# Patent

## Title
Diacylglycerol acyltransferases from flax

## Abstract

The invention relates to isolated diacylglycerol acyltransferases and polynucleotide sequences encoding the DGAT enzymes; polynucleotide constructs, vectors and host cells incorporating the polynucleotide sequences; and methods of producing and using same. Also provided are transformed cells and transgenic plants, especially Camelina sativa plant, with enhanced oil accumulation and quality.

## Background
PRIORITY
This application claims priority of U.S. provisional application No. 61/034,787 filed on Mar. 7, 2008


FIELD OF THE INVENTION
The present invention relates to isolated diacylglycerol acyltransferases and polynucleotide sequences encoding the DGAT enzymes; polynucleotide constructs, vectors and host cells incorporating the polynucleotide sequences; and methods of producing and using same.
BACKGROUND
Oils obtained from plant seeds are important sources of fatty acids for human consumption and for use as chemical feedstocks. These fatty acids include essential fatty acids, saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids. In plant seed oils, fatty acids are stored predominantly as triacylglycerols (TAGs). TAGs represent the most efficient form of stored energy in eukaryotic cells.
TAG biosynthesis occurs mainly in the endoplasmic reticulum (ER) of the cell using acyl-CoA and sn-glycerol-3-phosphate as primary substrates. Biosynthesis of TAG is effected through a biochemical process generally known as the Kennedy pathway (Kennedy, 1961) which involves the sequential transfer of fatty acids from acyl-CoAs to the glycerol backbone (acyl-CoA-dependent acylation). The pathway starts with the acylation of sn-glycerol-3-phosphate to form lysophosphatidic acid through the action of sn-glycerol-3-phosphate acyltransferase. The second acylation is catalyzed by lysophosphatidic acid acyltransferase, leading to the formation of phosphatidic acid which is dephosphorylated by phosphatidate phosphatase1 to form sn-1,2-diacylglycerol. The final acylation is catalyzed by diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) to form TAG. The DGAT enzyme catalyzes the transference of the acyl group from acyl-coenzymeA (acyl-CoA) donor to a sn-1,2-diacylglycerol, producing CoA and TAG. Previous research results suggest that the level of DGAT activity may have a substantial effect in the flow of carbon into seed oil (Ichihara and Noda, 1988; Perry and Harwood, 1993; Stobart et al., 1986; Settlage et al., 1998).
Two types of DGAT (DGAT1 and DGAT2) have been identified in animals and plants (Cases et al., 2001; Hobbs et al., 1999; Lardizabal et al., 2001; Kroon et al., 2006; Shockey et al., 2006). DGAT1 has been most studied and displays broad substrate specificity. DGAT1 null mutants in plants and animals have been shown to have substantially reduced levels of TAG (Routaboul et al., 1999; Smith et al., 2000). Furthermore, over-expression of DGAT1 in seeds of Arabidopsis thaliana results in increased seed weight and oil content (Jako et al., 2001). These results suggest that DGAT1 is the predominant type, although some studies indicate that DGAT2 might be more important for TAG biosynthesis in plants like castor bean (Kroon et al., 2006).
Flax is an oilseed that substantially accumulates α-linolenic acid (α-18:3) which is an omega-3 fatty acid. Other omega-3 fatty acids include eicosapentaenoic acid (EPA) and docosahexaneoic acid (DHA) which produce beneficial health effects in humans (Simopoulos, 2002). Flaxseed oil displays chemical attributes which are advantageous for industrial applications including, for example, the production of linoleum, preservation of concrete and as an ingredient in paints and varnishes. The enzymatic activity of DGAT has been studied in isolated ER of flax developing seeds (Sorensen et al., 2005). DGAT is able to incorporate polyunsaturated fatty acids (C18:3 n-3) at higher rates compared to monounsaturated (C18:1) fatty acids. In addition, flax microsomes incorporate EPA and DHA into TAGs (Sorensen et al., 2005), highlighting the usefulness of TAG biosynthetic enzymes such as DGAT as genetic tools for engineering vegetable oils. Over-expression of DGAT in oilseed plants could potentially increase TAG production or enhance seed oil content in plants. However, since numerous enzymatic activities occur within microsomes, it is difficult to evaluate the effect of DGAT in flax using a microsome-based system. Genetically modified organisms have not achieved widespread public acceptance; however, use of native flax DGAT genes for improving the oil content through biotechnology may more readily meet stringent controls.
SUMMARY OF THE INVENTION
The present invention relates to isolated diacylglycerol acyltransferases and polynucleotide sequences encoding the DGAT enzymes; nucleic acid constructs, vectors and host cells incorporating the polynucleotide sequences; and methods of producing and using same.
In one aspect, the invention provides an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from:
at least 300, at least 400 or at least 500 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 2 or of an amino acid sequence having at least 85% sequence identity therewith;
at least 300 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 4 or of an amino acid sequence having at least 85% sequence identity therewith; or
at least 300 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 6 or of an amino acid sequence having at least 85% sequence identity therewith.
In one embodiment, the invention provides an isolated polynucleotide, wherein the encoded polypeptide comprises the amino acid sequence depicted in SEQ ID NO: 2.
In one embodiment, the encoded polynucleotide comprises the nucleotide sequence depicted in SEQ ID NO: 1 from nucleotide 57 to nucleotide 1580.
In one embodiment, the encoded polypeptide comprises the amino acid sequence depicted in SEQ ID NO: 4.
In one embodiment, the polynucleotide comprises the nucleotide sequence depicted in SEQ ID NO: 3 from nucleotide 1 to nucleotide 1029.
In one embodiment, the encoded polypeptide comprises the amino acid sequence depicted in SEQ ID NO: 6.
In one embodiment, the polynucleotide comprises the nucleotide sequence depicted in SEQ ID NO. 5 from nucleotide 1 to nucleotide 1048.
In one embodiment, the encoded polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2.
In one embodiment, the encoded polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4.
In one embodiment, the encoded polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6.
In a further aspect, the invention provides a polynucleotide construct comprising any of the above polynucleotides operably linked to a promoter expressible in bacterial, yeast, fungal, mammalian or plant cells.
In a further aspect, the invention provides a vector comprising any of the above polynucleotides. In one embodiment, the invention provides a microbial cell comprising any of the above polynucleotides. In one embodiment, the microbial cell is Saccharomyces cerevisiae. 
In a further aspect, the invention provides a transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore, comprising any of the above polynucleotides. In one embodiment, the transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore is selected from a flax, canola, soybean, Camelina sativa, mouse-ear cress, castor, sunflower, linola, oat, wheat, triticale, barley, corn or Brachypodium distachyon plant, plant cell, plant seed, plant embryo, or microspore.
In another aspect, the invention provides a method for producing an oil, comprising the steps of growing the above transgenic plant and recovering oil which is produced by the plant. In one embodiment, the plant is selected from a flax, canola, soybean, Camelina sativa, mouse-ear cress, castor, sunflower, linola, oat, wheat, triticale, barley, corn or Brachypodium distachyon plant.
In yet another aspect, the invention provides a method for producing a transgenic plant comprising the steps of introducing into a plant cell or a plant tissue any of the above polynucleotides to produce a transformed cell or plant tissue, and cultivating the transformed plant cell or transformed plant tissue to produce the transgenic plant. In one embodiment, the plant is selected from a flax, canola, soybean, Camelina sativa, mouse-ear cress, castor, sunflower, linola, oat, wheat, triticale, barley, corn or Brachypodium distachyon plant.
Additional aspects and advantages of the present invention will be apparent in view of the description, which follows. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in which:
FIGS. 1A, 1B and 1C show a contig of LuDGAT1 obtained by assembling six isolated fragments (amplicon1 (SEQ ID NO: 28), amplicon2 (SEQ ID NO:29), RT-PCR (SEQ ID NO:30), 3′ RACEm (SEQ ID NO:33), 5′ RACE (SEQ ID NO:31) and 5′ RACEB (SEQ ID NO:32)).
FIG. 2 is a schematic drawing of the LuDGAT1 cDNA contig. The fragments obtained by PCR are represented by the rectangles. The 5′ and 3′ untranslated regions are designated by lines. The annealing position and orientation of the oligonucleotides are described on the top.
FIG. 3 shows the cDNA sequence of LuDGAT1 (SEQ ID NO:1) and the predicted polypeptide sequence (SEQ ID NO:2).
FIGS. 4A, 4B, 4C and 4D show an amino acid alignment of plant DGAT1 with the accession numbers and species indicated for each sequence, black highlight indicating identical residues and grey highlight indicating blocks of conserved residues.
FIG. 5 shows a phylogenetic tree of plant DGAT1 with the accession numbers indicated for each plant species and oilseed members of the Cruciferae family highlighted in grey.
FIG. 6 shows a hydropathy plot of LuDGAT1 polypeptide using the Kyte and Doolittle scale (Kyte and Doolittle, 1982).
FIG. 7 is a graph showing the specific activity of type-1 DGAT in microsomes from yeast expressing plant type-1 DGAT.
FIG. 8 is a schematic illustration of PCR-amplification of Brassica napus nap-A-promoter P-Bn-napA and Brassica napus napA-terminatorT-Bn-napA.
FIG. 9 illustrates construction of pGem7-P-Bn-napA construct.
FIG. 10 illustrates construction of pGem7-PT-Bn-napA construct.
FIG. 11 illustrates construction of pGem-PT-Bn-napA-LuDFGAT construct.
FIG. 12 illustrates construction of pC0300-PT-Bn-napA-LuDGAT construct.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
As will be apparent to those skilled in the art, various modifications, adaptations and variations of the foregoing specific disclosure can be made without departing from the scope of the invention claimed herein. The various features and elements of the described invention may be combined in a manner different from the combinations described or claimed herein, without departing from the scope of the invention.
To facilitate understanding of the invention, the following definitions are provided.
“Isolated” means that a substance or a group of substances is removed from the coexisting materials of its natural state.
A “polynucleotide” is a linear sequence of ribonucleotides (RNA) or deoxyribonucleotides (DNA) in which the 3′ carbon of the pentose sugar of one nucleotide is linked to the 5′ carbon of the pentose sugar of another nucleotide. The deoxyribonucleotide bases are abbreviated as “A” deoxyadenine; “C” deoxycytidine; “G” deoxyguanine; “T” deoxythymidine; “I” deoxyinosine. Some oligonucleotides described herein are produced synthetically and contain different deoxyribonucleotides occupying the same position in the sequence. The blends of deoxyribonucleotides are abbreviated as “W” A or T; “Y” C or T; “H” A, C or T; “K” G or T; “D” A, G or T; “B” C, G or T; “N” A, C, G or T.
A “polypeptide” is a linear sequence of amino acids linked by peptide bonds. The amino acids are abbreviated as “A” alanine; “R” arginine; “N” asparagine; “D” aspartic acid; “C” cysteine; “Q” glutamine; “E” glutamic acid; “G” glycine; “H” histidine; “I” isoleucine; “L” leucine; “K” lysine; “M” methionine; “F” phenylalanine; “P” proline; “S” serine; “T” threonine; “W” tryptophan; “Y” tyrosine and “V” valine.
“Downstream” means on the 3′ side of a polynucleotide while “upstream” means on the 5′ side of a polynucleotide.
“Expression” refers to the transcription of a gene into RNA (rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into a protein.
A “promoter” is a polynucleotide usually located within 20 to 5000 nucleotides upstream of the initiation of translation site of a gene. The “promoter” determines the first step of expression by providing a binding site to DNA polymerase to initiate the transcription of a gene. The promoter is said to be “inducible” when the initiation of transcription occurs only when a specific agent or chemical substance is presented to the cell. For instance, the GAL “promoter” from yeast is “inducible by galactose,” meaning that this GAL promoter allows initiation of transcription and subsequent expression only when galactose is presented to yeast cells.
A “coding sequence” or “coding region” or “open reading frame (ORF)” is part of a gene that codes for an amino acid sequence of a polypeptide.
A “complementary sequence” is a sequence of nucleotides which forms a duplex with another sequence of nucleotides according to Watson-Crick base pairing rules where “A” pairs with “T” and “C” pairs with “G.” For example, for the polynucleotide 5′-AATGCCTA-3′ the complementary sequence is 5′-TAGGCATT-3′.
A “cDNA” is a polynucleotide which is complementary to a molecule of messenger RNA mRNA. The “cDNA” is formed of a coding sequence flanked by 5′ and 3′ untranslated sequences.
“DGAT” is an enzyme of the class EC 2.3.1.20 which catalyzes the reaction: acyl-CoA+sn-1,2-diacylglycerol→CoA+triacylglycerol. Alternative names include: diacylglycerol O-acyltransferase, diacylglycerol acyltransferase, diglyceride acyltransferase and acylCoA:diacylglycerol acyltransferase.
A polypeptide having “DGAT activity” is a polypeptide that has, to a greater or lesser degree, the enzymatic activity of DGAT.
A “recombinant” polynucleotide is a novel polynucleotide sequence formed in vitro through the ligation of two DNA molecules.
A “construct” is a polynucleotide which is formed by polynucleotide segments isolated from a naturally occurring gene or which is chemically synthesized. The “construct” which is combined in a manner that otherwise would not exist in nature, is usually made to achieve certain purposes. For instance, the coding region from “gene A” can be combined with an inducible promoter from “gene B” so the expression of the recombinant construct can be induced.
“Transformation” means the directed modification of the genome of a cell by external application of a polynucleotide, for instance, a construct. The inserted polynucleotide may or may not integrate with the host cell chromosome. For example, in bacteria, the inserted polynucleotide usually does not integrate with the bacterial genome and might replicate autonomously. In plants, the inserted polynucleotide integrates with the plant chromosome and replicates together with the plant chromatin.
A “transgenic” organism is the organism that was transformed with an external polynucleotide. The “transgenic” organism encompasses all descendants, hybrids and crosses thereof, whether reproduced sexually or asexually and which continue to harbor the foreign polynucleotide.
A “vector” is a polynucleotide that is able to replicate autonomously in a host cell and is able to accept other polynucleotides. For autonomous replication, the vector contains an “origin of replication.” The vector may contain a “selectable marker” that confers the host cell resistance to certain environment and growth conditions. For instance, a vector that is used to transform bacteria usually contains a certain antibiotic “selectable marker” which confers the transformed bacteria resistance to such antibiotic.
Two polynucleotides or polypeptides are “identical” if the sequence of nucleotides or amino acids, respectively, in the two sequences is the same when aligned for maximum correspondence as described here. Sequence comparisons between two or more polynucleotides or polypeptides can be generally performed by comparing portions of the two sequences over a comparison window which can be from about 20 to about 200 nucleotides or amino acids, or more. The “percentage of sequence identity” may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of a polynucleotide or a polypeptide sequence may include additions (i.e., insertions) or deletions (i.e., gaps) as compared to the reference sequence. The percentage is calculated by determining the positions at which identical nucleotides or identical amino acids are present, dividing by the number of positions in the window and multiplying the result by 100 to yield the percentage of sequence identity. Polynucleotide and polypeptide sequence alignment may be performed by implementing specialized algorithms or by inspection. Examples of sequence comparison and multiple sequence alignment algorithms are: BLAST and ClustalW softwares. Identity between nucleotide sequences can also be determined by DNA hybridization analysis, wherein the stability of the double-stranded DNA hybrid is dependent on the extent of base pairing that occurs. Conditions of high temperature and/or low salt content reduce the stability of the hybrid, and can be varied to prevent annealing of sequences having less than a selected degree of homology. Hybridization methods are described in Ausubel et al. (1995).
The invention provides isolated DGAT1 and DGAT2 polynucleotides and polypeptides. DGAT1 and DGAT2 polynucleotides include, without limitation (1) single- or double-stranded DNA, such as cDNA or genomic DNA including sense and antisense strands; and (2) RNA, such as mRNA. DGAT1 and DGAT2 polynucleotides include at least a coding sequence which codes for the amino acid sequence of the specified DGAT polypeptide, but may also include 5′ and 3′ untranslated regions and transcriptional regulatory elements such as promoters and enhancers found upstream or downstream from the transcribed region.
In one embodiment, the invention provides a DGAT1 polynucleotide which is a cDNA comprising the nucleotide sequence depicted in SEQ ID NO: 1, and which was isolated from Linum usitatissimum. The cDNA is 1778 base pairs in length including a coding region of 1524 base pairs (SEQ ID NO: 1 from nucleotide 57 to nucleotide 1580) and untranslated 5′ and 3′ regions of 56 and 198 base pairs, respectively. The DGAT1 encoded by the coding region (designated as LuDGAT1, SEQ ID NO: 2) is a 507 amino acid polypeptide with a predicted molecular weight of 58,012 Daltons and an isoelectric point of 8.74.
In one embodiment, the invention provides a DGAT2 polynucleotide which is a coding region comprising the nucleotide sequence depicted in SEQ ID NO: 3, which was also isolated from Linum usitatissimum. The coding region is 1029 base pairs in length and the DGAT2 encoded by the coding region (designated as LuDGAT2A, SEQ ID NO: 4) is a 343 amino acid polypeptide with a predicted molecular weight of 38,201 Daltons and an isoelectric point of 9.28.
In one embodiment, the invention provides a DGAT2 coding region comprising the nucleotide sequence depicted in SEQ ID NO: 5 and which was isolated from Linum usitatissimum. The coding region is 1048 base pairs in length and the DGAT2 encoded by the coding region (designated here by LuDGAT2B, SEQ ID NO: 6) is a 349 amino acid polypeptide with a predicted molecular weight of 38,737 Daltons and an isoelectric point of 9.18.
Those skilled in the art will recognize that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for identical polypeptides. Accordingly, the invention includes polynucleotides of SEQ ID NOS: 1, 3 and 5, and variants of polynucleotides encoding polypeptides of SEQ ID NOS: 2, 4 and 6. In one embodiment, polynucleotides having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequences depicted in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 are included in the invention. Methods for isolation of such polynucleotides are well known in the art (see for example, Ausubel et al., 1995).
In one embodiment, the invention provides isolated polynucleotides which encode polypeptides having DGAT activity and which comprise amino acid sequences having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequences depicted in SEQ ID NO: 2; SEQ ID NO: 4 and SEQ ID NO: 6.
In one embodiment, the invention provides isolated polynucleotides which encode polypeptides having DGAT activity and which comprise amino acid sequences having a length of at least 300, at least 400 or at least 500 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 2. In one embodiment, the invention provides isolated polynucleotides which encode polypeptides having DGAT activity and which comprise amino acid sequences having a length of at least 300 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 4. In one embodiment, the invention provides isolated polynucleotides which encode polypeptides having DGAT activity and which comprise amino acid sequences having a length of at least 300 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 6.
The above described polynucleotides of the invention may be used to express polypeptides in recombinantly engineered cells including, for example, bacterial, yeast, fungal, mammalian or plant cells. In one embodiment, the invention provides polynucleotide constructs, vectors and cells comprising DGAT polynucleotides. Those skilled in the art are knowledgeable in the numerous systems available for expression of a polynucleotide. All systems employ a similar approach, whereby an expression construct is assembled to include the protein coding sequence of interest and control sequences such as promoters, enhancers, and terminators, with signal sequences and selectable markers included if desired. Briefly, the expression of isolated polynucleotides encoding polypeptides is typically achieved by operably linking, for example, the DNA or cDNA to a constitutive or inducible promoter, followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA. High level expression of a cloned gene is obtained by constructing expression vectors which contain a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator. Vectors may further comprise transit and targeting sequences, selectable markers, enhancers or operators. Means for preparing vectors are well known in the art. Typical vectors useful for expression of polynucleotides in plants include for example, vectors derived from the Ti plasmid of Agrobacterium tumefaciens and the pCaM-VCN transfer control vector. Promoters suitable for plant cells include for example, the nopaline synthase, octopine synthase, and mannopine synthase promoters, the caulimovirus promoters and seed specific promoters, such as Brassica napus napin promoter.
Those skilled in the art will appreciate that modifications (i.e., amino acid substitutions, additions, deletions and post-translational modifications) can be made to a polypeptide of the invention without eliminating or diminishing its biological activity. Conservative amino acid substitutions (i.e., substitution of one amino acid for another amino acid of similar size, charge, polarity and conformation) or substitution of one amino acid for another within the same group (i.e., nonpolar group, polar group, positively charged group, negatively charged group) are unlikely to alter protein function adversely. Some modifications may be made to facilitate the cloning, expression or purification. Variant DGAT polypeptides may be obtained by mutagenesis of the polynucleotides depicted in SEQ ID NOS: 1, 3 and 5 using techniques known in the art including, for example, oligonucleotide-directed mutagenesis, region-specific mutagenesis, linker-scanning mutagenesis, and site-directed mutagenesis by PCR (Ausubel et al., 1995). Variant DGAT polypeptides can be tested for DGAT activity by the assay described in Example 4.
Various methods for transformation or transfection of cells are available. For prokaryotes, lower eukaryotes and animal cells, such methods include for example, calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, electroporation, biolistics and microinjection. The transfected cells are cultured, and the produced DGAT polypeptides may be isolated and purified from the cells using standard techniques known in the art. Accordingly, in one embodiment, the invention provides methods for producing DGAT in yeast as described in Example 4. Various industrial strains of microorganisms including for example, Aspergillus, Pichia pastoris, Saccharomyces cerevisiae, E. coli, Bacillus subtilis) may be used to produce DGAT polypeptides. In one embodiment, the microbial cell is Saccharomyces cerevisiae. 
Methods for transformation of plant cells include for example, electroporation, PEG poration, particle bombardment, Agrobacterium tumefaciens- or Agrobacterium rhizogenes-mediated transformation, and microinjection. The transformed plant cells, seeds, callus, embryos, microspore-derived embryos, microspores, organs or explants are cultured or cultivated using standard plant tissue culture techniques and growth media to regenerate a whole transgenic plant which possesses the transformed genotype. Transgenic Camelina sativa plants are regenerated as is disclosed in U.S. patent application Ser. No. 12/290,379 and 12/228,791. Transgenic plants may pass polynucleotides encoding DGAT polypeptides to their progeny, or can be further crossbred with other species. Accordingly, in one embodiment, the invention provides methods for producing transgenic plants, plant cells, callus, seeds, plant embryos, microspore-derived embryos, and microspores comprising DGAT polynucleotides.
In one embodiment, the invention provides transgenic plants, plant cells, callus, seeds, plant embryos, microspore-derived embryos, and microspores comprising DGAT polynucleotides. Plant species of interest for transformation include, without limitation, crops used for commercial oil production such as, for example, flax (Linum spp.), canola, soybean (Glycine and Soja spp.), Camelina sativa, mouse-ear cress (Arabidopsis thaliana), castor, sunflower and linola. In one embodiment, the plant is a flax plant. In one embodiment, the plant is a canola plant. In yet another embodiment the plant is Camelina sativa plant. Transgenic Camelina sativa plants preferably do not contain selection markers. It will be appreciated by those skilled in the art that the plant species for transformation are not limited to crops grown for commercial oil production. Such additional plant species include, without limitation, oats, wheat, triticale, barley, corn and Brachypodium distachyon. 
The DGAT polynucleotides, polypeptides, and methods of the invention are useful in a wide range of agricultural, industrial and nutritional applications. Transgenic plants with increased seed oil content can be developed; for example, transgenic plants which have the unique preference of incorporating omega-3 fatty acids into TAGs. Co-expression of LuDGAT1 and LuDGAT2 with a delta-15 dessaturase gene to feed LuDGAT with a polyunsaturated fatty acid may be conducted in plant species which do not normally produce C18:3 fatty acid. Recombinant expression of DGAT may be achieved in plants which are typically not grown for commercial oil production, resulting in development of new cultivars which produce oil having a similar composition to flaxseed oil.
Further, the DGAT polynucleotides and polypeptides may be used in the industrial production and recovery of oil products using recombinant technology such as transformed bacterial, yeast or fungal cells. Transformed cells may be engineered to accumulate omega-3 fatty acids in TAGs.
The DGAT polynucleotides and polypeptides may be incorporated into human food and animal feed applications to provide healthier products or to improve the fat quality of products. For example, a healthier dietary oil having a fatty acid profile which reduces the risk of coronary heart disease and decreases plasma cholesterol may be developed for humans. Livestock are unable to convert n-6 fatty acids into n-3 fatty acids since they lack an n-3 fatty acid desaturase gene. However, co-expression of LuDGAT1 and/or LuDGAT2 with fat-1 desaturase gene in livestock may increase the amount of n-3 fatty acids.
The Examples provided below are not intended to be limited to these examples alone, but are intended only to illustrate and describe the invention rather than limit the claims that follow.
EXAMPLES
Example 1
Isolation of RNA from Flax Embryos
Flax plants (Linum usitatissimum L. cv AC Emerson) were grown in greenhouse conditions, irrigated at 2-3 day intervals and fertilized weekly with 1% Peters 20-20-20 general purpose fertilizer (Scotts, Marysville, Ohio). Flax embryos were isolated from developing seeds and RNA was obtained using 350 mg of embryos frozen in liquid nitrogen and ground with mortar and pestle. Ground embryos were transferred to a 2 ml tube and 500 μl of extraction buffer (50 mM TrisHCl pH 9.0, 200 mM NaCl, 1% Sarkosyl, 20 mM EDTA, and 5 mM DTT) was added, mixing with a vortex. 500 μl of phenol chloroform mixture (Sigma-Aldrich Ltd, Oakville, ON) was added and mixed with a vortex. The sample was centrifuged for 5 minutes at 12000 g at 4° C. The aqueous upper phase was transferred to a new tube and 1 ml of Trizol reagent (Gibco, Burlington, ON, Canada) was added, followed by addition of 250 μl of chloroform. The sample was mixed with a vortex and centrifuged for 5 minutes at 12000 g at 4° C. The aqueous upper phase (750 μl) was transferred to a new tube and 500 μl of chloroform was added and mixed in a vortex. The sample was centrifuged for 5 minutes at 12000 g at 4° C. The upper phase (600 μl) was transferred to a new tube and the RNA was precipitated with addition of 60 μl of sodium acetate (3M) and 1.2 ml of ethanol. The sample was incubated at −80° C. for 1 hour and centrifuged for 20 minutes at 14000 g at 4° C. The RNA pellet was washed with 70% ethanol, followed by brief centrifugation (2 minutes at 14000 g at 4° C.) and dried with a vacufuge (Ependorf, Westbury, N.Y., U.S.). The RNA pellet was diluted in 50 μl of water and centrifuged for 20 minutes at 14000 g at 4° C. Total RNA was quantified by using a NanodropTM spectrophotometer (NanoDrop Technologies, Wilmington, Del., U.S.).
Example 2
Isolation of LuDGAT1 cDNA
Recombinant DNA techniques such as digestion by restriction endonucleases, ligation and plasmid preparation were performed as described by Ausubel et al. (1995). First strand synthesis of complementary DNA (cDNA) was produced by reverse transcription. Five micrograms of flax embryo RNA were mixed with 50 pmoles of oligonucleotide 5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTTVN-3′ (oligodT adaptor; SEQ ID NO: 7) and 1 mM of dNTP in a total volume of 10 μl. The mixture was incubated at 65° C. for 5 minutes and immediately cooled on ice for 2 minutes. A total volume of 10 μl of cDNA synthesis mix was added. This mix consisted of 2× transcriptase buffer, 10 mM of MgCl2, 20 mM of DTT and 200 units of Superscript II (Invitrogen, Burlington, ON). The reaction was incubated at 50° C. for 50 minutes followed by enzyme inactivation at 85° C. for 5 minutes. The reaction was cooled to 37° C. and incubated at this temperature for 20 minutes in the presence of 4 units of RNAseH (Invitrogen) and 1 unit of RNAseT1 (Ambion, Austin, Tex., U.S.) to remove the mRNA strand from RNA-DNA duplexes and single-stranded RNA, respectively. The synthesized cDNA was stored at −20° C.
Polymerase chain reaction (PCR) was performed using 200 μM of each dNTP, 0.1 volumes of PCR reaction buffer, and varying amounts of oligonucleotide, polymerases, DNA template and MgSO4 or MgCl2, according to the application in a final volume of 50 μl. The general PCR thermal cycling conditions were: 2 minutes preheat at 94° C. followed by 30 cycles of 94° C. denaturing for 30 seconds, 55° C. annealing for 30 seconds and 72° C. or 68° C. extension for 1 to 2 minutes. After the final cycle, the PCR reactions were incubated for 10 minutes at 72° C. or 68° C. for further extension and cooled to 10° C. until used for analysis.
The degenerate oligonucleotides 5′-GARTTYTAYCANGAYTGGTGG-3′ (RS-007; SEQ ID NO: 8), 5′-GGNACNGCNATRCANARYTCRTG-3′ (RS-008; SEQ ID NO: 9), 5′-GARAANYTNATGAARTAYGG-3′ (RS-009; SEQ ID NO: 10) and 5′-TANTGYTCNATDATRAANCCCAT-3′ (RS-010; SEQ ID NO: 11) were designed based on several plant DGAT1 sequences available in public databases. Such oligonucleotides, which provide limited specificity to the template, were used on PCR amplification of two different segments of DGAT1 using cDNA previously described as a template. These two DNA segments were sequenced using BigDye Version 3.1 dye terminator cycle sequencing kit (Applied Biosystems, Streetsville, ON, Canada). Samples were analyzed in an automatic sequencer (Applied Biosystems 373A Sequencer) at the University of Alberta Molecular Biology Service Unit (MBSU). Sequencing chromatograms were trimmed and assembled using Contig Express and “BLASTed” (Altschul et al., 1995; www.ncbi.nlm.nih.gov/BLAST) against public DNA databases.
Sequences from these two DNA segments, here named amplicon1 and amplicon2 (FIGS. 1 and 2), were used to design specific oligonucleotides to LuDGAT1 cDNA. These specific oligonucleotides were used in rapid amplification of cDNA ends (RACE) and reverse transcription-PCR (RT-PCR) amplification reactions to obtain the full nucleotide sequence of DGAT1.
3′ RACE was obtained using the oligonucleotides 5′-GCCATATCTATTTCCCATGTCTGCGG-3′ (RS027; SEQ ID NO: 12) and 5′-GGCCACGCGTCGACTAGTAC-3′ (adaptor; SEQ ID NO: 13) using cDNA previously described as template. For 5′ RACE flax cDNA was produced using the oligonucleotide 5′-CACTGGAAGTGTTAGACAG-3′ (RS-039; SEQ ID NO: 14) using the same conditions described before. The cDNA and the 3′ end were tailed with dCTP using Terminal deoxynucleotidyl Transferase (TdT). 5′-RACE was amplified using the oligonucleotides 5′-GGCCACGCGTCGACTAGTACGGGGGGGGGGGGGGGGGN-3′ (oligo dG adaptor; SEQ ID NO: 15) and 5′-ACTGAACCAGAAGCCTGTC-3′ (RS-038; SEQ ID NO: 16). This reaction yielded a truncated 5′ RACE product. A second 5′-RACE (here called 5′RACEB) was performed by producing flax embryo cDNA with the oligonucleotide 5′-CGGAACTAAGCGGACTCTC-3′ (RS-057; SEQ ID NO: 17) and tailing with dCTP. 5′-RACEB was performed using 5′-GGCACGGAAGGGCGGTAAG-3′ (RS-056; SEQ ID NO: 18) and oligo dG adaptor. Sequences obtained from 5′ and 3′ RACE products were aligned with amplicon1 and amplicon2 sequences (FIG. 2).
RT-PCR of the DNA segment between amplicon1 and amplicon2 (FIG. 1) was performed using the oligonucleotides 5′-CAAGTTAGTAATATTTACAGGC-3′, (RS-054; SEQ ID NO: 19) and 5′-TCCACATTCTCCAGTATTCTTC-3′ (RS-055; SEQ ID NO: 20) (FIGS. 1 and 2) and a cDNA from flax embryos previously described. The RT-PCR product was sequenced and aligned with sequences from other LuDGAT1 segments previously obtained (FIG. 2).
The coding region of LuDGAT1 was obtained through RT-PCR using the oligonucleotides 5′-ATTAGGATCCGACCATGGGCGTGCTCGACACTCCTGACAATC-3′ (RS-100; SEQ ID NO: 21) and 5′-TTTAAGCTTGATTCCATCTTTCCCATTCCTG-3′ (RS-101; SEQ ID NO: 22) and flax embryo cDNA as template. The RT-PCR product of LuDGAT1 coding region was sequenced and analyzed.
Example 3
Analysis of LuDGAT1 Sequence
DNA sequences were analyzed using VectorNTiTM Advance 10.1.1 (Invitrogen) software package. Amino acid and DNA alignments were performed with AlignX, and phylogenetic trees were visualized using Tree ViewTM version 1.6.6.
The full sequence of DGAT1 cDNA has 1778 base pairs with an open reading frame of 1521 base pairs comprising 507 amino acids with a molecular weight of 58.02 kDa. The predicted LuDGAT1 polypeptide, obtained by analysis of the ORF of LuDGAT1 cDNA (FIG. 3), was compared to other DGAT polypeptides from plants available in public databases. This comparison showed that LuDGAT1 is 74% identical with Vernicia fordii (tung tree), 75% with Jatropha curcas, 73% with Euonymus alatus (burning bush) and 65% with Brassica napus but only 40% with Mus musculus and 39% with Homo sapiens. An alignment of LuDGAT1 with several other plant DGAT1 (FIG. 4) showed many similarities and also some unique features. When compared to DGAT1 from other plants, LuDGAT1 presents the polypeptide “APSAALNV” (SEQ ID NO: 23) in the region between positions 253 and 259, which is absent in DGAT1 from cruciferaceae (Arabidopsis and Brassica sp.). A phylogenetic tree obtained with the previous alignment (FIG. 5) shows higher similarity between LuDGAT1 and Vernicia fordii, Jatropha curcas and Ricinus communis, compared to Oryza sativa, Brassica napus and Arabidopsis thaliana. LuDGAT presents unique features such as the substitution of the aspartic acid with glycine at position 103 in the conserved motif “ESPLSSD” (SEQ ID NO: 24). In position 271, the motif “LAYF” (SEQ ID NO: 25) is modified to “LVYF” (SEQ ID NO: 26). In the conserved motif “MWNMPVH” (SEQ ID NO: 27) present in other plant DGATs, the conserved asparagine in position 395 is substituted by a serine. These variations could reflect unique characteristics of LuDGAT1 enzymatic activity and specificity.
LuDGAT presents a hydrophilic N-terminus and several hydrophobic regions (FIG. 6), which is typical in other DGAT1 proteins. The first 80 residues present much higher variability compared to the rest of the protein, which is a characteristic found in other DGATs. Nine transmembrane regions were predicted in LuDGAT1 using TMPRED (http://www.ch.embnet.org/) (TM1 from 127 to 148, TM2 from 172 to 191, TM3 from 204 to 225, TM4 from 230 to 252, TM 5 from 311 to 331, TM 6 from 360 to 380, TM7 from 432 to 457, TM8 from 461 to 477 and TM9 from 493 to 511). Motif searches revealed that LuDGAT1 has a membrane bound O-acyl transferase (MBOAT) motif (pfam03062.12) which is present in a variety of acyltransferase enzymes such as DGAT1.
Example 4
Expression of LuDGAT1 in Yeast
The LuDGAT1 coding region was subcloned into pYES2.1/V5-HIS vector under control of GAL1 promoter which is inducible by galactose. A yeast consensus sequence for initiation of translation, composed of 5-(′g/a) nnatgg-3′, was introduced and the second amino acid codon was changed from gcg (A) to ggg (G). The translation stop sequence 5′-tga-3′ was removed in order to fuse LuDGAT1 in frame with V5 and HIS tags. The recombinant plasmid, called pYES LuDGAT1, was introduced into Saccharomyces cerevisiae strain H1246. A single colony containing pYES LuDGAT1 was inoculated in medium containing 2% glucose and grown overnight. The expression of LuDGAT1 was induced with medium containing 2% galactose. The same procedure was performed for pYES BnDGAT1 which contains the cDNA encoding DGAT1 from Brassica napus (Nykiforuk et al., 2002). pYES BnDGAT1 was used to compare the activity of another plant DGAT to LuDGAT1. Microsomes were extracted from induced yeast cells as described by Urban et al. (1994) and DGAT activity was determined by measuring the incorporation of 14C-oleyl-CoA into TAG. As S. cerevisiae strain H1246 is deficient in TAG biosynthesis (Sandager et al., 2002), the DGAT activity observed results only from the recombinant DGAT expressed.
DGAT assays were performed according to Byers et al. (1999). The standard reaction mixture (60 μL) consisted of 0.2 M Hepes-NaOH buffer (pH 7.4) containing 0.15 mg BSA/mL, 20 mM MgSO4, 330 μM sn-1,2-diolein in 0.2% (wt/vol) TweenTM 20, 15 μM [1-14C] oleoyl-CoA (56 mCi/mmo) and microsomal protein (80-120 μg). The reaction was performed for 15 min at 30° C. Each reaction mixture was spotted directly onto a silica gel thin layer chromatography plate, which was developed in hexane/ether (80:20, vol/vol). Sections of silica containing TAG were scraped into scintillation vials, combined with 5 mL Ecolite(+) and assayed for radioactivity in a liquid scintillation counter. As observed in FIG. 7, LuDGAT1 has comparable DGAT specific activity to BnDGAT1.
Example 5
Transformation of Camelina sativa cv. Calena with Agrobacterium tumefaciens strain C58 harbouring the binary plasmid pC0301 containing rLuDGAT gene.
Vector Construction
Brassica napus napA-promoter (P-Bn-napA) is PCR-amplified from genomic DNA or from existing clone using high fidelity DNA polymerase. 5′-end of forward primer contains XbaI and PmeI restriction sites. 5′-end of reverse primer contains XmaI and BpiI restriction sites. P-Bn-napA amplification is shown in FIG. 8. The product is purified by EtOH precipitation, cut using XbaI and XmaI restriction enzymes, gel-purified and cloned into pGem-7Zf(+) vector, which is opened using XbaI and XmaI restriction enzymes, dephosphorylated with alkaline phosphatase and gel-purified. The construct is called pGem7-P-Bn-napA (FIG. 9).
Brassica napus napA-terminator (T-Bn-napA: 3′-end of the gene following STOP-codon) is PCR-amplified from genomic DNA or from existing clone using high fidelity DNA polymerase. 5′-end of forward primer contains XmaI and BpiI restriction sites. 5′-end of reverse primer contains BamHI and PmeI restriction sites. T-Bn-napA amplification is shown in FIG. 8. The product is purified by EtOH precipitation, cut using BamHI and XmaI restriction enzymes, gel-purified and cloned into pGem7-P-Bn-napA, which is opened using BamHI and XmaI restriction enzymes, dephosphorylated with alkaline phosphatase and gel-purified. This construct is called pGem7-PT-Bn-napA and shown in FIG. 10.
Flax DGAT cDNA is cloned by PCR-amplification from cDNA, representing developing seed mRNA of flax or from existing cDNA clone using phosphorylated primers and high fidelity DNA polymerase leaving the ends of the product blunt. The product is gel purified and cloned into pGem7-PT-Bn-napA, which is opened using BpiI restriction enzyme, made blunt using Klenow fragment of E. coli DNA polymerase I, dephosphorylated and gel-purified. BpiI is an outside-cutter, which is used to cut so that the obtained ends contain no extra nucleotides when compared with native promoter and terminator. The construct is called pGem7-PT-Bn-napA-LuDGAT and shown in FIG. 11.
The P-Bn-napA-LuDGAT-T-Bn-napA fragment is cut out from pGem7-PT-Bn-napA-LuDGAT using PmeI restriction enzyme, which leaves blunt ends and further cloned into pC0300 plant vector, which is opened using XmnI and PmeI restriction enzymes, dephosphorylated and gel-purified. pC0300 is a selection marker-free derivative of pCambia-1300. This construct is named pC0300-PT-Bn-napA-LuDGAT and shown in FIG. 12
The constructs are sequenced and electroporated into Agrobacter thumefaciens for plant transformation.
Camelina sativa Transformation
The seeds of Camelina sativa plant grown in greenhouse are sterilized by immersing them in 70% ethanol for 1 min and then treating for 10 min with Na-hypochlorite solution (3% active Cl.sup.−) with an addition of Tween-20 (1 drop per 100 ml). After sterilization the seeds are washed three times in sterile water and placed on solid Murashige and Skoog (MS) agar medium (Murashige and Skoog, Physiol. Plant. 15:472-493, 1962) without sugars for germination. Sterilized seeds are germinated and grown 12 days on solid Murashige and Skoog (MS) medium without hormones Green leaves serve as a source of explants for transformation procedure.
Agrobacterium tumefaciens strain c58 carrying plasmid pC0301 containing rLuDGAT1, rLuDGAT2A or rLuDGAT2B gene are grown overnight at 28° C. with shaking in liquid YEB medium (Lichtenstein and Draper, Gene Engineering of Plants. In: Glover D M (ed.) DNA Cloning—A Practical Approach, vol. 2. Oxford IRL, Oxford, pp 67-119, 1985) supplemented with 50 mg/l kanamycin and 12.5 mg/l rifampicin. Subsequently an aliquot of the culture ( 1/100 v/v) is inoculated in fresh YEB medium supplemented with 50 mg/l kanamycin and 12.5 mg/l rifampicin and the bacteria are grown overnight with shaking. Agrobacterium culture of OD.sub.600=1.0 is used in the transformation experiments.
Narrow leaves of in vitro grown Camelina sativa plants are cut only across the leaf blade, whereas large leaves are additionally cut in half along the central vein. The leaf segments are cultivated for 24 hours on Murashige and Skoog (MS) 0.7% agar medium supplemented with 1.5 mg/l 6-benzylaminopurine (BAP) and 1.0 mg/l .alpha.-naphthaleneacetic acid (NAA). All the Murashige and Skoog (MS) culture medium are supplemented with 2% sucrose and all in vitro cultures are kept at temperatures of 25° C. (day) and 18° C. (night) under the photoperiod of 16 h. The explants are immersed for 1-3 min in Murashige and Skoog (MS) solution inoculated with a dilution (e.g. 1/10 v/v) of the overnight culture of Agrobacterium tumefaciens. Redundant liquid on the leaf segments is removed using filter paper and the explants are placed on Murashige and Skoog (MS) agar medium supplemented with auxin and cytokinin for co-cultivation with bacteria for 2 days.
The explants are washed with water containing Ticarcillin 200 mg/l. The surfaces of the explants are dried on filter paper and the explants were placed on Murashige and Skoog (MS) medium supplemented with hormones [1.5 mg/l 6-benzylaminopurine (BAP), 1.0 mg/l alpha-naphthaleneacetic acid (NAA)] and 200 mg/l Ticarcillin.
Leaf explants are transferred after 7 days to new MS medium containing 1.5 mg/l BAP and 100 mg/l Ticarcillin. After another 7 days the leaf explants are transferred to new MS medium containing 1.5 mg/l BAP and 200 mg/l Ticarcillin.
Shoots developed in this medium in 7 to 14 days are cut and placed on regular or half strength Murasghige and Skoog (MS) medium solidified with 0.7% agar and supplemented with 100 mg/l Ticarcillin and 0.3 mg/l NAA. After root formation the shoots are transferred to soil and transgenic plants are grown in greenhouse conditions. The presence of the rLuDGAT transgene is confirmed from transgenic plants using PCR assay and Northern blot.
Seeds of the transgenic Camelina sativa plants are collected and oil content and composition of the seeds is analyzed.

## Claims

1. An isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from:
at least 300, contiguous residues of the amino acid sequence depicted in SEQ ID NO: 2 or of an amino acid sequence having at least 85% sequence identity therewith;
wherein said polypeptide has diacylglycerol acyltransferase activity.

2. The isolated polynucleotide of claim 1, wherein the encoded polypeptide consists of the amino acid sequence depicted in SEQ ID NO: 2.

3. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises the nucleotide sequence depicted in SEQ ID NO: 1 from nucleotide 57 to nucleotide 1580.

4. The isolated polynucleotide of claim 1, wherein the encoded polypeptide comprises an amino acid sequence having at least 90%, sequence identity to SEQ ID NO: 2.

5. A polynucleotide construct comprising a polynucleotide of claim 1 operably linked to a promoter expressible in bacterial, yeast, fungal, mammalian or plant cells.

6. A vector comprising a polynucleotide of claim 1.

7. The vector of claim 6, wherein the polynucleotide is operably linked to a seed specific promoter.

8. The vector of claim 7, wherein the promoter is Brassica napus napin promoter.

9. A microbial cell comprising a polynucleotide of claim 1.

10. The microbial cell of claim 9, wherein the cell is Saccharomyces cerevisiae.

11. A transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore, produced by transformation with the polynucleotide of claim 1.

12. The transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore of claim 11, which is selected from flax, canola, soybean, Camelina sativa, mouse-ear cress, castor, sunflower, linola, oats, wheat, triticale, barley, corn or Brachypodium distachyon plant, plant cell, plant seed, plant embryo, or microspore.

13. The transgenic plant of claim 12, wherein the plant is a Camelina sativa plant, comprising the polynucleotide of SEQ ID NO: 1 under control of Brassica napus napin promoter.

14. A method for producing oil, comprising the steps of:
growing a transgenic plant according to claim 11; and
recovering oil which is produced by the plant.

15. The method according to claim 14, wherein the plant is selected from flax, canola, soybean, Camelina sativa, mouse-ear cress, castor, sunflower, linola, oats, wheat, triticale, barley, corn or Brachypodium distachyon plant.

16. A method for producing a transgenic plant comprising the steps of:
introducing into a plant cell or a plant tissue a polynucleotide of claim 1 to produce a transformed cell or plant tissue; and
cultivating the transformed plant cell or transformed plant tissue to produce the transgenic plant.

17. The method of claim 16, wherein the polynucleotide is under control of seed specific promoter.

18. The method of claim 17, wherein the promoter is napin promoter.

19. The method of claim 16, wherein the plant is selected from flax, canola, soybean, Camelina sativa, mouse-ear cress, castor, sunflower, linola, oats, wheat, triticale, barley, corn or Brachypodium distachyon plant.