CN102250864B - Dual purposes of linoleate isomerase in aspects of dehydrogenation and isomerism - Google Patents
Dual purposes of linoleate isomerase in aspects of dehydrogenation and isomerism Download PDFInfo
- Publication number
- CN102250864B CN102250864B CN2011101126855A CN201110112685A CN102250864B CN 102250864 B CN102250864 B CN 102250864B CN 2011101126855 A CN2011101126855 A CN 2011101126855A CN 201110112685 A CN201110112685 A CN 201110112685A CN 102250864 B CN102250864 B CN 102250864B
- Authority
- CN
- China
- Prior art keywords
- sequence
- protein
- cell
- delta
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
The invention discloses dual purposes of linoleate isomerase in aspects of dehydrogenation and isomerism. A bifunctional enzyme PAI is used as delta-12 fattyaciddehydrogenase and linoleate isomerase or delta-12 fattyaciddehydrogenase; and the bifunctional enzyme PAI is protein a) or protein b), wherein the protein a) has amino acid sequences shown as a sequence 2 in a sequence table; and the protein b) has an amino acid residue sequence which is derived from the sequence 2 by substituting and/or losing and/or adding one or more amino acid residues, and has the activity of the delta-12 fattyaciddehydrogenase and linoleate isomerase or the delta-12 fattyaciddehydrogenase. The invention provides a basis and a method for further researching pai gene functions and performing related applicability research on the biosynthesis of t10c12-CLA (conjugated linoleic acid) by using the bifunctional enzyme PAI.
Description
Technical field
The present invention relates to the dual purpose of linoleate isomerase aspect dehydrogenation and isomery.
Background technology
Conjugated linolic acid, English name Conjugated linoleic acids is abbreviated as CLA, refers to linolic acid (C
18H
32O
2) one group of position and the general name of the isomers that forms of geometrical isomer.The characteristics of conjugated linolic acid are the carbon-carbon double bonds that molecule contains a pair of conjugation, and one is cis, and one is trans, and conjugated linolic acid has 28 kinds of isomerss at least.Wherein about cis-9, anti-form-1 1 (cis-9, trans-11; C9t11-CLA) and cis-10, anti-form-1 2 (trans-10, cis-12; T10c12-CLA) research of isomer is relatively many.C9t11-CLA has anticancer property, can reduce the sickness rate of cardiovascular disorder, improves immunologic function, opposing diabetes etc.; T10c12-CLA mainly participates in controlling the synthetic of body fat, can reduce the probability of occurrence of obesity, and the growth that bibliographical information t10c12-CLA can anticancer is also arranged in addition.2008, U.S. FDA was issued the food license licensed licenser licence, agreed CLA is used as foodstuff additive.At present, CLA by numerous food product and pharmacy corporation as healthcare products production and sale.
Natural CLA mainly from the milk of ruminating animal (ox, sheep etc.), is produced by rumen microorganism; The CLA content of one liter of milk mainly is c9t11-CLA and t10c12-CLA component about 1.4 grams, and the chances are 8～9: 1 for their accounting.Regrettably, there is the crowd of significant proportion can not eat milk-product, can't obtains the CLA useful to health.The CLA of industrial source is mainly generated by the linolic acid alkali isomerization, is the form of mixture.Wherein except the distinct c9t11-CLA and t10c12-CLA of biological activity, also contain the plurality of impurities isomer, the content of impurity can reach more than 30%, after these impurity components are digested and assimilated by the human or animal, derive the multiple conjugate class isomer that negative biological effect is arranged by arachidonic acid metabolic pathway easily.Therefore, be used for the CLA of foodstuff additive, preferably from biosynthetic one-component, rather than the synthetic complicated ingredient of industry.
Utilizing genetic engineering technique, produce single CLA component by microorganism, eukaryotic cell even breeding transgenic livestock are a large amount of, is the approach of a kind of more satisfactory biosynthesizing CLA.The key of this approach is to obtain to have bioactive functional gene.Know, mammiferous scd1 gene can be in reconstitution cell changes into t18:1 lipid acid c9t11-CLA component (Gou Kemian, a kind of method of biosynthesizing conjugated linoleic acid CLA, number of patent application: 201010200224.9, patent publication No.: CN101864463A).For the t10c12-CLA component, also there is not functional gene synthetic report of allos in zooblast or transgenic animal.
Summary of the invention
The purpose of this invention is to provide the dual purpose of linoleate isomerase aspect dehydrogenation and isomery.
A purposes provided by the present invention be bifunctional enzyme PAI as delta-12 fatty acid dehydrogenase and linoleate isomerase, or the application of delta-12 fatty acid dehydrogenase; Described bifunctional enzyme PAI be following a) or b) protein:
A) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;
B) aminoacid sequence with sequence in the sequence table 2 passes through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and has delta-12 fatty acid dehydrogenase and linoleate isomerase, or has delta-12 fatty acid dehydrogenase activity by the protein of a) deriving.
The aminoacid sequence shown in the sequence 2 is comprised of 425 amino-acid residues in the sequence table.
Albumen in above-mentioned in order to make (a) is convenient to purifying, label as shown in table 1 on N-terminal that can the protein that the aminoacid sequence shown in the sequence 2 forms in by sequence table or C-terminal connect.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned (b) but in the albumen synthetic, also can synthesize first its encoding gene, carry out again biological expression and obtain.The encoding gene of the albumen in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 1, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
Wherein, described linoleate isomerase can be the t10c12 conjugated linolic acid with the linolic acid isocatalysis, and described delta-12 fatty acid dehydrogenase can respectively be sloughed a H atom with the delta-12 of monounsaturated fatty acids and delta-13 position carbon atom and obtain polyunsaturated fatty acid; Two keys between the delta9 of described monounsaturated fatty acids and delta-10 position carbon atom.
The total number of carbon atoms of described monounsaturated fatty acids is 18, and described monounsaturated fatty acids specifically can be oleic acid.
Another purposes provided by the invention is a kind of method for preparing unsaturated fatty acids.
The method for preparing unsaturated fatty acids provided by the present invention, comprise the steps: the encoding gene of described bifunctional enzyme PAI is imported the reconstitution cell that obtains expressing said gene in the mammalian cell, described reconstitution cell is cultivated rear collecting cell in containing oleic acid and/or linoleic Zooblast culture medium, from collected cell, extract unsaturated fatty acids; Described unsaturated fatty acids is linolic acid, or is linolic acid and t10c12 conjugated linolic acid.
Described mammalian cell specifically can be stripped mammalian cell.
Described stripped Mammals carefully specifically can be the NIH/3T3 cell.
Described b) carboxyl terminal that protein specifically can be the 425th amino acids residue of sequence in the sequence table 2 connects 6 protein that histidine residues obtains.
The gene of described bifunctional enzyme PAI of encoding also belongs to protection scope of the present invention.
The encode gene of described bifunctional enzyme PAI is called for short pai, can be following 1) or 2) dna molecular:
1) its nucleotide sequence is the dna molecular of sequence 1 in the sequence table;
2) dna molecular that the codon of 6 Histidines of insertion obtains between the 1275th and 1276 of sequence 1 the in sequence table.
The recombinant vectors, expression cassette, transgenic cell line, recombinant bacterium or the recombinant virus that contain described dna molecular also belong to protection scope of the present invention.
The recombinant vectors that contains described dna molecular specifically can be above-mentioned 1) or 2) dna molecular insert expression that pIRES-AcGFP obtains above-mentioned a) or b) the recombinant expression of proteins carrier.
The encoding gene of described PAI is to express in host cell and/or animal individual by transfection or method for transformation.
The expression method of described bifunctional enzyme PAI, can comprise the steps: above-mentioned 1) or 2) the mammalian cell that import to exsomatize of dna molecular in obtain the reconstitution cell of expressing said gene, cultivate described reconstitution cell, express obtaining described bifunctional enzyme PAI.
The present invention with a kind of gene that comes from the linoleate isomerase of fungi carry out codon optimized after, be transfected in the zooblast, found that this enzyme has the activity of delta-12 desaturase, can increase linoleic content; Simultaneously, this enzyme also has the activity of cis-9 isomerase, linolic acid can be changed into the t10c12-CLA composition.The present invention illustrates first, and this gene has the dual biologic activity of desaturase and isomerase.Import the NIH/3T3 cell of this optimized gene, significantly (p＜0.05) increases the content of target product linolic acid and t10c12-CLA; Wherein linoleic acid content has improved 15.4～24.7%, the t10c12-CLA content of isomer improved 59.4～223.7%; The present invention is for further studying the pai gene function and utilizing the related application Journal of Sex Research of bifunctional enzyme PAI biosynthesizing t10c12-CLA that foundation and method are provided.
Description of drawings
Fig. 1 is that the RT-PCR of pai genetic transcription analyzes.
Fig. 2 is the detection signal of linolic acid (28.4min) and t10c12-CLA (31.6min) component in GFP cell and the pai clone F sample.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Embodiment 1, the stably express of external source pai gene in the NIH/3T3 cell
One, the structure of eukaryotic gene expression structure
According to the Propionibacterium linoleate isomerase pai original gene sequence (GenBank accession number AX062088) of publishing, by genetic engineering means, carried out the DNA original series codon optimized; Add in initiator codon ATG front simultaneously and be called the Kozak sequence oligonucleotides, such as GCCACC, the ATG back adds GNN, to satisfy the Kozak rule.Present embodiment adds 1 codon glycine GGA behind initiator codon ATG; Before terminator codon TAA, add histidine-tagged encoding sequence.The nucleotides sequence of this optimization classifies the codon that inserts 6 Histidines between the 1275th and 1276 of in sequence table sequence 1 as, at ATG (sequence 1 the 1st) dna sequence dna that front adding GCCACC obtains, wherein the 5th and 6 of sequence 1 the is respectively G and A.Insert in the codon of 6 Histidines, the codon of front 5 Histidines is CAT, the codon of the 6th Histidine is CAC.The nucleotide sequence encoding protein PAI of this optimization, the aminoacid sequence of this albumen are that wherein, the 2nd amino acids of sequence 2 is glycine with 6 sequences that histidine residues obtains of carboxyl terminal connection of the 425th amino acids residue of sequence in the sequence table 2.The pai gene of optimizing is connected on the pIRES-AcGFP plasmid (U.S. Clontech company) by the EcoRI restriction enzyme site, is built into the pPAI plasmid.Enzyme is cut evaluation and dna sequencing proves, the pPAI plasmid sequence is sudden change not.Conventional phenol--chloroform extracting and purifying pPAI plasmid are used for transfection NIH/3T3 cell (U.S. Sigma company, article No. 93061524).
Two, cell cultures and transfection
According to ordinary method, at 37 ℃, 5%CO
2Under the culture condition, the NIH/3T3 cell is cultivated with DMEM cell culture fluid (high glucose, Gibco company)+10% new-born calf serum (new calf serum, GIBCO).During passage, use 0.05% (w/v) trypsin (trypsinase)+0.04% (w/v) EDTA solution to get final product in 2 minutes 37 ℃ of digestion.
Inoculation NIH/3T3 cell (1 * 10 in the culture dish of diameter of phi 35-mm
5Individual/as ml), before the transfection when treating that degrees of fusion reaches 70%, transfection nutrient solution to be outwelled, with the nutrient solution rinsing that does not contain serum 2 times, add again the 1ml serum-free medium; With 10 μ l liposome (American I nvitrogen company, Lipofectamine
TM2000, article No. 11668-019) be diluted among the 250 μ l serum-free DMEM/F12, mixing gently, room temperature leaves standstill 5min, and the pPAI plasmid DNA with 4 μ g is diluted among another part 250 μ l serum-free DMEM/F12 simultaneously, mixing gently, room temperature leaves standstill 5min; Then with these two kinds of diluents totally 500 μ l mix, mixing gently, room temperature leaves standstill 20min; Subsequently, 500 μ l mixed solutions are splashed in the Φ 35-mm culture dish gently, gently mixing is rocked in front and back, puts into CO
2Incubator; Behind the 6h, change the standard serum nutrient solution into and continue to cultivate 24h, then digestion, and transfer in 24 orifice plates, with there being serum free culture system liquid to cultivate every hole 0.5ml; Cultivate behind the 24-48h according to cell density (about 1 * 10
5/ ml) add G418 nutrient solution (G418 final concentration 0.6mg/ml), screened 14 days.During this time, changed half solution in every 3-5 days; Use fluorescence microscope after 14 days, selection has cell strain digestion green fluorescence, normal growth and is inoculated into continuation cultivation in the culture dish, particularly, every strain cell uses a Φ 35-mm culture dish, and when growing into 70-80%, Φ 60-mm culture dish is transferred in digestion, subsequently, use Φ 100-mm culture dish, frozen corresponding clone is preserved or is carried out following analysis again.In the time of transfection target plasmid pPAI, go back in addition transfection pIRES-AcGFP plasmid, and obtain to express the clone of GFP fluorescence, as standard control.
Three, RT-PCR transcription analysis
Above-mentioned transfection, screening obtain positive pai cell and contrast GFP cell has many strains.Select at random wherein 9 strain pai clones and 1 strain GFP clone, after continuing to cultivate, digestion, centrifugal recovery degree of converging reach 70% cell, extract cell total rna according to standard method with RNAiso Plus (TaKaRa) reagent, and remove the DNA composition that mixes with DNase I (TaKaRa) digestion.Carry out reverse transcription with purifying RNA and obtain cDNA, use M-MLV ThermoScript II (Promega), the description operation that provides according to the manufacturer.In order to detect the pollution that whether has genomic dna, each RNA sample all has the negative control that does not add ThermoScript II.
Design and synthesize heterogeneic pcr amplification primer, particularly, pai upstream region of gene primer 5 '-gga ttc cacgat tac acc a-3 ', downstream primer 5 '-ggg tca tca gcc cat cta-3 ', expanding fragment length is 878-bp; Housekeeping gene hprt upstream primer 5 '-cct gct gga tta cat taa agc act g-3 ', downstream primer 5 '-gtc aag gca tat cca aca aca aac-3 ', expanding fragment length is 352-bp.With the cDNA template of above-mentioned reverse transcription acquisition and the amplimer of gene, with identical PCR condition, the specific fragment of two genes that increase respectively.The PCR condition is: 94 ℃, and 30s, 60 ℃, 30s, 72 ℃, 1min, totally 35 circulations.The PCR product carries out gel electrophoresis analysis and takes pictures.
RT-PCR the results show, the 9 strain pai cells of selecting are at random all expressed pai gene (express simultaneously the hprt housekeeping gene, and can both normal growth), and Fig. 1 has shown the wherein RT-PCR analytical results of 3 strain cell D, E, F.And the GFP cell does not detect the expression of pai gene.Compare with the GFP cell sample that the empty carrier transfection obtains with 3T3, cell strain D, E, F sample are all transcribed pai gene fragment (878-bp), illustrate that this three strains cell belongs to the pai positive cell.The all cells sample is all transcribed hprt housekeeping gene fragment (352-bp).Among Fig. 1,3T3 represents the NIH/3T3 cell, and GFP is the NIH/3T3 cell that turns pIRES-AcGFP, and D, E, F are the NIH/3T3 cell that 3 strains turn pPAI.
The CLA biosynthesizing that embodiment 2, determination of fatty acid and external source pai gene instruct
The positive pai clone of 9 strains that the 1 strain GPF control cells that 1 strain untransfected control cells NIH/3T3, embodiment 1 obtain, embodiment 1 obtain is respectively by every milliliter 2 * 10
5The density of individual cell is seeded in the Φ 100-mm culture dish, separately cultivates.
One, determination of fatty acid
Use the DMEM+10% new-born calf serum at 37 ℃ in above-mentioned 11 strain cells, 5%CO
2After cultivating 12h under the condition, change the above-mentioned fresh culture that adds 30 μ M linolic acid (Sigma, L1012) into, after continuing again to cultivate 48h, each strain cell harvesting to the glass with the tetrafluoroethylene bottle cap is methylated in the pipe, carry out the High-pressure gas chromatography analysis.Concrete steps are: the pipe inner cell is dissolved in first the 1.0ml normal hexane, then add the BF3/MeOH reagent (Sigma of 1.0ml 14%, B1252) mixing, in Glass tubing, be filled with nitrogen, naturally cool to room temperature behind the boiling water bath 1h, add the 1.0ml distilled water, centrifugal (3,000rpm, 1min) upper phase direct injection to the 100 meter long HP-88 type chromatographic column (J﹠amp that separates; The W112-88A7 model, Agilent, USA), use HP7890 full-automatic gas chromatography (Agilent) to analyze, concrete gas-chromatography program is: 120 ℃ of initial temperatures keep 1min; Speed with 10 ℃/min is warming up to 175 ℃, keeps 10min; Speed with 5 ℃/min is warming up to 210 ℃ again, keeps 5min; Speed with 5 ℃/min is warming up to 240 ℃ again, keeps finishing behind the 10min.Carrier gas is helium, and splitting ratio is 10: 1.With reference to lipid acid standard substance (Sigma, 47885-U and O5632) peak area, the signal that instrument is detected is with GC ChemStationSoftware (Agilent) software processes, calculate relative content, the experiment of cell cultures and determination of fatty acid repeats 4 times continuously.Under the above-mentioned condition, the t10c12-CLA in the lipid acid sample, linolic acid (18:2n6), stearic acid (18:0), oleic acid (18:1n9), arachidonic acid (20:4n6) retention time are respectively 31.6,28.4,25.5,26.7,35.0min.Fig. 2 only listed the NIH/3T3 cell (A) that turns pIRES-AcGFP and pai clone F sample (turning the NIH/3T3 cell of pPAI) (B) in the detection signal of linolic acid (28.4min) and t10c12-CLA (31.6min) component.X-coordinate among Fig. 2 is retention time minute, and ordinate zou is detection signal.* represent significant difference between same component (p＜0.05).
Two, as a result statistical study of determination of fatty acid
As can be seen from Table 2:
1) the pai gene of heterogenous expression can improve the content of t10c12-CLA in the cell, has the linoleate isomerase activity.
The pai clone sample of numbering D, E, F, their t10c12-CLA content is respectively 3.49 ± 0.45,3.66 ± 0.60 and 7.09 ± 1.98, significantly (p＜0.05) is higher than NIH/3T3 cell (1.78 ± 0.44) and GFP cell (2.19 ± 0.31) sample.That is to say that the heterogenous expression of linoleate isomerase PAI can synthesize t10c12-CLA effectively, with respect to the GFP control cells, t10c12-CLA content has improved 59.4% at least, has been up to 223.7%.
2) the pai gene of heterogenous expression can improve linolic acid 18:2n6 content in the cell, has the activity of delta-12 desaturase.
Linolic acid (18:2n6) is as the reaction substrate of isomerase PAI, accounting in three strain pai clones (numbering D, E, F) fatty acid total amount is respectively 15.54 ± 0.57,14.58 ± 0.09 and 14.38 ± 1.03, these data remarkable (p＜0.05) are higher than the linoleic acid content in NIH/3T3 cell (11.80 ± 0.81) and GFP cell (12.46 ± 0.71) sample; On the contrary, the stearic acid of pai groups of cells (18:0) content is between 11.85 ± 0.51 to 12.54 ± 0.15, and significantly (p＜0.05) is lower than 3T3 cell (14.13 ± 0.01) and GFP cell (14.07 ± 0.48) sample; In addition, the notable difference (p＞0.05) between oleic acid (18:1n9) content in the middle of stearic acid (C18:0) and the linolic acid does not but have to organize in metabolic process.Present knowledge is, in the zooblast, has the delta-9 desaturase, stearic acid can be generated oleic acid in the dehydrogenation of delta-9 position; But there is not oleic acid to be continued at delta-12 the delta-12 desaturase (vegetable cell and microorganism have this delta-12 desaturase) of dehydrogenation.Finding out that thus the pai gene also has the activity of delta-12 desaturase, can be linolic acid with the oleic acid biosynthesizing, and the oleic acid that reduces in the cell is transformed by stearic acid again, finally show as stearic acid and significantly reduce, and linolic acid significantly raises.
3) the pai gene of heterogenous expression can reduce arachidonic acid content in the cell effectively.
Compare with GFP cell (13.31 ± 0.77) with NIH/3T3 cell (13.35 ± 1.23), the arachidonic acid in the pai cell sample (20:4n6) composition significantly (p＜0.05) reduces, and only has 9.03 ± 0.85～11.13 ± 0.25.This presentation of results, the linoleate isomerase PAI of heterogenous expression can disturb arachidonic biosynthetic process or accelerate arachidonic metabolic process.
In sum, the pai gene has widely biologic activity, is embodied in the activity of linoleate isomerase activity and delta-12 desaturase, disturbs arachidonic biosynthesizing or metabolic process; And have obvious using value, can realize the biosynthesizing of linolic acid and t10c12-CLA.
The change of table 2pai cell partial fatty acid composition
| Fatty acid component | NIH/3T3 | GFP | D | E | F |
| t10c12-CLA | 1.78±0.44 | 2.19±0.31 | 3.49±0.45 * | 3.66±0.60 * | 7.09±1.98 * |
| C18:0 | 14.13±0.01 | 14.07±0.48 | 12.54±0.15 * | 11.85±0.51 * | 11.93±0.13 * |
| 18:1n9 | 18.00±1.21 | 18.45±2.00 | 18.40±0.81 | 18.65±0.31 | 17.35±0.55 |
| 18:2n6 | 11.80±0.81 | 12.46±0.71 | 15.54±0.57 * | 14.58±0.09 * | 14.38±1.03 * |
| 20:4n6 | 13.35±1.23 | 13.31±0.77 | 10.57±0.49 * | 11.13±0.25 * | 9.03±0.85 * |
Explanation of tables: (mean value ± SD) the corresponding fatty acid component of representative accounts for the per-cent of cell fatty acid total amount to each data in the table, from the result of 4 repeated experiments.Difference t-check analysis between three strain pai clones (D, E, F) and GFP and NIH/3T3 cell control cells system, * represents significant difference (p＜0.05).NIH/3T3 represents the NIH/3T3 cell, and GFP is the NIH/3T3 cell that turns pIRES-AcGFP, and D, E, F are the NIH/3T3 cell that 3 strains turn pPAI.
Claims (9)
1. following 1) or 2) or 3) dna molecular:
1) its nucleotide sequence is the dna molecular of sequence 1 in the sequence table;
2) in sequence table, insert the dna molecular that the codon of 6 Histidines obtains between the 1275th of sequence 1 the and 1276 Nucleotide;
3) in sequence table, insert between the 1275th and 1276 of sequence 1 the codon of 6 Histidines, before the 1st of sequence 1, add the dna molecular that GCCACC obtains.
2. the recombinant vectors, expression cassette, transgenic cell line or the recombinant virus that contain the described dna molecular of claim 1.
3. following protein expression method comprises the steps: dna molecular claimed in claim 1 is imported the reconstitution cell that obtains expressing said gene in the mammalian cell that exsomatizes, and cultivates described reconstitution cell, expresses obtaining described protein;
Described protein is the protein that is comprised of the aminoacid sequence shown in the sequence in the sequence table 2.
4. protein following 1) or 2) purposes:
1) as the application of delta-12 fatty acid dehydrogenase and linoleate isomerase;
2) as the application of delta-12 fatty acid dehydrogenase;
Described protein is the protein that is comprised of the aminoacid sequence shown in the sequence in the sequence table 2.
5. application according to claim 4, it is characterized in that: described linoleate isomerase can be the t10c12 conjugated linolic acid with the linolic acid isocatalysis, and described delta-12 fatty acid dehydrogenase can respectively be sloughed a H atom with the delta 12 of monounsaturated fatty acids and 13 carbon atoms and obtain polyunsaturated fatty acid; Two keys between the delta 9 of described monounsaturated fatty acids and 10 carbon atoms.
6. application according to claim 5 is characterized in that: the total number of carbon atoms of described monounsaturated fatty acids is 18.
7. application according to claim 6 is characterized in that: described monounsaturated fatty acids is oleic acid.
8. method for preparing unsaturated fatty acids, comprise the steps: the encoding gene of following protein is imported the reconstitution cell that obtains expressing said gene in the mammalian cell, described reconstitution cell is cultivated rear collecting cell in containing oleic acid and/or linoleic Zooblast culture medium, from collected cell, extract unsaturated fatty acids; Described unsaturated fatty acids is linolic acid, or is linolic acid and t10c12 conjugated linolic acid:
Described protein is comprised of the aminoacid sequence shown in the sequence in the sequence table 2.
9. arbitrary described application or method claimed in claim 8 according to claim 4-7 is characterized in that: the protein that the aminoacid sequence in the sequence table shown in the sequence 2 forms prepares in accordance with the method for claim 3; Or the encoding gene of protein described in the claim 8 is dna molecular claimed in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101126855A CN102250864B (en) | 2011-05-03 | 2011-05-03 | Dual purposes of linoleate isomerase in aspects of dehydrogenation and isomerism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101126855A CN102250864B (en) | 2011-05-03 | 2011-05-03 | Dual purposes of linoleate isomerase in aspects of dehydrogenation and isomerism |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102250864A CN102250864A (en) | 2011-11-23 |
| CN102250864B true CN102250864B (en) | 2013-03-20 |
Family
ID=44978434
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011101126855A Expired - Fee Related CN102250864B (en) | 2011-05-03 | 2011-05-03 | Dual purposes of linoleate isomerase in aspects of dehydrogenation and isomerism |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102250864B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146628B (en) * | 2013-03-01 | 2014-11-05 | 苟克勉 | Food-grade recombined lactococcal lactis capable of producing conjugated linoleic acid as well as construction method and application thereof |
| CN103233036B (en) * | 2013-04-09 | 2014-12-17 | 江南大学 | Method for optimizing conjugated linoleic acid synthesis by using Yarrowia lipolytica yeast recombinant strain based on genetic engineering strategy |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2108702A1 (en) * | 2008-04-07 | 2009-10-14 | Organo Balance GmbH | Synthesis of polyunsaturated fatty acids in yeast |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2004835A4 (en) * | 2006-03-15 | 2011-02-23 | Martek Biosciences Corp | Plant seed oils containing polyunsaturated fatty acids |
| DE102007014742A1 (en) * | 2007-03-23 | 2008-09-25 | Evonik Degussa Gmbh | Isoforms of pork liver esterase |
-
2011
- 2011-05-03 CN CN2011101126855A patent/CN102250864B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2108702A1 (en) * | 2008-04-07 | 2009-10-14 | Organo Balance GmbH | Synthesis of polyunsaturated fatty acids in yeast |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102250864A (en) | 2011-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Guo et al. | Improving docosahexaenoic acid production by Schizochytrium sp. using a newly designed high‐oxygen‐supply bioreactor | |
| CN1878785B (en) | Delta12 desaturases suitable for altering levels of polyunsaturated fatty acids in oleaginous yeast | |
| Tavares et al. | Metabolic engineering of Saccharomyces cerevisiae for production of eicosapentaenoic acid, using a novel Δ5-desaturase from Paramecium tetraurelia | |
| Muthuraj et al. | Evaluation of indigenous microalgal isolate Chlorella sp. FC2 IITG as a cell factory for biodiesel production and scale up in outdoor conditions | |
| Pei et al. | De novo transcriptomic and metabolomic analysis of docosahexaenoic acid (DHA)-producing Crypthecodinium cohnii during fed-batch fermentation | |
| CN101980595B (en) | Delta4 desaturase and its use in making polyunsaturated fatty acids | |
| ES2613553T3 (en) | CAR enzymes and improved production of fatty alcohols | |
| Ren et al. | Introduction of ω-3 desaturase obviously changed the fatty acid profile and sterol content of Schizochytrium sp. | |
| Takeno et al. | Development of fatty acid-producing Corynebacterium glutamicum strains | |
| Shi et al. | Production of eicosapentaenoic acid by application of a delta-6 desaturase with the highest ALA catalytic activity in algae | |
| US20190119689A1 (en) | Microbial oil containing fatty acids obtained from stramenopile and method of producing the same | |
| Kobayashi et al. | Increase of eicosapentaenoic acid in thraustochytrids through thraustochytrid ubiquitin promoter-driven expression of a fatty acid Δ5 desaturase gene | |
| Hong et al. | STUDY ON BIOLOGICAL CHARACTERISTICS OF HETEROTROPHIC MARINE MICROALGA—SCHIZOCHYTRIUM MANGROVEI PQ6 ISOLATED FROM PHU QUOC ISLAND, KIEN GIANG PROVINCE, VIETNAM 1 | |
| JP2021520821A (en) | Recombinant yeast strains that produce nervonic acid and their use | |
| JP2005510203A5 (en) | ||
| Shah et al. | Isolation, characterization and fatty acid analysis of Gilbertella persicaria DSR1: A potential new source of high value single-cell oil | |
| Silva et al. | Effect of n-dodecane on Crypthecodinium cohnii fermentations and DHA production | |
| CN102250864B (en) | Dual purposes of linoleate isomerase in aspects of dehydrogenation and isomerism | |
| Lassance et al. | Evolution of the codling moth pheromone via an ancient gene duplication | |
| CN101210234B (en) | Ocean micro-alga delta5 aliphatic acid desaturase and application thereof | |
| Zhang et al. | Optimal NaCl medium enhances squalene accumulation in thraustochytrium sp. ATCC 26185 and influences the expression levels of key metabolic genes | |
| Prabhakaran et al. | Whole genome analysis and elucidation of docosahexaenoic acid (DHA) biosynthetic pathway in Aurantiochytrium sp. SW1 | |
| Shi et al. | Determination of allosteric and active sites responsible for catalytic activity of delta 12 fatty acid desaturase from Geotrichum candidum and Mortierella alpina by domain swapping | |
| Zhang et al. | Effects of glycerol and glucose on docosahexaenoic acid synthesis in Aurantiochyrium limacinum SFD-1502 by transcriptome analysis | |
| CN100460506C (en) | Omega-3-aliphatic acid desaturase, and its encoding gene and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130320 Termination date: 20170503 |