CN104974944A - Schizochytrium limacinum genetic engineering strain for producing DHA (docosahexaenoic acid), and construction method and application thereof - Google Patents
Schizochytrium limacinum genetic engineering strain for producing DHA (docosahexaenoic acid), and construction method and application thereof Download PDFInfo
- Publication number
- CN104974944A CN104974944A CN201510417269.4A CN201510417269A CN104974944A CN 104974944 A CN104974944 A CN 104974944A CN 201510417269 A CN201510417269 A CN 201510417269A CN 104974944 A CN104974944 A CN 104974944A
- Authority
- CN
- China
- Prior art keywords
- schizochytrium limacinum
- genetic engineering
- schizochytrium
- dha
- malic enzyme
- 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.)
- Granted
Links
- 241000003595 Aurantiochytrium limacinum Species 0.000 title claims abstract description 54
- 238000010353 genetic engineering Methods 0.000 title claims abstract description 36
- 238000010276 construction Methods 0.000 title claims abstract description 21
- 235000020669 docosahexaenoic acid Nutrition 0.000 title description 26
- MBMBGCFOFBJSGT-KUBAVDMBSA-N all-cis-docosa-4,7,10,13,16,19-hexaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 title 4
- 229940090949 docosahexaenoic acid Drugs 0.000 title 2
- 241000894006 Bacteria Species 0.000 claims abstract description 70
- 239000012634 fragment Substances 0.000 claims abstract description 64
- 101710104378 Putative malate oxidoreductase [NAD] Proteins 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 21
- 230000006801 homologous recombination Effects 0.000 claims abstract description 14
- 238000002744 homologous recombination Methods 0.000 claims abstract description 14
- 239000001963 growth medium Substances 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 238000011218 seed culture Methods 0.000 claims abstract description 8
- 238000012216 screening Methods 0.000 claims abstract description 4
- 108090000848 Ubiquitin Proteins 0.000 claims description 26
- 102000044159 Ubiquitin Human genes 0.000 claims description 26
- 239000002609 medium Substances 0.000 claims description 16
- 238000011144 upstream manufacturing Methods 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 6
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 6
- 229940073490 sodium glutamate Drugs 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 108010083912 bleomycin N-acetyltransferase Proteins 0.000 claims description 2
- 230000010354 integration Effects 0.000 claims description 2
- 239000003550 marker Substances 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 abstract description 7
- 230000004151 fermentation Effects 0.000 abstract description 7
- 241000233671 Schizochytrium Species 0.000 abstract 5
- 238000010364 biochemical engineering Methods 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 34
- 239000013612 plasmid Substances 0.000 description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 26
- 238000006243 chemical reaction Methods 0.000 description 24
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 22
- 102000004190 Enzymes Human genes 0.000 description 19
- 108090000790 Enzymes Proteins 0.000 description 19
- 102000003960 Ligases Human genes 0.000 description 13
- 108090000364 Ligases Proteins 0.000 description 13
- 241000598397 Schizochytrium sp. Species 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 13
- 230000029087 digestion Effects 0.000 description 12
- 108091008146 restriction endonucleases Proteins 0.000 description 12
- 230000035939 shock Effects 0.000 description 12
- 238000012795 verification Methods 0.000 description 12
- 238000000246 agarose gel electrophoresis Methods 0.000 description 11
- 239000000284 extract Substances 0.000 description 11
- 239000003292 glue Substances 0.000 description 11
- 235000015097 nutrients Nutrition 0.000 description 11
- 239000000047 product Substances 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 108010084455 Zeocin Proteins 0.000 description 6
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 6
- 239000004519 grease Substances 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 4
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 108010006654 Bleomycin Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 229960001561 bleomycin Drugs 0.000 description 3
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 2
- 229940107700 pyruvic acid Drugs 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 101000942941 Arabidopsis thaliana DNA ligase 6 Proteins 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 102000013460 Malate Dehydrogenase Human genes 0.000 description 1
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000003987 high-resolution gas chromatography Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 235000020667 long-chain omega-3 fatty acid Nutrition 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a DHA-producing schizochytrium genetic engineering bacterium and a construction method and application thereof, belonging to the technical field of biochemical engineering. The DHA-producing Schizochytrium limacinum gene engineering strain is an engineering strain integrating an expression cassette of malic enzyme gene in a Schizochytrium limacinum genome, wherein the expression cassette of the malic enzyme gene sequentially comprises a promoter, the malic enzyme gene and a terminator. The construction method of the gene engineering bacteria of schizochytrium comprises the following steps: constructing a homologous recombination fragment containing an expression cassette of the malic enzyme gene; and introducing the homologous recombination fragments into schizochytrium limacinum for homologous recombination, and screening to obtain the schizochytrium limacinum genetically engineered bacteria. The invention also provides a method for preparing DHA by using the schizochytrium genetic engineering bacteria, which comprises the steps of culturing the schizochytrium genetic engineering bacteria by using a seed culture medium and then transferring the schizochytrium genetic engineering bacteria into a fermentation culture medium to prepare DHA. The Schizochytrium limacinum genetically engineered strain can produce DHA with high yield, and has the advantages of simple construction method, high efficiency and lower cost.
Description
Technical field
The invention belongs to technical field of biochemical industry, be specifically related to a kind of produce DHA schizochytrium limacinum genetic engineering bacterium and construction process and application.
Background technology
DHA is a kind of important omega-3 long chain polyunsaturated fatty acids, is the moiety of cytolemma in brain and retinal tissue, has vision enhancing, promotes the physiological function such as brain cell development and preventing hypertension, arteriosclerosis, cardiovascular disorder.Human body is difficult to meet the demand of body to DHA by self synthesis, needs to absorb from food.The source obtaining DHA from occurring in nature is very limited, and adopt industrialization schizochytrium limacinum fermentation to produce DHA, yield poorly, cost is high.
Summary of the invention
The object of the present invention is to provide the schizochytrium limacinum genetic engineering bacterium of a kind of high yield DHA.
Another object of the present invention is to provide the construction process of described schizochytrium limacinum genetic engineering bacterium, and the method is simple, and efficiency is high, and cost is lower.
Another object of the present invention is to provide the method adopting described schizochytrium limacinum genetic engineering bacterium to prepare DHA, and the method can obtain the DHA of high yield, and cost is lower.
Object of the present invention adopts following technical scheme to realize:
Producing a schizochytrium limacinum genetic engineering bacterium of DHA, is the engineering bacteria of the expression cassette being integrated with malic enzyme gene in schizochytrium limacinum genome, and the expression cassette of described malic enzyme gene is successively containing promotor, malic enzyme gene and terminator.
In preferred technical scheme, described promotor is ubiquitin promoter, and described malic enzyme gene is as shown in SEQ IDNO:1, and described terminator is ubiquitin tenninator.Described integration site is 18srDNA.
The present invention also provides the construction process of described schizochytrium limacinum genetic engineering bacterium, builds the homologous recombination fragment of the expression cassette containing malic enzyme gene; Described homologous recombination fragment is imported schizochytrium limacinum and carries out homologous recombination, screening obtains described schizochytrium limacinum genetic engineering bacterium.
In the present invention, be provided with the upstream and downstream homology arm of 18srDNA at the two ends of the expression cassette of described malic enzyme gene in described homologous recombination fragment, the upstream homology arm sequence of described 18srDNA is as shown in SEQ ID NO:2, and the downstream homology arm of 18srDNA is as shown in SEQ ID NO:3.
In preferred technical scheme, described homologous recombination fragment carries bleomycin resistance marker gene.
The present invention also provides and adopts described schizochytrium limacinum genetic engineering bacterium to prepare the method for DHA, adopts seed culture medium to cultivate schizochytrium limacinum genetic engineering bacterium, then transfers to prepare DHA into fermention medium; Described seed culture medium contains: glucose 40g/L, yeast extract paste 2g/L, Sodium Glutamate 10g/L, KH
2pO
44g/L, NaCl15g/L, MgCl
23g/L, CaCl
22H
2o 1g/L, KCl 2g/L, MgSO
47H
2o 5g/L, FeCl
30.1g/L; Described fermention medium contains: glucose 40g/L, yeast extract paste 2g/L, Sodium Glutamate 10g/L, KH
2pO
44g/L, NaCl 15g/L, MgCl
23g/L, (NH
4)
2sO
46g/L, KCl 2g/L, MgSO
47H
2o 5g/L, FeCl
30.1g/L.
Beneficial effect of the present invention: the expression cassette with strong promoter and malic enzyme gene is incorporated on schizochytrium limacinum genome by the present invention, builds the schizochytrium limacinum genetic engineering bacterium obtaining producing DHA.This genetic engineering bacterium can process LAN malic enzyme.In genetic engineering bacterium, malate dehydrogenase (malic acid dehydrogenase) can catalysis oxaloacetic acid be oxysuccinic acid, oxysuccinic acid can be reduced into pyruvic acid and generate a large amount of NADPH by the malic enzyme of process LAN, thus the circulating reaction of pyruvic acid → oxaloacetic acid → oxysuccinic acid is carried out, a large amount of NADPH is generated in this circulating reaction, have very large promoter action to the generation of grease, therefore genetic engineering bacterium of the present invention has the ability compared with high yield DHA.The construction process of genetic engineering bacterium of the present invention is simple, and efficiently, cost is low.The present invention adopts described schizochytrium limacinum genetic engineering bacterium to prepare the method for DHA, and output comparatively original strain significantly improves, and cost is lower.
Accompanying drawing explanation
Fig. 1: the construction step of plasmid pBZ-18S.
The construction step of Fig. 2 plasmid pBZ18S-PMT.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment is only limitted to the present invention is described, and also should can not limit the present invention described in detail in claims.
Biological material source in the present invention is as follows:
Schizochytrium limacinum (Schizochytrium sp.) HX-308, has been preserved in China typical culture collection center (CCTCC), and its deposit number is CCTCC No.M 209059;
Carrier pBlueScript II SK: commercial vector, is purchased from Invitrogen company;
Carrier pGAPZ α A: commercial vector, is purchased from Invitrogen company;
Carrier pMD19-T: commercial vector, is purchased from TaKaRa company;
Carrier pMD19-T (simple): commercial vector, is purchased from TaKaRa company.
The structure of embodiment 1 recombinant plasmid pBZ-18S
1. build recombinant vectors pBS-Zeo
By BamHI and EcoRI restriction enzyme site, Zeocin (bleomycin) resistance gene fragment in pGAPZ α A carrier is inserted pBlueScript II SK carrier, build recombinant vectors pBS-Zeo.
PGAPZ α A and pBlueScript II SK carrier carry out double digestion respectively.Enzyme cuts system (related reagent is purchased from TaKaRa): ddH
2o 60 μ L, EcoRI 2 μ L, BamHI 2 μ L, 10 × K buffer 10 μ L, carrier 26 μ L.Enzyme Qie Wendu: 30 DEG C.
Agarose gel electrophoresis with 0.8% detects digestion products, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is connected with T4 ligase enzyme and spend the night.
T4 ligase enzyme system: ddH
2o 11.5 μ L, T4 Ligase 1 μ L, T4 Ligase buffer 2.5 μ L, Zeocin resistance gene fragment 8 μ L, carrier 2 μ L.
Get connect spend the night after reaction solution 5 μ L add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 37 DEG C, cultivate 1h under 200rpm condition.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extracts positive colony plasmid and called after pBS-Zeo.
2. increase schizochytrium limacinum 18SrDNA upstream and downstream homology arm
(1) increase schizochytrium limacinum 18SrDNA upstream homology arm
Adopt the 18SrDNA fragment of fungi 18SrDNA universal primer NS1 and NS8 amplification schizochytrium limacinum (Schizochytrium sp.) HX-308, be connected in carrier T, obtain recombinant vectors pMD-18S.Wherein the sequence (SEQ ID NO:6) of NS1 is: the sequence (SEQID NO:7) of GTAGTCATATGCTTGTCTC, NS8 is: TCCGCAGCTTCACCTACGGA.
According to schizochytrium limacinum 18SrDNA fragment sequence design primer 18SupS and 18SupA amplification upstream homology arm (SEQ ID NO:2) obtained.18SupS (SEQ ID NO:8) sequence: 5 '-GGGTACCCGTAGTCATATGCTTGTCTC-3 ', 18SupA (SEQ ID NO:9) sequence: 5 '-CCTCGAGGATTTCACCTCTAGCGAC-3 '.
PCR reaction system (related reagent is purchased from TaKaRa):
hS (Premix) 25 μ L, 18SupS 0.3 μ L, 18SupA 0.3 μ L, pMD-18S 0.4 μ L, ddH
2o 24 μ L.PCR reaction conditions: 98 DEG C of 10s, 65 DEG C of 15s, 72 DEG C of 50s, circulate 30 times; 72 DEG C of 7min.
Agarose gel electrophoresis with 0.8% detects PCR primer, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is cloned on carrier pMD19-T.Clone's system: the fragment after 1 μ L pMD19-T carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, under 200rpm, 37 DEG C of conditions, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extracts positive colony plasmid and send sequence verification and called after pMD19-18Sup.
(2) increase schizochytrium limacinum 18SrDNA downstream homology arm
According to the schizochytrium limacinum 18SrDNA primers 18SdownS (SEQ ID NO:10) obtained and 18SdownA (SEQ ID NO:11) the downstream homology arm that increases (SEQ ID NO:3).18SdownS sequence: 5 '-CGGATCCGATGCCGACTAGAGATT-3 '; 18SdownA sequence: 5 '-GAGCTCTCCGCAGGTTCACCTACGGA-3 '.
PCR reaction system:
hS (Premix) 25 μ L, 18SdownS 0.3 μ L, 18SdownA 0.3 μ L, pMD-18S 0.4 μ L, ddH
2o 24 μ L.
PCR reaction conditions: 98 DEG C of 10s, 65 DEG C of 15s, 72 DEG C of 50s, circulate 30 times; 72 DEG C of 7min.
Agarose gel electrophoresis with 0.8% detects PCR primer, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is cloned on carrier pMD19-T.Clone's system: the fragment after 1 μ L pMD19-T carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, under 200rpm, 37 DEG C of conditions, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extracts positive colony plasmid and send sequence verification, the recombinant plasmid called after pMD19-18Sdown checking order correct.
3. build plasmid pBS-Zeo-18Sup
By SacI and BamHI restriction enzyme site, 18SrDNA upstream homology arm is inserted the upstream of Zeocin (bleomycin) resistance gene fragment in plasmid pBS-Zeo, build plasmid pBS-Zeo-18Sup.
PMD19-18Sup, pBS-Zeo carry out double digestion respectively.Enzyme cuts system: ddH
2o 65 μ L, SacI 2 μ L, BamHI 2 μ L, 10 × Kbuffer 5 μ L, carrier 26 μ L.Enzyme Qie Wendu: 30 DEG C.
Agarose gel electrophoresis with 0.8% detects digestion products, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is connected with T4 ligase enzyme and spend the night.T4 ligase enzyme system: ddH
2o 11.5 μ L, T4Ligase 1 μ L, T4Ligase buffer 2.5 μ L, fragment 8 μ L, carrier 2 μ L.
Get connect spend the night after reaction solution 5 μ L add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C of cultivation 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extracts positive colony plasmid and called after pBS-Zeo-18Sup.
4. build plasmid pBZ-18S
By KpnI and XhoI restriction enzyme site, 18SrDNA downstream homology arm is inserted the downstream of Zeocin (bleomycin) resistance gene fragment in plasmid pBS-Zeo-18Sup, build plasmid pBZ-18S.
PMD19-18Sdown, pBS-Zeo-18Sup carry out double digestion respectively.Enzyme cuts system: ddH
2o 60 μ L, KpnI 2 μ L, XhoI 2 μ L, 10 × M buffer 10 μ L, carrier 26 μ L.Enzyme Qie Wendu: 30 DEG C.
Agarose gel electrophoresis with 0.8% detects digestion products, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is connected with T4 ligase enzyme and spend the night.T4 ligase enzyme system: ddH
2o 11.5 μ L, T4Ligase 1 μ L, T4Ligase buffer 2.5 μ L, fragment 8 μ L, carrier 2 μ L.
Get connect spend the night after reaction solution 5 μ L add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, under 200rpm, 37 DEG C of conditions, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extracts positive colony plasmid and called after pBZ-18S.
The structure of embodiment 2 recombinant plasmid pBZ18S-PMT
1, the clone of ubiquitin promoter fragment, malic enzyme gene and ubiquitin tenninator fragment
1) clone of ubiquitin promoter fragment
Synthesize ubiquitin promoter fragment (SEQ ID NO:4) by Jin Weizhi company and be cloned into carrier T, forming recombinant plasmid Promoter-T.
According to the sequence of ubiquitin promoter, design and synthesis primer P1 (SEQ ID NO:12) and P2 (SEQ IDNO:13).P1 (band ClaI restriction enzyme site) sequence: 5 '-CCATCGATGG
TCGGTACCCGTTAGAACGCGTAAT-3 ', P2 (band SpeI restriction enzyme site) sequence: 5 '-GGACTAGTC TTCGTCTTATCCTCAGTCATGTTGG-3 '.
With Promoter-T plasmid for template, with primer P1 and P2, Takara high-fidelity enzyme
hS (Premix) pcr amplification ubiquitin promoter fragment.PCR reaction system:
hS (Premix) 25 μ L, upstream primer 0.3 μ L, downstream primer 0.3 μ L, Promoter-T plasmid 0.4 μ L, ddH
2o 24 μ L.PCR reaction conditions: 98 DEG C of 10s, 65 DEG C of 15s, 72 DEG C of 50s, circulate 30 times; 72 DEG C of 7min.
Agarose gel electrophoresis with 0.8% detects PCR primer, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is cloned on carrier pMD19-T.Clone's system: the fragment after 1 μ L pMD19-T carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, under 200rpm, 37 DEG C of conditions, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extracts positive colony plasmid and send sequence verification, by positive plasmid called after pMD19-P correct for sequence verification.
2) clone of malic enzyme gene
According to the sequence (SEQ ID NO:1) of malic enzyme gene, design and synthesis primer M1 (SEQ IDNO:14) and M2 (SEQ ID NO:15).M1 (with SpeI restriction enzyme site): 5 '-GGACTAGTCATGAGCGTACTGTTGGACACCAT-3 ', M2 (with SmaI restriction enzyme site): 5 '-TCCCCCGGGGGA TTAAAGACGGGTTTGCGGATAG-3 '.
With schizochytrium limacinum (Schizochytrium sp.) HX-308 genomic dna for template, with primer M1 and M2, Takara high-fidelity enzyme
hS (Premix) pcr amplification malic enzyme gene fragment.PCR reaction system:
hS (Premix) 25 μ L, upstream primer 0.3 μ L, downstream primer 0.3 μ L, schizochytrium limacinum (Schizochytrium sp.) HX-308 genomic dna 0.4 μ L, ddH
2o 24 μ L.PCR reaction conditions: 98 DEG C of 10s, 65 DEG C of 15s, 72 DEG C of 1min50s, circulate 30 times; 72 DEG C of 7min.
Agarose gel electrophoresis with 0.8% detects PCR primer, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is cloned on carrier pMD19-T (simple).Clone's system: the fragment after 1 μ LpMD19-T (simple) carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, under 200rpm, 37 DEG C of conditions, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extracts positive colony plasmid and send sequence verification.The positive plasmid called after pMD19s-M that sequence verification is correct.
3) clone of ubiquitin tenninator fragment
Synthesize ubiquitin tenninator fragment (SEQ ID NO:5) by Jin Weizhi company and be cloned into carrier T, forming recombinant plasmid Terminator-T.
According to the sequence of ubiquitin tenninator, design and synthesis primer T1 (SEQ ID NO:16) and T2 (SEQ IDNO:17).T1 (with ClaI, SmaI restriction enzyme site): 5 '-CCATCGATGGatagcagctgTCCCCCGGGGGACCAAGGCCAAGTCGGACTAAACT-3 ', T2 (with EcoRV restriction enzyme site): 5 '-GGAGATATCTCGGTACCACCGCGTAATACGAC-3 '.
Take Terminator-T as template, with primer T1 and T2, Takara high-fidelity enzyme
hS (Premix) pcr amplification ubiquitin tenninator fragment.PCR reaction system:
hS (Premix) 25 μ L, upstream primer 0.3 μ L, downstream primer 0.3 μ L, Terminator-T plasmid 0.4 μ L, ddH
2o 24 μ L.PCR reaction conditions: 98 DEG C of 10s, 65 DEG C of 15s, 72 DEG C of 40s, circulate 30 times; 72 DEG C of 7min.
Agarose gel electrophoresis with 0.8% detects PCR primer, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is cloned on carrier pMD19-T (simple).Clone's system: the fragment after 1 μ LpMD19-T (simple) carrier, 4 μ L purifying, mixes gently; After 25 DEG C of reaction 20min, add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, under 200rpm, 37 DEG C of conditions, cultivate 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extracts positive colony plasmid and send sequence verification.The recombinant plasmid called after pMD19s-T that sequence verification is correct.
2, enzyme is cut and is connected ubiquitin promoter and malic enzyme gene, carrier construction pMD19-PM.
Be connected ubiquitin promoter and malic enzyme gene by ClaI with SpeI restriction enzyme site, cut carrier pMD19-P, pMD19s-M by enzyme, carrier construction pMD19-PM.PMD19-P, pMD19s-M carry out double digestion respectively.Enzyme cuts system: ddH
2o 60 μ L, ClaI 2 μ L, SpeI 2 μ L, 10 × M buffer 10 μ L, carrier 26 μ L.Enzyme Qie Wendu: 37 DEG C.
Agarose gel electrophoresis with 0.8% detects digestion products, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is connected with T4 ligase enzyme and spend the night.T4 ligase enzyme system: ddH
2o 11.5 μ L, T4Ligase 1 μ L, T4Ligase buffer 2.5 μ L, fragment 8 μ L, carrier 2 μ L.
Get connect spend the night after reaction solution 5 μ L add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C of cultivation 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extracts positive colony plasmid and also checks order.By positive recombinant plasmid called after pMD19-PM correct for sequence verification.
3, enzyme is cut and is connected ubiquitin promoter, malic enzyme gene and ubiquitin tenninator, carrier construction pMD19-PMT.
By ClaI and SmaI restriction enzyme site, ubiquitin promoter, malic enzyme gene fragment are inserted the upstream of ubiquitin tenninator fragment in pMD19s-T, make ubiquitin promoter, malic enzyme gene fragment is connected successively with ubiquitin tenninator, carrier construction pMD19-PMT.Ubiquitin promoter, malic enzyme gene fragment and ubiquitin tenninator are connected to form the expression cassette of malic enzyme gene successively.
PMD19-PM, pMD19s-T carry out double digestion respectively.Enzyme cuts system: ddH
2o 60 μ L, ClaI 2 μ L, SpeI 2 μ L, 10 × T buffer 5 μ L, BSA (bovine serum albumin) 5 μ L, carrier 26 μ L.Enzyme Qie Wendu: 37 DEG C.
Agarose gel electrophoresis with 0.8% detects digestion products, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is connected with T4 ligase enzyme and spend the night.T4 ligase enzyme system: ddH
2o 11.5 μ L, T4Ligase 1 μ L, T4Ligase buffer 2.5 μ L, fragment 8 μ L, carrier 2 μ L.
Get connect spend the night after reaction solution 5 μ L add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C of cultivation 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extracts positive colony plasmid and also checks order.By positive recombinant plasmid called after pMD19-PMT correct for sequence verification.
4, carrier construction pBZ18S-PMT
By ClaI and EcoRV restriction enzyme site, the expression cassette of malic enzyme gene is inserted in pBZ-18S between 18SrDNA downstream homology arm and Zeocin resistance gene fragment, carrier construction pBZ18S-PMT.
PMD19-PMT, pBZ-18S carry out double digestion respectively.Enzyme cuts system: ddH
2o 60 μ L, ClaI 2 μ L, EcoRV 2 μ L, 10 × H buffer 10 μ L, carrier 26 μ L.Enzyme Qie Wendu: 37 DEG C.
Agarose gel electrophoresis with 0.8% detects digestion products, and uses Takara glue to reclaim kits object fragment, then the fragment of purifying is connected with T4 ligase enzyme and spend the night.T4 ligase enzyme system: ddH
2o 11.5 μ L, T4Ligase 1 μ L, T4Ligase buffer 2.5 μ L, fragment 8 μ L, carrier 2 μ L.
Get connect spend the night after reaction solution 5 μ L add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shock 30s, are placed in 2min on ice immediately.Add rapidly 250 μ L LB substratum, 200rpm, 37 DEG C of cultivation 1h.Get 200 μ L bacterium liquid and be coated with the LB flat board containing penbritin, after incubated overnight, the LB liquid nutrient medium that picking 10 positive bacterium colonies connect containing penbritin is cultivated, and extracts positive colony plasmid and also checks order.By positive recombinant plasmid called after pBZ18S-PMT correct for sequence verification.
The screening of the schizochytrium limacinum genetic engineering bacterium of embodiment 3 homologous recombination and product DHA
1) the 50mL polypropylene tube that 10mL schizochytrium limacinum (Schizochytrium sp.) HX-308 bacterium liquid puts into aseptic precooling is got, at 4 DEG C, the centrifugal 10min of 5000r/min, abandon supernatant, with the sterile water wash thalline twice of 10mL precooling, at 4 DEG C, the centrifugal 10min of 4472 × g.With the 1mol/L Sorbitol Solution USP of 10mL precooling, bacterial sediment is resuspended, 4 DEG C, the centrifugal 10min of 5000r/min, repeat 1 time.With the 1mol/L Sorbitol Solution USP of 10mL precooling, bacterial sediment is resuspended.
2) plasmid pBZ18S-PMT being joined 30 μ L steps 1) in pretreated schizochytrium limacinum (Schizochytrium sp.) HX-308, mix gently, ice bath leaves standstill 5min, then transfers in the electric shock cup of ice precooling, leaves standstill 10min.Shock parameters 0.75KV is set, 200 Ω, 50 μ F.1mL seed culture medium (with in embodiment 4) is added after electric shock, 30 DEG C, 200r/min recovery 1h, be coated on by the bacterium liquid of conversion on the flat board containing 1.5 μ g/mL Zeocin (seed culture medium adds 2% agar) subsequently, 28 DEG C of lucifuges are cultivated.
3), at resistant panel top sieve menu bacterium colony, after shake-flask culture, after extracting genomic dna, take genomic dna as template PCR amplifications object fragment, recombinant bacterium called after schizochytrium limacinum (Schizochytrium sp.) HX-ME that sequence verification is correct.
Embodiment 4 adopts genetic engineering bacterium schizochytrium limacinum (Schizochytrium sp.) HX-ME to produce DHA
Adopt original bacteria schizochytrium limacinum (Schizochytrium sp.) HX-308 and genetic engineering bacterium schizochytrium limacinum (Schizochytrium sp.) HX-ME fermentative production DHA respectively.
1. fermentation culture method
With the inoculum size of 10% (v/v) by three grades of seed liquor access fermentation tank culture medium.During cultivation, 5L fermentor tank liquid amount is 3L, needs supplementary glucose to make in fermented liquid grape concentration higher than 15g/L in fermenting process; Air flow is 1vvm, and mixing speed is 350rpm, and Initial sugar concentration is 40g/l, and leavening temperature is 30 DEG C.Fermentation time is 7 days.
Wherein, seed culture medium (g/L) consists of: glucose 40g/L, yeast extract paste 2g/L, Sodium Glutamate 10g/L, KH
2pO
44g/L, NaCl 15g/L, MgCl
23g/L, CaCl
22H
2o 1g/L, KCl 2g/L, MgSO
47H
2o 5g/L, FeCl
30.1g/L, sterilising conditions: 121 DEG C, 30min.
Fermention medium (g/L) consists of: glucose 40g/L, yeast extract paste 2g/L, Sodium Glutamate 10g/L, KH
2pO
44g/L, NaCl 15g/L, MgCl
23g/L, (NH
4)
2sO
46g/L, KCl 2g/L, MgSO
47H
2o5g/L, FeCl
30.1g/L, sterilising conditions: 121 DEG C, 30min.
2. detection method
During the fermentation, every day, sampling detected as follows:
(1) measuring method of fat content: the fermented liquid (100ml) getting certain volume, by it 50 DEG C of preheatings, by NaOH solution, pH is adjusted between 10-12, adds wall breaking enzyme in the ratio of 3 ‰ (g/l), stir at 50 DEG C and be incubated 2h; Add ethanol, normal hexane respectively, stirring, layering, extraction, continuous extraction 2-3 time in 1:1:1 (fermented liquid: ethanol: normal hexane) (v/v) ratio, get normal hexane phase, vacuum rotating solvent evaporated, obtain grease.Flask is put in an oven 105 DEG C dry to constant weight, weigh after cooling.
(2) DHA content detection method in grease: the grease adopting ordinary method (1) in the present embodiment title 2 to be extracted carries out esterification, then utilizes Japanese Shimadzu high resolution gas chromatography instrument to detect DHA content in grease.Gas phase analysis condition: chromatographic column: DB-23 (60m*0.25mm*0.25 μm); Detector: FID; Carrier gas: nitrogen; Splitting ratio: 30/1; Injector temperature: 250 DEG C; Detector temperature: 280 DEG C; Sample size: 1 μ l; Heating schedule: initial column temperature is 100 DEG C, first rises to 196 DEG C with the speed of 25 DEG C/min, then rises to 220 DEG C with the speed of 2 DEG C/min, keeps 12min.Column flow rate: 3.0ml/min; Tail wind drift speed: 30ml/min; Hydrogen flow rate: 40ml/min; Air velocity: 400ml/min.
(3) dry cell weight (DCW) measures: get 30mL fermented liquid, collected by centrifugation, supernatant discarded.Thalline dries to constant weight in 105 DEG C, measures and is dry cell weight.
(4) glucose concentration determination: get fermented liquid 1mL in 1.5mL centrifuge tube, under room temperature, the centrifugal 2min of 12000rpm, get 100 μ L supernatants to 10mL volumetric flask, 10mL is settled to WAHAHA water, then utilize the glucose concn in biosensor assay fermented liquid, the unit recording numerical value is g/L.
3. result
When fermentation ends, the oil offtake of original bacteria and genetic engineering bacterium reaches peak value.The peak oil production of original bacteria is 59.8g/L fermented liquid, and wherein DHA output only has 27.3g/L fermented liquid.The peak oil production of genetic engineering bacterium reaches 72.5g/L, and wherein DHA output is up to 32.6g/L fermented liquid.Can see, restructuring schizochytrium limacinum engineering bacteria bacterium is compared with original bacteria, and oil offtake adds 12%, and wherein DHA output adds 19.5%.
SEQUENCE LISTING
<110> Nanjing University of Technology
<120> mono-kind produces the schizochytrium limacinum genetic engineering bacterium of DHA and construction process thereof and application
<130> 201507151
<160> 17
<170> PatentIn version 3.3
<210> 1
<211> 1848
<212> DNA
<213> schizochytrium limacinum (Schizochytrium sp.) HX-308
<400> 1
atgagcgtac tgttggacac catgggtatc ttctcgcgca agagcaaggc cgccgacagc 60
aagcccgcag aggcgacgct ggcactcaaa gactcgagcc gcggcgcgga agagtcgaac 120
gtggagcggc tgcgaaaccc gcacttcaac aagggcacga gcttcacgca agaggagcgc 180
gcgcagtatg gcgtcctcgg cctcgtgccc agcgtcgagg agtccatgga gctccagacc 240
aagcgcgagc ttcagcacct gaggcgcaag acatcggaca tcgaaaagta cgagttcctt 300
atgggcctgc tcgacaggaa tgtccagctc ttctacaagc ttgtcactga gaacatctcc 360
gagtgcatgc ccctcgtcta cacgcccacc gtcggccagg catgccagga gttccacctc 420
atttacacgc agccgcgcgg tctctacgtc tcgctcaacg acctcggcaa cgtccaggcc 480
ctcgtcgaca actggcccga ggataacgtc accaccattg tcatgaccga cggtggccgc 540
attcttggtc ttggtgacct tggcgcgaat ggccttggca ttccccaggg caagctccaa 600
ctctactcgg cctgcgctgg catcccgcac catcagtgtc ttcccgtcat cctcgatgtc 660
ggcaccaaca acgagagcct cctcgaggac gaactctaca tgggtctccg ccagaagcgc 720
gagcgcggcg agacctacga ccaccttgtc aaggagttca tgcaggccgc gcagaagcgc 780
tggggccgct cgctcctcat ccagttcgag gactttgaca acaccaacgc ctttcgtctg 840
ctcgaagaga cccgccactc atacacgacc tttaacgacg acatccaagg caccgccgcc 900
gtctctctgg ccggcgttct cgcctcgctc cgcgtcacct ccgaggtcga tggcggcaag 960
aacaagcttc gcgaccacac ttttgtcttt ctcggcgctg gcgaggccgg taccggtatt 1020
gccaacctca ttgcccacgc catccaggaa gaggctgtcg acgatggcga ggagcccatc 1080
tcggaggctg aggcccgccg caagatctgg ctcgtcgatt ccaagggtct cgtcaccaag 1140
acgcgcaacg acaacggcga gctccagcac cacaagattg acttcgccca cgagatcacc 1200
gacgatctca ttgaagctgt caagggtacc gagtttgagg tcaaggacgg ccgcgtcact 1260
tcgctcgagc aggctgttca catgctcaag ccctcggcct tgattggcgt ctccgccatc 1320
ccgcgtacct ttacgcagag cattgttgag tacatggccg agatcaacga ggttccgctc 1380
atcttcgcgc ttagcaaccc gacctcacag gccgagtgca ccgccgagca ggcttacaac 1440
tggagcagtg gtcgcgccat ctttgtcagc ggatccccct ttgacccggt tgacgtcgag 1500
ctcgagtccg gcgaggttgt cacaaagtac ccaggccagg gcaacaacgc ctacatcttc 1560
cccggcctcg gccttggcgt tctcgccgcc aaggcgacca ccatccccaa cgagcttctc 1620
tacgtctccg cgcaggctct tgcagagcag gtggttgacg aggacctcga tggcggtcgc 1680
atgtaccccc atctcagcca cattcgcgag gtctctgcca agatcggtgt ccgcgtcgca 1740
gaccgcgcct tcaagcttgc tattgcatcg gcaaagcgac ccgaagacct tgacgcttac 1800
gtgcgctcct gcatggccaa gcccatctat ccgcaaaccc gtctttaa 1848
<210> 2
<211> 855
<212> DNA
<213> schizochytrium limacinum (Schizochytrium sp.) HX-308
<400> 2
aaatttatat tgtgaaactg cgaatggctc attaaatcag ttatgatcta cgtgacatat 60
tctttactac ttggataacc gtggtaattc tagagctaat acatgcaaaa aaacccaaac 120
ttacgaatgg gtgcacttat tagataaagc caacgctggg taaaaccagt ttcccttggt 180
gattcataat aattaagcgg atcgcatggc cttgtgctag cgacagtcca ctcgattttc 240
tgccctatca tggttgagat tgtaagatag aggcttacaa tgcctacaac gggtaacggg 300
gaattagggt tcgattccgg agagggagcc tgagaaacgg ctaccacatc caaggaaggc 360
agcaggcgcg caaattaccc aatcccgaca cggggaggta gtgacaataa ataacaatgc 420
agggccttta aggtcttgca attggaatga gtacaattta aatcccttaa cgaggatcaa 480
ttggagggca agtctggtgc cagcagccgc ggtaattcca gctccaatag cgtatattaa 540
agttgttgca gttaaaacgt ccgtagtcaa attttagtct ttagatgagg tggcctggtc 600
ttcattgatc aagctcgctt ttatcgagac tttttttctg gttatgctat gaatagcttc 660
ggttgtttat agtctctagc cagatgatta ccatgagcaa atcagagtgt ttaaagcagg 720
ctttcaagct tgaatgtgtt agcatggaat aatgaaatat gactttagtc cctatttcgt 780
tggttcagga acttaagtaa tgatgaatag aaacggttgg ggacatttgt atttggtcgc 840
tagaggtgaa attct 855
<210> 3
<211> 823
<212> DNA
<213> schizochytrium limacinum (Schizochytrium sp.) HX-308
<400> 3
actgcgaaag catttgatcc aggacgtttt cattgatcaa ggtctaaagt taagggatcg 60
aagacgatta gataccgtcg tagtcttaac cacaaactat gccgactaga gattgggctt 120
gtttattatg actagctcag catcttagcg aaagtaaagt ttttgggttc tggggggagt 180
atgggacgca aggctgaaac ttaaaggaat tgacggaagg gcaccaccag gagtggagcc 240
tgcggcttaa tttgactcaa cacggggaaa ctcaccaggt ccagacatag taaggattga 300
cagattgaaa gctctttcta gattctatgg gtggtggtgc atggccgttc ttagttcgtg 360
gagtgatttg tctggttaat tccgataacg aacgagacct tattctgcta aataggcagg 420
tcaacttttt agttgattaa tagatttatc tatctggctt cttagagaga ctatcggctt 480
caagccgaag gaagttttag gcaataacag gtctgtgatg cccttagatg ttctgggccg 540
cacgcgcgct acactgatga agtcagcgag tttataacct tagccggaag gtttgggtaa 600
acttttgaaa cttcatcgtg ctggggatag agcattgtaa ttattgctct tcaacgagga 660
attcctagta agcgcaagtc atcagcttgc gttgattacg tccctgccct ttgtacacac 720
cgcccgtcgc tactaccgat tgaatggtta tagtgagcat atgggatcag tagaattaga 780
ctggcaacag tctttctctg cagagaacta tggcaaacta ggc 823
<210> 4
<211> 812
<212> DNA
<213> artificial
<220>
<223> ubiquitin promoter
<400> 4
tcggtacccg ttagaacgcg taatacgact cactataggg agagtcgact gagcacaact 60
ctgctgcgag cgggcctcga gagcgtttgc ttcgagccgc ggagcaaggg ggatggatcg 120
ctcatgcggt cgtgcggccc tcggtcaccc ggtgggtcct gcactgacgc atctgttctg 180
atcagacaca cgaacgaaca aaccgaggag ccgcagcgcc tggtgcaccc gccgggcgtt 240
gttgtgtgct cttcttgcct ccgagagaga gagcggagcg gatgcatagg aaatcgggcc 300
acgcgggagg gccatgcgtt cgccccacac gccactttcc acgcccgctc tctctccggc 360
cggcaggcag cgcataactc tccgacgctg gcaggctggt agcaactggc agggacaact 420
cgcgcgcggg tcccggtcgt tcgatgtgcc aacccgagag aatccagcca gcagggcggt 480
tggcctcatc gcccacctgc tatggtgcag cgaaccaact cccgaagcgg ccggttctgc 540
gattccctct tctgaattct gaattctgaa ctgattccgg aggagaaccc tctggaagcg 600
cgggttgcct ctccagttct gccgaactag acaggggagt gagcagagag tgaccctgac 660
gcggagcgag ctggttgctg gaaaagtcgc gaacgctggg ctgtgtcacg cgtccacttc 720
gggcagaccc caaacgacaa gcagaacaag caacaccagc agcagcaagc gacctaagca 780
acactagcca acatgactga ggataagacg aa 812
<210> 5
<211> 614
<212> DNA
<213> artificial
<220>
<223> ubiquitin tenninator fragment
<400> 5
ccaaggccaa gtcggactaa actaagctat ctgtagtatg tgctatactc gaatcatgct 60
gccctgtacg tacctaccta tatctgattg agcgtgctgc gtcgaccata gacgcgggaa 120
cgcgggccag cctaccacgt tgccgccgcc ggtatccacg ggcacgccaa agcattggtc 180
gataacgctc tgcccagggc ttcctggcga ggacccgagg ccaacatgca tgcatgtgct 240
atcagcggtc atcatcgccc tcatcagcgc gcatcggcga gctcgcgcac gaacggcaag 300
cgcccaactc aactcactta ctcacactat ggtcttgccg ttggcggttg cttagctaat 360
gcgtgacgtc actctgcctc caacatcgcg aggcagagtc gcgagcagtg cagaggccac 420
ggcggacgcc aacaaagcgc caaccagcgc aacgcaccag cgggtctgtg ggcgtagctc 480
gagcgggcgt cttcaagagc cgccgtggag ccgacgcccc tgcgaagggc tcgagtgcaa 540
gcggggccgt tgagccgcgt ggtaggaaca actgcagtct ccctatagtg agtcgtatta 600
cgcggtggta ccga 614
<210> 6
<211> 19
<212> DNA
<213> artificial
<220>
<223> NS1
<400> 6
gtagtcatat gcttgtctc 19
<210> 7
<211> 20
<212> DNA
<213> artificial
<220>
<223> NS8
<400> 7
tccgcagctt cacctacgga 20
<210> 8
<211> 27
<212> DNA
<213> artificial
<220>
<223> 18SupS
<400> 8
gggtacccgt agtcatatgc ttgtctc 27
<210> 9
<211> 25
<212> DNA
<213> artificial
<220>
<223> 18SupA
<400> 9
cctcgaggat ttcacctcta gcgac 25
<210> 10
<211> 24
<212> DNA
<213> artificial
<220>
<223> 18SdownS
<400> 10
cggatccgat gccgactaga gatt 24
<210> 11
<211> 26
<212> DNA
<213> artificial
<220>
<223> 18SdownA
<400> 11
gagctctccg caggttcacc tacgga 26
<210> 12
<211> 34
<212> DNA
<213> artificial
<220>
<223> P1
<400> 12
ccatcgatgg tcggtacccg ttagaacgcg taat 34
<210> 13
<211> 34
<212> DNA
<213> artificial
<220>
<223> P2
<400> 13
ggactagtct tcgtcttatc ctcagtcatg ttgg 34
<210> 14
<211> 32
<212> DNA
<213> artificial
<220>
<223> M1
<400> 14
ggactagtca tgagcgtact gttggacacc at 32
<210> 15
<211> 34
<212> DNA
<213> artificial
<220>
<223> M2
<400> 15
tcccccgggg gattaaagac gggtttgcgg atag 34
<210> 16
<211> 55
<212> DNA
<213> artificial
<220>
<223> T1
<400> 16
ccatcgatgg atagcagctg tcccccgggg gaccaaggcc aagtcggact aaact 55
<210> 17
<211> 32
<212> DNA
<213> artificial
<220>
<223> T2
<400> 17
ggagatatct cggtaccacc gcgtaatacg ac 32
Claims (7)
1. producing a schizochytrium limacinum genetic engineering bacterium of DHA, is the engineering bacteria of the expression cassette being integrated with malic enzyme gene in schizochytrium limacinum genome, and the expression cassette of described malic enzyme gene is successively containing promotor, malic enzyme gene and terminator.
2. schizochytrium limacinum genetic engineering bacterium according to claim 1, it is characterized in that described promotor is ubiquitin promoter, described malic enzyme gene is as shown in SEQ ID NO:1, and described terminator is ubiquitin tenninator.
3. schizochytrium limacinum genetic engineering bacterium according to claim 2, is characterized in that described integration site is 18srDNA.
4. the construction process of the described schizochytrium limacinum genetic engineering bacterium of one of claim 1-3, is characterized in that the homologous recombination fragment of the expression cassette built containing malic enzyme gene; Described homologous recombination fragment is imported schizochytrium limacinum and carries out homologous recombination, screening obtains described schizochytrium limacinum genetic engineering bacterium.
5. the construction process of schizochytrium limacinum genetic engineering bacterium according to claim 4, it is characterized in that the upstream and downstream homology arm being provided with 18srDNA in described homologous recombination fragment at the two ends of the expression cassette of described malic enzyme gene, the upstream homology arm sequence of described 18srDNA is as shown in SEQ ID NO:2, and the downstream homology arm of 18srDNA is as shown in SEQ ID NO:3.
6. the construction process of schizochytrium limacinum genetic engineering bacterium according to claim 5, is characterized in that described homologous recombination fragment carries bleomycin resistance marker gene.
7. adopt the described schizochytrium limacinum genetic engineering bacterium of one of claim 1-3 to prepare the method for DHA, it is characterized in that adopting seed culture medium to cultivate schizochytrium limacinum genetic engineering bacterium, then transfer and prepare DHA into fermention medium; Described seed culture medium contains: glucose 40g/L, yeast extract paste 2g/L, Sodium Glutamate 10g/L, KH
2pO
44g/L, NaCl 15g/L, MgCl
23g/L, CaCl
22H
2o 1g/L, KCl 2g/L, MgSO
47H
2o 5g/L, FeCl
30.1g/L; Described fermention medium contains: glucose 40g/L, yeast extract paste 2g/L, Sodium Glutamate 10g/L, KH
2pO
44g/L, NaCl 15g/L, MgCl
23g/L, (NH
4)
2sO
46g/L, KCl 2g/L, MgSO
47H
2o 5g/L, FeCl
30.1g/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510417269.4A CN104974944B (en) | 2015-07-15 | 2015-07-15 | Schizochytrium limacinum genetic engineering strain for producing DHA (docosahexaenoic acid), and construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510417269.4A CN104974944B (en) | 2015-07-15 | 2015-07-15 | Schizochytrium limacinum genetic engineering strain for producing DHA (docosahexaenoic acid), and construction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104974944A true CN104974944A (en) | 2015-10-14 |
| CN104974944B CN104974944B (en) | 2018-05-25 |
Family
ID=54271911
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510417269.4A Active CN104974944B (en) | 2015-07-15 | 2015-07-15 | Schizochytrium limacinum genetic engineering strain for producing DHA (docosahexaenoic acid), and construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104974944B (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282250A (en) * | 2016-08-09 | 2017-01-04 | 中国科学院青岛生物能源与过程研究所 | A kind of method improving schizochytrium limacinum content of polyunsaturated fatty acid or yield |
| CN106947706A (en) * | 2017-05-10 | 2017-07-14 | 南京工业大学 | Schizochytrium limacinum strain, construction method and application thereof |
| WO2018205165A1 (en) * | 2017-05-10 | 2018-11-15 | 南京工业大学 | Schizochytrium limacinum strain, building method therefor and application thereof |
| CN109517834A (en) * | 2018-11-27 | 2019-03-26 | 昆明藻能生物科技有限公司 | A method of schizochytrium ATCC20888 grease and DHA content are improved by genetic modification |
| CN110846232A (en) * | 2019-12-04 | 2020-02-28 | 南京工业大学 | Strain capable of producing DHA through high-temperature fermentation and application thereof |
| CN112226453A (en) * | 2020-10-29 | 2021-01-15 | 南京工业大学 | Schizochytrium limacinum CRISPR/Cas9 gene editing system and application thereof |
| CN114426985A (en) * | 2021-11-23 | 2022-05-03 | 南京师范大学 | Method for transforming schizochytrium by agrobacterium and application |
| CN114480148A (en) * | 2022-01-07 | 2022-05-13 | 南京师范大学 | Schizochytrium limacinum genetic engineering strain for expressing EPA synthase gene, construction method and application thereof |
| CN114574373A (en) * | 2022-03-29 | 2022-06-03 | 陕西海斯夫生物工程有限公司 | Recombinant schizochytrium for producing tocopherol, construction method and application thereof |
| CN114958627A (en) * | 2022-05-05 | 2022-08-30 | 陕西海斯夫生物工程有限公司 | Construction method and application of recombinant schizochytrium limacinum engineering bacterium for high yield of tocopherol |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030180898A1 (en) * | 2000-01-28 | 2003-09-25 | Martek Biosciences Boulder Corporation | Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors |
| CN101575584A (en) * | 2009-06-18 | 2009-11-11 | 南京工业大学 | Vibrio schizogenum and method for producing DHA grease by using same |
| CN102373189A (en) * | 2011-11-08 | 2012-03-14 | 国家海洋局第三海洋研究所 | Fatty acid synthesis-related protein and encoding gene and application thereof |
-
2015
- 2015-07-15 CN CN201510417269.4A patent/CN104974944B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030180898A1 (en) * | 2000-01-28 | 2003-09-25 | Martek Biosciences Boulder Corporation | Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors |
| CN101575584A (en) * | 2009-06-18 | 2009-11-11 | 南京工业大学 | Vibrio schizogenum and method for producing DHA grease by using same |
| CN102373189A (en) * | 2011-11-08 | 2012-03-14 | 国家海洋局第三海洋研究所 | Fatty acid synthesis-related protein and encoding gene and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| LU-JING REN ET AL.: "Enhanced docosahexaenoic acid production by reinforcing acetyl-CoA and NADPH supply in Schizochytrium sp. HX-308", 《BIOPROCESS BIOSYST ENG》 * |
| LU-JING REN ET AL.: "Utilization of cane molasses for docosahexaenoic acid production by Schizochytrium sp. CCTCC M209059", 《KOREAN J. CHEM. ENG.》 * |
| 冯云等: "微生物发酵产二十二碳六烯酸代谢机理的研究进展", 《生物工程学报》 * |
| 庄小燕等: "基于同源重组的裂殖壶菌遗传转化体系的构建及验证", 《微生物学报》 * |
| 魏萍等: "强化乙酰辅酶A供应对裂殖壶菌合成二十二碳六烯酸的影响", 《中国生物工程杂志》 * |
| 魏萍等: "裂殖壶菌发酵生产DHA研究进展", 《食品工业科技》 * |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282250A (en) * | 2016-08-09 | 2017-01-04 | 中国科学院青岛生物能源与过程研究所 | A kind of method improving schizochytrium limacinum content of polyunsaturated fatty acid or yield |
| CN106947706B (en) * | 2017-05-10 | 2020-07-07 | 南京工业大学 | Schizochytrium limacinum strain, construction method and application thereof |
| CN106947706A (en) * | 2017-05-10 | 2017-07-14 | 南京工业大学 | Schizochytrium limacinum strain, construction method and application thereof |
| WO2018205165A1 (en) * | 2017-05-10 | 2018-11-15 | 南京工业大学 | Schizochytrium limacinum strain, building method therefor and application thereof |
| US11248244B2 (en) | 2017-05-10 | 2022-02-15 | Nanjing University Of Technology | Schizochytrium limacinum strain, building method therefor and application thereof |
| CN109517834B (en) * | 2018-11-27 | 2022-06-07 | 昆明藻能生物科技有限公司 | Method for improving contents of grease and DHA in schizochytrium ATCC20888 through genetic modification |
| CN109517834A (en) * | 2018-11-27 | 2019-03-26 | 昆明藻能生物科技有限公司 | A method of schizochytrium ATCC20888 grease and DHA content are improved by genetic modification |
| CN110846232B (en) * | 2019-12-04 | 2021-08-03 | 南京工业大学 | Strain capable of producing DHA through high-temperature fermentation and application thereof |
| CN110846232A (en) * | 2019-12-04 | 2020-02-28 | 南京工业大学 | Strain capable of producing DHA through high-temperature fermentation and application thereof |
| CN112226453A (en) * | 2020-10-29 | 2021-01-15 | 南京工业大学 | Schizochytrium limacinum CRISPR/Cas9 gene editing system and application thereof |
| CN112226453B (en) * | 2020-10-29 | 2024-01-30 | 南京工业大学 | Schizochytrium CRISPR/Cas9 gene editing system and application thereof |
| CN114426985A (en) * | 2021-11-23 | 2022-05-03 | 南京师范大学 | Method for transforming schizochytrium by agrobacterium and application |
| CN114426985B (en) * | 2021-11-23 | 2024-03-26 | 南京师范大学 | Method for transforming schizochytrium by agrobacterium and application |
| CN114480148A (en) * | 2022-01-07 | 2022-05-13 | 南京师范大学 | Schizochytrium limacinum genetic engineering strain for expressing EPA synthase gene, construction method and application thereof |
| CN114574373A (en) * | 2022-03-29 | 2022-06-03 | 陕西海斯夫生物工程有限公司 | Recombinant schizochytrium for producing tocopherol, construction method and application thereof |
| CN114958627A (en) * | 2022-05-05 | 2022-08-30 | 陕西海斯夫生物工程有限公司 | Construction method and application of recombinant schizochytrium limacinum engineering bacterium for high yield of tocopherol |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104974944B (en) | 2018-05-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104974944A (en) | Schizochytrium limacinum genetic engineering strain for producing DHA (docosahexaenoic acid), and construction method and application thereof | |
| CN103820335B (en) | Mortierella alpina, M. alpina genetic engineering strain of overexpression omega 3 desaturase gene and construction method of strain | |
| JP4124270B1 (en) | Glucose / Mannose / Xylose Parallel Fermentative Bacteria and Method for Producing Bioethanol Using the Same | |
| Bader et al. | Efficient saccharification of microalgal biomass by Trichoderma harzianum enzymes for the production of ethanol | |
| CN112941119B (en) | Method for increasing yield of fatty acid ethyl ester of saccharomyces cerevisiae engineering bacteria | |
| CN114561434B (en) | Method for producing EPA and DHA by schizochytrium limacinum fermentation | |
| CN101475914B (en) | Method for producing oligo-galactose by cyclic utilization of recombinant Saccharomyces cerevisiae | |
| CN107779423A (en) | Cofactor regeneration mycobacteria and its application in the fermentation of profit Two Liquid Phases | |
| CN104726505A (en) | Method for producing three-carbon compounds by using gene engineering cyanobacteria | |
| CN104805027B (en) | One kind restructuring Ye Shi solution fat yeast strains and its construction method and application | |
| CN104017738A (en) | High-yield engineering strain kmust-SQS for ganoderic acid | |
| CN103789282B (en) | The preparation method of a kind of high temperature mannase ManAHr and gene thereof and application | |
| CN104046586A (en) | Genetically engineered bacteria and application of genetically engineered bacteria to production of (2R, 3R)-2,3-butanediol | |
| CN104726477A (en) | Lipase coding gene and engineering strain thereof | |
| CN111088177B (en) | Construction and application of heat-resistant yeast engineering bacteria for producing glycerol under high-temperature aerobic condition | |
| Dai et al. | Bioconversion of inulin to 2, 3-butanediol by a newly isolated Klebsiella pneumoniae producing inulinase | |
| CN105296368B (en) | Recombination Mortierella alpine trichoderma strain, its construction method and its application in production EPA of one plant of heterogenous expression MpFADS6 gene | |
| CN105274041B (en) | One plant of recombination bacillus coli and its application in production 2- butanol | |
| CN102827780A (en) | Mortierella alpine strain for producing arachidonic acid | |
| CN105039385B (en) | T10, c12- conjugated linoleic acid engineered strain and its recombinant expression plasmid and construction method and application | |
| CN105176848B (en) | Mortierella alpine strain overexpressing 3-phosphoglycerol dehydrogenase gene(G3PD1), and construction method and application thereof | |
| CN113604459A (en) | Phosphoenol pyruvate carboxykinase and application thereof | |
| CN105368727A (en) | Glucose-6-phosphate dehydrogenase and malic enzyme coordinate-expressed recombinant mortierella alpine strain, constructing method thereof and application thereof | |
| CN104630079A (en) | Recombinant mortierella alpina strain for heterologous expression of linoleic acid isomerase gene and construction method thereof | |
| CN103122356A (en) | New linoleic acid isomerase gene, and vector and strain containing same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |