CN102839134A - Yeast strain for producing alpha-linolenic acid, culture method and application thereof - Google Patents
Yeast strain for producing alpha-linolenic acid, culture method and application thereof Download PDFInfo
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Abstract
The invention relates to a yeast strain for producing alpha-linolenic acid, a culture method and an application thereof. A saccharomyces cerevisiae strain Y-10 for producing the alpha-linolenic acid is preserved at the Common Micro-organism Center of China Microbial Culture Preservation Commission on July 31st, 2012, wherein the address is as follows: Microbiology Institute of Chinese Academy of Sciences, Yuan 3#, No.1, West Beichen Road, Chaoyang District, Beijing City, and the preservation number is: CGMCC No.6385. The saccharomyces cerevisiae strain Y-10 for producing the alpha-linolenic acid provided by the invention has the capability of converting oleic acid into the alpha-linolenic acid and is the yeast strain with a new function; the yeast strain can be directly used as food or feed; the production process is simplified; and the production cost is lowered, so that an excellent foundation for producing a yeast product for replenishing the alpha-linolenic acid is established.
Description
Technical field
The present invention relates to a strain and produce yeast strain and the cultural method and the application of alpha-linolenic acid, belong to microbial technology field.
Background technology
Alpha-linolenic acid (ALA) and linolic acid (LA) adhere to ω-3 and the serial pufas of ω-6 separately, and higher animal self can not be synthesized, and is essential fatty acid.ALA has very high nutritive value to the people.Discover; ALA has the effect that reduces serum total cholesterol, triglyceride level, low-density lipoprotein, vldl and rising serum high-density LP; Therefore the ability regulating blood fat prevents atherosclerosis, prevention and treatment cardiovascular and cerebrovascular disease; Improve heart blood supply, suppress thrombosis.ALA is for improving children's intelligence and preventing that the elderly's brain aging from all being essential, has the irreplaceable effect of other lipid acid.
ALA is present in some plant seeds, and particularly linseed oil, purple perilla, Semen Brassicae campestris, soybean, walnut, salvia hispanica L and hemp also are present in the leaf of broad leaved plant.In general fruit, vegetables, meat, the fish, also contain alpha-linolenic acid, but the content pettiness.People are fewer through the chance that food directly obtains alpha-linolenic acid, therefore the content wretched insufficiency of alpha-linolenic acid in most of human body.As far back as 1993, United Nations's health organization and world food tissue were just stressed in " about promoting the suggestion that replenishes alpha-linolenic acid in the daily ration " to humans and animals: in view of the importance of ALA and the situation that generally lacks, and the special additional ALA of suggestion.ALA is extensive in field demands such as medicine, food.But alpha-linolenic acid still can not synthetic up to now, can only depend on the limited natural resource.Therefore, because the restriction of resource and content problem can't be satisfied the demand of market to alpha-linolenic acid at present.
The biosynthesizing of ALA is to be raw material with oleic acid etc., through ω-6 fatty acid dehydrogenase (the synthetic LA of catalysis of ω-6FAD); (ω-3FAD) ALA is synthesized in catalysis through the omega-fatty acid desaturase again.ω-6FAD also is Δ 12-fatty acid dehydrogenase, is ω-3 and omega 6 polyunsaturated fatty acid synthetic rate-limiting enzyme.ω-6FAD only is present in plant and the mikrobe, in higher animal, does not exist, so human body self can not synthesize linolic acid.ω-3FAD also is Δ 15-fatty acid dehydrogenase, and it is substrate with LA, in the dehydrogenation of C15 position, forms ALA.The report of at present existing multiple biological ω-6 and ω-3FAD gene, this feasible lipid acid route of synthesis through genetic engineering modified mikrobe becomes possibility with synthetic ALA.
Yeast saccharomyces cerevisiae is the edible announced of U.S. food and Drug Administration and to unique yeast quasi-microorganism of person poultry harmless, rich in proteins and vitamin B group, and sophisticated heterologous gene expression system is arranged.At present, utilize biotechnology to process the yeast product of a series of tool special nutritioies.The yeast saccharomyces cerevisiae oleaginousness is about 30% of dry weight, and wherein oleic acid accounts for 30%; LA and ALA are very low.
Summary of the invention
The present invention is directed to the deficiency of prior art, provide a strain to produce recombinant Saccharomyces cerevisiae bacterial strain Y-10 and the cultural method and the application of alpha-linolenic acid.
Yeast saccharomyces cerevisiae (Saccharomycescerevisiae) the bacterial strain Y-10 of alpha-linolenic acid is produced in one strain; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 31st, 2012; The address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is: CGMCC No.6385.This yeast saccharomyces cerevisiae is that flax ω-6 fatty acid dehydrogenase gene and the purple perilla omega-fatty acid dehydrogenase gene that in the delicatessen food yeast, have imported plant origin are processed, and the content of human body beneficial's alpha-linolenic acid is accounted for 7.38% of all fatty acids.It is the Wine brewing yeast strain that a strain can be produced alpha-linolenic acid.Its morphological specificity meets the general aspects of yeast saccharomyces cerevisiae, is unicellular microorganism, and cell is an oval.Formed bacterium colony is an oyster white, the smooth surface protuberance.
The cultural method of the yeast saccharomyces cerevisiae of above-mentioned production alpha-linolenic acid (Saccharomyces cerevisiae) bacterial strain Y-10, step is following:
(1) the single colony inoculation of picking Wine brewing yeast strain Y-10 is in the YEPD liquid nutrient medium, under 28～32 ℃, 200～250r/min condition, cultivates 8～12h, makes seed liquor;
(2) get seed liquor that step (1) makes by volume per-cent 1～5% be inoculated in the YEPD liquid nutrient medium, under 28～32 ℃, 200～250r/min condition, cultivate 20～30h, centrifugal, get deposition, make the yeast thalline;
(3) get the yeast thalline that step (2) makes and 1～5% be inoculated in the YEPD liquid nutrient medium by weight percentage, under 28～32 ℃, 200～250r/min condition, cultivate 60～80h.
Preferred according to the present invention, every liter of component of said YEPD liquid nutrient medium is following:
Peptone 20g, yeast extract paste 10g, glucose 20g, water is settled to 1L, and pH 6.0.
The yeast saccharomyces cerevisiae of above-mentioned production alpha-linolenic acid (Saccharomyces cervisiae) bacterial strain Y-10 is rich in the application in linolenic acid food or the feed in preparation.
Beneficial effect
1, the Wine brewing yeast strain Y-10 of production alpha-linolenic acid according to the invention has the ability that oleic acid is converted into alpha-linolenic acid, is a kind of yeast strain with new function;
2, Wine brewing yeast strain Y-10 according to the invention is safe; To the person poultry harmless; Utilize this saccharomyces cerevisiae engineered yeast to carry out the fermentative prodn of alpha-linolenic acid, the alpha-linolenic acid in the yeast strain does not need purifying just can directly use as food or feed, has simplified production technique; Reduced production cost, thereby laid good basis for producing the yeast product that replenishes alpha-linolenic acid.
Description of drawings
The electrophorogram of Fig. 1, ω-6FAD gene;
Wherein: A figure: Lane 1: template M1, Lane 2:DNA Marker DL 2000; B figure: Lane 1: template M2, Lane 2:DNA Marker DL 2000; C figure: Lane 1:DNA Marker DL 2000Lane 2: ω-6FAD gene;
Fig. 2, pYB-6 plasmid double digestion result's electrophorogram;
Wherein: Lane 1:1Kb DNA Marker, Lane 2:pYB-6 plasmid double digestion;
Fig. 3, pYB-ω 6 transform daughter bacteria liquid PCR checking result's electrophorogram;
Wherein: Lane 1:DNA Marker DL 2000, Lane 2-6: transform daughter bacteria liquid PCR, Lane 7: be the negative control of template with the pYB-6 plasmid;
The electrophorogram of Fig. 4, the checking of pYB-ω 6 plasmid double digestions;
Wherein: Lane 1:pYB-ω 6 plasmids, Lane 2:1Kb DNA Marker, Lane 3:pYB-ω 6 plasmid double digestions;
Fig. 5, pUC57 plasmid and pYB-6 plasmid NotI enzyme are cut result's electrophorogram;
Wherein: A figure: Lane 1:DNA Marker DL2000, Lane 2:pUC57 plasmid single endonuclease digestion, Lane 3:pUC57 plasmid; B figure: Lane 1:1Kb DNA Marker, Lane 2:pYB-6 plasmid, Lane 3:pYB-6 plasmid single endonuclease digestion;
The electrophorogram of Fig. 6, the checking of pYB-ω 3 transformants;
Wherein: A figure: Lane 1:DNA Marker DL 2000, Lane 2-7: transform daughter bacteria liquid PCR, Lane 8: be the negative control of template with the pYB-6 plasmid; B figure: Lane 1:pYB-ω 3 plasmid single endonuclease digestions, Lane 2:1Kb DNA Marker, Lane 3:pYB-ω 3 plasmids;
Fig. 7, pYB-ω 6 and pYB-ω 3 plasmid linearization results' electrophorogram;
Wherein: Lane 1:pYB-ω 6 plasmid Hpa I enzymes are cut, Lane 2:pYB-ω 6 plasmids, and Lane 3:1Kb DNA Marker, Lane 4:pYB-ω 3 plasmids, Lane 5:pYB-ω 6 plasmid Hpa I enzymes are cut;
The electrophorogram of Fig. 8, Wine brewing yeast strain Y-10 bacterium liquid PCR checking;
Wherein: Lane 1/11: negative control, Lane 2-5: ω in the recon-3FAD gene verification, Lane 6:DNA Marker DL2000, Lane7-10: ω-6FAD gene verification in the recon corresponding with Lane 2/3/4/5.
The electrophorogram of Fig. 9, Wine brewing yeast strain Y-10 extracting genome DNA;
Wherein: Lane 1-3: recon genomic dna, Lane 4:1Kb DNA Marker;
The electrophorogram of Figure 10, URA3 fragment cloning;
Wherein: Lane 1:DNAMarker DL2000, the URA3 fragment that Lane 2-3:PCR amplification obtains;
Figure 11, former starting strain and Wine brewing yeast strain Y-10 growth curve.
Embodiment
Below in conjunction with embodiment technical scheme of the present invention is further specified, but institute of the present invention protection domain is not limited thereto.
Yeast saccharomyces cerevisiae described in the embodiment (Saccharomyces cerevisiae) bacterial strain Y-10; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 31st, 2012; The address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is: CGMCC No.6385.
The pUC57 plasmid is available from Jinan Isabel Coixet Science and Technology Ltd.; PYB-6 plasmid (is that 201010531234.0 described construction processs make up by the patent No.); The Trans-T1 competent cell is available from Beijing full formula gold bio tech ltd; High-fidelity PCR polysaccharase is available from Beijing full formula gold bio tech ltd; The T4DNA ligase enzyme available from the full formula in Beijing gold bio tech ltd, restriction endonuclease Not I, Xho I, Hpa I available from TaKaRa biotechnology (Dalian) ltd; The trace dna detector is available from Eppendorf company.
The acquisition of ω-6FAD gene and ω-3FAD gene
Genetic information is to transmit with the form of triplet codon by mRNA in proteinic transmittance process.Every seed amino acid can be encoded to a plurality of codons by 1, and different plant species exists the sub-preferences of different ciphers.The codon-bias of yeast saccharomyces cerevisiae and plant exists than big-difference; In order to make foreign gene ω-6FAD gene and ω-3FAD gene in yeast saccharomyces cerevisiae, obtain expressing more efficiently; Therefore according to yeast saccharomyces cerevisiae codon-bias table (as shown in table 1) and flax ω-6FAD gene (Genebank:EU660502.1) and purple perilla ω-3FD (Genebank:AF047039.1) nucleotide sequence and the aminoacid sequence found from NCBI, the nucleotide sequence that the sequence optimisation one-tenth of these two genes is adapted at expressing in the yeast saccharomyces cerevisiae.
Table 1 yeast saccharomyces cerevisiae codon-bias table
The same ω of the nucleotide sequence-6FAD gene after optimizing and the mRNA of ω-3FAD gene are carried out sequence alignment, and the result shows that two nucleotide sequences have only 73% and 77% homogeny respectively, explains that the codon preference of yeast saccharomyces cerevisiae and plant exists than big-difference.
(ω-6FAD and ω-3FAD) Not I restriction enzyme site has been introduced at two ends to nucleotide sequence after the optimization, entrusts the synthetic synthetic that carried out of Bo Shang Bioisystech Co., Ltd.The synthetic goal gene is inserted respectively in the pUC57 plasmid, make recombinant plasmid pUC57-ω-6FAD and recombinant plasmid pUC57-ω-3FAD.
The structure of embodiment 2 multi-copy integration expression vector pYB-ω 6
With the pYB-6 plasmid is template, is upstream primer with primer P1, and primer P2 is a downstream primer, utilizes high-fidelity PCR polysaccharase to carry out pcr amplification, obtains gene fragment PGKt (Figure 1A).The recombinant plasmid pUC57-ω-6FAD that makes with embodiment 1 is a template, is upstream primer with primer P3, and primer P4 is a downstream primer, utilizes high-fidelity PCR polysaccharase to carry out pcr amplification, gene fragment ω-6FAD (Figure 1B).With gene fragment PGKt and gene fragment ω-6FAD is pcr template; With primer P1 is upstream primer; Primer P3 is a downstream primer, utilizes high-fidelity PCR polysaccharase to carry out overlapping pcr amplification, the overlapping PCR product (Fig. 1 C) after making gene fragment PGKt and gene fragment ω-6FAD being connected.Primer sequence is following:
Primer P1:5'AATGCTGGTCGCTATACTGCT 3' (SEQ ID NO.1)
Primer P2:5'CATGGGAGATCCTAGCTAGATCCGCCGCTCATAACTTGTTGTACC 3' (SEQ ID NO.2)
Primer P3:5'GGTAACGCCAGGGTTTTCCCAGTCA 3' (SEQ ID NO.3)
Primer P4:5'GGTACAACAACAAGTTATGAGCGGCGGATCTAGCTAGCTAGGATCTCCCAT G 3' (SEQ ID NO.4)
Above-mentioned PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 30sec, cycle repeats 35 times; 72 ℃ of 10min.
Above-mentioned overlapping PCR product purification is reclaimed, carry out double digestion with restriction enzyme Not I and Xho I then, enzyme is cut the back purifying and recovering.Carry out double digestion pYB-6 plasmid (Fig. 2) with restriction enzyme Not I and Xho I simultaneously; Behind agarose gel electrophoresis; Cut glue and reclaim the big fragment of molecular weight, the PCR product of enzyme being cut the back purifying with the T4DNA ligase enzyme and enzyme are cut the big fragment of pYB-6 plasmid that glue recovery afterwards obtains and are connected.Connect product and transform the Trans-T1 competent cell, coating LB (100 μ g/ml penbritin) flat board, 37 ℃ of incubated overnight.
The picking transformant is that primer carries out bacterium liquid PCR checking with P7 and P8.Be that template is carried out PCR with the pYB-6 plasmid simultaneously, as negative control.The result is as shown in Figure 3, and positive transformant has obtained the PCR product of expection size (694bp), and negative control group does not have corresponding fragment.Extract the DNA that bacterium colony PCR identifies the positive colony that obtains, and carry out the double digestion checking with Not I and Xho I, electrophoresis result is as shown in Figure 4, and the result conforms to expection.Choose positive transformant with primer P5 and P6 order-checking, sequencing result shows that recombinant expression vector makes up successfully, and overlapping PCR product oppositely inserts pYB-6 in the carrier, will contain the recombinant expression vector called after multi-copy integration expression vector pYB-ω 6 of correct sequence.The relevant primer sequence is following:
Primer P7:5'CCCATGAATGCGGTCATCACGCT 3' (SEQ ID NO.5)
Primer P8:5'GCTTTGGTGGCTTCCATTGCGT 3' (SEQ ID NO.6)
Primer P5:5'CGGATAAGAAAGCAACACCTGGCA 3' (SEQ ID NO.7)
Primer P6:5'CGTAGTTTTTCAAGTTCTTAGATGCT 3' (SEQ ID NO.8)
The PCR reaction conditions is 94 ℃ of 5min; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 30sec, cycle repeats 35 times; 72 ℃ of 10min.
The structure of embodiment 3 multi-copy integration expression vector pYB-ω 3
After recombinant plasmid pUC57-ω-3FAD that embodiment 1 is made and pYB-6 plasmid carry out single endonuclease digestion with restriction enzyme Not I, carry out electrophoresis detection with the sepharose of 0.7wt%, the result is as shown in Figure 5.Cut the big fragment of molecular weight of glue recovery ω-3FAD gene (about 1184bp) and pYB-6 plasmid.Fragment purifying and recovering after terminal dephosphorylation is handled that pYB-6 plasmid molecule amount is big; Then; With the T4 dna ligase ω-3FAD gene that reclaims is connected with the big fragment of the molecular weight of pYB-6 plasmid; Transform the Trans-T1 competent cell, coating LB (containing 100 μ g/mL penbritins) flat board, 37 ℃ of incubated overnight.
The picking transformant is a upstream primer with primer P9, and primer P10 is a downstream primer, carries out the PCR checking.Be that template is carried out PCR with the pYB-6 plasmid simultaneously, as negative control.The result is shown in Fig. 6 A, and positive transformant has obtained the PCR product of expection size (604bp), and negative control group does not have corresponding product.Extraction positive transformant plasmid carries out enzyme with Not I and cuts checking, and the result is shown in Fig. 6 B figure.Choose positive transformant and use primer P5 to be upstream primer, use primer P6 to be downstream primer, check order, contain correct sequence plasmid called after multi-copy integration expression vector pYB-ω 3, goal gene ω-3FAD oppositely inserts plasmid pYB-6 in the plasmid.Primer sequence is following:
Primer P9:5'AAGTTTGACCCAGCCGCTCCAC 3' (SEQ ID NO.10)
Primer P10:5'TACACCCATAGCGGCCCAACAC 3' (SEQ ID NO.11)
The PCR reaction conditions is 94 ℃ of 5min; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 30sec, cycle repeats 35 times; 72 ℃ of 10min.
The linearization process of embodiment 4 plasmids and electric transformed saccharomyces cerevisiae
Multi-copy integration expression vector pYB-ω 3 plasmids that the multi-copy integration expression vector pYB-ω 6 that embodiment 2 is made up and embodiment 2 make up are cut with restriction enzyme Hpa I enzyme and are carried out linearization process.After cutting, enzyme obtains the fragment (as shown in Figure 7) of size respectively for 2348bp and 6791bp, 2348bp and 6833bp.Big fragment after enzyme cut is cut that glue reclaims and is measured the content and the purity of the linearization plasmid of recovery with the trace dna detector.
Join 80 μ l yeast saccharomyces cerevisiaes electricity transformed competence colibacillus cell after the two kinds big fragment balanced mix that recovery is obtained (each 5 μ g), utilize Gene pulser electroporation, at voltage 1.5KV, resistance 200 Ω shock by electricity under the electric capacity 25 μ F conditions.After electric shock finishes, transformant is coated with the YEPD flat board and under 30 ℃ of conditions, cultivates 2-3d.
The screening of embodiment 5 recombinant Saccharomyces cerevisiae bacterial strains
(I) PCR of recombinant Saccharomyces cerevisiae bacterial strain checking
The bigger recombinant Saccharomyces cerevisiae bacterial strain of bacterium colony is a template with these bacterium liquid on the picking YEPD flat board, carries out the PCR checking with the gene-specific primer (being respectively P7 and P8, P9 and P10) of ω-6FAD gene and ω-3FAD gene.Do template with unconverted blank bacterium simultaneously and carry out PCR, as negative control.Can be found out by Fig. 8: foreign gene is successfully recombinated in the genes of brewing yeast group.
(II) real-time fluorescence quantitative PCR detects the copy number of foreign gene in the recombinant Saccharomyces cerevisiae bacterial strain
(1) the single copy gene URA3 gene in the selected yeast saccharomyces cerevisiae is an internal control gene.The URA3 gene order (NM_001178836.1) and the target gene sequences of the yeast saccharomyces cerevisiae of announcing according to ncbi database, utilize Beacon Designer 7.92 software design primers following:
URA3-Sense：5'AAAGGCATTATCCGCCAAGTA?3'（SEQ?ID?NO.11）
URA3-Antisense：5'CCACATCATCCACGGTTCTAT?3'（SEQ?ID?NO.12）
ω-3-FAD-qPCR-Sense：5'ATGGCTGCTCTTATATCG?3'（SEQ?ID?NO.13）
ω-3-FAD-qPCR-Antisense：5'GACGGTCAACAACAATATG?3'（SEQ?ID?NO.14）
ω-6-FAD-qPCR-Sense：5'GACGGTCAACAACAATATG?3'（SEQ?ID?NO.15）
ω-6-FAD-qPCR-Antisense：5'CCACACGGTCCTATCTAT?3'（SEQ?ID?NO.16）。
(2) preparation of typical curve
1. extracting genes of brewing yeast group DNA (Fig. 9), is template with it, and URA3-Sense and URA3-Antisense are the upstream and downstream primer, carry out PCR, obtains one section sequence (Figure 10) of URA3 gene.With above-mentioned URA3 gene fragment and pEASY
TM-T1Cloning Vector connects.Above-mentioned connection product is transformed into the Trans-T1 competent cell.Use M13R and M13F to carry out PCR evaluation and order-checking as primer.The transformant that is verified as positive colony is carried out dna sequencing.The plasmid called after URA3-T1 that checks order correct.For foreign gene ω-6FAD and ω-3FAD, respectively with the integrating expression vector pYB-ω 6 that has these two genes and pYB-ω 3 as its standard plasmid.
2. each plasmid control sample is diluted successively and be about 10
3Individual/ml, 10
4Individual/ml, 10
5Individual/ml, 10
6Individual/ml, 10
7Individual/ml, be that template is carried out real-time fluorescence quantitative PCR with the plasmid of different concns after the dilution.
3. be internal control gene with URA3, utilize two calibration curve methods to detect the copy number of foreign gene in the recombinant Saccharomyces cerevisiae bacterial strain, as shown in table 2:
The copy number of foreign gene in the table 2 recombinant Saccharomyces cerevisiae bacterial strain
Can find out that by table 2 this research has successfully obtained containing the recombinant bacterial strain of a plurality of copy goal gene, choose the highest recombinant Saccharomyces cerevisiae bacterial strain of goal gene copy number, and called after yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain Y-10; This bacterial strain was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, address on July 31st, 2012: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is: CGMCC No.6385.This yeast saccharomyces cerevisiae is that flax ω-6 fatty acid dehydrogenase gene and the purple perilla omega-fatty acid dehydrogenase gene that in the delicatessen food yeast, have imported plant origin are processed, and the content of human body beneficial's alpha-linolenic acid is accounted for 7.38% of all fatty acids.It is the Wine brewing yeast strain that a strain can be produced alpha-linolenic acid.Its morphological specificity meets the general aspects of yeast saccharomyces cerevisiae, is unicellular microorganism, and cell is an oval.Formed bacterium colony is an oyster white, the smooth surface protuberance.
Produce the cultural method of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) the bacterial strain Y-10 of alpha-linolenic acid, step is following:
(1) the single colony inoculation of picking Wine brewing yeast strain Y-10 is in the YEPD liquid nutrient medium, under 30 ℃, 225r/min condition, cultivates 10h, makes seed liquor;
(2) get seed liquor that step (1) makes by volume per-cent 1% be inoculated in the YEPD liquid nutrient medium, under 30 ℃, 225r/min condition, cultivate 24h, the centrifugal 5min of 1000r/min gets deposition, makes the yeast thalline;
(3) get the yeast thalline that step (2) makes and 1% be inoculated in the YEPD liquid nutrient medium by weight percentage, under 30 ℃, 225r/min condition, cultivate 72h.
Preferred according to the present invention, every liter of component of said YEPD liquid nutrient medium is following:
Peptone 20g, yeast extract paste 10g, glucose 20g, water is settled to 1L, and pH 6.0.
Wine brewing yeast strain Y-10 growth characteristics detect:
The method of the drafting microbial growth curve that this research is adopted is a turbidimetry, promptly at cell concentration and OD
600In the linearity range that is directly proportional, utilize spectrophotometer, measure the OD of bacteria suspension
600Value is with OD
600The value representation biomass.
At first, original Wine brewing yeast strain and the Wine brewing yeast strain Y-10 mono-clonal bacterium colony of setting out of picking 30 ℃, cultivated 8-12h under the 225r/min condition, then three bottles of each enlarged culturing under the similarity condition in the YEPD of screening marker-free liquid nutrient medium respectively.Every is blank with the blank substratum of cultivating under the similarity condition at a distance from the 2h sampling once, detects the OD of each bacterium liquid
600And record, continuous monitoring 30h.
Afterwards, draw the growth curve (Figure 11) of original starting strain and Wine brewing yeast strain Y-10 respectively.Can find that by figure former starting strain is identical with the time that arrives stationary phase with the time that Wine brewing yeast strain Y-10 arrives logarithmic phase; Both growth characteristics are consistent basically, and the speed of growth not obviously influence of the insertion of foreign gene to bacterial strain is described.The initial inoculation amount of former starting strain and Wine brewing yeast strain Y-10 has small difference, so its growth curve is not to overlap fully.
The fatty acid content analysis of Wine brewing yeast strain Y-10
(I) fermentation of Wine brewing yeast strain Y-10: the single bacterium colony of picking Wine brewing yeast strain Y-10 at 30 ℃, under the 225r/min condition, is cultivated 96h in the YEPD liquid nutrient medium.
(II) Wine brewing yeast strain Y-10 fatty acid component is analyzed.
Dried Wine brewing yeast strain Y-10 thalline is placed mortar, fully be ground to Powderedly, collect the thalline powder; Send feed inspection center of the Ministry of Agriculture; Utilizing gas chromatographic analysis, Wine brewing yeast strain Y-10 is carried out the fatty acid component analysis, is contrast with the former yeast strain that sets out.The result is shown in table 3 and table 4:
The fatty acid component analysis of table 3 Wine brewing yeast strain Y-10
Table 4
Can draw following results by table 3 and table 4:
(1) Zoomeric acid and oleic content are the highest in the control group yeast saccharomyces cerevisiae lipid acid composition, are respectively 43.80% and 37.05%.Oleic acid content is the highest among the Wine brewing yeast strain Y-10, is 41.05%; Zoomeric acid content obviously descends, and reduces to 7.47% by 43.80% of control group; Simultaneously, linolic acid (C18:2n6c) content obviously increases: linoleic acid content is 0.08% in the control group, and linoleic percentage composition is 20.04% among the Wine brewing yeast strain Y-10, has increased more than 200 times.
Obvious variation has taken place in content that it should be noted that Zoomeric acid among the Wine brewing yeast strain Y-10, reduces to 7.47% by 43.80% of control group.ω-6 fatty acid dehydrogenase, i.e. Δ
12-fatty acid dehydrogenase can catalysis Zoomeric acid and oleic acid dehydrogenation, changes into palm diolefinic acid and linolic acid.The minimizing of Zoomeric acid and linoleic increase have proved that ω-6 fatty acid dehydrogenase has obtained efficiently expressing in yeast saccharomyces cerevisiae.
(2) in the control group bacterial strain, do not detect alpha-linolenic acid, the percentage composition of alpha-linolenic acid is up to 7.38% in Wine brewing yeast strain Y-10.Alpha-linolenic acid grows out of nothing, and explains and has expressed activated omega-fatty acid desaturase among the Wine brewing yeast strain Y-10.The intravital Zoomeric acid dehydrogenation of ω-6 fatty acid dehydrogenase catalysis yeast saccharomyces cerevisiae has generated the extremely low linolic acid of content in former starting strain in a large number.Afterwards, the further dehydrogenation of omega-fatty acid desaturase catalysis linolic acid has generated the alpha-linolenic acid that in former starting strain, does not have.
The synthetic of various lipid acid possibly exist complicated relation and delicate balance with katabolism in the body, adds new fatty acid dehydrogenase and broken original balance, and affected lipid acid maybe be a lot.
Linoleic acid content obviously increases among the Wine brewing yeast strain Y-10; Increase to 20.04% by 0.08% of control group, be higher than the linoleic acid content of plam oil (10%), Tallow Inedible Grade (1.8%), lard (6.0%), palm-kernel oil (2-3%), Oleum Cocois (2%), Viscotrol C (2.0-3.5%), haco oil and catfish wet goods.
Compare with control group; Alpha-linolenic acid grows out of nothing among the Wine brewing yeast strain Y-10; Percentage composition reaches 7.38%; Be higher than peanut oil (0.4%), Semen Maydis oil (0.6%), plam oil (0.4%) sweet oil (≤1.0%), til (0.8%), cream, tea oil (1.1%), cannabis oil (0.5%) and duck oil common greasy alpha-linolenic acid content such as (0.8), suitable with the alpha-linolenic acid content of hair scale fish oil, step fourth fish oil, mackerel oil and catfish wet goods; Compare with daily food, the alpha-linolenic acid content of bacterial strain is higher than Semen arachidis hypogaeae (2.0%), sunflower seeds (0.4%), Semen Cucurbitae (0.7%) and sesame (0.9%) etc.
ω-6PUFAs and ω-3PUFAs exists competition to suppress, so ω-6/ ω-3 ratio comes into one's own, WHO suggestion ω-6/ ω-3 ratio is 5-10: 1, and China's suggestion ω-6/ ω-3 ratio is 4-6: 1.And present China developed regions people take in the ratio of linolic acid and alpha-linolenic acid up to 20-30: 1, and both ratios are seriously unbalance, cause multiple chronic disease sickness rate constantly to rise.ω-6/ ω-3 ratio of the recombinant bacterial strain Y-10 that this research obtains is 2.7: 1, and alpha-linolenic acid is fit to produce the special yeast product that replenishes alpha-linolenic acid than great.
Alpha-linolenic acid has multiple important physical function as the unsaturated fatty acids of needed by human, and is therefore extensive in field demands such as medicine, food.But alpha-linolenic acid still can not synthetic up to now, can only depend on the limited natural resource, can't meet the need of market.In addition, in the grease leaching process, alpha-linolenic acid can lose a part, is 2.0% like the content of alpha-linolenic acid in the Semen arachidis hypogaeae, and in peanut oil, is merely 0.4%.This research uses the yeast saccharomyces cerevisiae to the person poultry harmless to be material, and the Wine brewing yeast strain Y-10 that contains linolic acid and alpha-linolenic acid that obtains does not need special extraction to eat together with yeast saccharomyces cerevisiae, has reduced the loss of beneficial fatty acids.
Claims (4)
1. yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) the bacterial strain Y-10 of alpha-linolenic acid is produced in a strain; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 31st, 2012; The address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is: CGMCC No.6385.
2. the cultural method of the yeast saccharomyces cerevisiae of the said production alpha-linolenic acid of claim 1 (Saccharomyces cerevisiae) bacterial strain Y-10, step is following:
(1) the single colony inoculation of picking Wine brewing yeast strain Y-10 is in the YEPD liquid nutrient medium, under 28～32 ℃, 200～250r/min condition, cultivates 8～12h, makes seed liquor;
(2) get seed liquor that step (1) makes by volume per-cent 1～5% be inoculated in the YEPD liquid nutrient medium, under 28～32 ℃, 200～250r/min condition, cultivate 20～30h, centrifugal, get deposition, make the yeast thalline;
(3) get the yeast thalline that step (2) makes and 1～5% be inoculated in the YEPD liquid nutrient medium by weight percentage, under 28～32 ℃, 200～250r/min condition, cultivate 60～80h.
3. cultural method as claimed in claim 2, and be characterised in that every liter of component of said YEPD liquid nutrient medium is following:
Peptone 20g, yeast extract paste 10g, glucose 20g, water is settled to 1L, and pH 6.0.
4. the yeast saccharomyces cerevisiae of the said production alpha-linolenic acid of claim 1 (Saccharomyces cerevisiae) bacterial strain Y-10 is rich in the application in linolenic acid food or the feed in preparation.
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