Enhanced acyltransferase polynucleotides, polypeptides and methods of use
11312971 · 2022-04-26
Assignee
Inventors
- Nicholas John Roberts (Feilding, NZ)
- Amy Christina Curran (San Diego, CA, US)
- Somrutai Winichayakul (Palmerston North, NZ)
- Marissa Roldan (Palmerston North, NZ)
- Richard William Scott (Palmerston North, NZ)
Cpc classification
C12N15/8247
CHEMISTRY; METALLURGY
C12N9/1029
CHEMISTRY; METALLURGY
C12P7/64
CHEMISTRY; METALLURGY
International classification
Abstract
The invention provides modified DGAT1 proteins that are modified in the N-terminal region upstream of the acyl-Co A binding site. The modified DGAT proteins show enhanced activity, without reduced protein accumulation when expressed in cells. The modified DGAT1 proteins of the invention can be expressed in cells to increase cellular lipid accumulation and/or modify the cellular lipid profile. The invention also provides polynucleotides encoding the modified DGAT1 proteins, cells and compositions comprising the polynucleotides or modified DGAT proteins, and methods using the modified DGAT1 proteins to produce oil.
Claims
1. A method for producing an improved plant DGAT1, the method comprising the steps: a) modifying a plant DGAT1 protein by truncation of 10 or more amino acids from the N-terminal end of an N-terminal region of the protein, wherein the N-terminal region is at least 13 amino acids upstream of a conserved motif ESPLSS (Glu-Ser-Pro-Leu-Ser-Ser) (SEQ ID NO: 112) in the acyl-CoA binding site, b) adding a flexible peptide linker having the sequence MGGGS (SEQ ID NO: 113) to the truncated N-terminal region to produce a modified DGAT1 protein with the flexible peptide linker at the N-terminus of the modified DGAT1 protein, c) testing the capacity the modified DGAT1 protein to produce lipid in a cell relative to that of the unmodified DGAT1 protein, and d) selecting the improved DGAT1 protein on the basis of its increased capacity to produce lipid in a cell relative to that of the unmodified DGAT1 protein.
2. The method of claim 1 in which the unmodified DGAT1 protein in a) has a sequence with at least 95% identity to any one of SEQ ID NO: 30 to 58.
3. The method of claim 1 in which the unmodified DGAT1 protein in a) has a sequence with at least 99% identity to any one of SEQ ID NO: 30 to 58.
4. The method of claim 1 wherein the modification is truncation of all of the N-terminal region of the DGAT1 protein.
5. The method of claim 1 wherein when the improved DGAT1 protein is expressed in the cell, it has altered substrate specificity relative to the unmodified DGAT1.
6. The method of claim 1 wherein the improved DGAT1 protein comprises a sequence with at least 95% identity to any one of SEQ ID NO: 59, 64 and 66, and includes the flexible peptide linker.
7. The method of claim 1 wherein the improved DGAT1 protein comprises a sequence with at least 99% identity to any one of SEQ ID NO: 59, 64 and 66, and includes the flexible peptide linker.
8. An improved plant DGAT1 protein that has: been modified by truncation of 10 or more amino acids from the N-terminal end of an N-terminal region of a plant DGAT1 protein, wherein the N-terminal region is at least 13 amino acids upstream of a conserved motif ESPLSS (Glu-Ser-Pro-Leu-Ser-Ser) (SEQ ID NO: 112) in the acyl-CoA binding site, and to which a flexible peptide linker having the sequence MGGGS (SEQ ID NO: 113) has been added to the truncated N-terminal region, and wherein the improved DGAT1 has increased capacity to produce lipid in a cell relative to that of the unmodified DGAT1 protein, wherein the improved DGAT1 protein comprises a sequence with at least one of: a) at least 95% identity to any one of SEQ ID NO: 59, 64 and 66, or b) at least 99% identity to any one of SEQ ID NO: 59, 64 and 66 and wherein the improved DGAT1 protein includes the flexible peptide linker.
9. A cell that expresses the improved plant DGAT1 protein of claim 8.
10. The cell of claim 9 that produces more lipid than does a control cell that does not express the improved plant DGAT1 protein.
11. The cell of claim 10 which also has an altered lipid profile relative to a control cell that does not comprise the construct.
12. The cell of claim 9 which is also transformed to express at least one of: an oleosin, steroleosin, caloleosin, polyoleosin, and an oleosin including at least one artificially introduced cysteine.
13. A plant that expresses the improved plant DGAT1 protein of claim 8.
14. The plant of claim 13 that produces more lipid, in at least one of its tissues or parts, or as a whole, than does a control plant that does not comprise the improved plant DGAT1 protein.
15. The plant of claim 13 that has an altered lipid profile, in at least one of its tissues or parts, or as a whole, relative to a control plant that does not comprise the improved plant DGAT1 protein.
16. The plant of claim 13 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.
17. A part, propagule or progeny of the plant of claim 13, wherein the part, propagule or progeny expresses the improved DGAT1 protein.
18. The part, propagule or progeny of claim 17 that produces more lipid than does a part, propagule or progeny of a control plant that does not comprise the improved DGAT1 protein.
19. The part, propagule or progeny of claim 17 that has an altered lipid profile relative to a part, propagule or progeny of a control plant that does not comprise the improved DGAT1 protein.
20. An animal feedstock or biofuel feedstock comprising at least one cell of claim 9.
21. A biofuel feedstock or animal feedstock comprising at least one cell of claim 9.
22. A method for producing lipid, the method comprising expressing the improved DGAT1 protein in the cell of claim 9 or in a plant comprising the cell.
23. The method of claim 22 wherein expressing the improved DGAT1 protein in the cell, or plant, leads to production of the lipid in the cell or plant.
24. The method of claim 22 wherein the method includes the step of transforming the cell or plant with a polynucleotide encoding the improved DGAT1 protein.
25. The method of claim 22 which includes the step of extracting the lipid from the cell or plant.
26. A method for producing lipid, the method comprising extracting lipid from at least one of a cell of claim 9 or from a plant, plant part, propagule and progeny comprising the cell, wherein the cell, plant, plant part, propagule or progeny expresses the improved DGAT1 protein.
27. The method of claim 22 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)
(2)
(3)
EXAMPLES
Example 1: Plant DGAT1 Sequence Selection and Splice Site Prediction
(4) The majority of nucleic acid sequences and peptide 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™ 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
(5) TABLE-US-00001 TABLE 1 PROTEIN DGAT1 DNA accession SEQ accession #s SEQ Species #s & 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: Modification of DGAT1 Proteins in the Region Upstream of the Acyl CoA Binding Site
(6)
(7) As a starting point for their experiments the applicants used the DGAT1 sequences of SEQ ID NO: 30, 34, 39, 41, 42 and 44 as summarized in the Table 2 below. These DGAT1s were modified by replacing the sequence 13 residues upstream of the beginning of the N-terminal acyl-CoA binding region (Weselake et al. 2006) with Met-Gly-Gly-Gly-Ser (MGGGS) (Table 2, Region 1 specific modifications/truncation constructs for expression in Saccharomyces cerevisiae). This meant their truncated N-termini were approximately 9 residues longer than the native N-terminus of the Selaginella moellendolffii native DGAT1 (SEQ ID NO: 46). Furthermore this placed the N-terminal truncations 18 residues upstream of the 84 amino acid truncation performed by McFie et al., (2010, JBC., 285:37377-37387) on the mouse DGAT1 which resulted in a large increase in activity but substantial drop in both accumulation of recombinant DGAT1 and its ability to oligomerise. Thus the N-terminal truncations shown in SEQ ID NO: 59, 60, 62, 63, 64 and 65, left 32 residues of the original N-terminal putative cytoplasmic domains intact. In addition we generated a number of other truncated forms of AtDGAT1 in which repeat residues from OsL-DGAT1 were added (Table 2).
(8) Sequences with modifications were synthesised either by GENEART AG (Germany) or GeneScript (U.S.A). Sequences were optimised for expression in Saccharomyces cerevisiae and flanked with appropriate restriction sites to enable the cloning into the pYES2.1 vector (Invitrogen).
(9) TABLE-US-00002 TABLE 2 Starting Modified Sequence Muta- Sequence SEQ ID N-terminal tion SEQ ID NO: Species modification # NO: 30 A. MGGGS 59 thaliana (SEQ ID NO: 113) 30 A. MAPPPGGGSPQQQQGGGSQ 60 thaliana QQQGGGS (SEQ ID NO: 121) 30 A. Multiple individual 61 thaliana additions within N-terminus 34 T. MGGGS 62 majus (SEQ ID NO: 113) 42 O. MGGGS 63 sativa-S (SEQ ID NO: 113) 41 O. MGGGS 64 sativa-L (SEQ ID NO: 113) 44 Z. MGGGS 65 mays-S (SEQ ID NO: 113) 39 Z. MGGGS 66 mays-L (SEQ ID NO: 113)
Example 3: Expression of Modified DGAT1 Sequences in Cells
(10) Expression of Constructs in S. cerevisiae
(11) The parent DGAT1 constructs and modified 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 names of the modified constructs, and the number of their corresponding peptide sequences, are shown in Table 3.
(12) The Saccharomyces cerevisiae quadruple mutant (H1246) in which all four neutral lipid biosynthesis genes have been disrupted (Sandager et al., 2002, The Journal of Biological Chemistry, 277:6478-6482) was transformed as per Elble (1992, BioTechniques 13, 18-20) and selected by the ability to grow in the absence of uracil. Routinely, yeast cells were grown aerobically overnight in a synthetic medium with 0.67% YNB, without uracil (SC-U) and containing 2% glucose. Cells from overnight culture were used to inoculate 200 mL of induction medium (SC-U containing 2% galactose and 1% raffinose) to an initial OD.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.
(13) Lipid Analysis of S. cerevisiae
(14) 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).
(15) 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 μg 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.
(16) 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).
(17) After preconditioning with methanol and equilibrating the Silica column with heptanes the 1 mL TAG extract (including 50 μg 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.
(18) FAMEs of Extracted TAG
(19) To the TAG residue above 10 μL 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-dimethoxypropane (as water scavenger) were added.
(20) 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.
(21) From the top heptane layer, 100 μL was collected and transferred to a flat-bottom glass insert fitted into a vial for FAMES GC/MS analysis.
(22) Protein Extraction and Trypsin Digestion
(23) 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 μL 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.
(24) Trypsin was added to give a final concentration of 25 μg/mL to 50 μL 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 μg/μL. 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) which serves as a control to demonstrate the presence of intact microsomes.
Example 4: Truncation of the N-Terminal Cytoplasmic Region—Region 1 of Plant DGAT1s—Enhances Lipid Production in Saccharomyces cerevisiae
(25) The N-terminal cytoplasmic region can be truncated to raise the lipid yield. Table 3 shows the lipid yields of a variety of DGAT1s in which the N-terminal cytoplasmic region has been truncated. The lipid yields are presented both as grams of lipid produced per litre of (which therefore compensates for any differences in growth rate) as well as normalized as a percentage of the lipid yield of the corresponding unmodified parent DGAT1.
(26) A comparison of A. thaliana, T. majus, O. sativa-S, O. sativa-L, Z. mays-S and Z. mays-L and their N-terminal cytoplasmic region truncated counterparts are shown in Table 3. The lipid yields are presented as grams of lipid per litre of culture at 32 and 48 hours of culture as well as a percentage of the lipid yield obtained with the corresponding native (non-truncated) DGAT1 parent isolated at the same time.
(27) TABLE-US-00003 TABLE 3 Lipid yield Lipid yield Lipid as % of Lipid as % of SEQ yield native yield native Construct ID @ 32 hr parent @ 48 hr parent Description NO: (g FA/L) @ 32 hr (g FA/L) @ 48 hr native 67 0.25 100 0.25 100 A. thaliana N-truncated 59 0.36 147.55 0.37 148.91 A. thaliana Native 68 0.34 100 0.40 100 T. majus N-truncated 62 0.32 95.95 0.37 93.81 T. majus Native 69 0.40 100 0.47 100 O. sativa-S Truncated 63 0.32 79.72 0.35 74.24 O. sativa-S Native 70 0.44 100 0.52 100 O. sativa-L Truncated 64 0.54 122.01 0.53 101.79 O. sativa-L Native 71 0.30 100 0.31 100 Z. mays-S Truncated 65 0.25 80.53 0.25 81.27 Z. mays-S Native 72 0.50 100 0.54 100 Z. mays- L Truncated 66 0.54 108.44 0.68 125.60 Z. mays- L
Example 5: Expression of Modified DGAT1 in Brassica napus
(28) The strategy above can also be used to generate a variety of modified DGAT1 constructs for expression in the seeds of Brassica napus. The parent DGATs and their modified forms can be transferred into the Gateway®-compatible binary vector pMD107 (courtesy of Dr Mark Smith, NRC Saskatoon, SK, Canada, S7N 0W9) to place them under the control of the seed-specific napin promoter (Josefsson et al., 1987, J Biol Chem. 262(25):12196-201; Ellerström et al., 1996, Plant Molecular Biology, Volume 32, Issue 6, pp 1019-1027).
(29) Plant Transformation
(30) B. napus (cv. DH12075) can be transformed via Agrobacterium tumefaciens (GV3101) using the cotyledon co-cultivation method (adapted from that of Maloney et al., 1989, Plant Cell Reports Vol 8, No 4, pg 238-241). Control lines may contain an empty-vector, and when identified, null sibling lines may be subsequently used as true controls.
(31) Approximately 200 T.sub.0 transformed lines may be produced and their corresponding T.sub.1 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 (8 lines).
(32) A total of approximately T.sub.1 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.
Example 6: Expression of Modified DGAT1 in Camelina sativa
(33) The strategy above can also be used to generate a variety of modified DGAT1 constructs for expression in the seeds of Camilina sativa and other plants
(34) Sequences with modifications were synthesised either by GENEART AG (Germany) or GeneScript (U.S.A). Sequences were optimised for expression in Brassica species and included an intron (SEQ ID NO:73) from Arabidopsis thaliana DGAT1—intron 3. Each sequence was flanked with appropriate attL recombination sites to enable the cloning Gateway® adapted vectors.
(35) TABLE-US-00004 TABLE 3 Starting Modified seq N-terminal C-terminal Additional Type of SEQ ID ID # Species modification modification information sequence NO 39 Z. mays-L none V5-His tag + intron NUCLEIC 74 39 Z. mays-L none V5-His tag ORF only NUCLEIC 75 39 Z. mays-L none V5-His tag PEPTIDE 76 39 Z. mays-L MGGGS V5-His tag + intron NUCLEIC 77 39 Z. mays-L MGGGS V5-His tag ORF only NUCLEIC 78 39 Z. mays-L MGGGS V5-His tag PEPTIDE 79
(36) 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:80).
(37) Camelina sativa Transformation
(38) 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).
(39) 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, selfed seed populations can also be screened by immuno blot for the presence of the V5 eptiope.
(40) 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.