT, control yeast strain 1 hosting the native
BnDGAT-1; VEC,
control yeast strain 2 hosting an empty yeast expression vector.
parental strain 111246 strain
S/N code Clone# average stdev average
stdev AA substitutions
1 PHY0001 1447F 0.54 0.18 0.38 0.08 1447F
L29H / L136F /
2 PHY0018 G07 0.53 0.03 0.19 0.06 V341L
3 PHY0032 F01 0.49 0.09 0.31 0.03 F302/C
A66S / C184Y /
4 PHY0005 E01 0.44 0.02 0.30 0.04 D328E / L493*
R51Q / L164F /
PHY0006 D07 0.44 0.02 0.44 0.15 Y386F
6 PHY0007 D09 0.40 0.07 0.36 0.04 1287V / L441P
C286G / F302L /
7 PHY0008 1108 0.36 0.15 0.33 0.16 R388S
L224V / K322E /
8 PHY0009 D08 0.32 0.05 0.37 0.14 L422F

CA 2959039 2017-02-27
S33T /R51Q / L164F
9 PHY0010 A02 0.31 0.03 0.40 0.07 / Y386F
PHY0011 H05 0.29 0.01 0.36 0.06 FL136F / V341L
11 PHY0012 G08 0.29 0.02 0.40 0.07 G290S / 1314M
S32Y / V56I / E7OK /
S74F / L1361 /
12 PHY0013 CO3 0.28 0.01 0.33 0.05 N248Y / V486E
V52D / F3021/
13 PHY0014 B12 0.64 0.19 0.01 0.02 F308L / Y386F
14 PHY0015 G2 0.59 0.05 0.23 0.02 S112R / F302L
15 PHY0016 Gll 0.51 0.07 0.17 0.04 F473L
16 PHY0017 H06 0.51 0.07 0.27 0.10 N3431
17 PHY0002 B10 0.49 0.01 0.06 0.11 L441P
18 PI-1Y0019 CO1 0.47 0.08 0.28 0.01 S54T / F449L
19 PI-1Y0020 C07 0.46 0.02 0.04 0.02 L441P
A172G /N2481/
PHY0021 B01 0.36 0.11 0.26 0.02 K326N / L441P
V41L / All4D /
21 PHY0022 B08 0.35 0.02 0.09 0.12 K183R / K322E
22 PHY0023 Fl 1 0.32 0.00 0.01 0.02 L224F
T1ON /1143F / T2001
23 PHY0024 B09 0.30 0.04 0.16 0.02 / Y386F / M392K
24 PHY0025 H12 0.29 0.05 0.07 0.06 T421 / L193F / F473L
V1250 / M161K /
PHY0026 Bll 0.29 0.04 0.03 0.01 R409S / L441P
26 PHY0027 GO1 0.29 0.02 0.17 0.02 R51Q
27 PHY0028 D02 0.29 0.01 0.24 0.03 G332V
28 PHY0029 A07 0.29 0.04 0.30 0.05 V125F
29 PHY0030 A03 0.24 0.04 0.41 0.18 1143V
30 PHY0031 All 0.23 0.01 0.04 0.02 L441V
31 PHY0003 C04 0.12 0.02 0.19 0.02 E70G / L453M
32 PHY0033 A01 0.03 0.06 0.35 0.02 K322E / L4381
33 PHY0034 CO2 0.44 0.05 0.32 0.01 L438H
34 PHY0035 H01 0.67 0.02 0.36 0.02 N410K / L441P
PHY0036 H07 0.52 0.06 0.48 0.09 L136F / V341L
A1l4P/M199T/
C286Y / G332A /
L438H / L445V /
36 PHY0037 E07 0.47 0.03 0.51 0.07 F473L
37 PHY0038 H09 0.39 0.16 0.24 0.05 F4731
38 PHY0039 C08 0,33 0.08 0.23 0.02 F4731
39 PHY0040 1102 0.33 0.05 0.40 0.06 A46P / 1108T
PHY0041 D04 0.30 0.00 0.47 0.04 K11ON / L441P
31

CA 2959039 2017-02-27
Gl6S /D21G/
41 PHY0042 E08 029 0.03 0.40 0.04 R437H / F473L
42 PHY0043 F09 0.29 0.02 0.36 0.03 F449C / S501*
1(27R / E7OK / N81D
/L116I/K326Q /
43 PHY0044 E05 0.28 0.01 0.31 0.04 N458D / M4841
44 PHY0045 A10 0.27 0.02 0.19 0.03 M484L
45 PHY0046 F08 0.27 0.01 0.37 0.09 K289N
46 PI-1Y0047 C10 0.24 0.01 0.39 0.01 A107T
G35E / C286S /
47 PHY0048 E02 0.24 0.02 0.34 0.06 M484V
E82V / D100E /
1202V / M336V /
48 PHY0049 A04 0.20 0.01 0.42 0.04 F386Y / K467M
49 PHY0050 012 0.19 0.07 0.23 0.03 E7OV
V52I / E201V /
50 PHY0052 E10 0.18 0.01 0.19 0.01 S262T / Q450H
negative
control
(VEC) 0.12 0.03 0.01 0.01
native
BnDGAT-
PHY0051 1 (WT) 0.14 0.03 0.13 0.03
[00101] Example 2. Identification of 12 single site mutants that accumulate
higher oil in
yeast that are not naturally occurring in plants
[00102] To further explore the contribution of amino acid substitution in DGAT-
1 activity,
all 50 DGAT-1 variants were sequenced and the substitution positions were
compared. Totally
82 amino acid substitutions were identified (Table 1, Figure 2). Twenty-six of
the mutations
were the result of single site substitutions (Table 2). These mutations were
expressed in the
knock out yeast strain H1246, and the yeast strains were cultured to early
stationary phase for
oil analysis. As shown in Table 2, 19 single site mutants resulted in higher
oil accumulation,
32

,, ..
CA 2959039 2017-02-27
in which 12 substitutions do not occur naturally in plants, and are not
believed to have been
previously reported.
1001031 Table 2. Twenty-six amino acid sites selected for single site
mutagenesis. WT,
control yeast strain 1 hosting the native BnDGAT-1; VEC, control yeast strain
2 hosting an
empty yeast expression vector.
system name SSM s/n Amino acid deltaTAG/0D600 Unique?
substitution
PHY0016 29 F473L 2.16 yes
PHY0002 25 L441P 2.15 yes
PHYT0059 9 Al 14P 2.04 yes
PHYT0058 8 S112R 1.74 yes
P11YT0056 6 1108T 1.65 yes
PHYT0073 10 L136F ' 1.53
PHYT0065 17 F302C 1.51
PHYT0062 14 C286Y 1.47
PHY0001 27 I447F 1.45 yes
PHYT0064 16 F302L 1.43 yes
PHYT0070 28 F449L 1.43
PHYT0071 30 L493* 1.38
PHYT0067 19 G332A 1.26
PHYT0068 20 V341L 1.15
PHYT0069 22 Y386F 1.13 yes
PHYT0054 3 S54T 1.12 yes
PHY0017 21 N343I 1.11 yes
PHYT0074 11 L164F 1.08 yes
PHYT0072 4 A46P 1.08 yes
PHYT0051 32 WT 1.00
PHYT0061 13 M199T 0.99
PHYT0055 5 A66S 0.98
PHYT0057 7 Kl1ON 0.90
PHYT0066 18 D328E 0.83
33

CA 2959039 2017-02-27
PHY0027 2 R51Q 0.75
PHYT0063 15 1287V 0.75
P11Y0034 24 L438H 0.74
PHYT0076 26 L445V 0.64
PHYT0053 31 VEC 0.00
[00104] Example 3. Detailed Characterization of selected single site mutations

[00105] Four single site mutants, include three boosting oil content (SSM s/n
17, 25, 27) and
one down-regulating TAG biosynthesis in yeast (SSM s/n 15), were selected for
further
characterization. Yeast strains with an empty vector (s/n 31) and native
BnDGAT-1 (s/n 32),
respectively, were used as controls. Yeast strains were cultured in 200 ml of
liquid medium in
500 ml flasks for a better growth condition. As shown in Figure 3, all strains
showed a similar
growth curve, except that SSM25 has a slightly slower growth rate. SSM 17, 25,
and 27 # had
higher TAG content than native DGAT-1 at all time points, whereas SSM15# had
lower TAG
content (Figure 4), which is consistent with the data in Table 2. Furthermore,
cells were
harvested at 52 hours for lipid analysis by GC/MS. As shown in Figure 5, the
results were
consistent with Nile red assay.
[00106] Yeast cells were also harvested at different OD values for
measurements of gene
expression, protein expression and enzyme activity of the DGAT-1 variants. As
shown in
Figures 6 and 7, all mutants and the native DGAT-1 (31#) had very high
transcript levels in
yeast at all measured time points, as well as high protein levels.
34

CA 2959039 2017-02-27
[00107] As shown in Figure 8, calculation of specific enzyme activity of
mutants SSM 17,
25, and 27# gives a higher activity than native DGAT-1 in general, whereas SSM
15# is
lower.
[00108] Example 4¨ Expression in N. benthamiana
[00109] DGAT1 mutants were tested in a N. benthamiana system (Vanhercke 2013),
All
genes were subcloncd first in a 35S vector backbone. Wildtype and mutant
DGATls were
cloned together with Arabidopsis WRI1 to maximize TAG response. Arab
WRI1+AtDGAT1
combination was also included as a control. Due to smaller leaf sizes, the
infiltration
experiment was divided in two parts. Comparisons between the two infiltration
groups should
be treated may be considered indicative only.
[00110] At least three mutants do result in increased TAG levels in leaf
tissue compared to
the wildtype BnDGAT1 controls. Two single mutants (1447F and L441P) gave the
highest
TAG yields and outperform the AtDGAT1 control.
[00111] The results are shown in Figure 9 and Tables 3and 4 below.

'
,
Table 3
!-
L ,Samples. ___ . , ,
1C1401C1601161*C161r116:3 1180 :C18:1 1C1810 C18:2 ---1-
61834C20:01C201-/C2- 0:24C20:34C220C2401mg/100 mg DW -1
,
r8D0 P19 ad zasi 031 a; 3.9 4.81E 3.01 0.5-1-1i;L:::i1...A.
1&8[. 37.7 0.91 r 0.21 0.11 0.1' 0.3; all o.o81 aTii--- P19+ AtDGAT1+
AtWRI ad 23.71 at az! Q21 5.31.1111 KE.--11 -- 0.4 7.t.,,a741 4, 14 I
.3 tal air-o 2--1 ! 01 td 0-61.. .---
. - ' '
: - - vAtor.181
,
r802 P19+AtWRI+pOIL346 0.1 2331 al a3i azi 4.6';
:-__:-,f_ill'il__ as] ,...i.61 14.2 L8, Q2; az at 1.z a7t_ _
_ _7:049_1
53". P19+AtWRI+p0IL347 ad 24.91 ciii- 0-31 0.2:1, 4.8, ' 17-2.M.1
0.51 1-1_1111119-211 15.1 171 0.21, 0.21 01 11 0 61
8
111/711171
1.,--- -1 1-- - õ
_____________________
04 P19+AtWRI+pOIL348
0.011 24.7 at az 0.24.611111111.M.91 as]1111MgilliK-1.71 14.4 161
02102.az zo[ 0.61 ,... 231
'805 P19 031 2.5.81 03! 03; 0.41 6.7 igi 1.09
05 -23 22.7 2.01. 021 0.31 at 11 asil 0.06!
'8-06 P19+ AtDGAT1+ AtWRI 0.01 24.21 al 0.3 6.2' - 4.4'
:-YI4T.9 a6! .2/51 3i 14.5 1.91 0 2; 0 i 0 1 - 1.4! 0 51-
-111.rit'-1201
r8-0/1- P19+ALWRI+pOIL346 al 23--3.f- 0.21 - 0.2r 0.3.1. 4.-
811111.14E93.31 oil.7:.:MiS 5 14.61 1.81- O2 ai ail 1.14111111
: Qt. -- 1.92
-1- : -4
= .
ii-- P19+AtWRI+p011_347
00 23.41 0.11 021 0.21 4.8',1111ffilW5-.2 0.41 _1.7 ' 1 '3 14.81 LEI az a;
at 121 a6-111111 1.71,
.,_ .
610'.9 P19+AtWRI+pOIL348 ad, 23.51 tiara* a3i 421
_Oil .1T 0.51 __ L 7_5_3-casi 15.01 1.7i az 0.21 at tz 0-.7
till
r810 P19 0.31 26.31 0.31 0.31 0.41 7.6 -311 931 _ 0.4
_______________________ i 21.111111 3..81_ 0..9_1_ 0.6j _ 0.06!
:
-
ri-1.1 P19+ AtDGAT1+ AtWR1 0.0 24.8 0.21 4. aij 4.5.õ
_ .0 as _754,iiign"21 3.4.71 tZ 0-.2j0.2 01 10, aO
r5-1-2-- P19+AtWRI+p0IL346 QO _24.94_ Oil 0.3.1___
0.21.._ 431 _ .. __Lig-al..._ 06 ...,_ _ .... ' 1,;=-= ___...2_ 14.91 1.81-
a; _ az_ad_ 1.21 aiLM11111000t-
;813 P19+AtWRI+p0IL347
0.01 24.0 0.2 03; 0.2; 4.81111111W1-.6 0.51.9.5 14.611 17 0.2 0.2
AL 101 0.51 _ -4c,--61 1.88 .
u,
1'81- 4 P19+AtWRI+pOIL348
01] 23.4 az 031 azi, 4.31- - . 7.J.:-...i. -1-11-. ---.1
0.51 -- - -. .i...1.,,Y&IS =.9 14.81 17 ----02 0.-211 01 1.0,.-06i---- 7:- 1---
::iiiii----ii-o- u,
i 1 I :
, !
i- -.4.-----; ! --i ________ .
,.,
w
'Sample 1C14:01C16:0116:1J C16:11163 118:0
1618:1 1C18:1d1C18:2 IC18:3iC20:01C20:11C20:24,C20:1,C2201C2401mg/100 mg
OW
,
. ,. c;
1-;
r815 P19 031 24.7 as, 0.31 0.41 7.21 " YENII 3Ø6! 04 1111-181 22.8!
1.9 al a31 4 av, 0.51 006] ...]
--1 1
r816 P19+ AtDGAT1+ AtWR1 ao M.& 0.21 0.3 031 4.71W. - T 10.7;-- 0.5
111111M111:77--.61 14.4 17 0.2! 0.2, 0.11 1.11 0.5 : 4 ..-11!,.:111A111111
1_67!
1
riiii P19+AtWRI+pOIL372
0.1 20.70.1_ 0.2 031 443 31 (16111-; Z.5j iaz 2.11 03! aZ
0.3.1 1.81 0.8 2.141
..,
r818 P19+AtWRI+pOIL373 0-151 20.6 0.11 0.2, 0.41 4Ø_
...r.1;Y:13_,81 0.5111.71 14.1 2.21 0.31 0.21 0.1; 1.91 0.6,_
819
1 819 P19 011 24.1 0.41 03i 0.41 7.04 111r:11.11111,_L 7/1 Q41E EF7--
'1w.. - 212.2' 25.41 1.61 Q21 Q31 0.21 Q8I 0.41 _ 0.05
9+
1
---- 1 ' 1 -1- 1.--
1-- t
111-20 P1 AtDGAT1+ AtWRI ao 22.7, 0...1_ 031 Q21
4.51 :.:'''...F; 111.3i 0.5 .1 22.7 13.61 1.71 0.2 0_Z 0.1 LO 0_1
.5_ i'1'-1 -
1821 P19+AtWRI+p011_372
0.1 2Q4 021 021 a31-__ 4.4iffifE077-481 0.61101ffigiVW---1,-;
Ire; 12.21 2.1 0.3 a; 0.1; 17, asIIENIMIEF-7-:-... 1.w!
i.- , 1
1822 P19+ALWRI+pOIL373
0.0 21.2 0.1 -0.21 0.41 4.21 .11:11)&4116 = - as1,.:-
;;Siiall,:', .:,, 24 1 14.21 2.2, 0.31 0.21 0.1 1.91 0.6111111bri1t11111'11'
21531
r823 P19 0.01 23.41 041 03I 0.41 6.81:::14
4.9 0.411///fUE-7171-7Th0.61 28.11 131 0.24.1_0.3i 0.211
0.7 0311 . 0.031
r---
-=
1824 P19+ AtDGAT1+ A1WRI ao 22.81 0.1 Q3; az 4.4*,-,..4:,- i2.6 06
.' -&-Ti izsl 161 az! az at 0.9 ii?,--0=ffi=3 1.81
r- -I
1---
:825 P19+AtWRI+pOIL372
00 219 al 034_! a34_ 4.41PW,Tio 0.61111111k125.7.1_ 12.1 2.1 03 o.z
QV 16 0.711.2C 2..2, 91
11.3-26 P19+AIWRI+p0I1373 0.0 20.9 0.1; 0.21 0.41 --4-14---
1111 as.L,,'Y s 13.91 2.2 0.3 0.21 0.11 L . 0.61111=e4i; '1-'1
i..2-01
_ _ _
.
, .
= ,
._
36

Table 4 .
AVERAGE 1 1 ,C14:0,C16:0116:1WC16:1
. õ . 1163 118:0 :C18:1 C18:11 C18:2iC18:3I
C20:0I C20:1IC20:21C20:3n3 1C22.-0 1C240 . i 1 1 i 1 ',TAG (% leaf
OW)
P19 ! J..._ 02' 24.21 (13, 0.31
1.61 6.4 7.7 0.51 2L7r 27.1 161 0.2 0.2!
..,..
al.! I
0.8.
' ,
aa i
= . , , al
-- :
,....___J--........i....4.4 4-___________
P19+AtWR11+A(DGAT1 i .1. : 0.0242 0.11 0.3
0.21 4.71 150 0.5/- ! 21514.51 11 0.2 0.21 0.1i 1.1, 0.6. i i
I = i . 2.2
P19+AtVIIR11+13nDGAT1 (G70E+L453M) : ao, 23.34 al! 0.31
0.21 4.6, 15.3Ø5, 20.81 14.6i 1.8 0.2 0.1 0.11r ill 03 .
, ii 1 1 2.2
, i ,
P19+AtWRIl+BnDGAT11 . ' ! (10, 24 1' af 03
azt 48147 0.51 19,4. 14.8.4.,...___}! 171 azt az; 0.1i
Li
' -t----i- 1 ---"----"t- ! -- .....
1. , 1.- --f-ri-hr .--i.------- ---
P19+AtiNR11+HIS-BnDGAT1 : 1 . 002 3.9! at. 03
az 4.41 117. asi zaz1 147! 171 0.21 az! 01! .1. 064
. 7"-----i' .
1 - i 1-1-1-- -
_____________________________________________________________________ "
1
.
=-t--",--'....4...q...1.4.....4.______ _
P19 --1--. --'4-
0.1' 24.1r 0.4
--.. a3 0.4!
----t---- i 7.01.. 73 OA 211.. 25.41_ L61 0.4. 0,31 0.2!

: 0.81-- --,-
P19+AtWRI1+AtDGAT1 i L_ i .... 0.0: 22.81 0.11 _________ 031
0.21 4.6 115, 052 2.6 i36,_161 0.1 0.21 1.01
......._
L7
P19+AlWRI1+13nDGAT1 (I447F) 1.... r 0.0 210, al! 0._431
0.3 44 14.7 0.61 25 3. 17-51 2.11 0.31 0.21

. . , ,
00..1114 0-5_,'....i_f_li

1.71_
a8: 1_1.4 , . , 2.1
P19+AtWRI1+BnDGAT1 (L441P) 1 T aa 20.91 all azi 0.4
4.3: 15.91 as! 24.L 14.1l 2.21 0.3f 0.2! 0.11 191
0.6 i 1 1 ! ! ,
2.4
: ' I = ' ' F 1 '
f i I . i I ' I i
- ' I 1'- .
-...e-
' -1- - 1 . , -1"-
1 L i
! _________ i i .. i 1 1
,
STDEV 1 1 I ! C140 , C16:0116:1 vil Ct 61
163 1180 1C18:1 C18:14C18:21C18:31C2001C20:11C20:2iC20:3n3
I 020 10240 I 1 1 1 1 : ,TAG (% leaf OW)
P19 4.. _4' 1, Loz 3.31 aol aol
2.0! 1.4! 42! 0.0! 21:: 9.1l 061 col al aol az. a4.1 . 1
I
I- . . :
'
I '
,
, . , , ao P
P19+AtWRIl+AtDGAT1 1 I l
tRI . - 0.0, 0.6 0.01 ao; ao _______________ o_si
0.1 at. as azj al o.o, ao ao, 0.21 0.1' :
0.1 I _ "
P19+AW1+BnDGAT1(G70E+L453M)
Iv
: 0.0: 0.91 0.0 0.0 0.0
0.2 2.1 0.1! 0 71 0.4' 0.0 0.01 0.01 0.01 0.11 0.0' ..i..44.4
Li_ . _ 0.3
r}-= , . .
, (T.,
P19+AtWRI1+13nDGAT1 ! 1 ! 0.0: 0.81 0.0 0.0
0.0 0.0 16 0.0!0.2' 0.3l 0.0 o.ol o.ol o.o o.i.: 0.0- ! 1
i 1 : _ 03, ..
. ' -,----t- . . = -_.
P-19+AtWRI1+1-11S-BnDGAT1 ' 1 1 0.0 07 1 aot . ao
001 az, 11 _ 0.0, as, 0.41 0.0 0.0 o.o! 00! all al , i kr-I
as ..
i - --",- , T . .
I I -4-, . 1-1-=
:
" tRI . i-- - -tI-- , , -
0.,1
-
..jI7. .I1.711._....-...________. I.
P19 1 alØ- 0.0 0.01 280
162.70.30.010.01 0.01 0.1 0.0
P19+A00211+AtDGAT1 1 0.0 aoao! 0.0 i
al! 1-0.0 al1 o.s1 o.i ao, o.ol al ao, 0.1
I 0---1 4;
P19+AW1+BnOGAT1(447F 0.0 as 0.0! co 00 00 a61.
0 o al_ao 02 .-1..v..1'
....!
P19+AtWRIl+BnDGAT1 (L441P) 1 -1- ma as! aol ao co;
as, as ao as, 021 0.0' ao 001 o.o. ail ac T 1 I I, , . az
:
. . . .
. .
37
-
--.

CA 2959039 2017-02-27
Definitions and Interpretation
[00112] The description of the present invention has been presented for
purposes of
illustration and description, but it is not intended to be exhaustive or
limited to the invention
in the form disclosed. Many modifications and variations will be apparent to
those of ordinary
skill in the art without departing from the scope and spirit of the invention.
Embodiments
were chosen and described in order to best explain the principles of the
invention and the
practical application, and to enable others of ordinary skill in the art to
understand the
invention for various embodiments with various modifications as are suited to
the particular
use contemplated.
[00113] The corresponding structures, materials, acts, and equivalents of all
means or steps
plus function elements in the claims appended to this specification are
intended to include any
structure, material, or act for performing the function in combination with
other claimed
elements as specifically claimed.
[00114] References in the specification to "one embodiment", "an embodiment",
etc., indicate
that the embodiment described may include a particular aspect, feature,
structure, or
characteristic, but not every embodiment necessarily includes that aspect,
feature, structure, or
characteristic. Moreover, such phrases may, but do not necessarily, refer to
the same
embodiment referred to in other portions of the specification. Further, when a
particular
aspect, feature, structure, or characteristic is described in connection with
an embodiment, it is
within the knowledge of one skilled in the art to affect or connect such
aspect, feature,
structure, or characteristic with other embodiments, whether or not explicitly
described. In
38

CA 2959039 2017-02-27
other words, any element or feature may be combined with any other element or
feature in
different embodiments, unless there is an obvious or inherent incompatibility
between the
two, or it is specifically excluded.
[00115] It is further noted that the claims may be drafted to exclude any
optional element. As
such, this statement is intended to serve as antecedent basis for the use of
exclusive
terminology, such as "solely," "only," and the like, in connection with the
recitation of claim
elements or use of a "negative" limitation. The terms "preferably,"
"preferred," "prefer,"
"optionally," "may," and similar terms are used to indicate that an item,
condition or step
being referred to is an optional (not required) feature of the invention.
[00116] The singular forms "a," "an," and "the" include the plural reference
unless the
context clearly dictates otherwise. The term "and/or" means any one of the
items, any
combination of the items, or all of the items with which this term is
associated.
[00117] The term "and/or" means any one of the items, any combination of the
items, or all
of the items with which this term is associated. The phrase "one or more" is
readily
understood by one of skill in the art, particularly when read in context of
its usage.
[00118] As will be understood by the skilled artisan, all numbers, including
those expressing
quantities of reagents or ingredients, properties such as molecular weight,
reaction conditions,
and so forth, are approximations and are understood as being optionally
modified in all
instances by the term "about." These values can vary depending upon the
desired properties
sought to be obtained by those skilled in the art utilizing the teachings of
the descriptions
herein. It is also understood that such values inherently contain variability
necessarily
resulting from the standard deviations found in their respective testing
measurements.
39

CA 2959039 2017-02-27
[00119] The term "about" can refer to a variation of 5%, 10%, 20%, or
25% of the
value specified. For example, "about 50" percent can in some embodiments carry
a variation
from 45 to 55 percent. For integer ranges, the term "about" can include one or
two integers
greater than and/or less than a recited integer at each end of the range.
Unless indicated
otherwise herein, the term "about" is intended to include values and ranges
proximate to the
recited range that are equivalent in terms of the functionality of the
composition, or the
embodiment.
[00120] As will be understood by one skilled in the art, for any and all
purposes, particularly
in terms of providing a written description, all ranges recited herein also
encompass any and
all possible sub-ranges and combinations of sub-ranges thereof, as well as the
individual
values making up the range, particularly integer values. A recited range
(e.g., weight percents
or carbon groups) includes each specific value, integer, decimal, or identity
within the range.
Any listed range can be easily recognized as sufficiently describing and
enabling the same
range being broken down into at least equal halves, thirds, quarters, fifths,
or tenths. As a
non-limiting example, each range discussed herein can be readily broken down
into a lower
third, middle third and upper third, etc.
[00121] As will also be understood by one skilled in the art, all language
such as "up to", "at
least", "greater than", "less than", "more than", "or more", and the like,
include the number
recited and such terms refer to ranges that can be subsequently broken down
into sub-ranges
as discussed above. In the same manner, all ratios recited herein also include
all sub-ratios
falling within the broader ratio. Accordingly, specific values recited for
radicals, substituents,

CA 2959039 2017-02-27
and ranges, are for illustration only; they do not exclude other defined
values or other values
within defined ranges for radicals and substituents.
[001221 One skilled in the art will also readily recognize that where members
are grouped
together in a common manner, such as in a Markush group, the invention
encompasses not
only the entire group listed as a whole, but each member of the group
individually and all
possible subgroups of the main group. Additionally, for all purposes, the
invention
encompasses not only the main group, but also the main group absent one or
more of the
group members. The invention therefore envisages the explicit exclusion of any
one or more
of members of a recited group. Accordingly, provisos may apply to any of the
disclosed
categories or embodiments whereby any one or more of the recited elements,
species, or
embodiments, may be excluded from such categories or embodiments, for example,
as used in
an explicit negative limitation.
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