Acyltransferase polynucleotides, polypeptides and methods of use

Abstract

The invention provides a novel DGAT1 protein with improved properties over known DGAT proteins, particularly known DGAT1 proteins from plants. The novel DGAT1 protein of the invention can be expressed in cells to increase cellular lipid accumulation. Expression of the DGAT1 protein of the invention in cells results in a higher level of lipid than any of several other plant DGAT1 proteins tested by the applicants. The invention provides polynucleotides encoding the novel DGAT1 protein of SEQ ID NO:39, constructs, cells, plant, plant parts and progeny comprising the polynucleotides, and methods of use of the polynucleotides and polypeptides of the invention.

Claims

1. A cell transformed with a polynucleotide encoding a diacylglycerol acyltransferase 1 (DGAT1) polypeptide comprising at least one of: a) a sequence with at least 99% identity to the sequence of SEQ ID NO:39 b) the sequence of SEQ ID NO:39 c) a sequence comprising at least 450 contiguous amino acids of the sequence of a) or b), wherein the polynucleotide is heterologous to the cell.

2. The cell of claim 1 wherein the polypeptide has DGAT1 activity.

3. The cell of claim 1, wherein the DGAT1 polypeptide, when expressed in the cell has at least one of: a) higher DGAT1 activity than a DGAT1 protein having the amino acid sequence of SEQ ID NO: 44, and b) altered substrate specificity relative to a DGAT1 protein having the amino acid sequence of SEQ ID NO: 44.

4. A genetic construct comprising a polynucleotide encoding a DGAT1 polypeptide comprising at least one of: a) a sequence with at least 99% identity to the sequence of SEQ ID NO:39 b) the sequence of SEQ ID NO:39 c) a sequence comprising at least 450 contiguous amino acids of the sequence of a) or b), wherein the polynucleotide is operably linked to a promoter, and wherein the polynucleotide and the promoter are not found operably linked in nature.

5. A cell comprising a genetic construct of claim 4.

6. The cell of claim 1 wherein the DGAT1 polypeptide has increased DGAT1 activity relative to a DGAT1 protein having the amino acid sequence of SEQ ID NO: 44.

7. The cell of claim 1 wherein the DGAT1 polypeptide has altered substrate specificity relative to a DGAT1 protein having the amino acid sequence of SEQ ID NO: 44.

8. The cell of claim 1 that produces more lipid than does a control cell that is not transformed with the polynucleotide.

9. The cell of claim 1 that has an altered lipid profile relative to a control cell that is not transformed with the polynucleotide.

10. The cell of claim 1 that is a plant cell.

11. The cell of claim 1 that is also transformed to express at least one of: an oleosin, steroleosin, caloleosin, polyoleosin, and an oleosin including at least one artificially introduced cysteine.

12. A plant comprising the cell of claim 1.

13. A plant comprising a genetic construct of claim 4.

14. A plant comprising a plant cell of claim 10.

15. The plant of claim 12 wherein the plant, or its predecessor, has been transformed or genetically modified to express the polynucleotide or encoded polypeptide.

16. The plant of claim 12 wherein the polypeptide expressed by the polynucleotide has increased DGAT1 activity relative to a DGAT1 protein having the amino acid sequence of SEQ ID NO: 44.

17. The plant of claim 12 wherein the plant produces more lipid, in at least one of its tissues or parts, or as a whole, than does a control plant that is not transformed with the polynucleotide.

18. The plant of claim 12 wherein the plant has an altered lipid profile, in at least one of its tissues or parts, or as a whole, relative to a control plant that is not transformed with the polynucleotide.

19. The plant of claim 12 wherein the plant is also transformed to express at least one of: an oleosin, a steroleosin, a caloleosin, a polyoleosin, and an oleosin including at least one artificially introduced cysteine.

20. A part, propagule or progeny of the plant of claim 12, wherein the part, propagule or progeny comprises the cell.

21. A part, propagule or progeny of the plant of claim 13 wherein the part, propagule or progeny comprises the construct.

22. The part, propagule or progeny of claim 20 wherein at least one of the following applies: the part, propagule or progeny produces more lipid than does a control part, propagule or progeny, or part, propagule or progeny of a control plant, and the part, propagule or progeny has an altered lipid profile relative to a control part, propagule or progeny, or part, propagule or progeny of a control plant, wherein the control part, propagule or progeny, or part propagule or progeny of the control plant is not transformed to comprise the polynucleotide.

23. The part, propagule or progeny of claim 21, wherein at least one of the following applies: the part, propagule or progeny produces more lipid than does a control part, propagule or progeny, or part, propagule or progeny of a control plant, and wherein the part, propagule or progeny has an altered lipid profile relative to a control part, propagule or progeny, or part, propagule or progeny of a control plant, wherein the control part, propagule or progeny, or part propagule or progeny of the control plant does not comprise at least one of: a) a sequence with at least 99% identity to the sequence of SEQ ID NO:39 b) the sequence of SEQ ID NO:39 c) a sequence comprising at least 450 contiguous amino acids of the sequence of a) or b).

24. An animal or biofuel feedstock comprising a cell transformed with a polynucleotide encoding a polypeptide comprising at least one of: a) a sequence with at least 99% identity to the sequence of SEQ ID NO:39 b) the sequence of SEQ ID NO:39 c) a sequence comprising at least 450 contiguous amino acids of the sequence of a) or b), wherein the polynucleotide is heterologous to the cell.

25. An animal feedstock or biofuel feedstock comprising at least one plant part, propagule or progeny as defined in claim 22.

26. A method for producing a lipid, the method comprising growing a cell, plant cell or plant that is transformed, or genetically modified, with a polynucleotide encoding a diacylglycerol acyltransferase 1 (DGAT1) polypeptide comprising at least one of: a) a sequence with at least 99% identity to the sequence of SEQ ID NO:39 b) the sequence of SEQ ID NO:39 c) a sequence comprising at least 450 contiguous amino acids of the sequence of a) or b), wherein the cell, plant cell, or plant produces oil through the activity of the expressed polypeptide, and wherein the polynucleotide is heterologous to the cell.

27. The method of claim 26 wherein the cell, plant cell or plant produces the lipid as a result of the DGAT1 activity of the polypeptide.

28. A method for producing lipid, the method comprising extracting lipid from at least one cell of claim 14.

29. The method of claim 26 wherein the lipid is triacylglycerol (TAG).

30. The method of claim 26 wherein the lipid is processed into at least one of: a) a fuel, b) an oleochemical, c) a nutritional oil, d) a cosmetic oil, e) a polyunsaturated fatty acid (PUFA), and f) a combination of any of a) to e).

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the nucleic acid sequence and three frame translation of the Arabidopsis thaliana DGAT1 transcribed region (SEQ ID NO:116). Exon coding sequences are shown in bold face, underlined, grey blocks.

(2) FIG. 2 shows the nucleic acid sequence and three frame translation of the Zea mays short DGAT1 transcribed region (SEQ ID NO:117). This genomic sequence has F469 deleted and Q67 added compared to the cDNA (EU039830) and peptide (ABV91586) sequences actually used in this patent. Exon coding sequences are shown in bold face, underlined, grey blocks.

(3) FIG. 3 shows the nucleic acid sequence and three frame translation of the Zea mays long DGAT1 transcribed region (SEQ ID NO:118) derived from CHORI-201 Maize B73 BAC Library (available from the World Wide Web at http://www.ncbi.nlm.nih.gov/nuccore/AC204647; http://bacpac.chori.org/maize201.htm). Exon coding sequences are shown in bold face, underlined, grey blocks.

(4) FIG. 4 shows the peptide sequence of the N-terminal cytoplasmic region of a number of plant DGAT1s including both long and short versions from the grasses as well as examples from dicotyledonous species. Left hand box represents acyl-CoA binding site (Nykiforuk et al., 2002, Biochimica et Biophysica Acta 1580:95-109). Right hand box represents first transmembrane region (McFie et al., 2010, JBC., 285:37377-37387). Left hand arrow represents boundary between exon 1 and exon 2. Right hand arrow represents boundary between exon 2 and exon 3. The sequences are AtDGAT1 (SEQ ID NO:119), BjDGAT1 (SEQ ID NO:120), BnDGAT1-AF (SEQ ID NO:121), BjDGAT1 (SEQ ID NO:122), TmajusDGAT1 (SEQ ID NO:123), EpDGAT1 (SEQ ID NO:124), VgDGAT1 (SEQ ID NO:125), NtDGAT1 (SEQ ID NO:126), PfDGAT1 (SEQ ID NO:127), ZmL (SEQ ID NO:128), SbDGAT1 (SEQ ID NO:129), OsL (SEQ ID NO:130), OsS (SEQ ID NO:131), SbDGAT1 (SEQ ID NO:132), ZmS (SEQ ID NO:133), PpDGAT1 (SEQ ID NO:132), SmDGAT1 (SEQ ID NO:135), EaDGAT1 (SEQ ID NO:136), VvDGAT1 (SEQ ID NO:137), GmDGAT1 (SEQ ID NO:138), GmDGAT1 (SEQ ID NO:139), LjDGAT1 (SEQ ID NO:140), MtDGAT1 (SEQ ID NO:141), JcDGAT1 (SEQ ID NO:142), VfDGAT1 (SEQ ID NO:143), RcDGAT1 (SEQ ID NO:144), PtDGAT1 (SEQ ID NO:145), Pt DGAT1 (SEQ ID NO:146).

(5) FIG. 5 shows the line-bond structures of the amino acid residues lysine (K) and arginine (R).

EXAMPLES

Example 1

Identification of the DGAT1 Sequence of the Invention

(6) Several nucleic acid sequences and polypeptide sequences for the plant type 1 DGATs can be found by accession number in public domain libraries (Table 1). For creating initial alignments we used ClustalW (Thompson et al., 1994, Nucleic Acids Res., 22, 4673-4680); these were manually edited and used to create the models to search the DGAT sequences, using the HMMER2 package (HMMER 2.3.2 (October 2003) Copyright? 1992-2003 HHMI/Washington University School of Medicine, available from the World Wide Web at http://hmmer.org). Initial matching of protein sequences against genomic DNA with splice prediction was performed with the GeneWise package (Birney et al., 2004, Genome Res. 14: 988-995). Some of the sequences retrieved appeared to have errors; in particular incorrectly predicted splice sites which would result in internal deletions that would likely result in non-functional proteins. While both dicotyledonous and monocotyledonous type 1 DGATs have 16 exons there are some differences in the position of the splicing. Exon 8 in the dicotylendonous DGAT1 gene corresponds to exons 8 and 9 in the monocotyledonous DGAT1 gene, while exon 14 in the monocotyledonous gene corresponds to exons 13 and 14 in the dicotyledonous gene. We have found that the most accurate method for determining the likely genuine coding sequence from genomic data has been to use Vector NTI Advance (TM) 11.0 (? 2008 Invitrogen Corporation) to translate the genome in the three forward reading frames and align these with demonstrated functional DGAT1s from dicotyledonous or monocotyledous species as appropriate (for example A. thaliana cDNA NM_127503, protein NP_179535 and Z. mays cDNA EU039830, protein ABV91586). The genomic sequence and corresponding exon/intron boundary positions for Arabidopsis thaliana encoding NP_179535 and Zea mays encoding ABV91586 that can be used as a template for determining other plant DGAT coding regions are shown in FIG. 1 and FIG. 2, respectively.

(7) Using this method, the applicants have assembled/identified a novel DGAT1 sequence from Z. mays DGAT1 (SEQ ID NO: 10 and SEQ ID NO: 39 [FIG. 3]). To the best of the applicant's knowledge, a functional portion of sequence is not present in any public cDNA database, which may indicate that the functional protein is not expressed in naturally occurring plants.

(8) The applicants designated this sequence Zea mays DGAT-Long (ZmDGAT1-L or Zm-L DGAT1) because the encoded polypeptides is longer than the known Zea mays of SEQ ID NO: 44 (referred to as Zea mays DGAT1 -short or ZmDGAT1 -S or Zm-S DGAT1) as indicated in FIG. 4.

(9) A similar relationship exists between Oryza sativa DGAT1 -short, or OsDGAT1-S, or Os-S DGAT1 (SEQ ID NO:41, and Oryza sativa DGAT1 -long, or OsDGAT1-L, or Os-L DGAT1 (SEQ ID NO:42).

(10) TABLE-US-00001 TABLE 1 PROTEIN DGAT1 DNA accession #s SEQ accession #s SEQ Species & ID & ID Source BAC # NO: BAC # NO: A. thaliana NM_127503 1 NP_179535 30 B. juncea AF164434 2 AAY40784 31 B. napus AF164434_1 3 AAD45536.1 32 B. juncea DQ016107 4 AAY40785 33 T. majus AY084052 5 AAM03340 34 E. pitardii FJ226588 6 ACO55635 35 V. galamensis EF653276 7 ABV21945 36 N. tabacum AF129003_1 8 AAF19345.1 37 P. frutescens AF298815_1 9 AAG23696.1 38 Z. mays From: CHORI-201 10 From: CHORI-201 39 Maize B73 BAC Maize B73 BAC S. bicolor XM_002439374 11 XP_002439419 40 O. sativa Os05g0196800 12 NP_001054869 41 O. sativa From: AP003714.1 13 From: AP003714.1 42 S. bicolor XM_002437120.1 14 XP_002437165 43 Z. mays EU039830 15 ABV91586 44 P. patens XM_001770877.1 16 XP_001770929 45 S. XM_002964119 17 XP_002964165 46 moellendorffii E. alatus AY751297 18 AAV31083 47 V. vinifera XM_002279309 19 XP_002279345 48 G. max AY496439 20 AAS78662 49 G. max AB257590 21 BAE93461 50 L. japonicus AY859489 22 AAW51456 51 M. truncatula AC174465.2 23 ABN09107 52 J. curcas DQ278448.1 24 ABB84383 53 V. fordii DQ356680.1 25 ABC94472 54 V. galamensis EF653276.1 26 ABV21945 55 R. communis XM_002514086.1 27 XP_002514132 56 P. trichocarpa XM_002308242.1 28 XP_002308278 57 P. trichocarpa XM_002330474.1 29 XP_002330510 58

Example 2

The DGAT1 Sequence of the Invention has Surprisingly High Activity in Increasing Cellular Lipid Content, and Fragments of the DGAT1 Sequence of the Invention are Useful in Conferring Increased Activity to Other DGAT1 Proteins

(11) Summary

(12) The applicants compared the activity of the DGAT1 sequence of the invention to other known DGAT1 sequences. Surprisingly the DGAT1 sequence of the invention showed higher activity, in increasing cellular lipid content, than any of the other DGAT1 sequences tested.

(13) Furthermore the applicants have shown that fragments of the DGAT1 protein of the invention are useful in conferring increase activity on at least parts of other DGAT proteins.

(14) Materials and Methods

(15) Nucleic acid constructs encoding the amino acid sequences, SEQ ID NO: 30 (A. thaliana DGAT1), 34 (T. magus DGAT1), 39 (Zea mays DGAT1-L), 41 (O. sativa DGAT1-S), 42 (O. sativa DGAT1-L) and 44 (Table 1) were optimised for expression in Saccharomyces cerevisiae by GeneArt AG (Germany). These were engineered to have an internal XhoI site within exon 1 encoding the conserved N-terminal acyl-Co binding region (identified by Lung and Weselake, 2006, Lipids. December 2006; 41(12):1073-88) without altering the amino acid sequence leucine-serine-serine (LSS). FIG. 4 shows alignment of a number of DGAT1 sequences from plants. The left box shows the position of the Acyl-CoA binding site.

(16) An EcoRI site was engineered upstream of the 5 coding sequence while an XbaI site was placed downstream of the 3 stop codon. The internal XhoI and flanking EcoRI and XbaI sites were used to generate chimeras between the DGAT1 sequence of the invention and each of the other DGAT1 clones; essentially this fused the N-terminal reputed cytoplasmic region (based on Lung and Weselake, 2006, Lipids. December 2006; 41(12):1073-88 and McFie et al., 2010, JBC., 285:37377-37387) from one DGAT1 with the C-terminal ER luminal region of a different DGAT1. In some combinations this resulted in one amino acid change in the remaining cytoplasmic region downstream of the engineered XhoI site. The putative acyl-Co binding region the A. thaliana DGAT1, T. majus DGAT1, Z. mays-L DGAT1 and O. sativa-L DGAT1 have an identical amino acid sequence down stream of the XhoI site (LSSDAIFKQSHA). While in the Z. mays-S DGAT1 and O. sativa-S DGAT1 the lysine (K) residue is replaced by an arginine (R) residue (LSSDAIFRQSHA). Since the position of this residue is located 3 to the Xho I site encoded by LLS then chimeras deriving from one parent containing the lysine and one parent containing the arginine residue will effectively result in a substitution of this residue. This was considered to be a minimal disruption since both lysine and arginine are large, positively charged, hydrophilic, basic amino acids containing a free amine or guanidinium group, respectively at the end of an aliphatic side chain (FIG. 5). The N-terminal region/C-terminal region domain swapping constructs, and the parent constructs (highlighted in bold) are shown in Table 2, with their corresponding SEQ ID NOs.

(17) TABLE-US-00002 TABLE 2 DGAT1 DGAT1 N-terminal C-terminal C-terminal SEQ parent parent Tail Fusion ID NO: A. thaliana A. thaliana V5-6xHis 59 A. thaliana Z. mays-L V5-6xHis 63 O. sativa-S O. sativa-S V5-6xHis 65 O. sativa-S Z. mays-L V5-6xHis 69 O. sativa-L O. sativa-L V5-6xHis 71 O. sativa-L Z. mays-L V5-6xHis 75 Z. mays-S Z. mays-S V5-6xHis 77 Z. mays-S Z. mays-L V5-6xHis 81 Z. mays-L Z. mays-L Y5-6xHis 83 Z. mays-L Z. thaliana V5-6xHis 84 Z. mays-L O. sativa-S V5-6xHis 85 Z. mays-L O. sativa-L V5-6xHis 86 Z. mays-L Z. mays-S V5-6xHis 87 Z. mays-L T. majus V5-6xHis 88 T. majus T. majus V5-6xHis 89 T. majus Z. mays-L V5-6xHis 94

(18) Sequences were synthesised either by GENEART AG (Germany) or GeneScript (USA). Sequences were optimised for expression in Saccharomyces cerevisiae and flanked with appropriate incorporated appropriate restriction sites to facilitate the cloning into the pYES2.1 vector (Invitrogen).

(19) Expression of Constructs in S. cerevisiae

(20) The parent DGAT1 constructs and chimeric DGAT1 constructs were placed into the galactose-inducible yeast expression vector pYES2.1/V5-His TOPO? (Invitrogen). This resulted in the addition of an inframe C-terminal V5 epitope and 6? histidine tag. The chimeric constructs and the number of their corresponding polypeptide sequences are shown in Table 2 above.

(21) The Saccharomyces cerevisiae quadruple mutant (H1246) in which all four neutral lipid biosynthesis genes have been disrupted (Sandager et at, 2002, The Journal of Biological Chemistry, 277:6478-6482) was transformed as per Elble (1992, BioTechniques 13, 18-20) and selected by the ability to grow in the absence of uracil. Routinely, yeast cells were grown aerobically overnight in a synthetic medium with 0.67% YNB, without uracil (SC-U) and containing 2% glucose. Cells from overnight culture were used to inoculate 200 mL of induction medium (SC-U containing 2% galactose and 1% raffinose) to an initial OD.sub.600 of 0.4. Cells were allowed to further grow at 30? C., with shaking at 200 rpm until late stationary phase, normally 48 h. Cells were harvested by centrifugation at 1500?g for 5 min, then cell pellets were washed with distilled water and either used immediately for subsequent analysis or kept in ?80? C. until required. Cell pellets for neutral lipid extraction were freeze-dried for 48 h and stored in ?20? C. freezer until required.

(22) Lipid Analysis of S. cerevisiae

(23) Approximately 10 mg of freeze-dried yeast cell material was accurately weighed then disrupted using glass beads by vortexing for 1 minute. This lysate was extracted in hot methanolic HCl for fatty acid methyl ester (FAME) analysis (Browse et al., 1986, Anal. Biochem. 152, 141-145).

(24) For FA profile analysis approximately 50 mg freeze dried yeast was placed in a 13-mm screw cap tube, and an equal volume of glass beads added before vortexing at high speed in 3?1 min bursts. Following addition of 50 ?g of 19:0 TAG internal standard, 2.4 mL of 0.17 M NaCl in MeOH was added and the mixture vortexed for 15 sec followed by the addition of then 4.8 mL of heptane and the entire contents mixed.

(25) The solution was then incubated in 80? C. water bath for 2 h without shaking. After incubation, the solution was cooled to room temperature. After cooling, the upper phase (lipidic phase) was transferred to fresh screw-cap tube and evaporated to dryness under stream of nitrogen gas. The dried residue was then dissolved in 1 mL heptane and mixed thoroughly for TAG SPE separation using Strata Si-1 Silica column (Phenomenwx, 8B-S012-EAK).

(26) After preconditioning with methanol and equilibrating the Silica column with heptane the 1 mL TAG extract (including 50 ?g 17:0 TAG Internal Standard was passed through the pre-equilibrated column, followed by 1.2 mL of heptane and then 2 mL of chloroform:heptane (1:9 v/v/) and the eluate collected. The total eluate collected was evaporated to dryness under the stream of N gas and the residue used for FAMEs extraction.

(27) FAMEs of Extracted TAG

(28) To the TAG residue above 10 ?L of internal standard 15:0 FA (4 mg/mL dissolved in heptane) and 1 mL of methanolic HCl (1N) reagent containing 5% of 2,2-dimeethoxypropane (as water scavenger) were added.

(29) The tube was then flushed with N gas, then sealed immediately with Teflon-lined cap, and heated at 80? C. in a water bath for 1 h. After cooling down, 0.6 mL heptane and 1.0 mL of 0.9% (w/v) NaCl was added, the mixture vortexed then spun at 500 rpm for 1 min.

(30) From the top heptane layer, 100 ?L was collected and transferred to a flat-bottom glass insert fitted into a vial for FAMES GC/MS analysis.

(31) Protein Extraction and Trypsin Digestion

(32) Yeast cell pellets were washed with lysis buffer (50 mM sodium phosphate, pH 7.4, 1 mM EDTA, 5% glycerol, 1 mM PMSF) then resuspended in 500 ?L lysis buffer, glass beads were added and cells disrupted by vortexing 2? at medium speed for 30 seconds. Cell debris was pelleted by centrifugation at 1000?g for 5 min, the supernatant transferred to fresh tubes and total cellular membranes pelleted by ultracentrifugation at 100,000?g for 1 h. Membrane proteins were resuspended in lysis buffer with or without detergent (1% Dodecyl maltoside) and quantified in a Qubit Fluorometer using the Qubit IT Quantitation Kit.

(33) Trypsin was added to give a final concentration of 25 ?g/mL to 50 ?L of protein extract and the mixture incubated at 30? C. for 30 min. The reaction was terminated by addition of Trypsin inhibitor from Glycine max (Sigma-Aldrich catalogue #T6414) to a final concentration of 0.4 ?g/?L. After addition of trypsin inhibitor, 4?SDS loading dye and 10? reducing agent (Invitrogen) were added, and the protein incubated at 70? C. for 10 min prior to SDS-PAGE followed by immunoblotting. The blot was probed with either Anti V5-HRP antibody (Cat #R96125, Invitrogen) at 1:2500 dilution, or anti Kar2 (y-115) antibody produced in rabbit (SC-33630, Santa Cruz Biotechnology) at 1:200 dilution. Anti Kar2 was used to detect the yeast protein Kar2, an ER luminaly-located protein (Rose et al., 1989, Cell 57, 1211-1221) which serves as a control to demonstrate the presence of intact microsomes.

(34) Expression of Chimeric DGAT1 in Brassica napus

(35) The same strategy, as described above, was used to generate a variety of chimeric DGAT1 constructs for expression in the seeds of Brassica napus. This included the parent DGAT1s of T. majus DGAT1, Z. mays-L DGAT1 and Z. mays-S DGAT1 (amino acid SEQ ID NO: 34, 39 and 44 respectively, Table 1) optimised for expression in Brassica napus by GeneArt AG. The T. majus construct was engineered to contain a single point mutation S.sub.197A (Xu et al., 2008, Plant Biotechnology Journal, 6:799-818). All constructs were engineered to have an optimised Kozak, Arabidopsis thaliana UBQ10 intron, and tetranucleotide stop codon as per Scott et al., (2010, Plant Biotechnology Journal, 8:912-917) as indicated in Table 3 below.

(36) TABLE-US-00003 TABLE 3 DGAT1 Kozak, Parent intron, stop Residue SEQ Species codon modification ID NO: T. majus yes S197A 95 Z. mays-S yes none 96 Z. mays-L yes none 97

(37) The same digestion pattern used to generate the chimeras for expression in S. cerevisiae was used on the B. napus-optimised constructs to generate the chimeras Tm-ZmL and ZmL-Tm(S189A); resulting in the peptide sequences listed in Table 4.

(38) TABLE-US-00004 TABLE 4 DGAT1 DGAT1 N-terminal C-terminal Residue SEQ parent parent modification ID NO: T. majus T. majus S197A 98 Z. mays-L Z. mays-L none 99 T. majus Z. mays-L none 100 Z. mays-L T. majus S189A 101

(39) The parent DGATs and their chimeras were transferred into the Gateway?-compatible binary vector pMD107 (courtesy of Dr Mark Smith, NRC Saskatoon, SK, Canada, S7N 0W9) which placed them under the control of the seed-specific napin promoter (Ellerstr?m et al., 1996, Plant Molecular Biology, Volume 32, Issue 6, pp 1019-1027).

(40) Plant Transformation

(41) B. napus (cv. DH12075) was transformed via Agrobacterium tumefaciens (GV3101) using the cotyledon co-cultivation method (adapted from that of Maloney et al., 1989, Plant Cell Rep. 8, 238-242). Control lines contained an empty-vector, and when identified, null sibling lines were subsequently used as true controls.

(42) Approximately 200 T.sub.0 transformed lines were produced and their corresponding T.sub.1 selfed seeds were analysed for oil content by GC. Approximately 50 individual transgenic lines (including control lines) were selected for the next generation (10 plants/line) based on their oil content, or seed weight (8 lines).

(43) A total of approximately T.sub.1 plants were grown and screened by PCR for copy number and identification of null sibling lines. T.sub.2 seeds were analysed in triplicate for oil content by NMR.

(44) Expression of Z. mays-L and T. majus DGAT1 in Camelina sativa

(45) The strategy above can also be used to generate a variety of chimeric DGAT1 constructs for expression in the seeds of Camelina sativa and other plants.

(46) Sequences with modifications were synthesised either by GENEART AG (Germany) or GeneScript (USA). Sequences with modifications were synthesised either by GENEART AG (Germany) or GeneScript (USA). Sequences were optimised for expression in Brassica species and included an intron (SEQ ID NO:102) from Arabidopsis thaliana DGAT1 -intron 3. Each sequence was flanked with appropriate attL recombination sites to enable the cloning Gateway? adapted vectors.

(47) TABLE-US-00005 TABLE 5 DGAT1 DGAT1 Residue Additional SEQ N-terminal C-terminal modifi- C-terminal infor- Type of ID parent parent cation mod mation sequence NO: T. majus T. majus S197A V5-His tag + intron NUCLEIC 103 T. majus T. majus S197A V5-His tag ORF only NUCLEIC 104 T. majus T. majus S197A V5-His tag PEPTIDE 105 Z. mays-L Z. mays-L None V5-His tag + intron NUCLEIC 106 Z. mays-L Z. mays-L None V5-His tag ORF only NUCLEIC 107 Z. mays-L Z. mays-L None V5-His tag PEPTIDE 108 T. majus Z. mays-L None V5-His tag + intron NUCLEIC 109 T. majus Z. mays-L None V5-His tag ORF only NUCLEIC 110 T. majus Z. mays-L None V5-His tag PEPTIDE 111 Z. mays-L T. majus S189A V5-His tag + intron NUCLEIC 112 Z. mays-L T. majus S189A V5-His tag ORF only NUCLEIC 113 Z. mays-L T. majus S189A V5-His tag PEPTIDE 114

(48) The parent DGATs and their modified forms were transferred into the Gateway?-compatible binary pRSh1 Gateway adapted binary vector (Winichayakul et al., 2009, Biotechnol. Appl. Biochem. 53, 111-122) modified by replacement of the CaMV35S promoter replaced with the Brassica napus Napin promoter (SEQ ID NO:115).

(49) Camelina sativa Transformation

(50) C. sativa (cf. Calena) were transformed via Agrobacterium tumefaciens (GV3101) using the floral dip method (adapted from that of Clough and Bent, 1998, Plant J. 16(6):735-745). Essentially seeds were sown in potting mix in 10 cm pots in a controlled environment, approximately 6 weeks after planting the flowers were dipped for 5-14 minutes under vacuum (70-80 inch Hg) in an overnight culture of appropriated Agrobacterium GV3101 cells re-suspended in a floral dip buffer. After vacuum-transformation, plants were kept for 24 h under low light conditions by partly covering with a black plastic sheet. Vacuum transformations can be repeated three times at approximately 10-12 days intervals, corresponding to the flowering duration. Plants were grown in potting mix in a controlled environment (16-h day length, 21-24? C., 65-70% relative humidity).

(51) The T.sub.1 seeds produced can be collected and screened for transformants by germinating and growing seedlings at 22? C. with continuous light on a half-strength MS medium (pH 5.6) selection plate containing 1% (w/v) sucrose, 300 mg/L Timentin, and 25 mg/L DL-phosphinothricin to select for herbicide resistance. T.sub.2 selfed seed populations can also be screened by immuno blot for the presence of the V5 eptiope.

(52) T.sub.2 selfed seeds may be analysed for oil content by GC. Approximately 50 individual transgenic lines (including control lines) may be selected for the next generation (10 plants/line) based on their oil content, or seed weight. T.sub.2 plants may be grown and screened by PCR for copy number and identification of null sibling lines. T.sub.2 seeds may be analysed in triplicate for oil content by NMR or GC/MS.

(53) Results

(54) Addition of Fragments of the DGAT1 Polypeptide of the Invention to other DGAT1 Sequences Enhance Lipid Production in Saccharomyces cerevisiae Relative to that with the other DGAT1 Sequences Alone.

(55) Tables 1-8 show the lipid yields of a variety of chimeric DGAT1s in which the N-terminal or C-terminal region has been derived from the DGAT1 sequence of the invention while the remainder of the protein has been derived from another plant DGAT1. The lipid yields are presented either as grams of lipid produced per liter (which therefore compensates for any differences in growth rate) or have been normalised as a percentage of the lipid yield of the corresponding unmodified parent DGAT1.

(56) A comparison of parent DGAT1s with each other, and with each of the chimeric DGAT1s made using one donor parent for the N-terminal region, and a different donor parent for the C-terminal region are shown in Table 6. The parent DGAT1 sequences are highlighted in bold. Surprisingly the DGAT1 sequence of the invention shows higher activity, in lipid yield production, than any of the other sequences tested.

(57) The lipid yields at 32 hr have been normalised against the highest lipid-producing parent (Z. mays-L) and are presented in ascending order.

(58) TABLE-US-00006 TABLE 6 N-terminal region DGAT1 C-terminal region SEQ Lipid yield Parent DGAT1parent ID NO: as % Z. mays-L Vector only Vector only N/A 31.96 A. thaliana Z. mays-L 63 38.28 A. thaliana A. thaliana 59 64.69 T. majus T. majus 89 77.62 Z. mays-S Z. mays-S 77 81.79 Z. mays-L T. majus 88 83.39 O. sativa-S O. sativa-S 65 84.76 O. sativa-L O. sativa-L 71 88.33 O. sativa-S Z. mays-L 69 95.81 Z. mays-L O. sativa-L 86 96.17 Z. mays-L A. thaliana 84 97.53 Z. mays-L Z. mays-L 83 100.00 T. majus Z. mays-L 94 100.71 O. sativa-L Z. mays-L 75 104.29 Z. mays-L O. sativa-S 85 105.02

(59) The results also show that addition of the Z. mays-L N-terminal region to the C-terminal region of the A. thaliana DGAT1 parent results in increased lipid yield over the full-length A. thaliana DGAT1 sequence (see SEQ ID NO: 84 versus SEQ ID NO: 59).

(60) The results also show that addition of the Z. mays-L N-terminal or C-terminal region to the C-terminal or N-terminal region respectively, of the T. majus DGAT1 sequence results in increased lipid yield over the full-length T. majus DGAT1 sequence (see SEQ ID NO: 88 and 94 versus SEQ ID NO: 89).

(61) The results also show that addition of the Z. mays-L N-terminal or C-terminal region to the C-terminal or N-terminal region respectively, of the O. sativa-S DGAT1 sequence results in increased lipid yield over the full-length O. sativa-S DGAT1 sequence (see SEQ ID NO: 85 and 69 versus SEQ ID NO: 65).

(62) The results also show that addition of the Z. mays-L N-terminal or C-terminal region to the C-terminal or N-terminal region respectively, of the O. sativa-L DGAT1 sequence results in increased lipid yield over the full-length O. sativa-L (see SEQ ID NO: 86 and 75 versus SEQ ID NO: 71).

(63) The results also show that addition of the Z. mays-L N-terminal or C-terminal region to C-terminal or N-terminal region respectively, of the Z. mays-S sequence results in increased lipid yield over the full-length Z. mays-S sequence (see SEQ ID NO: 87 and 81 versus SEQ ID NO: 77).

(64) In summary addition of fragments (either the N-terminal region or C-terminal region) of the Z. mays-L polypeptide of the invention to another DGAT1 sequence can increase the cellular lipid yield attainable by the combined sequence over that of the other DGAT1 sequence.

(65) Addition of Fragments of the DGAT1 Polypeptide of the Invention to other DGAT1 Sequences Enhance Lipid Production in Brassica napus Relative to that with the other DGAT1 Sequences Alone.

(66) Fragments (N-terminal region or C-terminal region) of the Z. mays-L polypeptide of the invention can also be combined with fragments of other plant DGAT1s to raise the oil content in Brassica napus seeds. Tables 7-8 show the seed oil contents from a variety of transgenic plants expressing such chimeric DGAT1s. In Table 7 the seed oil contents are presented both as a % of Dry Matter (DM) and as a normalised percentage of the seed oil content of the corresponding unmodified DGAT1 parents.

(67) TABLE-US-00007 TABLE 7 Oil Increase Oil Increase Oil Increase as % of as % of Transgenic as % of N-terminal C-terminal Construct plant Seed Oil as % Vector DGAT1 DGAT1 description ID # DM Control Parent Parent Vector CV 37.99 0.00 N/A N/A control T. majus N2 39.07 2.84 N/A N/A Z. mays-L N6 38.96 2.55 N/A N/A Tm-ZmS 182-38-4 44.66 17.56 14.31 10.96 Tm-ZmL 183-60-6 44.47 17.06 13.82 14.14 ZmL-Tm 185-24-5 45.27 19.16 16.20 15.87 ZmL-Tm 185-24-9 45.14 18.82 15.86 15.54 ZmL-Tm 185-22-1 44.23 16.43 13.53 13.21 ZmL-Tm 185-22-4 43.20 13.71 10.88 10.57 ZmL-Tm 185-22-9 43.49 14.48 11.63 11.31 ZmL-Tm 185-14-10 44.77 17.85 14.91 14.59 ZmL-Tm 185-9-9 43.73 15.11 12.24 11.93 ZmL-Tm 185-8-4 44.02 15.87 12.99 12.67 ZmL-Tm 185-8-7 45.11 18.74 15.79 15.46 ZmL-Tm 185-8-8 44.62 17.45 14.53 14.21 ZmL-Tm 185-8-9 43.48 14.45 11.60 11.29

(68) In Table 8 the oil contents are presented both on a % of DM basis and as a normalised percentage of the seed oil content of the corresponding segregating null sibling.

(69) TABLE-US-00008 TABLE 8 Oil Increase as % of Construct Transgenic Seed Oil as Null description ID # % DM Sibling Tm-ZmL 183-17-10 43.8 29.43 Tm-ZmL 183-17-4 33.84 N/A Null Sib ZmL-Tm 185-24-5 45.27 19.41 ZmL-Tm 185-24-9 45.14 19.07 ZmL-Tm 185-24-10 37.91 N/A Null Sib ZmL-Tm 185-22-1 44.23 30.09 ZmL-Tm 185-22-4 43.2 27.06 ZmL-Tm 185-22-9 43.49 27.91 ZmL-Tm 185-22-2 34 N/A Nun sib ZmL-Tm 185-9-9 43.73 15.60 ZmL-Tm 185-9-8 37.83 N/A Null Sib

(70) Together these results show that addition of fragments (N-terminal or C-terminal) of the Z. mays DGAT1-L polypeptide of the invention can be added to parts of the T. majus DGATS1 sequence to increase oil yield relative to that produced by the full length T. majus DGAT1.

DISCUSSION

(71) The applicants have thus shown that the novel Z. mays DGAT1-L protein of the invention can be used to manipulate cellular lipid accumulation. The DGAT1 of the invention also has higher activity in increasing cellular lipid content than any other DGAT1 proteins tested by the applicants. The applicants have also shown that subsequences, or fragments, of the DGAT1 of the invention can be combined with parts of other DGAT1 to increase activity over that shown over the other DGAT1 sequences.