USES OF NOVEL FATTY ACID DESATURASES AND ELONGASES AND PRODUCTS THEREOF

Abstract

The invention provides isolated nucleic acid molecules which encode novel fatty acid desaturases and elongases from the organism Emiliana huxleyi. The invention also provides recombinant expression vectors containing desaturase or elongase nucleic acid molecules, host cells into which the expression vectors have been introduced, and methods for large-scale production of long chain polyunsaturated fatty acids (LCPUFAs), e.g. arachidonic acid (ARA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).

Claims

1. A polynucleotide comprising a nucleic acid sequence elected from the group consisting of: a) a nucleic acid sequence having a nucleotide sequence as shown in SEQ ID NOs: 1, 3, 5, 7 or 9; b) a nucleic acid sequence encoding a polypeptide having an amino acid sequence as shown in SEQ ID NOs: 2, 4, 6, 8 or 10; c) a nucleic acid sequence being at least 70% identical to the nucleic acid sequence of a) or b), wherein said nucleic acid sequence encodes a polypeptide having desaturase or elongase activity; d) a nucleic acid sequence encoding a polypeptide having desaturase or elongase activity and having an amino acid sequence which is at least 82% identical to the amino acid sequence of any one of a) to c); and e) a nucleic acid sequence which is capable of hybridizing under stringent conditions to any one of a) to d), wherein said nucleic acid sequence encodes a polypeptide having desaturase or elongase activity.

2. The polynucleotide of claim 1, wherein said polynucleotide further comprises an expression control sequence operatively linked to the said nucleic acid sequence.

3. The polynucleotide of claim 1, wherein said polynucleotide further comprises a terminator sequence operatively linked to the nucleic acid sequence.

4. A vector comprising the polynucleotide of claim 1.

5. A host cell comprising the polynucleotide of claim 1.

6. A method for the manufacture of a polypeptide comprising a) cultivating the host cell of claim 5 under conditions which allow for the production of the said polypeptide; and b) obtaining the polypeptide from the host cell of step a).

7. A polypeptide encoded by the polynucleotide of claim 1.

8. A non-human transgenic organism comprising the polynucleotide of claim 1.

9. The non-human transgenic organism of claim 8, which is a plant, plant part, or plant seed.

10. A method for the manufacture of polyunsaturated fatty acids comprising: a) cultivating the host cell of claim 5 under conditions which allow for the production of polyunsaturated fatty acids in said host cell; and b) obtaining said polyunsaturated fatty acids from the said host cell.

11. A method for the manufacture of polyunsaturated fatty acids comprising: a) cultivating the non-human transgenic organism of claim 8 under conditions which allow for the production of polyunsaturated fatty acids in said non-human transgenic organism; and b) obtaining said polyunsaturated fatty acids from the said non-human transgenic organism.

12. The method of claim 10, wherein said poly-unsaturated fatty acid is arachidonic acid (ARA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).

13. The method of claim 10, further comprising formulating the polyunsaturated fatty acid as oil, lipid or fatty acid composition.

14. The method of claim 13, wherein said oil, lipid or fatty acid composition is feed, a foodstuff, a cosmetic or a medicament.

15. An Antibody or a fragment thereof which specifically recognizes the polypeptide of claim 7.

Description

FIGURES

[0076] FIG. 1 shows a schematical figure of the different enzymatic activities leading to the production of ARA, EPA and DHA.

[0077] FIG. 2 shows a yeast expression experiment with feeding of 22:5n-3 in the prescence (A) and absence (B) of d4Des(Eh).

[0078] FIG. 3 shows a yeast expression experiment with feeding of 20:3n-3 in the prescence (A) and absence (B) of d8Des(Eh).

[0079] FIG. 4 shows a yeast expression experiment with feeding of 18:3n-3 in the prescence (A) and absence (B) of d9Elo(Eh).

[0080] FIG. 5 shows a yeast expression experiment with feeding of 18:3n-6 (GLA) and 18:4n-3 (SDA) in the prescence (A) and absence (B) of d5Elo(Eh).

[0081] FIG. 6 shows a yeast expression experiment with feeding of 20:4n-6 (ARA) and 20:5n-3 (EPA) in the prescence (A) and absence (B) of d5Elo(Eh).

[0082] FIG. 7 shows the expression of d9Elo(Eh) in seeds of two Arabidopsis events. As control seeds not expression d9Elo(Eh) are shown (WT).

[0083] FIG. 8 shows the Acyl-CoA analysis of mature Arabidopsis seeds from both events expressing the d9Elo(Eh) in comparison to seeds not expressing d9Elo(Eh) (Col0)).

[0084] FIG. 9 shows the expression of d9Elo(Eh), d8Des(Eh) and d5Des(Eh) in seeds of various Arabidopsis events.

[0085] FIG. 10 shows gas chromatographic analysis of mature Arabidopsis seeds transformed with the construct OstELO5EmD4. Peaks were quantified and listed in the table below. The products of d5Elo(Ot) and d4Des(Eh) activity are 22:6n-3 (DHA).

[0086] FIG. 11 is a comparison between two d4-desaturases (Tc and Eh) showing that d4Des(Eh) is different from known d4-desaturases in producing a high ratio of DHA:DPA.

[0087] FIG. 12 shows the expression of d5Elo(Eh) in seeds of various Arabidopsis events.

[0088] FIG. 13 is a comparison between three different d6-desaturases and the substrate specificity of d5Des(Eh).

[0089] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the figures, are incorporated herein by reference.

EXAMPLES

Example 1: Organism and Culture Conditions

[0090] Emiliana huxleyi was grown as described in Sciandra et al. (2003) Marine Ecology Progress Series 261:111-122 with following conditions:

[0091] Growth in 50 ml inconical flasks using K/2 medium (Keller et al. (1987) Journal of Phycology 23:633-638). The flasks were placed in a growth chamber at a temperature of 17±0.1° C. under 14L:10D irradiance. Light was provided by fluorescent lamps giving a photon fluxdensity (400 to 700 nm) of 170 μmol photon m-2 s-1.

Example 2: Cloning of Novel Desaturase and Elongase Sequences

[0092] RNA from cells grown as described under Example 1 was extracted using the RNA-extraction Kit from Qiagen, a RACE-library was generated using the RACE-Kit from Clontech. From the RACE-library sequences for desaturase and elongases were amplified with PCR using following primer pairs and PCR conditions.

[0093] PCR reaction (50 μl):

[0094] 5.00 μl Template cDNA

[0095] 5.00 μl 10× Puffer (Advantage-Polymerase)+25 mM MgCl.sub.2

[0096] 5.00 μl 2 mM dNTP

[0097] 1.25 μl je Primer (10 pmol/μL)

[0098] 0.50 μl Advantage-Polymerase

[0099] The Advantage polymerase mix from Clontech was used.

[0100] Reaction conditions of the PCR:

[0101] Annealing: 1 min 55° C.

[0102] Denaturation: 1 min 94° C.

[0103] Elongation: 2 min 72° C.

[0104] Cycles: 35

[0105] Primer pairs used in PCR:

TABLE-US-00002 Name Primer pair (5′ orientation) SEQ ID NO. Eh4ff CCATGGGAGGCGCCGGCGCGAG 11 Eh4rv CTAGTCCGCCTTGAGGTTCTC 12 Eh5ff ACCATGTGCAAGGCGAGCGGCCT 13 Eh5rv TCACCAATCATGAGGAAGGT 14 Eh8ff CCATGGGCAAGGGCGGCAACGC 15 Eh8rv GGGCAGAGATGCCGCACTAG 16 Eh9ff ACCATGCTCGATCGCGCCTCGTC 17 Eh9rv TCACAGCGCCTTGCGGGTAGC 18

[0106] The PCR reactions resulted in following polynucleotide sequences:

TABLE-US-00003 Gene Activity Length in bp SEQ ID NO. D4Des(Eh) D4-desaturase 1280 5 D8Des(Eh) D8-desaturase 1256 1 D9Elo(Eh) D9-elongase  804 3 D5Elo(Eh) Multi-elongase  921 7

[0107] A list of identified full-length coding sequences is shown in Table 1.

TABLE-US-00004 TABLE 1 List of full-length coding sequences and deduced amino acid sequences SEQ ID NO: Gene Coding sequence in bp Amino acid sequence 1 D8Des(Eh) 1254 417 3 D9Elo(Eh) 801 266 5 D4Des(Eh) 1278 425 7 D5Elo(Eh) 918 305

[0108] Open reading frames as shown in Table 1 were cloned into the pESC(Leu) vector from Stratagene according to manufactures reaction conditions. Reactions were transformed into E. coli DH5a and plasmid DNA was isolated. The plasmids pESC-d4Des(Eh), pESC-d8Des(Eh), pESC-d9Elo(Eh), pESC-d5Elo(Eh) were then used for yeast transformation.

Example 3: Yeast Transformation and Growth Conditions

[0109] S. cerevisiae strain INVSC from Invitrogen was transformed with the constructs pESC-d4Des(Eh), pESC-d8Des(Eh), pESC-d9Elo(Eh), pESC-d5Elo(Eh) and pESC using the S. C. EasyComp Transformation Kit (Invitrogen, Carlsbad, Calif.) with selection on leucine-deficient medium.

[0110] Yeast were grown after transformation in complete medium containing all amino acids and nucleotides. Then yeast were plated on different medium containing either the complete medium (SD) or the complete medium lacking leucine (SD-Leu). Only yeast containing pESC-d4Des(Eh), pESC-d8Des(Eh), pESC-d9Elo(Eh), pESC-d5Elo(Eh) or pESC vector can grow on this medium.

Example 4: Functional Expression of Desaturases and Elongases in Yeast and Gas Chromatographic Analysis

[0111] Yeast cells containing the respective pESC plasmids as prepared above were incubated 12 h in liquid DOB-U medium at 28° C., 200 rpm inkubiert and than additional 12 h in induction medium (DOB-U+2% (w/v) galactose+2% (w/v) raffinose). To the induction medium 250 μM of the respecitve fatty acids were added to check for enzyme activity and specificity.

[0112] Yeast cells were analyzed as following:

[0113] Yeast cells from induction medium were harvested by centrifugation (100×g, 5 min, 20° C.) and washed with 100 mM NaHCO.sub.3, pH 8,0, to remove residual fatty acids. From the yeast pellet a total extract of fatty acid methylesters (FAME) was generated by adding 2 ml 1 N methanolic sulfuric acid and 2% (v/v) Dimethoxypropan for 1 h at 80° C. FAME were extracted two times with Petrolether (PE). Not derivased fatty acids were removed by washing with 2 ml 100 mM NaHCO.sub.3, pH 8.0 and 2 ml Aqua dent. The PE-phases were dried with Na.sub.2SO.sub.4 and eluted in 100 μl PE. The samples were then separated with a DB-23-column (30 m, 0.25 mm, 0.25 μm, Agilent) in a Hewlett-Packard 6850-machine with FID using following conditions: oven temperature 50° C. to 250° C. with a rate of 5° C./min and finally 10 min at 250° C.

[0114] The identification of the fatty acids was done using the retention times of known fatty acid standards (Sigma). The method is described e.g. in Napier and Michaelson, 2001, Lipids. 36(8):761-766; Sayanova et al., 2001, Journal of Experimental Botany. 52(360):1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218.

Example 5: Functional Characterization of d4Des(Eh)

[0115] As described above d4Des(Eh) was functionally characterized in yeast. The result of the analysis is shown in FIG. 2. Yeast transformed with pESC-d4Des(Eh) was compared to yeast transformed with pESC (control) while feeding the yeast cells with the fatty acid DPA 22:5n-3. Based on this comparison pESC-d4Des(Eh) exhibits d4-desaturase activity as in the control no 22:6 is observed. Therefore d4Des(Eh) is a functional d4-desaturase.

Example 6: Functional Characterization of d8Des(Eh)

[0116] As described above d8Des(Eh) was functionally characterized in yeast. The result of the analysis is shown in FIG. 3. Yeast transformed with pESC-d8Des(Eh) was compared to yeast transformed with pESC (control) while feeding the fatty acid 20:3n-3. Based on this comparison a new fatty acid was formed compared to the control, which is 20:4n-3. The formation of this fatty acid proves that d8Des(Eh) was functionally expressed and has d8-desaturase activity. The conversion rate of 20:3n-3 to 20:4n-3 was 5%.

Example 7: Functional Characterization of d9Elo(Eh)

[0117] As described above d9Elo(Eh) was functionally characterized in yeast. The result of the analysis is shown in FIG. 4. Yeast transformed with pESC-d9Elo(Eh) was compared to yeast transformed with pESC (control) while feeding the fatty acids 18:3n-3 (ALA) or 18:2 (LA). Based on this comparison a new fatty acid was formed compared to the control, which is 20:3n-3 or 20:2n-6, respectively. The formation of these fatty acids proves that d9Elo(Eh) was functionally expressed and has d9-elongase activity. The conversion rate of 18:3n-3 to 20:3n-3 was 17%, the conversion rate of 18:2n-6 to 20:2n-6 was 49%.

Example 8: Functional Characterization of d5Elo(Eh)

[0118] As described above d5Elo(Eh) was functionally characterized in yeast. The result of the analysis is shown in FIGS. 5 and 6. Yeast transformed with pESC-d5Elo(Eh) was compared to yeast transformed with pESC (control) while feeding the fatty acids 18:3n-6 (GLA), 18:4 (SDA) or 20:4n-6 (ARA), 20:5n-3 (EPA), respectively. Based on this comparison new fatty acids formation was observed when compared to the control, which is 20:3n-6 or 20:4n-3 when fed GLA or SDA and 22:4n-6 or 22:5n-3 when fed ARA or EPA, respectively. The formation of these fatty acids proves that d5Elo(Eh) was functionally expressed and has d5-elongase activity. The conversion rate of GLA was 13%, the conversion rate of 18:4n-3 was 30%, the conversion rate of ARA was 38% and the conversion rate of EPA was 30%. Surprisingly the elongase used a wide variety of substrates of elongation. The specification indicates a multifunctional elongase activity with higher specificities for omega3 fatty acids.

Example 9: Expression of Novel Elongases from Emiliana huxleyi in Plants

[0119] The novel desaturases and elongases were cloned into a plant transformation vector as described in WO2003/093482, WO2005/083093 or WO2007/093776. Exemplary suitable combinations of genes are described in Table 2, 3 and 4.

TABLE-US-00005 TABLE 2 Gene combinations for the production of ARA. Gene Activity SEQ ID NO: D6Des(Ot) Δ6-Desaturase 19 D6Elo(Pp) Δ6-Elongase 21 D5Des(Eh) Δ5-Desaturase 9 D12Des(Ps) Δ12-Desaturase 23 D6Elo(Tp) Δ6-Elongase 25 D8Des(Eh) Δ8-Desaturase 1 D9Elo(Eh) Δ9-Elongase 3

TABLE-US-00006 TABLE 3 Gene combinations for the production of EPA. Gene Activity SEQ ID NO: D6Des(Ot) Δ6-Desaturase 19 D5Elo(Eh) Δ5-Elongase 7 D5Des(Eh) Δ5-Desaturase 9 D12Des(Ps) Δ12-Desaturase 23 D6Elo(Tp) Δ6-Elongase 25 o3-Des(Pi) Omega 3-Desaturase 27 D15Des(Cp) Δ15-Desaturase 29 D8Des(Eh) Δ8-Desaturase 1 D9Elo(Eh) Δ9-Elongase 3

TABLE-US-00007 TABLE 4 Gene combinations for the production of DHA. Gene Activity SEQ ID NO: D6Des(Ot) Δ6-Desaturase 19 D5Elo(Eh) Δ5-Elongase 7 D5Des(Eh) Δ5-Desaturase 9 D12Des(Ps) Δ12-Desaturase 23 D6Elo(Tp) Δ6-Elongase 25 ω3-Des(Pi) Omega 3-Desaturase 27 D15Des(Cp) Δ15-Desaturase 29 D5Elo(Ot) Δ5-elongase 31 D4Des(Eh) Δ4-desaturase 5 D8Des(Eh) Δ8-Desaturase 1 D9Elo(Eh) Δ9-Elongase 3

[0120] Based on the gene combinations as described in Table 2, Table 3 or Table 4 following combinations were designed: [0121] AP2: LuCnl-d5Des(Eh)_LuCnl-d8Des8Eh)_Napin-o3Des(Pi)_Napin-d12Des(Ps)_LuCnl-d9Elo(Eh) [0122] OstELO5EmD4: VfUSP-d6Elo(Pp)_LuCnl-d5Des8Tc)_VfSBP-d6Des(Ot)_Napin-o3Des(Pi)_Napin-d12Des(Ps)_LuCnl-d5Elo(Ot)_LuCnl-d4Des(Eh) [0123] OstELO5TcD4: VfUSP-d6Elo(Pp)_LuCnl-d5Des8Tc)_VfSBP-d6Des(Ot)_Napin-o3Des(Pi)_Napin-d12Des(Ps)_LuCnl-d5Elo(Ot)_LuCnl-d4Des(Tc)

[0124] Transgenic rapeseed lines were generated as described in Deblaere et al, 1984, Nucl. Acids. Res. 13, 4777-4788 and seeds of transgenic rapeseed plants are analyzed as described in Qiu et al. 2001, J. Biol. Chem. 276, 31561-31566.

[0125] Transgenic Arabidopsis plants were generated as described in Bechtholdt et al. 1993 C. R. Acad. Sci. Ser. III Sci. Vie., 316, 1194-1199. Seeds of transgenic Arabidopsis plants expressing d9Elo(Eh) by using the seed-specific promoter Glycinin from soybean (Lelievre et al. (1992) Plant Physiol 98:387-391) were analyzed by gas chromatography (FIG. 7). Compared to non-transgenic control plants (WT) there are changes in the fatty acid profile, proving that d9Elo(Eh) was functionally expression in seeds. The major shifts in the fatty acid profile is directed to a 10 fold increase in the fatty acid 20:2n-6 and 20:3n-3 (FIG. 7). Therefore d9Elo(Eh) exhibits a Δ9-elongase activity, which is consistent with the yeast characterization. Further, the levels of 18:2 and ALA in the transgenic events expressing d9Elo(Eh) are lowered compared to WT, as these fatty acids are direct substrates for the d9Elo(Eh). Further, the endogenous elongation system in the plant is unchanged as levels of 20:1 and 22:1 are similar between transgenic plants expression d9Elo(Eh) and WT control. This indicates that the expression of d9Elo(Eh) does not disturb endogenous elongation process, but delivers additional activity.

[0126] To further prove the activity of d9Elo(Eh) expressed in seeds of Arabidopsis thaliana AcylCoA-measurements were done. Substrates and products of the d9Elo(Eh) elongation reaction are AcylCoA-esters, which are then further incorporated into triacylglycerides (oil). The analysis of the acylCoA-pool reveals the formation and flux of the elongation reaction.

[0127] FIG. 8 summarizes the AcylCoA measurements for Arabidopsis event expressing d9Elo(Eh) in comparison to controls not expressing d9Elo(Eh) (Col0). The change in the chromatogram is indicated by a star. At this position a massive amount of 20:2n-6 is detected, which is much lower in the control. The conditions for separation of the fatty acid CoA-esters does not allow the detection of 20:3n-3 as this CoA ester is not separated from 18:3CoA.

[0128] The massive occurrence of 20:2n-6-CoA proves the expression of d9Elo(Eh) as this is the direct product of its enzymatic activity.

[0129] Further, transgenic Arabidopsis lines have been generated to validate the activity of d8Des(Eh) and d5Des(Eh). Vector AP2 has been constructed according to standard molecular biology steps as described in WO2003/093482, WO2005/083093, WO2007/093776 or WO2009/016202 and transformed into Arabidopsis thaliana as described above. Analysis of transgenic seeds is shown in FIG. 9. The products of d9Elo(Eh) are 20:2 and 20:3n-3.

[0130] Further, transgenic Arabidopsis lines have been generated to validate the activity of d4Des(Eh). Construct OstELO5EmD4 was transformed into Arabidopsis as described above and seeds of a number of individual lines have been analyzed by gas chromatography (FIG. 10). The activity of d4Des(Eh) is demonstrated by the formation of DHA 22:6 (last column). All lines show the production of DHA with levels of up to 4.7%. Of special interest is the ratio of DHA to DPA. Surprisingly the ratio of d4Des(Eh) is much higher than in d4-desaturases known in the art. A comparison against the d4-desaturase from Thraustochytrium ssp. of WO2002/026946 is shown in FIG. 11. The enzyme from Thraustochytrium ssp. showed so far highest levels of DHA (WO2005/083093), but with an unfavorable ratio of DPA to DHA. A high ratio of DHA:DPA is for the commercial use of such oils of importance.

[0131] Further, transgenic Arabidopsis lines have been generated to validate the activity of d5Elo(Eh). Construct EmELO5TcD4 was transformed into Arabidopsis as described above and seeds of a number of individual lines have been analyzed by gas chromatography (FIG. 12). The activity of d5Elo(Eh) is demonstrated by the formation of DPA 22:5 and DHA 22:6. Most lines show the production of these two fatty acids, proofing that d5Elo(Em) is functionally expressed in the seeds.

[0132] Further, transgenic Arabidopsis lines have been generated to validate the activity and substrate specificity of d5Des(Eh). For this purpose two Δ6-desaturases were selected based on their different substrate specificity. The borageΔ6 is expected to use phosphatidylcholin-18:2 as substrate (WO96/21022), whereas the Ostreococcus Δ6 (OstrΔ6) uses Acyl-CoA ester (WO2005/012316). In combination with the d6-elongase from Physcomitrella patens (WO2001/059128) both d6-desaturases produce DGLA or 20:4n-3, respectively. The ratio of ARA to EPA is for the borageΔ6 2.9, for the OstrΔ6 2.3. It is noted that the use of OstrΔ6 results in 3-4 times higher levels of products compared to the borageΔ6. The further combination of the d5Des(Eh) resulted in the production of ARA and EPA, demonstrating the functionality of the d5Des(Eh). The conversion of d5Des(Eh) of DGLA to ARA is 29% (borageΔ6) or 47% (OstrΔ6). For 20:4n-3 to EPA it is 33% (borageΔ6) or 26% (OstrΔ6).

[0133] Based on these results it is concluded that for Acyl-CoA substrates d5Des(Eh) is specific for the omega6 fatty acid DGLA. This is a novel substrate specificity not observed in the state of the art d5-desaturases.

REFERENCE LIST

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[0146] All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.