CN109517834A - A method of schizochytrium ATCC20888 grease and DHA content are improved by genetic modification - Google Patents
A method of schizochytrium ATCC20888 grease and DHA content are improved by genetic modification Download PDFInfo
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Abstract
A kind of method that the present invention discloses schizochytrium ATCC20888 mutanted strain that high grease and DHA content are obtained by genetic modification.Comprise the following steps: (1) malate dehydrogenase being overexpressed from hidden dinoflagellate improves schizochytrium ATCC20888 reducing power, obtains the mutanted strain of high Lipid-producing；(2) algae obtained with previous step is algae of setting out, it is overexpressed the extension enzyme for deriving from Mortierella alpine mould, it is 18 carbon by 16 carbon conversions, release the inhibiting effect to acetyl-CoA carboxylase, improve precursor supply, while further increasing fat content, the ratio that DHA accounts for dry cell weight is improved.Genetic modification is carried out to schizochytrium algae using method of the invention, the schizochytrium mutanted strain of acquisition is compared with algae of setting out, and grease accounts for dry cell weight ratio and improves 48.63%, and content reaches the 71.68% of frond dry weight；DHA accounts for dry cell weight ratio and improves 38.69%, and content reaches the 26.70% of frond dry weight.
Description
Technical field
The present invention discloses a kind of schizochytrium ATCC20888 mutation that high grease and DHA content are obtained by genetic modification
The method of algae, and in particular to a kind of preparation method of the engineering algae strain of high grease and DHA content and application.It is micro- to belong to industry
Biological field.
Background technique
Docosahexaenoic acid (Docosahexaenoic acid, DHA) is that human body itself cannot synthesize, but to human body
A kind of healthy very favorable essential fatty acid, the in recent years always hot spot of scientific research.Clinical test shows that DHA is mind
A kind of main component for growing and maintaining through system cells, is the important composition ingredient of brain and retina, in human brain skin
Content is up to 20% in layer, and proportion is maximum in eye retina, accounts for about 50%, therefore, for infant's nervous system
Play the role of with visual acuity very important, while also having prevents cardiovascular disease from occurring and anti-inflammatory, antitumor etc. making
With.It is reported that 2000 tons of DHA at least sell every year in Dutch DSM N. V., nearly 3.17 hundred million dollars of gross sales amount, therefore have very
Wide market application prospect.
The source of DHA is mainly marine fishes, due to that can accumulate plurality of heavy metal and organic contamination in marine fishes body
Object, it is extremely unhealthy to human body, therefore in recent years, permitted on finding sustainable, high-quality DHA substitution source
Multiplexing is made.Wherein, a main alternative has been considered as it using marine microalgae heterotrophic fermentation production DHA.Schizochytrium
It (Schizochytrium) is a kind of microalgae kind rich in DHA, and most widely used in DHA fermenting and producing at present
Algae strain.Advantage of the schizochytrium in terms of DHA production is: the speed of growth is very fast, and final biomass is higher, and DHA yield is opposite
It is higher, report that peak is culture 90-100 hours in the world, frond dry weight 200g/L, DHA 40-45g/L, the current country is permitted
More unit screenings obtain excellent schizochytrium algae, realize high density fermentation and pilot scale expanding test, push entire industry
Fast development.
Currently, there are grease and DHA content are relatively low for the schizochytrium of production application in practical schizochytrium fermentation process
Problem.The character of algae is easy to degenerate during the fermentation.Therefore, using effective technological means to schizochytrium
It is transformed, the excellent species for obtaining high yield and inheritance stability are to the sustainable development for promoting schizochytrium production DHA industry
It is highly important.
It is reported that an important sources of NADPH are malate dehydrogenases in frond.It is reported that by cultivating hidden dinoflagellate
The vigor that malate dehydrogenase inhibitor inhibits malate dehydrogenase is added in culture medium, grease yield is remarkably decreased, illustrates from hidden first
The malate dehydrogenase of algae plays the role of very important in terms of oil synthesis.
Summary of the invention
The object of the present invention is to provide a kind of methods for improving schizochytrium grease and DHA content.The present invention provides one
Kind improves its activity, the method for being quickly obtained the excellent schizochytrium algae of character by being overexpressed critical sites enzyme.
The present invention obtains the side of the schizochytrium ATCC20888 mutanted strain of high grease and DHA content by genetic modification
Method, specific steps include:
1. expand malic enzyme gene from hidden dinoflagellate cDNA, expanded from p1301 plasmid CaMV35s promoter and
CaMV poly (A) terminator merges after forming expression cassette with neomycin resistance gene expression cassette, forms malic enzyme gene
Convert expression cassette.
2. cultivating schizochytrium ATCC20888 in enriched medium, the seed of exponential phase is chosen, is ground using zirconium oxide
Grind thallus, be then utilized respectively 2% sea salt and 50mM sucrose washing cell, by 10 μ L malic enzyme genes convert expression cassette (>
500ng/ μ l) it is added in the 100 processed cells of μ L, DNA is sent into cell using electroporation, electricity turns condition: 2000V field strength
With 50 μ F capacitors, electroporation under 200 Ω resistance.Cell after electricity turns is added in 0.6mL enriched medium, and at 25 DEG C and
It is cultivated 12 hours under 180rpm.
3. 200 μ L cells are coated on the basal medium plate that neomycin concentration is 100 μ g/ml, cultivated at 25 DEG C
4 days.
Screening obtains transformant in initial medium, is transferred again into 600 μ g/ml neomycin basal medium plates
In, it cultivates 4 days.Screening obtains dependent conversion, and passes through primer 1 (CCTGCCCATTCGACCACCAAGCGAAACATCGC)
PCR, which is carried out, with primer 2 (CCTTCAGGAGAGTGCCGTAGTAGACCTCTGGG) Identifies and adopts primer 3
(GGCTCCCCGCAGCCTTTCTCTTC) and primer 4 (TTTGGACCTCGGCCACTACAGCTGTC) carries out RT-PCR confirmation.
4. the comparison of growth rate, oil and fat accumulation and fatty acid composition between schizochytrium mutant strain and wild strain: by institute
It obtains and is cultivated in schizochytrium mutant strain and wild strain access enriched medium, identify the variation of mutant strain grease and DHA content.
5. from synthesis source in the extension enzyme of Mortierella alpine mould, expanded from p1301 plasmid CaMV35s promoter and
Nos terminator merges after forming expression cassette with bleomycin resistance expression casette, and composition extends enzyme gene conversion expression
Box.
6. the algae obtained with previous step is algae of setting out, is cultivated in enriched medium, chooses the seed of exponential phase,
Using zirconium oxide abrasive thallus, it is then utilized respectively 2% sea salt and 50mM sucrose washing cell, 10 μ L are extended into enzyme gene and are turned
Change expression cassette (> 500ng/ μ l) to be added in the 100 processed cells of μ L, DNA be sent into cell using electroporation, electricity turns condition:
2000V field strength and 50 μ F capacitors, electroporation under 200 Ω resistance.Cell after electricity turns is added in 0.6mL enriched medium, and 25
DEG C and 180rpm under cultivate 12 hours.200 μ L cells are coated on the basal medium that bleomycin concentration is 50 μ g/ml to put down
On plate, cultivated 4 days at 25 DEG C.Screening obtains transformant in initial medium, is transferred again into and wins Lay containing 150 μ g/ml
In mycin basal medium plate, cultivate 4 days.Screening obtains dependent conversion, and passes through primer 5
(CATCATCATCCTCAAGGGCCGCCGCTCGTC) it is carried out with primer 6 (AGTCCTGCTCCTCGGCCACGAAGTGCACG)
PCR Identifies and adopts primer 7 (CTGCCGGCATCCCCTTCCCTGAGTACTA) and primer 8
(CTGGGCCTTCTTCTGAGCGGCGATG) RT-PCR confirmation is carried out.
7. the comparison of grease and DHA content between schizochytrium mutant strain and starting strain: by gained schizochytrium mutant strain
It is cultivated with starting strain access enriched medium, identifies the variation of mutant strain grease and DHA content accumulation.
Step 2, enriched medium described in step 7 is by 2~80g of glucose, 0.5~20g of yeast extract, peptone
0.25~10g, 1~40g of sea salt add water to 1L and form, pH 7.0；Optimal is 40 g of glucose, yeast extract 10g, peptone
5g, sea salt 20g add water to 1L and form, pH 7.0；
The step 3, basal medium described in step 8 are by 0.45~18g of glucose, 0.1~4 g of yeast extract, sea
1.25~50g of salt, 1~40g of agar add water to 1L and form, pH 7.0；Optimal is glucose 9g, yeast extract 2g, sea salt
25g, agar 20g add water to 1L and form, pH 7.0；
The step 2, it is 20-200rpm that frond condition of culture, which is shaking table setting parameter, in step 7, and temperature is 25 DEG C；
Laboratory research of the present invention discovery, NADPH and acetyl coenzyme A be a large amount of Synthetic Oils of frond and DHA it is important before
Body substance.The first step of oil synthesis is that acetyl coenzyme A is converted to propionyl coenzyme under the action of acetyl-CoA carboxylase
A.Feedback inhibition of the discovery acetyl-CoA carboxylase by downstream final product, the acetyl of schizochytrium in many biologies
Feedback inhibition of the CoA carboxylase enzyme by 16 carbon fatty acids or derivatives thereof.Therefore, 16 carbon fatty acids are converted
For 18 carbon fatty acids, the inhibiting effect to acetyl-CoA carboxylase can be released, the supply of precursor substance is improved.
The schizochytrium mutanted strain obtained by means of the present invention, grease account for dry cell weight ratio and improve 48.63%,
Its content reaches the 71.68% of frond dry weight；DHA accounts for dry cell weight ratio and improves 38.69%, and content reaches frond dry weight
26.70%.
Schizochytrium is transformed using the method for orientation genetic modification provided by the invention, is educated compared to traditional
Kind of method, have the characteristics that can efficiently, be quickly obtained grease and algae strain that DHA content improves, in DHA industrialization field tool
There is important application prospect.
Detailed description of the invention
Expression derives from the malic enzyme gene of hidden dinoflagellate in Fig. 1 schizochytrium ATCC20888；In figure: A) malic acid
Enzyme gene expression box.The upstream 1:18s；2:CaMV 35s promoter；3: neomycin resistance gene；4: CaMV poly(A)
Terminator；5:CaMV 35s promoter；6: malic enzyme gene；7:CaMV poly (A) terminator；Under 8:18s
Trip.B) PCR verifying is carried out using primer 1 and primer 2.M: 1kb marker；1: water；2: wild type；3: being overexpressed
Malic enzyme gene transformant.C) RT-PCR verifying is carried out using primer 3 and primer 4.M: 1kb marker；1: water；2:
Wild type；3: being overexpressed malic enzyme gene transformant.
The wild algae of Fig. 2 and the grease (a) and DHA(b for being overexpressed malate dehydrogenase algae) content analysis；Wherein fragmentation pot sets out
Algae and be overexpressed malic enzyme gene transformant grease (a) and DHA content (b) compare (fat content improve 39.26%,
16.94%) DHA content improves；DHA content/frond dry weight (%).
Fig. 3 extends enzyme gene expression box and transformant verifies picture, is overexpressed expression in malic enzyme gene algae and derives from height
The extension enzyme gene of mountain mortierella sp；Wherein A) Mortierella alpine mould extension enzyme gene expression box.1:CaMV 35s starting
Son；2: extending enzyme gene；3:Nos terminator；4: α tubulin promoter；5: bleomycin resistance gene； 6:
CYC1 terminator.B) PCR verifying is carried out using primer 5 and primer 6.M: 1kb marker；1: while being overexpressed malic acid
Enzyme gene and extension enzyme gene transformant；2: being overexpressed malic enzyme gene transformant.C it) is carried out using primer 7 and primer 8
RT-PCR verifying.M: 1kb marker；1: being overexpressed malic enzyme gene transformant；2: while being overexpressed malic enzyme gene
With extension enzyme gene transformant.
Specific embodiment
The following examples are to make those skilled in the art more fully understand the present invention but be not limited to this
Invention.
Enzyme of the present invention using specific function is transformed schizochytrium frond and obtains high Lipid-producing and DHA
The algae of content, the enzyme of overexpression are malate dehydrogenase and extension enzyme.Wherein malate dehydrogenase derives from hidden dinoflagellate, extends enzyme source
In Mortierella alpine mould.
The growth of schizochytrium mutanted strain, fat content and the analysis of DHA component characteristic, obtained schizochytrium mutation
Body is cultivated in basal medium.Selected when inoculation OD660nm for 1 fresh cells 1mL be added to 20mL fresh enrichment training
It supports in base, every group is done three parallel, the cell progress grease of selection 72 hours and the composition analysis of DHA content.Grease analysis side
Method: 3500 × g be centrifuged 10 minutes collection cells, and with PBS buffer solution (NaCl 137mmol/L, KCl 2.7mmol/L,
Na2HPO410mmol/L, KH2PO42mmol/L) washing cell is primary, and is frozen into dry powder.Using contain 0.01% fourth hydroxyl first
The chloroform/methanol solution (2:1, v/v) of benzene extracts 25mg algae powder, extracts 3 to 4 times in total, then utilizes the 1.0M KCl of 1mL
With distillation water washing, is finally drained with vacuum concentration instrument, obtain total fat content.DHA content measurement: about 25mg algae is weighed
98 DEG C of 4mL solution (2mL methanol+2mL chloroform+0.6mL sulfuric acid) reaction 2 hours is added in powder, after cooling, addition 2mL water, and 7400
× g is centrifuged 10 minutes, collects lower layer's organic phase.Utilize GC/MS Instrument measuring DHA content, GC/MS instrument model: GC7890-
MSD5975.Column model: HP-5MS capillary column (30m × 250mm id) identifies DHA using NIST/EPA/NIH mass spectral database
Peak figure.
Invention is further described in detail by way of example and in conjunction with the accompanying drawings.
Embodiment 1
It is overexpressed malic enzyme gene
1. expand malic enzyme gene from hidden dinoflagellate cDNA, expanded from p1301 plasmid CaMV35s promoter and
CaMV poly (A) terminator merges after forming expression cassette with neomycin resistance gene expression cassette, forms malic enzyme gene
It converts expression cassette (Fig. 1).
2. cultivating schizochytrium ATCC20888 in enriched medium, the seed of exponential phase is chosen, is ground using zirconium oxide
Grind thallus, be then utilized respectively 2% sea salt and 50mM sucrose washing cell, by 10 μ L malic enzyme genes convert expression cassette (>
500ng/ μ l) it is added in the 100 processed cells of μ L, DNA is sent into cell using electroporation, electricity turns condition: 2000V field strength
With 50 μ F capacitors, electroporation under 200 Ω resistance.Cell after electricity turns is added in 0.6mL enriched medium, and at 25 DEG C and
It is cultivated 12 hours under 180rpm.
3. 200 μ L cells are coated on the basal medium plate that neomycin concentration is 100 μ g/ml, cultivated at 25 DEG C
4 days.
Screening obtains transformant in initial medium, is transferred again into 600 μ g/ml neomycin basal medium plates
In, it cultivates 4 days.Screening obtains dependent conversion, and passes through primer 1 (CCTGCCCATTCGACCACCAAGCGAAACATCGC)
PCR, which is carried out, with primer 2 (CCTTCAGGAGAGTGCCGTAGTAGACCTCTGGG) Identifies and adopts primer 3
(GGCTCCCCGCAGCCTTTCTCTTC) and primer 4 (TTTGGACCTCGGCCACTACAGCTGTC) carries out RT-PCR confirmation.
4. the comparison of growth rate, oil and fat accumulation and fatty acid composition between schizochytrium mutant strain and wild strain: by institute
It obtains and is cultivated in schizochytrium mutant strain and wild strain access enriched medium, identify the variation of mutant strain grease and DHA accumulation
(Fig. 2).
The present invention improves the vigor in Key Metabolic site by orientation expression key enzyme, and can be quickly obtained has expectation special
The microorganism of sign realizes the directional transformation of algae, is the current improvement most promising method of industrial microorganism performance.Crucial position
Point directional transformation belongs to the scope of design of molecular breeding, has very extensive application value.
Embodiment 2
The present embodiment is substantially the same manner as Example 1, and the initial algae only used is the obtained mutation algae of embodiment 1
Kind, expression cassette is the extension enzyme (Fig. 3) from Mortierella alpine mould, and PCR verifying uses primer 5
(CATCATCATCCTCAAGGGCCGCCGCTCGTC) and primer 6 (AGTCCTGCTCCTCGGCCACGAAGTGCACG)；It carries out
RT-PCR confirmation uses primer 7 (CTGCCGGCATCCCCTTCCCTGAGTACTA) and primer 8
(CTGGGCCTTCTTCTGAGCGGCGATG).Be overexpressed malic enzyme gene transformant and be overexpressed malic enzyme gene and
The grease and DHA content for extending enzyme gene transformant, which compare, sees Fig. 4.
By above-mentioned two embodiment, the schizochytrium mutanted strain of acquisition, compared with algae of setting out, grease accounts for cell
Dry ratio improves 48.63%, and content reaches the 71.68% of frond dry weight；DHA accounts for the raising of dry cell weight ratio
38.69%, content reaches 26.70% (Fig. 5) of frond dry weight.
Claims (7)
1. a kind of method for improving schizochytrium ATCC20888 grease and DHA content by genetic modification, it is characterised in that: base
In the function by expression certain enzyme, the supply of intracellular reducing power and oil synthesis precursor is improved, high Lipid-producing and DHA are obtained
The algae of content, concrete operations include:
(1) overexpression box of the building containing the efficient malic enzyme gene from hidden dinoflagellate；
(2) schizochytrium ATCC20888 algae will be entered containing the conversion of efficient malic enzyme gene expression cassette, in enriched medium
Middle its grease of test and DHA content；
(3) overexpression box of the building from the efficient extension enzyme gene of Mortierella alpine mould；
(4) algae obtained with previous step is algae of setting out, and will enter in frond containing the expression and conversion that extend enzyme gene, obtains
Obtain final mutanted strain；
(5) detection in enriched medium to final mutanted strain progress grease and DHA content is obtained, identification mutanted strain oil
Rouge and DHA content situation.
2. the method according to claim 1, wherein the algae is schizochytrium category；The height being overexpressed is living
Property encoding gene is derived from the malic enzyme gene of hidden dinoflagellate and the extension enzyme gene from Mortierella alpine mould；(including
Schizochytrium but be not limited to schizochytrium).
3. the method according to claim 1, wherein enriched medium described in step 2 be by 2~80g of glucose,
0.5~20g of yeast extract, 0.25~10g of peptone, 1~40g of sea salt add water to 1L and form, pH 7.0.
4. the method according to claim 1, wherein malic enzyme gene described in step 1 is started by CaMV35s
Son and the control of CaMV poly (A) terminator.
5. the method according to claim 1, wherein extension enzyme gene described in step 3 is by CaMV35s promoter
It is controlled with Nos terminator.
6. a kind of method of schizochytrium genetic modification, it is characterised in that based on function (including the malic acid for being overexpressed certain enzyme
Enzyme and extension enzyme, but be not limited only to malate dehydrogenase and extend enzyme), intracellular reducing power and the supply of oil synthesis precursor are improved, is obtained
The method for obtaining high Lipid-producing and the schizochytrium algae strain of DHA.
7. a kind of method of schizochytrium genetic modification according to claim 6, it is characterised in that the certain enzyme of overexpression
Encoding gene is that schizochytrium is imported by the method for electroporation into the cell.
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