CA2959039A1 - Plant dgat-1 variants - Google Patents

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.

Description

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).

[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.

[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 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.

[0016] Figure 2. Eighty-two amino acid substitutions were identified from the 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).

[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.

[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 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"

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 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 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.

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 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 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 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.) [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 "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 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", _ 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 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 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 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 [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).

[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 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 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), [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 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.

[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 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

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

4 PHY0005 E01 0.44 0.02 0.30 0.04 D328E / L493*

PHY0006 D07 0.44 0.02 0.44 0.15 Y386F
6 PHY0007 D09 0.40 0.07 0.36 0.04 1287V / L441P

7 PHY0008 1108 0.36 0.15 0.33 0.16 R388S

8 PHY0009 D08 0.32 0.05 0.37 0.14 L422F

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

12 PHY0013 CO3 0.28 0.01 0.33 0.05 N248Y / V486E

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

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

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

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/

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

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

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

47 PHY0048 E02 0.24 0.02 0.34 0.06 M484V

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

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, ,, ..

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 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.

[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 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] ...]

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!

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,_ 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-- 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' 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

: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 _ _ _ .
, .
= , ._ 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-- -_____________________________________________________________________ "

.
=-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 ......._ 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 :
. . . .
. .

---.

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 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.

[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, 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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Claims (12)

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.

.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.

.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.

.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.

.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.

.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 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 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.