Source: https://patents.google.com/patent/US8143383
Timestamp: 2018-04-21 06:20:42
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US8143383B2 - Δ-6 desaturase and uses thereof - Google Patents
Δ-6 desaturase and uses thereof Download PDF
US8143383B2
US8143383B2 US12878807 US87880710A US8143383B2 US 8143383 B2 US8143383 B2 US 8143383B2 US 12878807 US12878807 US 12878807 US 87880710 A US87880710 A US 87880710A US 8143383 B2 US8143383 B2 US 8143383B2
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EXAMPLE 1 Design of Degenerate Oligonucleotides for the Isolation of Desaturases from Fungi and cDNA Library Construction
T. aureum (ATCC 34304) cells were grown in BY+ Media (Difco #790) at room temperature for 4 days, in the presence of light, and with constant agitation (250 rpm) to obtain the maximum biomass. These cells were harvested by centrifugation at 5000 rpm for 10 minutes and rinsed in ice-cold RNase-free water. These cells were then lysed in a French press at 10,000 psi, and the lysed cells directly collected into TE buffered phenol. Proteins from the cell lysate were removed by repeated phenol:chloroform (1:1 v/v) extraction, followed by a chloroform extraction. The nucleic acids from the aqueous phase were precipitated out at −70° C. for 30 minutes using 0.3M (final concentration) sodium acetate (pH 5.6) and one volume of isopropanol. The precipitated nucleic acids were collected by centrifugation at 15,000 rpm for 30 minutes at 4° C., vacuum-dried for 5 minutes and then treated with DNaseI (RNase-free) in 1× DNase buffer (20 mM Tris-Cl, pH 8.0; 5 mM MgCl2) for 15 minutes at room temperature. The reaction was quenched with mM EDTA (pH 8.0) and the RNA further purified using the Qiagen RNeasy Maxi kit (Qiagen, Valencia, Calif.) as per the manufacturer's protocol.
mRNA was isolated from total RNA from each organism using oligo dT cellulose resin. The pBluescript II XR library construction kit (Stratagene, La Jolla, Calif.) was then used to synthesize double stranded cDNA which was then directionally cloned (5′ EcoRI/3′ XhoI) into pBluescript II SK(+) vector. The S. diclina and T. aureum libraries contained approximately 2.5×106 clones each with an average insert size of approximately 700 bp. Genomic DNA from PUFA producing cultures of S. diclina and T. aureum was isolated by crushing the culture in liquid nitrogen and purified using Qiagen Genomic DNA Extraction Kit (Qiagen, Valencia, Calif.).
Primer RO753
Primer RO754
EXAMPLE 2 Isolation of Δ6-Desaturase Nucleotide Sequences from Saprolegnia diclina (ATCC 56851)
To isolate the Δ6-desaturase gene, various permutations and combinations of the above mentioned degenerate oligonucleotides were used in PCR reactions. Of the various primer sets tried, the only primers to give distinct bands were RO834/RO838. PCR amplification was carried out in a 100 μl volume containing: 2 μl of the first strand cDNA template, 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 μm each deoxyribonucleotide triphosphate and 2 pmole of each primer. Thermocycling was carried out at two different annealing temperatures, 42° C. and 45° C., and these two PCR reactions were combined, resolved on a 1.0% agarose gel, and the band of −1000 bp was gel purified using the QiaQuick Gel Extraction Kit (Qiagen, Valencia, Calif.). The staggered ends on these fragments were ‘filled-in’ using T4 DNA polymerase (LifeTechnologies, Rockville, Md.) as per manufacturer's specifications, and these DNA fragments were cloned into the PCR-Blunt vector (Invitrogen, Carlsbad, Calif.). The recombinant plasmids were transformed into TOP10 supercompetent cells (Invitrogen, Carlsbad, Calif.), and clones were sequenced.
a. Clone#20-2 was partially sequenced and the deduced amino acid sequence from 702 bp showed 30.2% identity with Δ6-desaturase from Mortierella alpina as the highest scoring match in a TfastA search.
b. Clone #30-1 was partially sequenced, and the deduced amino acid sequence of 687 bp showed 48.5% amino acid identity with Mortierella alpina's Δ6-desaturase as the highest scoring match in a TfastA search. These two sequences also overlapped each other indicating they belonged to a single putative Δ6-desaturase from S. diclina. This novel Δ6-desaturase sequence was then used to design primers to retrieve the 3′- and the 5′-end of the full-length Δ6-desaturase gene from the cDNA library generated from the mRNA of S. diclina.
To isolate the 3′-end, PCR amplification was carried out using plasmid DNA purified from the cDNA library as the template and oligonucleotides RO923 (SEQ ID NO:7) (5′-CGGTGCAGTGGTGGAAGAACAAGCACAAC-3′) and RO899 (SEQ ID NO:8) (5′-AGCGGATAACAATTTCACACAGGAAACAGC-3′). Oligonucleotide RO923 was designed based on the #20-2 fragment of this putative Δ6-desaturase, and oligonucleotide RO899 corresponded to sequence from the pBluescript II SK(+) vector used for preparation of the cDNA library. Amplification was carried out using 10 pmols of each primer and the Taq PCR Master Mix (Qiagen, Valencia, Calif.). Samples were denatured initially at 94° C. for 3 minutes, followed by 30 cycles of the following: 94° C. for 1 minute, 60° C. for 1 minute, 72° C. for 2 minutes. A final extension cycle at 72° C. for 10 minutes was carried out before the reaction was terminated. The PCR fragments were resolved on a 0.8% agarose gel and gel purified using the Qiagen Gel Extraction Kit. The staggered end on these fragments were ‘filled-in’ using T4 DNA polymerase (LifeTechnologies, Rockville, Md.) as per manufacturer's specifications, and these DNA fragments were cloned into the PCR-Blunt vector (Invitrogen, Carlsbad, Calif.). The recombinant plasmids were transformed into TOP10 supercompetent cells (Invitrogen, Carlsbad, Calif.), and clones were sequenced. Clone sd2-2 contained a 958 bp insert which was identified to contain the 3′-end of the putative Δ6-gene based on sequence homology with known Δ6-desaturases and the presence of the ‘TAA’ stop codon and Poly A tail.
To isolate the 5′-end of this Δ6-desaturase from Saprolegnia diclina, the oligonucleotide RO939 (SEQ ID NO:9) (5′-CGTAGTACTGCTCGAGGAGCTTGAGCGCCG-3′) was designed based on the sequence of the #30-1 fragment identified earlier. This oligonucleotide was used in combination with RO898 (SEQ ID NO:10) (5′-CCCAGTCACGACGTTGTAAAACGACGGCCAG-3′) (designed based on the sequence of from the pBluescript SK(+) vector) to PCR amplify the 5′-end of the Δ6-desaturase from the cDNA library. In this case, the Advantage-GC cDNA PCR kit (Clonetech, Palo Alto, Calif.) was used to overcome PCR amplification problems that occur with GC rich regions, predicted to be present at the 5′-end of this β6-desaturase. PCR thermocycling conditions were as follows: The template was initially denatured at 94° C. for 1 minute, followed by 30 cycles of [94° C. for 30 seconds, 68° C. for 3 minutes], and finally an extension cycle at 68° C. for 5 minutes. The PCR products thus obtained were cloned into the PCR-Blunt vector (Invitrogen, Carlsbad, Calif.) following the same protocol as described above. Clone sd21-2 was thus obtained that contained a 360 bp insert that contained the putative ‘ATG’ start site of the novel Δ6-desaturase. The deduced amino acid sequence of this fragment, when aligned with known Δ6-desaturases showed 37-45% identity.
(5′-ATACGTAAGCTTTTACATGGCGGGAAACTCCTTGAAGAACTCGAT
CG-3′)
PCR amplification was carried out using 200 ng of the cDNA library plasmid template, 10 pmoles of each primer and the Taq PCR Master Mix (Qiagen, Valencia, Calif.), or 200 ng of genomic DNA, 10 pmoles of each primer, and the Advantage-GC cDNA PCR kit (Clonetech, Palo Alto, Calif.). Thermocycling conditions were as follows: the template was initially denatured at 94° C. for 1 minute, followed by 30 cycles of [94° C. for 30 seconds, 68° C. for 3 minutes], and finally an extension cycle at 68° C. for 5 minutes. The PCR product thus obtained was digested with EcoRI/HindIII and cloned into the yeast expression vector pYX242 (Invitrogen, Carlsbad, Calif.) to generate clones pRSP1 (genomic DNA-derived) and pRSP2 (library-derived) which were then sequenced and used for expression studies.
EXAMPLE 3 Isolation of Δ5-Desaturase Nucleotide Sequences from Saprolegnia diclina (ATCC 56851)
Saprolegnia diclina (ATCC 56851) produces both arachidonic acid (ARA, 20:4 n-6) and eicosapentanoic acid (EPA, 20:5 n-3); thus, it was thought to have, perhaps, a Δ5-desaturase which can convert dihomo-gamma-linolenic acid (DGLA, 20:3n-6) to arachidonic acid (ARA, 20:4 n-6). As with the Δ6-desaturase isolation, for the β5-desaturase isolation from S. diclina, various combinations of the degenerate primers were used in PCR reactions, using first strand cDNA as the template. The primer combination, RO753 and RO754, generated a distinct band of 588 bp using the following PCR conditions: 2 μl of the first strand cDNA template, 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 μm each deoxyribonucleotide triphosphate, 2 pmole of each primer and 1 U cDNA polymerase (Clonetech, Palo Alto, Calif.), in a final reaction volume of 50 μl. Thermocycling was carried out as follows: an initial denaturation at 94° C. for 3 minutes, followed by 35 cycles of: denaturation at 94° C. for 30 seconds, annealing at 60° C. for 30 seconds and extension at 72° C. for 1 minute. This was followed by a final extension at 72° C. for 7 minutes, and the reaction was terminated at 4° C. This fragment thus generated was cloned (clone #18-1), sequenced and, when translated, showed 43% amino acid identity with Mortierella alpina Δ5-desaturase (Genbank accession # AF067654) and 38.7% identity with Dictyostelium discoideum Δ5-desaturase (Genbank accession # AB029311). The second PCR fragment was identified using Primers RO834 and RO838 in the reaction described in Example 2. This fragment, of approximately 1000 bp in length, was cloned (Clone #20-8) and the deduced amino acid sequence derived from 775 bp showed 42% identity with Δ5-desaturase from Dictyostelium discoideum Δ5-desaturase (Genbank accession # AB029311). These two sequences, #18-1 and #20-8, overlapped each other indicating they belonged to a single putative Δ5-desaturase from S. diclina. These sequences were then used to design primers to retrieve the 3′- and the 5′-end of the novel Δ5-desaturase gene from the cDNA library generated from the mRNA of S. diclina.
To isolate the 3′-end of this putative Δ5-desaturase, PCR amplification was carried out using plasmid DNA purified from the cDNA library, as the template and oligonucleotides RO851 (SEQ ID NO:15) (5′-CCATCAAGACGTACCTTGCGATC-3′) and RO899 (SEQ ID NO:8) (5′-AGCGGATAACAATTTCACACAGGAAACAGC-3′). Oligonucleotide RO851 was designed based on the #18-1 fragment of this putative Δ5-desaturase, and oligonucleotide RO899 corresponded to sequence from the pBluescript II SK(+) vector. Amplification was carried out using 200 ng of template plasmid DNA, 10 pmoles of each primer and the Tag PCR Master Mix (Qiagen, Valencia, Calif.). Samples were denatured initially at 94° C. for 3 minutes, followed by 35 cycles of the following: 94° C. for 30 seconds, 60° C. for 30 seconds, 72° C. for 1 minutes. A final extension cycle at 72° C. for 7 minutes was carried out before the reaction was terminated. The PCR fragments were cloned into the PCR-Blunt vector (Invitrogen, Carlsbad, Calif.) as per the protocol described in Example 2. The recombinant plasmids were transformed into TOP10 supercompetent cells (Invitrogen, Carlsbad, Calif.), and clones were sequenced. Clone sd12-11 contained a 648 bp insert which contained the 3′-end of the putative Δ5-gene based on sequence homology with known Δ5-desaturases and the presence of the ‘TAA’ stop codon and polyA tail.
The 5′-end of this Δ5-desaturase from Saprolegnia diclina was isolated using primers RO941 and RO898. The oligonucleotide RO941 (SEQ ID NO:16) (5′-GCTGAACGGGTGGTACGAGTCGAACGTG-3′) was designed based on the sequence of the #20-8 fragment identified earlier. This oligonucleotide was used in combination with RO898 (SEQ ID NO:10) (5′-CCCAGTCACGACGTTGTAAAACGACGGCCAG-3′) (designed based on the sequence of from the pBluescript II SK(+) vector) in a PCR amplification reaction using the cDNA library plasmid DNA as the template. Here the Advantage-GC cDNA PCR kit (Clonetech, Palo Alto, Calif.) was used as per the manufacturer's protocol, and the thermocycling conditions were as follows: an initial denaturation was carried out at 94° C. for 1 minute, followed by 30 cycles of [denaturation at 94° C. for 30 seconds, annealing and extension 68° C. for 3 minutes], and a final extension cycle at 68° C. for 5 minutes. These PCR products were purified, cloned into the PCR-Blunt vector (Invitrogen, Carlsbad, Calif.), and sequenced as described above. Clone sd24-1 was identified to contain a 295 bp insert that contained the putative ‘ATG’ start site of the novel Δ5-desaturase. Analysis of the deduced amino acid sequence of this fragment showed regions of high homology with known Δ5-desaturases and also the presence of a cytochrome b5 domain.
a. RO 953
(5′-ACGAGAGAATTCATGGCCCCGCAGACGGAGCTCCGCCAGCGC-3′)
that contained sequence from the 5′ end of clone sd24-1 as well as an EcoRI site (underlined) to facilitate cloning into a yeast expression vector; and
b. RO956
(5′-AAAAGACTCGAGTTAGCCCATGTGGATCGTGGCGGCGATGCCCTG
C-3′)
that contained sequence from the 3′ end of clone sd12-11 including the stop codon as well as a XhoI site (underlined) for cloning in an expression vector.
EXAMPLE 4 Expression of S. diclina Desaturase Genes in Baker's Yeast
a. Stearic acid (18:0) (conversion to oleic acid would indicate Δ9-desaturase activity)
b. Oleic acid (18:1) (conversion to linoleic acid would indicated Δ12-desaturase activity)
c. Linoleic acid (18:2 n-6) (conversion to alpha-linolenic acid would indicate Δ15-desaturase activity and conversion to gamma-linolenic acid would indicate Δ6-desaturase activity)
d. Alpha-linolenic acid (18:3 n-3) (conversion to stearidonic acid would indicate Δ6-desaturase activity)
e. Dihomo-gamma-linolenic acid (20:3 n-6) (conversion to arachidonic acid would indicate Δ5-desaturase activity).
The negative control strain was S. cerevisiae 334 containing the unaltered pYX242 vector, and these were grown simultaneously. The cultures were vigorously agitated (250 rpm) and grown for 48 hours at 24° C. in the presence of 50 μm (final concentration) of the various substrates. The cells were pelleted and vortexed in methanol; chloroform was added along with tritridecanoin (as an internal standard). These mixtures were incubated for at least an hour at room temperature or at 4° C. overnight. The chloroform layer was extracted and filtered through a Whatman filter with 1 gm anhydrous sodium sulfate to remove particulates and residual water. The organic solvents were evaporated at 40° C. under a stream of nitrogen. The extracted lipids were then derivitized to fatty acid methyl esters (FAME) for gas chromatography analysis (GC) by adding 2 ml of 0.5 N potassium hydroxide in methanol to a closed tube. The samples were heated to 95° C.-100° C. for 30 minutes and cooled to room temperature. Approximately 2 ml of 14% borontrifluoride in methanol were added and the heating repeated. After the extracted lipid mixture cooled, 2 ml of water and 1 ml of hexane were added to extract the FAME for analysis by GC. The percent conversion was calculated by dividing the product produced by the sum of (the product produced+the substrate added) and then multiplying by 100.
EXAMPLE 5 Co-Expression of S. diclina Desaturases with Elongases
EXAMPLE 6 Isolation of Δ5-Desaturase Nucleotide Sequences from Thraustochvtrium aureum (ATCC 34303)
To isolate putative desaturase genes, total RNA was Isolated as described in Example 2. Approximately 5 μg was reverse transcribed using the SuperScript Preamplification system (LifeTechnologies, Rockville, Md.) as shown in Example 2 to produce first strand cDNA. Using the degenerate primers RO834 (SEQ ID NO:1) and 838 (SEQ ID NO:4) designed with the block maker program in a 50 μl reaction, the following components were combined: 2 μl of the first strand cDNA template, 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 μm each deoxyribonucleotide triphosphate, 2 pmole final concentration of each primer and cDNA polymerase (Clonetech, Palo Alto, Calif.). Thermocycling was carried out as follows: an initial denaturation at 94° C. for 3 minutes, followed by 35 cycles of denaturation at 94° C. for 30 seconds, annealing at 60° C. for 30 seconds and extension at 72° C. for 1 minute. This was followed by a final extension at 72° C. for 7 minutes. Two faint bands of approximately 1000 bp were separated on a 1% agarose gel, excised, and purified with the QiaQuick Gel Extraction Kit (Qiagen, Valencia, Calif.). The ends were filled in with T4 DNA polymerase and the blunt-end fragments cloned into PCR Blunt as described in Example 2. Sequencing of the obtained clones identified the partial sequence of 680 bp from clone 30-9 whose translation of 226 amino acids had 31.5% identity with Δ6-desaturase from adult zebrafish (Genbank accession number AW281238). A similar degree of amino acid (29.6%-28.7%) homology was found with human Δ6-desaturase (Genbank accession number AF126799), Physcomitrella patens (moss) Δ6-desaturase (Genbank accession number AJ222980), Brassica napus (canola) Δ8-sphingolipid desaturase (Genbank accession number AJ224160), and human Δ5-desaturase (ATCC accession number 203557, Genbank accession number AF199596). Since there was a reasonable degree of amino acid homology to known desaturases, a full-length gene encoding a potential desaturase was sought to determine its activity when expressed in yeast.
To isolate the 3′ end of the gene, 10 pmol of primer RO936 (SEQ ID NO:23) (5′-GTCGGGCAAGGCGGAAAAGTACCTCAAGAG-3′) and vector primer RO899 (SEQ ID NO:8) were combined in a reaction with 100 ng of purified plasmid from the T. aureum cDNA library in reaction volume of 100 μl in Taq PCR Master Mix (Qiagen, Valencia, Calif.). Thermocycling conditions were as follows: an initial melt at 94° C. for 3 minutes followed by 30 cycles of 94° C. for 1 minute, 60° C. for 1 minute, and 72° C. for 2 minutes. This was followed by an extension step of 10 minutes at 72° C. Several bands, including the predicted size of 1.2 kb, were separated on a 1% agarose gel and purified as stated earlier. Also as described earlier, the ends of the fragments were blunt ended, cloned into PCR Blunt and sequenced. Fragment #70-2 of approximately 1.2 kb was sequenced and contained an open reading frame and a stop codon, which overlapped fragment 30-9.
To isolate the 5′ end of the gene, RO937 (SEQ ID NO:24) (5′-AAACCTGTAGACAATGTGGAGGGGCGTGGG-3′) and RO 899 (SEQ ID NO:8) were used in a 50 μl PCR reaction with Advantage-GC cDNA PCR kit (Clonetech, Palo Alto, Calif.), as per the manufacturer's protocol, with 100 ng of purified plasmid DNA from the library and 10 pmol of each primer. The thermocycling conditions were as follows: An initial denaturation was carried out at 94° C. for 1 minute, followed by 30 cycles of [denaturation at 94° C. for 30 seconds, annealing and extension 68° C. for 3 minutes], and a final extension cycle at 68° C. for 5 minutes. A band of approximately 500 bp, in the range of the expected size, was gel purified, blunt ended and cloned into PCR Blunt as previously described. Clone 95-2 contained an open reading frame with a start codon. This fragment also overlapped with clone 30-9, indicating that they were indeed pieces of the same gene.
EXAMPLE 7 Expression of T. aureum Desaturase Gene in Baker's Yeast
The clone pRTA4 containing the full-length gene was transformed into the yeast host S. cerevisiae 334 and plated on selective media as described in Example 4. The cultures were grown at 24° C. for 48 hours in minimal media lacking leucine with 50 μm of exogenous free fatty acid added as a substrate as shown in Table 5. The only conversion of a substrate was DGLA (20:3n-6) to ARA (20:4n-6). The conversion of 23.7% of the added DGLA indicates that this gene encodes for a Δ5-desaturase.
EXAMPLE 8 Co-Expression of T. aureum Desaturase Gene with Elongases
Fatty Acids % Total g/8 fl oz* 9/L*
The high-fat and low-carbohydrate content of Oxepa is designed to minimize carbon dioxide (C02) production. High C02 levels can complicate weaning in ventilator-dependent patients. The low level of carbohydrate also may be useful for those patients who have developed stress-induced hyperglycemia.
5. The isolated nucleic acid molecule of claim 4, wherein said fungus is Saprolegnia diclina.
6. An isolated nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to SEQ ID NO:14, wherein said nucleic acid sequence encodes a functionally active Δ6-desaturase.
9. The isolated nucleic acid sequence of claim 6, wherein said nucleic acid sequence is isolated from Saprolegnia diclina.
10. A method of producing a desaturase in a host cell in vitro comprising the steps of:
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