CN1283230A - Desaturated enzyme - Google Patents
Desaturated enzyme Download PDFInfo
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- CN1283230A CN1283230A CN98812505A CN98812505A CN1283230A CN 1283230 A CN1283230 A CN 1283230A CN 98812505 A CN98812505 A CN 98812505A CN 98812505 A CN98812505 A CN 98812505A CN 1283230 A CN1283230 A CN 1283230A
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- fatty acid
- polypeptide
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
This invention relates to cDNA sequences encoding DELTA5-fatty acid desaturases comprising the sequences shown in SEQ.1 and SEQ.2.
Description
The present invention relates to the to encode dna sequence dna of △ 5-fatty acid desaturase, the △ 5-fatty acid desaturase of coding, and the application of △ 5-fatty acid desaturase.
Polyunsaturated fatty acid is very important on neuropharm (neutraceutically), because they have unique sanatory activity; They are also very important on biomedicine, because they are having the potential pharmaceutical use aspect the treatment specified disease situation.
Polyunsaturated fatty acid is the precursor of two class major metabolite-prostanoids (comprising prostaglandin(PG) and thromboxane) and leukotriene one.△ 5-fatty acid desaturase comes catalysis two height (dihomo) gamma linolenic acid (DHL) to be transformed into arachidonic acid (AA) by the two keys of introducing on each substrate △ 5 carbon and catalysis eicosatetraenoic acid (ETA) is transformed into timnodonic acid (EPA), and it exists so that endoplasmic reticulum is conjugated protein in native state.
Arachidonic acid is 20 carbochains with 4 two keys, and it is extremely important in body metabolism, because it is to contain one 5 yuan isocyclic prostaglandin(PG)s-20-chain fatty acid synthetic precursor.Prostaglandin(PG) is the conditioning agent of hormonal action, and its potential effect comprises stimulates inflammation, regulate the blood flow, the control ion transportation that flow into certain organs passes through some film and regulate the cynapse transmission.Prostaglandin(PG) also may be used as contraceptive bian, because they can suppress the secretion of progesterone.Therefore, by control polyunsaturated fatty acid precursor synthetic expression level regulate prostaglandin(PG) synthetic ability medically with commercial all be very important.
The increase of polyunsaturated fatty acid importance in food and pharmaceutical industries has caused the increase of its demand to surpass present production level, therefore needs additional source high-quality, low-cost polyunsaturated fatty acid.
The commercial source of polyunsaturated fatty acid comprises spermatophyte, marine fishes and the Mammals through selecting through selecting at present, and traditional processing technology of extracting polyunsaturated fatty acid from these sources comprises solvent extraction, condensation, formation urea adduct and distillation.Yet present source has following shortcoming: production level and quality lack available plant and fish source with season and climate change, and the cost height of refining inferior grade oil.Expensively add that insufficient production level has hindered the development of polyunsaturated fatty acid in the coml utility value.
People have done other source that polyunsaturated fatty acid is developed in many effort, now its biosynthetic constitutive gene and proteins encoded are studied.For example, in oil grain, have many advantages with the biosynthesis gene of polyunsaturated fatty acid is engineered, for example can be used to a large amount of gamma linolenic acid esters of scale operation (GLA), dihomo-gamma-linolenic acid ester (DHGLA), arachidonic acid (AA), timnodonic acid (EPA) and docosahexenoic acid (DHA).Its practicality by Borrago officinalis △ 6 delta 8 desaturase genes in tobacco, express cause producing GLA and therapic acid (18:4, people such as Soyanova, (1997), PNAS 94,9411-9414) are described.Obtain more polyunsaturated fatty acid synthetic biosynthesis gene, this has opened up the possibility of producing GLA, AA, EPA and DHA in oil grain at least thereupon, and can control the lipid type of assembling.The benefit that can obtain from these crops comprises: the required polyunsaturated fatty acid that continues to provide cheapness on a large scale, the characteristic of customization polyunsaturated fatty acid to be satisfying special nutritional needs, and produces the unusual lipid acid with predeterminated level and unsaturated position in fine chemistry industry.
The another kind of method of producing polyunsaturated fatty acid is to utilize the biosynthesis ability of unicellular lower eukaryote, and these unicellular lower eukaryotes for example are algae, bacterium, fungi (comprising phycomycete), and they can synthesize all polyunsaturated fatty acids, and can grow with technical scale.These biologies are carried out gene transformation, might develop excessive type of production bacterial strain, and can handle the distribution pattern of polyunsaturated fatty acid by genetic engineering modified approach.
Now cloned △ 5 and △ 6 fatty acid desaturases of fungi, their sequence is disclosed among WO98/46763, WO98/46764 and the WO98/46764.
The metabolism of polyunsaturated fatty acid is of paramount importance in the body metabolism.These acid play an important role to suitably keeping homeostasis by the eicosane class, and relevant with pathologic, physiologic syndrome with serious physiological.
The present inventor is grown the dna sequence dna that separates and identified zygomycota Mortierella alpina (Mortierella alpina) encoding function △ 5-fatty acid desaturase the filamentous fungus from soil astoundingly.
In addition, the present inventor also separates and has identified the dna sequence dna of encoding function △ 5-fatty acid desaturase among the nematode Caenorhabditis elegans astoundingly.Think the relation of this dna sequence dna and people △ 5-fatty acid desaturase of encoding function △ 5-fatty acid desaturase, may be all nearer than separating any △ 5-fatty acid desaturase gene order that obtains so far.
Except dna sequence dna encoded polypeptide of the present invention has potential human advantage, perhaps, dna sequence dna of the present invention can be cloned people's gene of equal value, thereby make human DNA sequence's excessive production become easily, and make it can on biomedicine, be used for the treatment of some human diseases.
Plant and fungi desaturase mainly are the membrane polypeptides of integrating, and this makes them be difficult to come purifying and evaluation with ordinary method.Therefore, for studying lipid metabolism better, adopted the molecular engineering that comprises mutant and transgenic plant.
First aspect of the present invention provides a kind of isolating animal △ 5-fatty acid desaturase and functional part thereof.
Second aspect of the present invention provides a kind of isolating C.elegans △ 5-fatty acid desaturase.
Third aspect present invention provides the dna sequence dna of code book invention first or second aspect, it comprises at least a portion of sequence shown in the SEQ.2, with with the sequence of this sequence or this sequence part equivalence, their rely on genetic code degeneracy encoding function △ 5-fatty acid desaturase.Preferable, this dna sequence dna is from the acquisition of deriving of Caenorhabditselegans dna sequence dna.
Preferable, by the long 1341bp of encoding gene of the △ 5-fatty acid desaturase of clone gene coding.Its protein length is 447 amino acid, and the molecular weight of estimation is 57kDa.
Perhaps, the functional △ 5-of this dna sequence encoding fatty acid desaturase, and comprise sequence shown in the SEQ.1 at least a portion and with the sequence of this sequence or this sequence part equivalence, their rely on genetic code degeneracy encoding function △ 5-fatty acid desaturase.Preferable, this dna sequence dna is from the acquisition of deriving of Mortierella alpina dna sequence dna.
Preferable, by the long 1338bp of encoding gene of the △ 5-fatty acid desaturase of clone gene coding.Long 446 amino acid of its protein, the molecular weight of estimation is 57kDa.
Preferable, in Mammals, have function according to the described dna sequence dna of third aspect present invention.
Preferable, this dna sequence dna is expressed in Mammals.
Preferable, this dna sequence dna is expressed in human body.
Preferable, this dna sequence dna is to obtain by the functional natural gene of modifying coding △ 5-fatty acid desaturase.
Preferable, this modification comprises with chemistry, physics or biological method modifies and the catalytic activity of not removing the enzyme of its coding.
Preferable, this modification has improved the catalytic activity of the enzyme of its coding.
Preferable, biology is modified recombinant DNA method and the forced evolution technology of comprising.
Preferable, the forced evolution technology is DNA reorganization.
The 4th aspect of the present invention provides the polypeptide by the dna sequence encoding of third aspect present invention.
Preferable, at least a portion of this polypeptide has the functional equivalent sequence of sequence shown in the SEQ.3 or this sequence or its part.Perhaps, at least a portion of this polypeptide has the functional equivalent sequence of sequence shown in the SEQ.4 or this sequence or its part.
Preferable, these polypeptide catalysis two high gamma linolenic acid are transformed into arachidonic acid.
Preferable, this polypeptide does not lose the catalytic activity of coded polypeptide through modifying.
Preferable, this polypeptide is introduced the substrate saturation ratio of specified level through the modification of mode like this with activation certain specific location in the molecular structure of substrate.
The 5th aspect of the present invention provides the carrier of any a part of dna sequence dna of the dna sequence dna that contains third aspect present invention.
The 6th aspect of the present invention provides a kind of method of producing polyunsaturated fatty acid, and this method comprises makes substrate contact with the △ 5-fatty acid desaturase of the present invention first or second aspect or the polypeptide of fourth aspect present invention.
The 7th aspect of the present invention provides a kind of two high gamma linolenic acid is transformed into arachidonic method, and wherein this transformation reaction is by the △ 5-fatty acid desaturase of the present invention first or second aspect or the polypeptide of fourth aspect present invention comes catalysis.
The 8th aspect of the present invention provides a kind of biology, and this biology is through the genetic engineering modified polypeptide that can produce fourth aspect present invention high-levelly.
The 9th aspect of the present invention provides a kind of biology, this biology through genetic engineering modified can produce high-levelly a kind of by of the present invention first or the catalytic reaction product of the polypeptide of the △ 5-fatty acid desaturase of second aspect or fourth aspect present invention.
Preferable, this biology process is engineered can be in order to implement the method for sixth aspect present invention or the 7th aspect.
Preferable, this biology is a microorganism.
Preferable, this microorganism is selected from algae, bacterium and fungi.
Preferable, this fungi comprises phycomycete.Perhaps, this microorganism is a yeast.
In addition, this biology is a plant.Preferable, this plant is selected from oil seed plant.
Preferable, this oil seed plant is selected from rape, Sunflower Receptacle, cereal (comprising corn), tobacco, pod (comprising peanut and soybean), safflower, oil palm, coconut and other palm, cotton, sesame, leaf mustard, linseed oil, castor-oil plant, Borrago officinalis and root of Redsepal Eveningprimrose.
Tenth aspect present invention provides from the seed of the biologically-derived acquisition of ninth aspect present invention or other fertile material.
Preferable, this biology is a Mammals.
The 11 aspect of the present invention provides a kind of isolating multienzyme approach, and wherein this approach comprises the △ 5-fatty acid desaturase of the present invention first or second aspect.
The 12 aspect of the present invention provides a kind of compound that produces by substrate conversion, and wherein said conversion is by the △ 5-fatty acid desaturase catalysis of the present invention first or second aspect.
The 13 aspect of the present invention provides a kind of intermediate compound, and this compound is produced by the catalytic reaction of the △ 5-fatty acid desaturase of the present invention first or second aspect.
The 14 aspect of the present invention provides a kind of food or dietary supplement, and it contains the polyunsaturated fatty acid of the method generation of useful sixth aspect present invention.
The 15 aspect of the present invention provides a kind of pharmaceutical preparation, and said preparation contains the polyunsaturated fatty acid of the method generation of useful sixth aspect present invention.
The 16 aspect of the present invention provides by biosynthetic pathway synthetic prostaglandin(PG), and this biosynthetic pathway comprises the catalytic activity of the △ 5-fatty acid desaturase of the present invention first or second aspect.
The 17 aspect of the present invention provides a kind of adjusting prostaglandin(PG) synthetic method, and this method is the expression level of the dna sequence dna of control third aspect present invention.
The 18 aspect of the present invention provides a kind of probe, and this probe comprises all or part of dna sequence dna of third aspect present invention or the RNA sequence of equivalence.
Nineteen of the present invention aspect provides a kind of probe, and this probe comprises all or part of of △ 5-fatty acid desaturase polypeptide of fourth aspect present invention.
The 20 aspect of the present invention provides a kind of method of separating △ 5-fatty acid desaturase with the probe of the present invention's nineteen aspect.
Gene of the present invention can be transformed in people's cell and be used for the vivo gene treatment with suitable level, with the enzyme that constant basis is provided the intravital lipid acid of patient is transformed into polyunsaturated fatty acid.For example hypercholesterolemia patient or suffer from other medical science symptom and give among the patient that polyunsaturated fatty acid has the diseases prevention advantageous effects, this may be a kind of effective prophylactic treatment means.
In addition, DAN sequence of the present invention all or part of, or all or part of search probe that can be used as of peptide sequence of the present invention is used for research or diagnostic purpose.
To the present invention be described by embodiment and referring to accompanying drawing Fig. 1 to 4 and SEQ.1 to SEQ.4 now, wherein:
SEQ.1 is the cDNA sequence of coding Mortierella alpina △ 5-fatty acid desaturase;
SEQ.2 is the cDNA sequence of coding C.elegans △ 5-fatty acid desaturase;
SEQ.3 is the peptide sequence that the gene order translation of SEQ.1 obtains;
SEQ.4 is the peptide sequence that the gene order translation of SEQ.2 obtains;
Fig. 1 is the sequence contrast of Mortierella alpina △ 5-fatty acid desaturase and various △ 6 desaturases and △ 12 desaturases of this genes encoding;
Fig. 2 is the sequence contrast of △ 5 desaturases of the △ 5-fatty acid desaturase of this genes encoding and C.elegans △ 6 desaturases and fungi Mortierella alpina;
Fig. 3 be transformed Mortierella alpina △ 5-fatty acid desaturase gene through inductive yeast cell transformant and without the gas-chromatography trajectory of the fatty acid methyl ester of inductive yeast cell transformant;
Fig. 4 be transformed C.elegans △ 5-fatty acid desaturase gene through inductive yeast cell transformant and without the gas-chromatography trajectory of the fatty acid methyl ester of inductive yeast cell transformant.
The clone and the order-checking of the △ 5-fatty acid desaturase gene of Mortierella alpina
Dna sequence encoding △ 5-fatty acid desaturase of the present invention has been cloned this sequence with round pcr in conjunction with cDNA library template and specially designed primer.By in yeast, expressing the function (being about to two high gamma linolenic acid (DHL) is transformed into arachidonic acid (AA) and eicosatetraenoic acid (ETA) is transformed into timnodonic acid (EPA)) that corresponding cDNA confirms this dna sequence dna.
CDNA with Mortierella alpina makes template, according to the plant △ 12 and △ 15 desaturases first and the 3rd Histidine base (Shanklin, J.Whittle, the EJ ﹠amp that have been identified by Shanklin; Fox, BG.Biochemistry.33,12787-12794 (1994)), design the Oligonucleolide primers (DP) (as follows) of degeneracy especially, cloned the △ 5-fatty acid desaturase of Mortierella alpina with polymerase chain reaction (PCR).
The Oligonucleolide primers of degeneracy (DP)
5′-GCGAATTA(A/T)TIGGICA(T/C)GA(T/C)TG(T/C)GICA-3′
5′-GCGAATTC?ATIT(G/T)IGG(A/G)AAIA(G/A)(A/G)TG(A/G)TG-3′
Wherein I represents inosine, and EcoRI represents with underscore in the site.
Fully with ordinary method in the amplification of the enterprising performing PCR of thermal cycler, program be 94 ℃ 2 minutes, 94 ℃ of-55 ℃ of-72 ℃ of circulations 32 in 1 minute in 1 minute in 45 seconds are taken turns then, and then extend 10 minutes in 72 ℃.On 1% sepharose, separate pcr amplification product.
The product that the PCR product scope that obtains from Mortierella alpina cDNA template amplification comprises 660bp carries out gel-purified to it, is cloned into pGEM-T (Promega
RTM) in, and be transformed among the escherichia coli expression host DH5 α.
According to the product sequences Design primer of 660bp, as template, carry out the fragment PCR amplification according to 660bp product sequences Design sequence specific primers (P) with the 660bp fragment of cloning.
△ B forward primer
5′-GATGCGTCTCACTTTTCA-3′
△ B reverse primer
5′GTGGTGCGCACAGCCTGGTAGTT-3′
This pcr amplification product of gel-purified, and screen Mortierella alpina cDNA library as probe.Fragment probe and 3.5 * 10
525 hybridization in the individual phage clone, a clone shows to have the expectation size of 1.5kb through restriction analysis.Selecting this clone (called after L11) further analyzes.
L11 is carried out sequential analysis, and the result discloses that a length is arranged is the open reading frame of 1.338kb, its 446 amino acid whose polypeptide of encoding.When with GCG 8 programs (people such as Devereux J., Nucleic Acids.Res..12, when 387-395 (1984)) analyzing in protein and genome database, L11 demonstrates with the homogeny of △ 6 delta 8 desaturase genes of synechocystis PCC6803 very low, has only 20% (Fig. 1).
In Fig. 1, carry out the correlated sequence of sequence and have following preserving number:
S54259 △ 12 Spirullina preserving number: X86736
S54809 △ 6 Spirullina preserving number: X87094
The sphingolipid desaturase preserving number that S68358 infers: X87143
S35157 △ 6 synechocystis preserving number: L11421
PBOR6 △ 6 Borrago officinalis preserving number: U79010
FU2 △ 5 desaturase preserving number: AF054824
In addition, although there are all three Histidine box features of desaturase in the sequence of translation, the 3rd the Histidine box that is positioned at this sequence 1159bp position contains the QXXHH of variation.The N end of this translation sequences also contains cytochrome b
5-sample of blood red pigment binding domains, this structural domain comprises the EHPGG motif, and former of this feature is observed at the C of other fungi desaturase end.
The Southern trace of genomic dna
The clone's of coding △ 5-fatty acid desaturase Mortierella alpina expression of gene
For the △ 5-fatty acid desaturase of having confirmed the L11 sequence encoding, with the cDNA subclone to Invitrogen
TMAmong the Yeast expression carrier pYES2 that provides, make it under the control of GAL4 polymerase promoter, produce plasmid pYES2/L11.Use Promega
TMLink coupled is transcribed and translation system in-vitro transcription-translation pYES2/L11, checks the expression of L11.Produce the translation product of 35S methionine(Met) mark, it is carried out SDS PAGE, and show by the radioautograph exposure.The estimation molecular weight of this product is 55-60kD, and does not have the control plasmid pYES2 of inset can not produce any markd translation product.
Construction pYES2/L11 is transformed in the yeast saccharomyces cerevisiae, on the YCA substratum of uridylic disappearance, grows.But rely on the existence of the URA3 selective marker that pYES2/L11 carries to select transformant, adding semi-lactosi to ultimate density is 1%mM, induces L11 to express.Make culture grow overnight in the presence of 0.5mM two high gamma linolenic acid esters, washing composition (1%tergitolNP-40) and 2% raffinose.At 0,4 and 16 hour results equal portions.GC by methyl esters analyzes the zymic total fatty acids.The 1M HCl that joins with methyl alcohol in 80 ℃ to the inductive sample and not the lipid of inductive control sample carried out transmethylaseization 1 hour.With hexane extraction fatty acid methyl ester (FAMES).With the Hewlett Packard 58804 serial gas-chromatographies that 25M * 0.32mm RSL-500bp bonded capillary column and flame ionization detector are housed FAME being carried out GC analyzes.
When to carry plasmid pYes2/L11 and the yeast of growth in the presence of semi-lactosi and two high gamma linolenic acid in isolated total fatty acids methyl esters carry out GC when analyzing, observe extra peak (see figure 3) in addition.This extra peak has and the identical residence time of arachidonic acid standard substance (Sigma), and this shows that transgenic yeast can make 5 dehydrogenations of △ of two high gamma linolenic acid.And in control sample (transforming), do not observe such peak (Fig. 3) with pYes2.Confirm the identity at this extra peak with GCMS (Kratos MS80RFA operates under the ionization voltage of 70eV, and sweep limit is 500-40 dalton), it is arachidonic acid that the result clearly identifies this compound.
The coded functional polypeptide of dna sequence dna of this proof Mortierella alpina has participated in arachidonic synthetic in the presence of semi-lactosi and two high gamma linolenic acid esters.
The clone and the order-checking of C.elegans △ 5-fatty acid desaturase gene
The present inventor has identified earlier and different fungi △ 5 and the △ 6-fatty acid desaturases of identifying in the past from the plant and animal kind of microsome desaturase.Difference is to exist one and electron donor albuminous cell pigment b5 homologous N-end extension sequence.
To fungi (Mortierella alpina) △ 5-fatty acid desaturase and C.elegans △ 6-fatty acid desaturase (in clay WO8D2 (preserving number Z70271)) when carrying out specificity analysis, the present inventor identifies the relevant sequence fatty acid desaturase of may encoding also containing on the clay T13F2.1 of C.elegans DNA (preserving number Z81122).
The analysis of this sequence (discloses clay WO8D2 and T13F2 contains overlapping areas with Genefinder program (Wilson, people such as R., (1994) Nature, 368.32-38)).In addition, find that clay T13F2 contains an open reading frame (ORF) (called after T13F2.1), it contains three " Histidine box " features of N terminal cell pigment b5 structural domain (determining by His-Pro-Gly-Gly diagnostic motif) and all microsome desaturases.In addition, this desaturase of inferring contains the 3rd Histidine box of variation, and the displacement of H → Q takes place first Histidine in the His-X-X-His-His motif.The displacement of this L-glutamic acid be present in the plant and animal △ 6-fatty acid desaturase and the fungi △ 5-fatty acid desaturase of Mortierella alpina in.
Can determine the desaturase ORF that infers by overlapping between clay T13F2 and the WO8D2, the proximity of T13F2.1 and △ 6-fatty acid desaturase, the result discloses these two sequences arranged in series in karyomit(e) V, 990 bases of being separated by from the expectation terminator codon of T13F2.1 to the initial methionine triplet of △ 6-fatty acid desaturase.
Owing to be dispersed with a plurality of introns among the sequential analysis indication T13F2.1 ORF, so the allos functional expression of genomic dna is infeasible.Therefore, with the partial cDNA Cloning of polymerase chain reaction (PCR) amplification corresponding to the big expectation exon of T13F2.1 ORF 5 ' end, PCR adopts following primer CEFOR and CEREV:
CEFOR
5′-ATGGTA?TTACGA?GAGCA?AGA-3′
CEREV
5′-TCTGGG?ATCTCT?GGTT?CTTG-3′
94 ℃ of initial sex change after 2 minutes, followingly carry out 32 and take turns the round-robin amplification: 94 ℃ 45 seconds, 55 ℃ 1 minute, 72 ℃ 1 minute, at last again 72 ℃ extended 10 minutes.
Amplification obtains having the correct dna fragmentation (shown on 1% sepharose) of estimating size, downcuts the gel band, and purify DNA is directly connected among the pGEM-T (Promega), and the gained plasmid is transformed in the e.colidh5.Check order with Qiagen QIAprep micropreparation test kit plasmid DNA purification, the nucleotide sequence of inset is carried out the automatization order-checking with ABI-377 dna sequencing instrument.
In order to separate complete coding region, screen the mix stages C.eleganscDNA library that in λ Zap II, makes up by Prof Yuji Kohara-Mishima (Japan) with the fragment of this isolating 233bp PCR-amplification corresponding to ORF T13F2.1.As probe, adopt standard technique people (1989) " molecular cloning experiment guide " such as () Sambrook to screen with clone's PCR product.With Ready to Go DNA-labeled reactant mixture (Pharmacia), usefulness α [
32P] the dCTP labeled dna fragment.In screening and 1.4 * 10 of 233bp fragment hybridization
5Among the pfu, 5 plaques provide positive signal, and they are downcut from agar plate, are diluted in the SM damping fluid.Carry out pcr amplification with CEFOR and CEREV, whether gained phage suspension liquid exists T13F2.1.With the isolating 233bp fragment of PCR under 65 ℃ to a clone (called after L4) carry out extra 2 take turns the inoculation and screening by hybridization give purifying.From λ clone L4, discharge plasmid L4 by cutting, the cDNA that inserts is checked order on two chains with Perkin Elmer AB1-377 dna sequencing instrument.
The gained dna sequence dna is presented among the SEQ.2, and the aminoacid sequence of expectation is presented among the SEQ.4.
The functional selection of L4 in the yeast
With the complete coding region (447 amino acid of encoding) of pcr amplification L4, pcr amplification adopts primer YCEDFor and TCEDRev as follows, and these primers have also been introduced the HindIII and the BamHI site of side joint:
YCEDFor：
5′-GCGAAGCTT?AAAATGG?TATTACG?AGAGCAA?GAGC-3′
(representing with runic that with the annealing of initial methionine the HindIII restriction site is represented with underscore)
YCEDRev：
5′-GCGGGAT?CCAAT?CTAGGC?AATCTT?TTTAG?TCAA-3′
(representing with runic that with the annealing of terminator codon complementary sequence the BamHI restriction site is represented with underscore)
Utilize HindIII and BamHI site (enzyme is provided by Boehringer Mannheim), the pcr amplification product that will contain the L4 complete coding region is connected to the downstream of GAL1 promotor among the Yeast expression carrier pYES2 (Invitrogen).(called after pYES2/L4) is transformed in the intestinal bacteria with the gained construction, transcribes/translate the fidelity of reproduction of the inset that PCR produces in external affirmation plasmid pYES2/L4 by the coupling that utilizes TNT system (Promega).With
35S methionine(Met) mark gained translation product shows with the SDS-PAGE separation and by radioautograph.
The molecular weight of the translation product that obtains from pYES2/L4 is about 57000, and does not have the control vector pYES2 of inset not produce translation product.
For functional selection is carried out in the L4 coding region, with lithium acetate method (Elble R. (1992) Bio Techniques13 18-20) with recombinant plasmid transformed in yeast saccharomyces cerevisiae DBY746.Containing raffinose as carbon source and be added into linolic acid (18: 2 △
9.12) or two-Gao-gamma-linolenic acid (C20: 3 △
8,13,14) substratum in the presence of the 1%tergitol tensio-active agent culturing cell spend the night (described in people such as Napier (1998) Bioehem.J.330 611-614).Do not have these lipid acid in the yeast saccharomyces cerevisiae, but these lipid acid are the specific substrates of △ 6 or △ 5 desaturases respectively.Add semi-lactosi to 1%, induce the L4 coding region to begin to express from the GAL1 promotor of carrier.Made the culture continued growth 16 hours, and took out equal portions then lipid acid is carried out the GC analysis.Analyze the total fatty acids from yeast culture, extract with methyl esters gas-chromatography (GC).The 1M HCl that joins with methyl alcohol makes lipid in 80 ℃ of transmethylaseizations 1 hour, then with hexane extraction fatty acid methyl ester (FAME).With Hewlett Packard 58804 series gas chromatograph that 25M * 0.32mm RSL-500bp bonded capillary column and flame ionization detector are housed FAME being carried out GC analyzes.Compare by residence time, identified lipid acid with FAME standard substance (Sigma).Estimate the relative percentage ratio of lipid acid from peak area.Identify arachidonic acid with GC-MS (use Kratos MS80RFA, operate, sweep limit is 500-40 dalton) under the ionization voltage of 70eV.Fig. 4 shows the GC analytical results of the fatty acid methyl ester of transformed yeast bacterial strain.Compare with the empty carrier contrast, an extra peak is obviously arranged from the track that pYES2/L4 obtained of induced growth in the presence of two-Gao-gamma-linolenic acid.This peak is not present in the not inductive culture of growing on two-Gao-gamma-linolenic acid yet, importantly should also be noted that, the pYES2/L4 of growth does not produce any new peak in the presence of linolic acid, and this shows that this kind lipid acid is not the substrate of the enzyme of C.elegans cDNA coding.The residence time at this extra peak is with methyl-the arachidonic acid standard substance are identical reliably.With GCMS (gaschromatographic mass spectrometry method) lipid acid that two-Gao-gamma-linolenic acid produces is made further specificity analysis, be accredited as arachidonic acid.Therefore, the result shows that plasmid pYES2/L4 transformed yeast cells has obtained functional △ 5-desaturase activity, now can be from the synthetic arachidonic acid of substrate two-Gao-gamma-linolenic acid.It seems that the △ 5-desaturase in this transformed yeast be a kind of effective catalyzer.
This proved C.elegans dna sequence encoding a kind of functional polypeptide, this polypeptide can participate in synthetic arachidonic acid in the presence of semi-lactosi and two-Gao-gamma-linolenic acid.
SEQ.4 coding C.elegans △
5The aminoacid sequence of the expectation of the gene of fatty acid desaturase
Claims (46)
1. an isolating animal △ 5-fatty acid desaturase and functional part thereof.
2. isolating C.elegans △ 5-fatty acid desaturase.
3. dna sequence dna of claim 1 or 2 described △ 5-fatty acid desaturases of encoding.
4. dna sequence dna according to claim 3, it comprises at least a portion of sequence shown in the SEQ.2 and the sequence equivalent with the part of this sequence or this sequence, and their rely on genetic code degeneracy encoding function △ 5-fatty acid desaturase.
5. dna sequence dna according to claim 4, this sequence is from the acquisition of deriving of Caenorhabditis elegans dna sequence dna.
6. dna sequence dna according to claim 3, a kind of functional △ 5-fatty acid desaturase of this dna sequence encoding, and comprise at least a portion of sequence shown in the SEQ.1, with with the sequence of this sequence or sequence part equivalence, their rely on genetic code degeneracy encoding function △ 5-fatty acid desaturase.
7. dna sequence dna according to claim 6, this dna sequence dna is from the acquisition of deriving of Mortierella alpina dna sequence dna.
8. according to each described dna sequence dna in the claim 3 to 7, wherein this dna sequence dna has function in Mammals.
9. dna sequence dna according to claim 8, wherein this dna sequence dna is expressed in Mammals.
10. dna sequence dna according to claim 9, wherein this dna sequence dna is expressed in human body.
11. dna sequence dna that obtains by the functional natural gene of modifying coding claim 1 or 2 described △ 5-fatty acid desaturases.
12. dna sequence dna according to claim 11, wherein modification comprises with chemistry, physics or biological method and modifies and do not lose the catalytic activity of the enzyme of its coding.
13. dna sequence dna according to claim 12, wherein this modification has improved the catalytic activity of the enzyme of its coding.
14. according to claim 12 or 13 described dna sequence dnas, wherein biology is modified recombinant DNA method and the forced evolution technology of comprising.
15. dna sequence dna according to claim 14, wherein the forced evolution technology is DNA reorganization.
16. polypeptide by each described dna sequence encoding in the claim 3 to 15.
17. polypeptide according to claim 16, wherein at least a portion of this polypeptide has the functional equivalent sequence of the sequence shown in the SEQ.3 or this sequence or its part.
18. polypeptide according to claim 16, wherein at least a portion of this polypeptide has the functional equivalent sequence of the sequence shown in the SEQ.4 or this sequence or its part.
19. according to each described polypeptide in the claim 16 to 18, wherein these polypeptide catalysis two high gamma linolenic acid are transformed into arachidonic acid.
20. according to each described polypeptide in the claim 16 to 19, wherein this polypeptide does not lose the catalytic activity of coded polypeptide through modifying.
21. polypeptide according to claim 20, wherein this polypeptide is modified through mode like this, so that specific location is introduced the substrate saturation ratio of specified level in the molecular structure of substrate.
22. a carrier, it contains any part of each described dna sequence dna in the claim 3 to 15 or this dna sequence dna.
23. a method of producing polyunsaturated fatty acid, this method comprise suitable substrate and claim 1 or 2 described △ 5-fatty acid desaturases or the described polypeptide of claim 16 to 21 are contacted.
24. one kind is transformed into arachidonic method with two high gamma linolenic acid, wherein said transformation reaction comes catalysis by each described polypeptide or modified polypeptides in the △ 5-fatty acid desaturase of claim 1 or 2 or the claim 16 to 21.
25. a biology, this biology can produce each described polypeptide in the claim 16 to 21 through genetic engineering modified high-levelly.
26. a biology, this biology through genetic engineering modified can produce by claim 1 or 2 described △ 5-fatty acid desaturases or claim 16 to 21 high-levelly in the catalytic reaction product of each described polypeptide.
27. a biology, this biology is through genetic engineering modified claim 23 or the 24 described methods implemented.
28. according to claim 26 or 27 described biologies, wherein this biology is a microorganism.
29. biology according to claim 28, wherein this microorganism is selected from algae, bacterium and fungi.
30. biology according to claim 29, wherein fungi comprises phycomycete.
31. biology according to claim 28, wherein said microorganism is a yeast.
32. according to each described biology in the claim 25 to 27, wherein this biology is a plant.
33. biology according to claim 32, wherein this plant is selected from oil seed plant and tobacco.
34. biology according to claim 33, wherein said oil seed plant are selected from rape, Sunflower Receptacle, comprise the cereal of corn, tobacco, the pod that comprises peanut and soybean, safflower, oil palm, coconut and other palm, cotton, sesame, leaf mustard, linseed oil, castor-oil plant, Borrago officinalis and root of Redsepal Eveningprimrose.
35. an Accessory Right requires seed or other fertile material of 33 or 34 described biologically-derived acquisitions.
36. according to each described biology in the claim 25 to 27, wherein this biology is a Mammals.
37. an isolating multienzyme approach, wherein this approach comprises each described polypeptide in claim 1 or 2 described △ 5 desaturases or the claim 16 to 21.
38. a compound that produces by the substrate transformation, wherein said transformation reaction comes catalysis by each described polypeptide in claim 1 or 2 described △ 5 desaturases or the claim 16 to 21.
39. an intermediate compound, this compound is produced by the catalytic reaction of each described polypeptide in claim 1 or 2 described △ 5 desaturases or the claim 16 to 21.
40. food or dietary supplement, it contains the polyunsaturated fatty acid that useful claim 23 or 24 described methods produce.
41. a pharmaceutical preparation, it contains the polyunsaturated fatty acid of useful claim 23 or 24 described methods generations.
42. by biosynthetic pathway synthetic prostaglandin(PG), this biosynthetic pathway comprises the catalytic activity of each described polypeptide in claim 1 or 2 described △ 5 desaturases or the claim 16 to 21.
43. regulate prostaglandin(PG) synthetic method for one kind, this method is the expression level of each described dna sequence dna in the control claim 3 to 15.
44. it is all or part of that a probe, this probe comprise in the claim 3 to 15 each described dna sequence dna, or the RNA sequence of equivalence.
45. diagnosis or research use probe, this probe to comprise all or part of of each described polypeptide in claim 1 or 2 described △ 5 desaturases or the claim 16 to 21.
46. method with claim 44 or 45 described probe separates △ 5 desaturases.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9727256.1A GB9727256D0 (en) | 1997-12-23 | 1997-12-23 | Desaturase gene |
| GB9727256.1 | 1997-12-23 | ||
| GB9814034.6 | 1998-06-29 | ||
| GBGB9814034.6A GB9814034D0 (en) | 1998-06-29 | 1998-06-29 | Desaturase gene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1283230A true CN1283230A (en) | 2001-02-07 |
Family
ID=26312844
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN98812505A Pending CN1283230A (en) | 1997-12-23 | 1998-12-23 | Desaturated enzyme |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP1042485A2 (en) |
| JP (1) | JP2002508932A (en) |
| KR (1) | KR20010033517A (en) |
| CN (1) | CN1283230A (en) |
| AU (1) | AU1774899A (en) |
| BR (1) | BR9814434A (en) |
| CA (1) | CA2315297A1 (en) |
| EE (1) | EE200000372A (en) |
| HU (1) | HUP0101153A3 (en) |
| PL (1) | PL344868A1 (en) |
| WO (1) | WO1999033958A2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100412197C (en) * | 2002-12-19 | 2008-08-20 | 布里斯托尔大学 | Novel method for the production of polyunsaturated fatty acids |
| CN1922311B (en) * | 2004-02-17 | 2012-03-14 | 约克大学 | Desaturase enzymes |
| CN101376020B (en) * | 2007-08-29 | 2013-05-01 | 益生生技开发股份有限公司 | Application of fungal immunomodulatory protein for inhibiting delta 5-desaturated enzyme |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8791327B2 (en) | 1998-12-07 | 2014-07-29 | Washington State University Research Foundation | Desaturases and methods of using them for synthesis of polyunsaturated fatty acids |
| WO2001092489A2 (en) * | 2000-05-26 | 2001-12-06 | Washington State University Research Foundation | Palmitate desaturase gene |
| PT1322752E (en) | 2000-09-28 | 2010-05-06 | Bioriginal Food & Science Corp | Fad4, fad5, fad5-2, and fad6, fatty acid desaturase family members and uses thereof |
| GB0107510D0 (en) | 2001-03-26 | 2001-05-16 | Univ Bristol | New elongase gene and a process for the production of -9-polyunsaturated fatty acids |
| US7807849B2 (en) | 2004-04-22 | 2010-10-05 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
| CA2884237C (en) | 2004-04-22 | 2020-09-08 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
| CA2568689A1 (en) | 2004-06-04 | 2005-12-15 | Fluxome Sciences A/S | Metabolically engineered cells for the production of polyunsaturated fatty acids |
| EP1780275B1 (en) * | 2004-07-12 | 2010-11-24 | Suntory Holdings Limited | Polypeptide having a delta 5 fatty acid unsaturating activity, polynucleotide coding for the polypeptide and use thereof |
| GB2431158A (en) | 2005-10-13 | 2007-04-18 | Rothamsted Res Ltd | Process for the production of arachidonic and/or eicosapentaenoic acid |
| BRPI0716075A2 (en) | 2006-08-29 | 2013-08-06 | Commw Scient Ind Res Org | Fatty Acid Synthesis |
| EP3260544A1 (en) | 2008-11-18 | 2017-12-27 | Commonwealth Scientific and Industrial Research Organisation | Enzymes and methods for producing omega-3 fatty acids |
| CA2876519C (en) | 2012-06-15 | 2020-09-15 | Commonwealth Scientific And Industrial Research Organisation | Production of long chain polyunsaturated fatty acids in plant cells |
| SG11201604871VA (en) | 2013-12-18 | 2016-07-28 | Commw Scient Ind Res Org | Lipid comprising long chain polyunsaturated fatty acids |
| CA2953008C (en) | 2014-06-27 | 2024-03-19 | Nuseed Pty Ltd | Lipid comprising docosapentaenoic acid |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5972664A (en) * | 1997-04-11 | 1999-10-26 | Abbott Laboratories | Methods and compositions for synthesis of long chain poly-unsaturated fatty acids |
-
1998
- 1998-12-23 PL PL98344868A patent/PL344868A1/en unknown
- 1998-12-23 EE EEP200000372A patent/EE200000372A/en unknown
- 1998-12-23 HU HU0101153A patent/HUP0101153A3/en unknown
- 1998-12-23 EP EP98962620A patent/EP1042485A2/en not_active Withdrawn
- 1998-12-23 WO PCT/GB1998/003895 patent/WO1999033958A2/en not_active Application Discontinuation
- 1998-12-23 AU AU17748/99A patent/AU1774899A/en not_active Abandoned
- 1998-12-23 CA CA002315297A patent/CA2315297A1/en not_active Abandoned
- 1998-12-23 KR KR1020007007019A patent/KR20010033517A/en not_active Application Discontinuation
- 1998-12-23 CN CN98812505A patent/CN1283230A/en active Pending
- 1998-12-23 JP JP2000526616A patent/JP2002508932A/en active Pending
- 1998-12-23 BR BR9814434-0A patent/BR9814434A/en not_active IP Right Cessation
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100412197C (en) * | 2002-12-19 | 2008-08-20 | 布里斯托尔大学 | Novel method for the production of polyunsaturated fatty acids |
| CN1922311B (en) * | 2004-02-17 | 2012-03-14 | 约克大学 | Desaturase enzymes |
| CN101376020B (en) * | 2007-08-29 | 2013-05-01 | 益生生技开发股份有限公司 | Application of fungal immunomodulatory protein for inhibiting delta 5-desaturated enzyme |
Also Published As
| Publication number | Publication date |
|---|---|
| HUP0101153A3 (en) | 2006-03-28 |
| HUP0101153A2 (en) | 2001-08-28 |
| WO1999033958A2 (en) | 1999-07-08 |
| WO1999033958A3 (en) | 1999-09-02 |
| PL344868A1 (en) | 2001-11-19 |
| JP2002508932A (en) | 2002-03-26 |
| CA2315297A1 (en) | 1999-07-08 |
| EP1042485A2 (en) | 2000-10-11 |
| EE200000372A (en) | 2001-12-17 |
| AU1774899A (en) | 1999-07-19 |
| BR9814434A (en) | 2001-10-23 |
| KR20010033517A (en) | 2001-04-25 |
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