CN104974944B - A kind of schizochytrium limacinum genetic engineering bacterium for producing DHA and its construction method and application - Google Patents
A kind of schizochytrium limacinum genetic engineering bacterium for producing DHA and its construction method and application Download PDFInfo
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Abstract
The present invention provides a kind of schizochytrium limacinum genetic engineering bacterium for producing DHA and its construction method and application, belongs to technical field of biochemical industry.The schizochytrium limacinum genetic engineering bacterium of the production DHA, is the engineering bacteria for the expression cassette that malic enzyme gene is integrated in schizochytrium limacinum genome, and the expression cassette of the malic enzyme gene contains promoter, malic enzyme gene and terminator successively.The construction method of the schizochytrium limacinum genetic engineering bacterium, including：Build the homologous recombination segment of the expression cassette containing malic enzyme gene；The homologous recombination segment is imported into schizochytrium limacinum and carries out homologous recombination, screening obtains the schizochytrium limacinum genetic engineering bacterium.The present invention also provides the methods for preparing DHA using the schizochytrium limacinum genetic engineering bacterium, using seed culture medium culture schizochytrium limacinum genetic engineering bacterium, then transfer and prepare DHA into fermentation medium.Schizochytrium limacinum genetic engineering bacterium of the present invention can high yield DHA, construction method is simple, efficient, cost is relatively low.
Description
Technical field
The invention belongs to technical field of biochemical industry, and in particular to it is a kind of produce DHA schizochytrium limacinum genetic engineering bacterium and its
Construction method and application.
Background technology
DHA is a kind of important omega-3 long-chain polyunsaturated fatty acids, is cell membrane in brain and retinal tissue
Constituent has enhancing eyesight, promotes the physiology work(such as brain cell development and preventing hypertension, artery sclerosis, angiocardiopathy
Effect.Human body is difficult to synthesize meet the needs of body is to DHA, it is necessary to be absorbed from food by itself.It is obtained from nature
The source of DHA is very limited, and using industrialization schizochytrium limacinum fermentation production DHA, low output is of high cost.
The content of the invention
It is an object of the invention to provide a kind of schizochytrium limacinum genetic engineering bacteriums of high yield DHA.
It is a further object of the present invention to provide the construction methods of the schizochytrium limacinum genetic engineering bacterium, and this method is simple, effect
Rate is high, and cost is relatively low.
Another object of the present invention is to provide the method for preparing DHA using the schizochytrium limacinum genetic engineering bacterium, this method
The DHA of high yield can be obtained, cost is relatively low.
The purpose of the present invention adopts the following technical scheme that realization：
A kind of schizochytrium limacinum genetic engineering bacterium for producing DHA, is to be integrated with malic enzyme gene in schizochytrium limacinum genome
Expression cassette engineering bacteria, the expression cassette of the malic enzyme gene contains promoter, malic enzyme gene and terminator successively.
In preferred technical solution, the promoter is ubiquitin promoter, the malic enzyme gene such as SEQ ID NO:1
Shown, the terminator is ubiquitin tenninator.The integration site is 18srDNA.
The present invention also provides the construction methods of the schizochytrium limacinum genetic engineering bacterium, build the table containing malic enzyme gene
Up to the homologous recombination segment of box；The homologous recombination segment is imported into schizochytrium limacinum and carries out homologous recombination, is split described in screening acquisition
Grow chytrid genetic engineering bacterium.
In the present invention, it is equipped in the homologous recombination segment at the both ends of the expression cassette of the malic enzyme gene
The upstream and downstream homology arm of 18srDNA, the upstream homology arm sequence such as SEQ ID NO of the 18srDNA:Shown in 2,18srDNA's
Downstream homology arm such as SEQ ID NO:Shown in 3.
In preferred technical solution, the homologous recombination segment carries bleomycin resistance marker gene.
The present invention also provides the methods for preparing DHA using the schizochytrium limacinum genetic engineering bacterium, are trained using seed culture medium
Schizochytrium limacinum genetic engineering bacterium is supported, then transfers and prepares DHA into fermentation medium；The seed culture medium contains：Glucose
40g/L, yeast extract 2g/L, sodium glutamate 10g/L, KH2PO44g/L、NaCl15g/L、MgCl23g/L、CaCl2·2H2O 1g/L、
KCl 2g/L、MgSO4·7H2O 5g/L、FeCl30.1g/L；The fermentation medium contains：Glucose 40g/L, yeast extract 2g/
L, sodium glutamate 10g/L, KH2PO44g/L、NaCl 15g/L、MgCl23g/L、(NH4)2SO46g/L、KCl 2g/L、MgSO4·
7H2O 5g/L、FeCl30.1g/L。
Beneficial effects of the present invention：Expression cassette with strong promoter and malic enzyme gene is integrated into fragmentation by the present invention
On chytrid genome, structure obtains the schizochytrium limacinum genetic engineering bacterium of production DHA.The genetic engineering bacterium can be overexpressed malic acid
Enzyme.In genetic engineering bacterium, malic dehydrogenase can be catalyzed oxaloacetic acid as malic acid, and the malate dehydrogenase of overexpression can incite somebody to action
Malic acid is reduced into pyruvic acid and generates substantial amounts of NADPH, so that the circular response of pyruvic acid → oxaloacetic acid → malic acid
It can carry out, substantial amounts of NADPH is generated in this circular response, there is very big facilitation, therefore the present invention to the generation of grease
Genetic engineering bacterium has the ability compared with high yield DHA.The construction method of genetic engineering bacterium of the present invention is simple, efficiently, at low cost.This hair
The bright method for preparing DHA using the schizochytrium limacinum genetic engineering bacterium, yield are significantly improved compared with original strain, and cost is relatively low.
Description of the drawings
Fig. 1：The construction step of plasmid pBZ-18S.
The construction step of Fig. 2 plasmids pBZ18S-PMT.
Specific embodiment
According to following embodiments, the present invention may be better understood.It is however, as it will be easily appreciated by one skilled in the art that real
It applies the described content of example to be only limitted to illustrate the present invention, without sheet described in detail in claims should will not be limited
Invention.
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), deposit number is CCTCC No.M 209059；
Carrier pBlueScript II SK：Commercial vector is purchased from Invitrogen companies；
Carrier pGAPZ α A：Commercial vector is purchased from Invitrogen companies；
Carrier pMD19-T：Commercial vector is purchased from TaKaRa companies；
Carrier pMD19-T (simple)：Commercial vector is purchased from TaKaRa companies.
The structure of 1 recombinant plasmid pBZ-18S of embodiment
1. build recombinant vector pBS-Zeo
By BamHI and EcoRI restriction enzyme sites by Zeocin (bleomycin) resistant gene piece in pGAPZ α A carriers
Section insertion pBlueScript II SK carriers, structure recombinant vector pBS-Zeo.
PGAPZ α A and pBlueScript II SK carriers carry out double digestion respectively.(related reagent is purchased from digestion system
TaKaRa)：ddH260 22 μ L, 10 × K buffer of μ L, BamHI of μ L, EcoRI of O 10 μ L, 26 μ L of carrier.Digestion temperature：30
℃。
Digestion products are detected with 0.8% agarose gel electrophoresis, and use Takara plastic recovery kits purifying purpose
Segment, then the segment of purifying T4 ligases are connected overnight.
T4 connection enzyme systems：ddH211.5 μ L, T4 Ligase of O, 1 μ L, T4 Ligase buffer 2.5 μ L, Zeocin
8 μ L of resistance gene fragment, 2 μ L of carrier.
The 5 μ L of reaction solution after connection overnight is taken to add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shocks
30s is immediately placed on 2min on ice.250 μ L LB culture mediums are rapidly added, 1h is cultivated under the conditions of 37 DEG C, 200rpm.Take 200 μ L
Bacterium solution applies the LB tablets containing ampicillin, and after being incubated overnight, the positive bacterium colony of picking 10 connects the LB liquid containing ampicillin
Medium culture extracts positive colony plasmid and is named as pBS-Zeo.
2. expand schizochytrium limacinum 18SrDNA upstream and downstream homology arm
(1) schizochytrium limacinum 18SrDNA upstreams homology arm is expanded
Schizochytrium limacinum (Schizochytrium sp.) HX-308 is expanded using fungi 18SrDNA universal primers NS1 and NS8
18SrDNA segments, be connected in carrier T, obtain recombinant vector pMD-18S.The wherein sequence of NS1 (SEQ ID NO:
6) it is:Sequence (the SEQ ID NO of GTAGTCATATGCTTGTCTC, NS8:7) it is:TCCGCAGCTTCACCTACGGA.
Primer 18SupS and 18SupA amplification upstream homology arm are designed according to the schizochytrium limacinum 18SrDNA fragment sequences of acquisition
(SEQ ID NO:2).18SupS(SEQ ID NO:8) sequence：5 '-GGGTACCCGTAGTCATATGCTTGTCTC-3 ',
18SupA(SEQ ID NO:9) sequence：5’-CCTCGAGGATTTCACCTCTAGCGAC-3’.
PCR reaction systems (related reagent is purchased from TaKaRa)：25 0.3 μ of μ L, 18SupS of HS (Premix)
L, 18SupA 0.3 μ L, pMD-18S 0.4 μ L, ddH2O 24μL.PCR reaction conditions：98 DEG C of 10s, 65 DEG C of 15s, 72 DEG C of 50s,
Xun Huan 30 times；72℃7min.
PCR product is detected with 0.8% agarose gel electrophoresis, and uses Takara plastic recovery kits purifying purpose piece
Section, then the segment of purifying is cloned on carrier pMD19-T.Clone's system：1 μ L pMD19-T carriers, the pieces of 4 μ L after purification
Section, gently mixing；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,
It is immediately placed on 2min on ice.250 μ L LB culture mediums are rapidly added, 1h is cultivated under the conditions of 200rpm, 37 DEG C.Take 200 μ L bacterium solutions
The LB tablets containing ampicillin are applied, after being incubated overnight, the positive bacterium colony of picking 10 connects the LB Liquid Cultures containing ampicillin
Base culture, extraction positive colony plasmid send sequence verification and are named as pMD19-18Sup.
(2) schizochytrium limacinum 18SrDNA downstreams homology arm is expanded
According to schizochytrium limacinum 18SrDNA primers 18SdownS (the SEQ ID NO of acquisition:And 18SdownA 10)
(SEQ ID NO:11) downstream homology arm (SEQ ID NO are expanded:3).18SdownS sequences：5’-
CGGATCCGATGCCGACTAGAGATT-3’；18SdownA sequences：5’-GAGCTCTCCGCAGGTTCACCTACGGA-3’.
PCR reaction systems：25 0.3 0.3 μ L of μ L, 18SdownA of μ L, 18SdownS of HS (Premix),
PMD-18S 0.4 μ L, ddH2O 24μL。
PCR reaction conditions：98 DEG C of 10s, 65 DEG C of 15s, 72 DEG C of 50s are cycled 30 times；72℃7min.
PCR product is detected with 0.8% agarose gel electrophoresis, and uses Takara plastic recovery kits purifying purpose piece
Section, then the segment of purifying is cloned on carrier pMD19-T.Clone's system：1 μ L pMD19-T carriers, the pieces of 4 μ L after purification
Section, gently mixing；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,
It is immediately placed on 2min on ice.It is rapidly added 250 μ L LB culture mediums, 200rpm, cultivates 1h under the conditions of 37 DEG C.200 μ L bacterium solutions is taken to apply
LB tablets containing ampicillin, after being incubated overnight, the positive bacterium colony of picking 10 connects the LB fluid nutrient mediums containing ampicillin
Culture, extraction positive colony plasmid send sequence verification, correct recombinant plasmid are sequenced and is named as pMD19-18Sdown.
3. build plasmid pBS-Zeo-18Sup
It is by SacI and BamHI restriction enzyme sites that Zeocin in 18SrDNA upstreams homology arm insertion plasmid pBS-Zeo is (rich
Lay mycin) resistance gene fragment upstream, structure plasmid pBS-Zeo-18Sup.
PMD19-18Sup, pBS-Zeo carry out double digestion respectively.Digestion system：ddH2O 65 μ L, SacI 2 μ L, BamHI
2 μ L, 10 × Kbuffer 5 μ L, 26 μ L of carrier.Digestion temperature：30℃.
Digestion products are detected with 0.8% agarose gel electrophoresis, and use Takara plastic recovery kits purifying purpose
Segment, then the segment of purifying T4 ligases are connected overnight.T4 connection enzyme systems：ddH211.5 1 μ L of μ L, T4Ligase of O,
2.5 μ L of T4Ligase buffer, 8 μ L of segment, 2 μ L of carrier.
The 5 μ L of reaction solution after connection overnight is taken to add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shocks
30s is immediately placed on 2min on ice.It is rapidly added 250 μ L LB culture mediums, 200rpm, 37 DEG C of culture 1h.The painting of 200 μ L bacterium solutions is taken to contain
The LB tablets of ampicillin, after being incubated overnight, the positive bacterium colony of picking 10 connects the LB fluid nutrient mediums training containing ampicillin
It supports, extract positive colony plasmid and is named as pBS-Zeo-18Sup.
4. build plasmid pBZ-18S
It will be in 18SrDNA downstreams homology arm insertion plasmid pBS-Zeo-18Sup by KpnI and XhoI restriction enzyme sites
The downstream of Zeocin (bleomycin) resistance gene fragment, structure plasmid pBZ-18S.
PMD19-18Sdown, pBS-Zeo-18Sup carry out double digestion respectively.Digestion system：ddH260 2 μ of μ L, KpnI of O
2 μ L, 10 × M buffer of L, XhoI 10 μ L, 26 μ L of carrier.Digestion temperature：30℃.
Digestion products are detected with 0.8% agarose gel electrophoresis, and use Takara plastic recovery kits purifying purpose
Segment, then the segment of purifying T4 ligases are connected overnight.T4 connection enzyme systems：ddH211.5 1 μ L of μ L, T4Ligase of O,
2.5 μ L of T4Ligase buffer, 8 μ L of segment, 2 μ L of carrier.
The 5 μ L of reaction solution after connection overnight is taken to add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shocks
30s is immediately placed on 2min on ice.It is rapidly added 250 μ L LB culture mediums, 200rpm, cultivates 1h under the conditions of 37 DEG C.Take 200 μ L bacterium
Liquid applies the LB tablets containing ampicillin, and after being incubated overnight, the positive bacterium colony of picking 10 connects the LB liquid training containing ampicillin
Base culture is supported, extract positive colony plasmid and is named as pBZ-18S.
The structure of 2 recombinant plasmid pBZ18S-PMT of embodiment
1st, the clone of ubiquitin promoter segment, malic enzyme gene and ubiquitin tenninator segment
1) clone of ubiquitin promoter segment
Ubiquitin promoter segment (SEQ ID NO are synthesized by Jin Weizhi companies:4) and carrier T is cloned into, forms recombinant plasmid
Promoter-T。
According to the sequence of ubiquitin promoter, design synthetic primer P1 (SEQ ID NO:And P2 (SEQ ID NO 12):13).
P1 (band ClaI restriction enzyme sites) sequence：5’-CCATCGATGG
TCGGTACCCGTTAGAACGCGTAAT-3 ', P2 (band SpeI restriction enzyme sites) sequence：5’-GGACTAGTC
TTCGTCTTATCCTCAGTCATGTTGG-3’。
Using Promoter-T plasmids as template, with primer P1 and P2, Takara high-fidelity enzymeHS
(Premix) PCR amplification ubiquitin promoter segment.PCR reaction systems：25 μ L of HS (Premix), sense primer
0.3 μ L, 0.3 μ L, Promoter-T plasmid of anti-sense primer 0.4 μ L, ddH2O 24μL.PCR reaction conditions：98 DEG C of 10s, 65 DEG C
15s, 72 DEG C of 50s are cycled 30 times；72℃7min.
PCR product is detected with 0.8% agarose gel electrophoresis, and uses Takara plastic recovery kits purifying purpose piece
Section, then the segment of purifying is cloned on carrier pMD19-T.Clone's system：1 μ L pMD19-T carriers, the pieces of 4 μ L after purification
Section, gently mixing；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,
It is immediately placed on 2min on ice.It is rapidly added 250 μ L LB culture mediums, 200rpm, cultivates 1h under the conditions of 37 DEG C.200 μ L bacterium solutions is taken to apply
LB tablets containing ampicillin, after being incubated overnight, the positive bacterium colony of picking 10 connects the LB fluid nutrient mediums containing ampicillin
Culture, extraction positive colony plasmid send sequence verification, the correct positive plasmid of sequence verification are named as pMD19-P.
2) clone of malic enzyme gene
According to sequence (the SEQ ID NO of malic enzyme gene:1) synthetic primer M1 (SEQ ID NO, are designed:And M2 14)
(SEQ ID NO:15).M1 (carries SpeI restriction enzyme sites)：5 '-GGACTAGTC ATGAGCGTACTGTTGGACACCAT-3 ',
M2 (carries SmaI restriction enzyme sites)：5’-TCCCCCGGGGGA TTAAAGACGGGTTTGCGGATAG-3’.
Using schizochytrium limacinum (Schizochytrium sp.) HX-308 genomic DNAs template, with primer M1 and M2,
Takara high-fidelity enzymesHS (Premix) PCR amplification malic enzyme gene segment.PCR reaction systems：25 μ L of HS (Premix), 0.3 μ L of sense primer, 0.3 μ L of anti-sense primer, schizochytrium limacinum
(Schizochytrium sp.) HX-308 genomic DNAs 0.4 μ L, ddH2O 24μL.PCR reaction conditions：98 DEG C of 10s, 65 DEG C
15s, 72 DEG C of 1min50s are cycled 30 times；72℃7min.
PCR product is detected with 0.8% agarose gel electrophoresis, and uses Takara plastic recovery kits purifying purpose piece
Section, then the segment of purifying is cloned on carrier pMD19-T (simple).Clone's system：1 μ L pMD19-T (simple) are carried
Body, the segments of 4 μ L after purification, gently mixing；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, is immediately placed on 2min on ice.It is rapidly added 250 μ L LB culture mediums, 200rpm, trains under the conditions of 37 DEG C
Support 1h.200 μ L bacterium solutions is taken to apply the LB tablets containing ampicillin, after being incubated overnight, the positive bacterium colony of picking 10 connects the green grass or young crops of benzyl containing ammonia
The LB fluid nutrient medium cultures of mycin, extraction positive colony plasmid send sequence verification.The correct positive plasmid name of sequence verification
For pMD19s-M.
3) clone of ubiquitin tenninator segment
Ubiquitin tenninator segment (SEQ ID NO are synthesized by Jin Weizhi companies:5) and carrier T is cloned into, forms recombinant plasmid
Terminator-T。
According to the sequence of ubiquitin tenninator, design synthetic primer T1 (SEQ ID NO:And T2 (SEQ ID NO 16):17).
T1 (carries ClaI, SmaI restriction enzyme site)：5’-CCATCGATGGatagcagctgTCCCCCGGGGGACCAAGGCCAAGTCGGA
CTAAACT-3 ', T2 (carry EcoRV restriction enzyme sites)：5’-GGAGATATC TCGGTACCACCGCGTAATACGAC-3’.
Using Terminator-T as template, with primer T1 and T2, Takara high-fidelity enzymeHS
(Premix) PCR amplification ubiquitin tenninator segment.PCR reaction systems：25 μ L of HS (Premix), sense primer
0.3 μ L, 0.3 μ L, Terminator-T plasmid of anti-sense primer 0.4 μ L, ddH2O 24μL.PCR reaction conditions：98 DEG C of 10s, 65 DEG C
15s, 72 DEG C of 40s are cycled 30 times；72℃7min.
PCR product is detected with 0.8% agarose gel electrophoresis, and uses Takara plastic recovery kits purifying purpose piece
Section, then the segment of purifying is cloned on carrier pMD19-T (simple).Clone's system：1 μ L pMD19-T (simple) are carried
Body, the segments of 4 μ L after purification, gently mixing；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, is immediately placed on 2min on ice.It is rapidly added 250 μ L LB culture mediums, 200rpm, trains under the conditions of 37 DEG C
Support 1h.200 μ L bacterium solutions is taken to apply the LB tablets containing ampicillin, after being incubated overnight, the positive bacterium colony of picking 10 connects the green grass or young crops of benzyl containing ammonia
The LB fluid nutrient medium cultures of mycin, extraction positive colony plasmid send sequence verification.The correct recombinant plasmid name of sequence verification
For pMD19s-T.
2nd, digestion connection ubiquitin promoter and malic enzyme gene, carrier construction pMD19-PM.
Ubiquitin promoter and malic enzyme gene are connected by ClaI with SpeI restriction enzyme sites, passes through digestion carrier pMD19-
P, pMD19s-M, carrier construction pMD19-PM.PMD19-P, pMD19s-M carry out double digestion respectively.Digestion system：ddH2O 60μ
22 μ L, 10 × M buffer of μ L, SpeI of L, ClaI 10 μ L, 26 μ L of carrier.Digestion temperature：37℃.
Digestion products are detected with 0.8% agarose gel electrophoresis, and use Takara plastic recovery kits purifying purpose
Segment, then the segment of purifying T4 ligases are connected overnight.T4 connection enzyme systems：ddH211.5 1 μ L of μ L, T4Ligase of O,
2.5 μ L of T4Ligase buffer, 8 μ L of segment, 2 μ L of carrier.
The 5 μ L of reaction solution after connection overnight is taken to add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shocks
30s is immediately placed on 2min on ice.It is rapidly added 250 μ L LB culture mediums, 200rpm, 37 DEG C of culture 1h.The painting of 200 μ L bacterium solutions is taken to contain
The LB tablets of ampicillin, after being incubated overnight, the positive bacterium colony of picking 10 connects the LB fluid nutrient mediums training containing ampicillin
It supports, extract positive colony plasmid and is sequenced.The correct positive recombinant plasmid of sequence verification is named as pMD19-PM.
3rd, digestion connection ubiquitin promoter, malic enzyme gene and ubiquitin tenninator, carrier construction pMD19-PMT.
Ubiquitin promoter, malic enzyme gene segment are inserted into ubiquitin in pMD19s-T by ClaI and SmaI restriction enzyme sites
The upstream of sub-piece is terminated, is sequentially connected ubiquitin promoter, malic enzyme gene segment and ubiquitin tenninator, carrier construction
pMD19-PMT.Ubiquitin promoter, malic enzyme gene segment and ubiquitin tenninator are sequentially connected the table to form malic enzyme gene
Up to box.
PMD19-PM, pMD19s-T carry out double digestion respectively.Digestion system：ddH260 22 μ of μ L, SpeI of μ L, ClaI of O
L, 10 × T buffer 5 μ L, BSA (bovine serum albumin(BSA)) 5 μ L, 26 μ L of carrier.Digestion temperature：37℃.
Digestion products are detected with 0.8% agarose gel electrophoresis, and use Takara plastic recovery kits purifying purpose
Segment, then the segment of purifying T4 ligases are connected overnight.T4 connection enzyme systems：ddH211.5 1 μ L of μ L, T4Ligase of O,
2.5 μ L of T4Ligase buffer, 8 μ L of segment, 2 μ L of carrier.
The 5 μ L of reaction solution after connection overnight is taken to add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shocks
30s is immediately placed on 2min on ice.It is rapidly added 250 μ L LB culture mediums, 200rpm, 37 DEG C of culture 1h.The painting of 200 μ L bacterium solutions is taken to contain
The LB tablets of ampicillin, after being incubated overnight, the positive bacterium colony of picking 10 connects the LB fluid nutrient mediums training containing ampicillin
It supports, extract positive colony plasmid and is sequenced.The correct positive recombinant plasmid of sequence verification is named as pMD19-PMT.
4th, carrier construction pBZ18S-PMT
The expression cassette of malic enzyme gene is inserted into 18SrDNA downstreams in pBZ-18S by ClaI and EcoRV restriction enzyme sites
Between homology arm and Zeocin resistance gene fragments, carrier construction pBZ18S-PMT.
PMD19-PMT, pBZ-18S carry out double digestion respectively.Digestion system：ddH260 22 μ of μ L, EcoRV of μ L, ClaI of O
10 μ L of L, 10 × H buffer, 26 μ L of carrier.Digestion temperature：37℃.
Digestion products are detected with 0.8% agarose gel electrophoresis, and use Takara plastic recovery kits purifying purpose
Segment, then the segment of purifying T4 ligases are connected overnight.T4 connection enzyme systems：ddH211.5 1 μ L of μ L, T4Ligase of O,
2.5 μ L of T4Ligase buffer, 8 μ L of segment, 2 μ L of carrier.
The 5 μ L of reaction solution after connection overnight is taken to add in 50 μ L DH5 α competent cells, ice bath 30min, 42 DEG C of heat shocks
30s is immediately placed on 2min on ice.It is rapidly added 250 μ L LB culture mediums, 200rpm, 37 DEG C of culture 1h.The painting of 200 μ L bacterium solutions is taken to contain
The LB tablets of ampicillin, after being incubated overnight, the positive bacterium colony of picking 10 connects the LB fluid nutrient mediums training containing ampicillin
It supports, extract positive colony plasmid and is sequenced.The correct positive recombinant plasmid of sequence verification is named as pBZ18S-PMT.
The screening of the schizochytrium limacinum genetic engineering bacterium of 3 homologous recombination of embodiment and production DHA
1) 10mL schizochytrium limacinums (Schizochytrium sp.) HX-308 bacterium solutions is taken to be put into the 50mL poly- third of sterile precooling
In alkene pipe, in 4 DEG C, 5000r/min centrifugation 10min, abandon supernatant, with the sterile water wash thalline of 10mL precoolings twice, 4 DEG C,
4472 × g centrifuges 10min.Bacterial sediment is resuspended with the 1mol/L sorbitol solutions of 10mL precoolings, 4 DEG C, 5000r/min centrifugations
10min is repeated 1 times.Bacterial sediment is resuspended with the 1mol/L sorbitol solutions of 10mL precoolings.
2) plasmid pBZ18S-PMT is added to the 30 pretreated schizochytrium limacinum (Schizochytrium of μ L steps 1)
Sp.) in HX-308, gently mixing, ice bath stand 5min, are then transferred into the electric shock cup of ice precooling, stand 10min.It sets
Shock parameters 0.75KV, 200 Ω, 50 μ F.1mL seed culture mediums (in embodiment 4), 30 DEG C, 200r/min are added in after electric shock
The bacterium solution of conversion is then coated on the tablet containing 1.5 μ g/mL Zeocin (seed culture medium 2% fine jade of addition by recovery 1h
Fat), 28 DEG C are protected from light culture.
3) single bacterium colony is screened in resistant panel, after Shaking culture, after extracting genomic DNA, using genomic DNA as template
PCR amplification target fragment, the correct recombinant bacterium of sequence verification are named as schizochytrium limacinum (Schizochytrium sp.) HX-ME.
Embodiment 4 produces DHA using genetic engineering bacterium schizochytrium limacinum (Schizochytrium sp.) HX-ME
Original bacteria schizochytrium limacinum (Schizochytrium sp.) HX-308 and genetic engineering bacterium schizochytrium limacinum is respectively adopted
(Schizochytrium sp.) HX-ME fermenting and producings DHA.
1. fermentation culture method
Three-level seed liquor is accessed in fermentation tank culture medium with the inoculum concentration of 10% (v/v).During culture, the canned liquid of 5L fermentations
It measures as 3L, requires supplementation with glucose in fermentation process so that grape concentration is higher than 15g/L in zymotic fluid；Throughput is 1vvm, is stirred
Mix rotating speed is 350rpm, and Initial sugar concentration 40g/l, fermentation temperature is 30 DEG C.Fermentation time is 7 days.
Wherein, seed culture medium (g/L) composition is：Glucose 40g/L, yeast extract 2g/L, sodium glutamate 10g/L,
KH2PO44g/L、NaCl 15g/L、MgCl23g/L、CaCl2·2H2O 1g/L、KCl 2g/L、MgSO4·7H2O 5g/L、
FeCl30.1g/L, sterilising conditions：121℃、30min.
Fermentation medium (g/L) forms：Glucose 40g/L, yeast extract 2g/L, sodium glutamate 10g/L, KH2PO44g/L、
NaCl 15g/L、MgCl23g/L、(NH4)2SO46g/L、KCl 2g/L、MgSO4·7H2O5g/L、FeCl30.1g/L, sterilize item
Part：121℃、30min.
2. detection method
During the fermentation, sampling is detected as follows daily：
(1) assay method of fat content：The zymotic fluid (100ml) of certain volume is taken, it at 50 DEG C is preheated, uses NaOH
Between pH is adjusted to 10-12 by solution, wall breaking enzyme is added in the ratio of 3 ‰ (g/l), is stirred at 50 DEG C and keeps the temperature 2h；By 1:1:1
(zymotic fluid：Ethyl alcohol：N-hexane) (v/v) ratio adds ethyl alcohol, n-hexane respectively, stirring, layering, extraction, continuous extraction 2-3
It is secondary, n-hexane phase is taken, vacuum rotating solvent evaporated obtains grease.Flask is put and is dried to constant weight for 105 DEG C in an oven, after cooling
It weighs.
(2) DHA content detection method in grease：Using conventional method by the present embodiment title 2 (1) extract grease into
Then row esterification utilizes DHA content in Japanese Shimadzu high resolution gas chromatography instrument detection grease.Gas phase analysis condition：Chromatographic column：
DB-23(60m*0.25mm*0.25μm)；Detector：FID；Carrier gas：Nitrogen；Split ratio：30/1；Injector temperature：250℃；Inspection
Survey device temperature：280℃；Sample size：1μl；Temperature program：Initial column temperature is 100 DEG C, first rises to 196 with the speed of 25 DEG C/min
DEG C, then 220 DEG C are risen to the speed of 2 DEG C/min, keep 12min.Column flow rate：3.0ml/min；Tail wind drift speed：30ml/min；Hydrogen
Gas velocity：40ml/min；Air velocity：400ml/min.
(3) dry cell weight (DCW) measures:30mL zymotic fluids are taken, are collected by centrifugation, supernatant discarding.Thalline is dried to perseverance in 105 DEG C
Weight, it is dry cell weight to measure.
(4) glucose concentration determination：Zymotic fluid 1mL is taken in 1.5mL centrifuge tubes, at room temperature, 12000rpm centrifugation 2min,
100 μ L supernatants is taken 10mL to be settled to Wahaha water, then using in biosensor assay zymotic fluid to 10mL volumetric flasks
Concentration of glucose, the unit for measuring numerical value are g/L.
3. result
In fermentation ends, the oil production of original bacteria and genetic engineering bacterium reaches peak value.The peak crude flow of original bacteria is
59.8g/L zymotic fluids, wherein DHA yield only have 27.3g/L zymotic fluids.The peak crude flow of genetic engineering bacterium reaches 72.5g/L,
Wherein DHA yield is up to 32.6g/L zymotic fluids.It can be seen that schizochytrium limacinum engineering bacteria bacterium is recombinated compared with original bacteria, oil production
12% is added, wherein DHA yield adds 19.5%.
SEQUENCE LISTING
<110>Nanjing University of Technology
<120>A kind of schizochytrium limacinum genetic engineering bacterium for producing DHA and its construction method 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 segment
<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 (5)
1. a kind of schizochytrium limacinum genetic engineering bacterium for producing DHA, is to be integrated with malic enzyme gene in schizochytrium limacinum genome
The engineering bacteria of expression cassette, the expression cassette of the malic enzyme gene contain promoter, malic enzyme gene and terminator successively；Institute
Promoter is stated as ubiquitin promoter, the malic enzyme gene such as SEQ ID NO:Shown in 1, the terminator terminates for ubiquitin
Son；The integration site is 18srDNA.
2. the construction method of schizochytrium limacinum genetic engineering bacterium described in claim 1, it is characterised in that structure contains malate dehydrogenase base
The homologous recombination segment of the expression cassette of cause；The homologous recombination segment is imported into schizochytrium limacinum and carries out homologous recombination, screening obtains
The schizochytrium limacinum genetic engineering bacterium.
3. the construction method of schizochytrium limacinum genetic engineering bacterium according to claim 2, it is characterised in that the homologous recombination piece
The upstream and downstream homology arm of 18srDNA is equipped in section at the both ends of the expression cassette of the malic enzyme gene, the 18srDNA's
Upstream homology arm sequence such as SEQ ID NO:Shown in 2, the downstream homology arm such as SEQ ID NO of 18srDNA:Shown in 3.
4. the construction method of schizochytrium limacinum genetic engineering bacterium according to claim 3, it is characterised in that the homologous recombination piece
Section carries bleomycin resistance marker gene.
5. the method that DHA is prepared using schizochytrium limacinum genetic engineering bacterium described in claim 1, it is characterised in that using seed culture
Then base culture schizochytrium limacinum genetic engineering bacterium transfers and prepares DHA into fermentation medium；The seed culture medium contains：Grape
Sugared 40g/L, yeast extract 2g/L, sodium glutamate 10g/L, KH2PO4 4g/L、NaCl 15g/L、MgCl2 3g/L、CaCl2·
2H2O 1g/L、KCl 2g/L、MgSO4·7H2O 5g/L、FeCl30.1g/L；The fermentation medium contains：Glucose
40g/L, yeast extract 2g/L, sodium glutamate 10g/L, KH2PO4 4g/L、NaCl 15g/L、MgCl2 3g/L、(NH4)2SO46g/
L 、KCl 2g/L、MgSO4·7H2O 5g/L、FeCl3 0.1g/L。
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| US11248244B2 (en) | 2017-05-10 | 2022-02-15 | Nanjing University Of Technology | Schizochytrium limacinum strain, building method therefor and application thereof |
| CN106947706B (en) * | 2017-05-10 | 2020-07-07 | 南京工业大学 | Schizochytrium limacinum strain, construction method 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 |
| 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 |
| CN112226453B (en) * | 2020-10-29 | 2024-01-30 | 南京工业大学 | Schizochytrium CRISPR/Cas9 gene editing system and application thereof |
| 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 |
| CN114574373B (en) * | 2022-03-29 | 2022-11-01 | 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 |
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