CN109593811A - A kind of red phaffia rhodozyma high yield 3S, 3`S astaxanthin method and its application of transgenosis - Google Patents
A kind of red phaffia rhodozyma high yield 3S, 3`S astaxanthin method and its application of transgenosis Download PDFInfo
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Abstract
The present invention provides a kind of red phaffia rhodozyma high yield 3S of transgenosis, the preparation method of 3`S astaxanthin, belong to technical field of bioengineering, by the way that the plasmid integration of beta carotene ketone group enzyme and beta carotene hydroxylase expression vector will be contained into red Phaffia Rhodozyma mutant strain, it obtains that 3S, the transgenic yeast of 3'S astaxanthin can be produced；Then it provides above-mentioned transgenosis red phaffia rhodozyma, provides a kind of culture medium for the above-mentioned yeast of fermented and cultured, and extract 3S from tunning, 3'S astaxanthin.Preparation method provided by the invention can protect exogenous enzymes expression activity, and foreign gene and carrier usage amount are few, and joint efficiency, yield and the product stability of expression vector are high; gained yeast exogenous gene expression amount is high; antiviral antibiotic ability is high, and long service life is applied widely；The method of production astaxanthin is avoided that byproducts build-up, improves astaxanthin yield and yield, production cost and low energy consumption, for producing 3S, 3'S configuration astaxanthin.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of red phaffia rhodozyma high yield 3S of transgenosis, 3 ' S shrimps
Green element method and its application.
Background technique
Astaxanthin (3,3 '-dihydroxy -4,4 ' diketo-β, β ' carrotene) is a kind of keto-acid carotenoid, naturally
Taking on a red color is also the highest level product of carotenogenesis, is known as singing the praises of for " king of carotenoid ".Astaxanthin is initial
Be used as the feed addictive of aquatic products industry, later pharmacology and Physiologic Studies show astaxanthin have it is extremely strong it is anti-oxidant, be quenched
The function of free radical, and the generation of antibody can be promoted, enhance the immune function of host, delay senility, reduces diabetes and cancer
The function of onset risk is absorbed in oxygen radical and is quenched in single line oxygen ability, and the potentiality of astaxanthin are much higher than other class Hu trailing plants
Bu Su has broad application prospects in fields such as cultivation, food, medicine and cosmetics, has very high economic value.
Currently, the production of astaxanthin has artificial synthesized and biological acquisition two ways.The process of chemical synthesis astaxanthin is multiple
It is miscellaneous, and product is mostly cis-structure, is not natural transconfiguration, therefore bioavailability is lower, while in its stabilization
The effect of property, coloring, safety and biological value etc. is also poor.Artificial synthesized astaxanthin is not only expensive, but also
With natural astaxanthin structure, function, application and in terms of significant difference.Currently, a kind of most promising producer
Formula is microbial fermentation, wherein studying most commonly used is red phaffia rhodozyma and haematococcus pluvialis.The astaxanthin of haematococcus pluvialis
For content up to the 3% of dry cell weight, redder phaffia rhodozyma yield is high, still, since its condition of culture is harsh and extracts difficult, limit
Its production application is made.
Red phaffia rhodozyma (Phaffia rhodozyma) is the yeast that uniquely can naturally produce astaxanthin, trans- astaxanthin
Ratify in acquisition FDA in 2000, be used for food additives, it be mycota, Eumycota, Deuteromycotina, Cryptococcaceae,
Unique kind of red phaffia rhodozyma category.As the production bacterium of astaxanthin, red phaffia rhodozyma have many advantages: using it is a variety of sugar into
The quick heterotrophism metabolism of row, incubation time is short, and cell density is big, be not required to illumination and High Density Cultivation may be implemented etc..It is naturally occurring
Red Astaxanthin from Xanthophyllomyces dendrorhous during Storage content it is extremely low (0.16~1.1mg/g stem cell), can not be competed with chemical synthesis, Wu Faman
Sufficient industrialized production, also, since the astaxanthin that red phaffia rhodozyma generates is 3R, 3 ' R configurations, oxidation resistance is lower, unfavorable
In the utilization as nutriment, its use on a commercial scale is limited, brings obstacle to its biological utilisation.Cause
This, to construct high yield 3S, the engineering bacteria of 3 ' S structure astaxanthins, by by the beta carotene ketone group enzyme of external source and β-carrot
Plain '-hydroxylase gene is transferred in red Producing Strain Phaffia rhodozyma beta carotene mutant strain, to the rDNA of red phaffia rhodozyma mutant strain
Conversion integration is carried out, the red phaffia rhodozyma transformant of the astaxanthin of purpose product 3S, 3'S configuration has been obtained, in transformant inner product
Tired 3S, the natural astaxanthin of 3'S configuration change red Astaxanthin from Xanthophyllomyces dendrorhous during Storage configuration with this, accumulate 3S in red phaffia rhodozyma,
The astaxanthin of 3'S configuration reaches the purpose of red phaffia rhodozyma high yield 3S, the 3'S astaxanthin of transgenosis, solves 3R, and 3'R astaxanthin is only
The problem of special structure and bioavailability brings obstacle.
Summary of the invention
Exogenous enzymes expression activity, foreign gene and carrier usage amount can be protected one of the objects of the present invention is to provide a kind of
It is few, the preparation method of the joint efficiency of expression vector, yield and the high red phaffia rhodozyma of transgenosis of product stability, the yeast
3S can be produced, 3 ' S configuration astaxanthins, exogenous gene expression amount is high, and antiviral antibiotic ability is high, and long service life is applied widely.
The second object of the present invention is to provide a kind of accumulation for being avoided that by-product, improves astaxanthin yield and yield,
Reaction condition and equipment requirement are simple, production cost and the red phaffia rhodozyma high yield 3S of the transgenosis that low energy consumption, 3 ' S astaxanthins
Method, this method can be used for producing 3S, the astaxanthin of 3'S configuration.
The technical solution that the present invention is taken to achieve the above object are as follows:
A kind of red phaffia rhodozyma high yield 3S of transgenosis, the preparation method of 3 ' S astaxanthins, including,
A kind of red phaffia rhodozyma of transgenosis is provided, is integrated in the red phaffia rhodozyma cell of the transgenosis containing beta carotene
The plasmid of ketone group enzyme and beta carotene hydroxylase expression vector；
A kind of culture medium, the red phaffia rhodozyma of the above-mentioned transgenosis of fermented and cultured are provided；And
3S, the astaxanthin of 3 ' S configurations are extracted from tunning.
Preferably, the red phaffia rhodozyma of transgenosis is by that will contain beta carotene ketone group enzyme bkt and beta carotene
The plasmid integration of hydroxylase crtZ expression vector is into host cell, to obtain beta carotene ketone group enzyme bkt and β-Hu Luo
Bu Su hydroxylase crtZ is able to produce 3S in the host cell inner expression, the transgenic yeast of 3 ' S astaxanthins；The place
Chief cell is red Producing Strain Phaffia rhodozyma beta carotene mutant strain.Foreign gene is introduced red Producing Strain Phaffia rhodozyma β-carrot by this method
In plain mutant strain, energy successful expression goes out to recombinate astaxanthin albumen, synthesizes and accumulate 3S, 3 ' S configurations using beta carotene as substrate
Astaxanthin, the expression quantity of the transgenosis phaffiafhodozyma of acquisition is high, yield and yield are high, and antiviral antibiotic ability is strong, uses the longevity
Life extends, and is conducive to practical metaplasia and produces, has good prospects for commercial application.
Further preferably, integration is using electroporated；Electric shock condition be 5~6KV/cm of voltage, electric shock medium be containing
The STM buffer of 0.08~0.12 μ L DMAC N,N' dimethyl acetamide and 0.02~0.05 μ L ethyl lactate.Contain foreign gene
Vector plasmid can be integrated under electric current stimulation with the rDNA of phaffiafhodozyma, and N in the medium that shocks by electricity, N- dimethylacetamide
The presence of amine and ethyl lactate, on the one hand the two can be replaced with the hydrogen of DNA insertion point intramolecular base centering, improved and inserted
Extensibility at angle of striking slows down the active force for moving and colliding between medium intermediate ion, so that outside in target gene insertion
The copy number of source gene insertion increases, so that the ability of thallus antiviral antibiotic is enhanced, so that transgenosis thallus is in antibiosis prime ring
Service life under border extends, and has effectively expanded the scope of application of transgenosis thallus, is conducive to practical metaplasia and produces, another aspect energy
The damage to enzymatic activity is reduced to the maximum extent, and protects the expression activity of exogenous enzymes, can especially promote hydroxylase in ketone group
Hydroxyl group is added on No. 3 carbon of enzyme product, to effectively convert astaxanthin for intermediate material, improves and avoid ketone group class
The accumulation of carrotene by-product, while improving the yield of astaxanthin.
Further preferably, plasmid concentration is that cell is resuspended in 0.65~0.8 μ g/ μ L；Above-mentioned resuspension cell be with 45~
50mmol/L concentration dithiothreitol (DTT) processing, the red Producing Strain Phaffia rhodozyma beta carotene mutant strain in logarithmic growth phase it is thin
Born of the same parents.Phaffia rhodozyma cell in mid log phase grows vigorous, cell wall structure of the cell wall structure compared with stationary phase cell
It is loose, it is more suitable as the acceptor material of transgenosis, dithiothreitol (DTT) can be acted on-S-S- the key of loose protein with it
Yeast cells can be such that cell wall generates loose, be conducive to voltage and act on yeast cell wall, form Micro-v oid, promote external source
Plasmid DNA enters cell.
Further preferably, expression vector containing beta carotene ketone group enzyme bkt and beta carotene hydroxylase crtZ
Plasmid preparation step is as follows: expression vector pPRcDNA bkt being connected on pMON carrier by pcr amplification product, then again
It is secondary to be reacted using PCR, and product is connected on pMON carrier, target fragment Pgpd+bkt+Tgpd is obtained, is then used simultaneously
Hind III single endonuclease digestion target fragment Pgpd+bkt+Tgpd and carrier pPRcDNA crtZ, then connected with T4DNA ligase
It is reversed should be to get the plasmid of pPRcDNA crtZ-bkt expression vector.
Still further preferably, PCR reaction system are as follows: 10 × Buffer, 5 μ L, MgCl2 3μL、dNTP(25mM) 4μL、
0.5 μ L of DNA profiling, Taq Polymerase (5u/ μ L) 0.25 μ L, on draw 1 μ L, under draw 1 μ L, then be added sterile water be assigned to 50
μL.Pcr amplification reaction program are as follows: 94 DEG C of initial denaturations 4min, 94 DEG C of denaturation 30s, 50~60 DEG C of annealing 30s, 72 DEG C of extension 30s,
30 circulations, last 72 DEG C extend 10~15min eventually, are cooled to 4 DEG C of end.
Still further preferably, digestion system are as follows: 10 × enzyme cutting buffering liquid, 2 μ L, ddH2It is 16 μ L of O, 0.3 DNA μ L, restricted
0.2~0.8 μ L of restriction endonuclease, then addition sterile water is assigned to 20 μ L, then system is incubated to 1~2h at 16~20 DEG C, above-mentioned
When system is double digestion system, restriction enzyme is added with the ratio of 1:1.
Still further preferably, the coupled reaction system of T4DNA ligase are as follows: 10 × T4buffer, 2 μ L, ddH2O 17μL、
0.5 μ L of T4DNA ligase, 0.05~0.1 μ L of fatty diglycollic amide, 0.03~0.06 μ L of isobutyl acetate, segment Pgpd
4 μ L of+bkt+Tgpd and carrier pPRcDNA crtZ, distilled water add to 25ul；Above-mentioned Pgpd+bkt+Tgpd segment and carrier
The weight ratio of pPRcDNA crtZ is 1.5~2.5:1, after above-mentioned connection reaction mixes, is used after incubating 2h at 23 DEG C.Connection
Fatty diglycollic amide and the special presence of isobutyl acetate, are integrated on the viscous terminal residue of carrier surface in reaction system,
It prevents to attract each other between carrier, avoids carrier from being formed from even causing active site occupied, the usage amount of carrier can be reduced, increased
Add the joint efficiency and yield of expression vector, while the stability of vector plasmid can be improved, and then guarantees the red hair husband's ferment of transgenosis
Mother can succeed, high efficient expression goes out chemical activators albumen, increase the yield and yield of astaxanthin.
Still further preferably, specific preparation process is as follows for the red phaffia rhodozyma of transgenosis:
Step 1: extracting the total serum IgE of red phaffia rhodozyma with TRizol, then carried out total serum IgE with Reverse Transcriptase kit inverse
It is transcribed into cDNA template, obtains the plasmid gene group of red phaffia rhodozyma, and as template, carries out the building of expression vector；
Step 2: expression vector of the building containing beta carotene ketone group enzyme bkt and beta carotene hydroxylase crtZ, it will
It after carrier is connect with pMON, is transformed into genome of E.coli, the positive colony filtered out, as pPRcDNA crtZ-bkt
The plasmid of expression vector；
Step 3: after being activated to red phaffia rhodozyma with YEPD culture medium and expand culture, collecting and be in logarithmic growth phase
Thallus after, with containing 45~50mmol/L dithiothreitol (DTT) kaliumphosphate buffer be resuspended thallus, vibrate 10~15min after,
It is centrifuged 5~10min, discards supernatant liquid, then with after STM buffer washing thalline 2~3 times, thallus is placed in containing 0.08~
It in the STM buffer of 0.12 μ L n,N-dimethylacetamide and 0.02~0.05 μ L ethyl lactate, then is placed in and saves on ice, i.e.,
It obtains phaffiafhodozyma competence and cell is resuspended；
Step 4: taking the plasmid of pPRcDNAcrtZ-bkt expression vector, using sfiI single endonuclease digestion plasmid, keep plasmid linear
Change, the plasmid after linearisation is more convenient on the rDNA for being inserted into red phaffia rhodozyma；
Step 5: taking phaffiafhodozyma competence that cell is resuspended and contain expression vector pPRcDNA crtZ- with what is linearized
The Plasmid DNA of bkt mixes, and the plasmid concentration in mixed system is that cell is resuspended in 0.65~0.8 μ g/ μ L, is subsequently placed in ice pre-cooling
Electrocution device in, set voltage as 5~6KV/cm, after carrying out electroporated 1~2min, the YEPD culture of ice pre-cooling be added
Bacteria suspension after mixing, is cultivated at 20~25 DEG C 2~3h, is then transferred on YEPD solid medium, in 20~25 DEG C by base
48~72h of lower culture, screening collect and save thallus to get the red phaffia rhodozyma of transgenosis.
Preferably, beta carotene ketone group enzyme source is in haematococcus pluvialis；Beta carotene hydroxylase derives from Irving
Family name bacterium.Assimilation and hydroxylating betide the later period of carotenogenesis in red phaffia rhodozyma chemical activators approach,
Two kinds of foreign genes, which are added, can form facilitation to assimilation and hydroxylating, resist Metopirone and piperonyl butoxide prawn
The inhibiting effect of green element synthesis increases yield to accumulate and be formed more astaxanthin products.
Preferably, it is 3~4:1 that the total carbon source concentration of culture medium, which is 15~20g/L, wherein glucose and sucrose weight ratio,；
The total nitrogen source of culture medium is peptone and (NH4)2SO4, the method fermented respectively using single nitrogen source；It further include 4 in above-mentioned culture medium
The malt juice extract of~6g/L.
Preferably, fermentation culture conditions are as follows: temperature be 20~22 DEG C, 6~10L/h of aeration quantity, intensity of illumination be 500~
800Lux.Control fermentation temperature is conducive to the growth metabolism and pigment accumulation of red phaffia rhodozyma, with the glucose in cellobiose
It is used as carbon source with sucrose collaboration, it is the most advantageous to thalli growth, while during fermented and cultured, keep enough aeration quantitys extremely
It closes important, is to guarantee to produce higher yield and fine quality with this because assimilation and hydroxylating require the participation of oxygen
Astaxanthin.
A kind of application of the preparation method of red phaffia rhodozyma high-yield astaxanthin of above-mentioned transgenosis is also disclosed in the present invention, i.e., should
The red phaffia rhodozyma of transgenosis produce astaxanthin is configured as 3S, 3'S configuration.
The invention has the benefit that
1) transgenic technology is utilized in the present invention, and conversion integration is carried out to the rDNA of red phaffia rhodozyma, has obtained mesh
Product 3S, 3'S configuration astaxanthin red phaffia rhodozyma transformant, be converted into the configuration of product astaxanthin by 3R, 3 ' R with this
3S, 3'S configuration, and achieve the purpose that high yield；
2) vector plasmid of foreign gene and the rDNA of phaffiafhodozyma, operation are integrated in the present invention using electric shock
Easily-controllable, reaction condition and equipment requirement are simple, and gained transgenosis phaffiafhodozyma expression quantity is high, yield is high, antiviral antibiotic ability
By force, service life extends, applied widely, and production is rapidly and efficiently；
3) two target gene are constructed on an expression vector in the present invention, the optimization to building process, so that inserting
Extensibility enhancing at angle of striking, the damage to enzymatic activity reduce, avoid carrier from being formed from connecting, reduce the usage amount of carrier, increase
Add the joint efficiency and yield of expression vector, the stability of vector plasmid is high, so that the two expression condition having the same, improves
With avoid the accumulation of ketone group carotenoid by-product, while improving the yield of astaxanthin, reduce production cost.
Present invention employs above-mentioned technical proposals to provide a kind of red phaffia rhodozyma high yield 3S of transgenosis, 3 ' S astaxanthin methods
And its application, compensate for the deficiencies in the prior art, reasonable design, easy operation.
Specific embodiment
Technical solution of the present invention is described in further detail below in conjunction with specific embodiment:
STM buffer: 0.25mol/L sugarcane is warded off, 10mM TrisHCl, 0.5mmol/LMgCl2, 10mmol/L EDTA adds
Distilled water prepares 50mL, PH8.0.
YEPD culture medium: yeast powder 10g, peptone 20g, glucose 20g, ethyl alcohol 3g, glycerol 3g, distilled water add to
1000mL, pH6.0, in 115 DEG C of moist heat sterilization 20min, 4 DEG C of preservations.
YPD seed culture medium: yeast extract 1%, peptone 2%, glucose 2%, dipotassium hydrogen phosphate 0.2%, sodium acetate
0.5%, if solid medium processed, agar powder 2% is added, distilled water adds to 1000mL, after 115 DEG C of sterilizing 15min, 4 DEG C of guarantors
It deposits.
Embodiment 1:
A kind of red phaffia rhodozyma high yield 3S of transgenosis, the preparation method of 3 ' S astaxanthins, including,
A kind of red phaffia rhodozyma of transgenosis is provided, is integrated in the red phaffia rhodozyma cell of the transgenosis containing beta carotene
The plasmid of ketone group enzyme and beta carotene hydroxylase expression vector；
A kind of culture medium, the red phaffia rhodozyma of the above-mentioned transgenosis of fermented and cultured are provided；And
3S, the astaxanthin of 3 ' S configurations are extracted from tunning.
The red phaffia rhodozyma of transgenosis is by that will contain beta carotene ketone group enzyme bkt and beta carotene hydroxylase
The plasmid integration of crtZ expression vector is into host cell, to obtain beta carotene ketone group enzyme bkt and β-carrotene hydroxyl
Change enzyme crtZ in the host cell inner expression, and is able to produce 3S, the transgenic yeast of 3 ' S astaxanthins；The host cell
For red Producing Strain Phaffia rhodozyma beta carotene mutant strain.Foreign gene is introduced red Producing Strain Phaffia rhodozyma beta carotene and is mutated by this method
In strain, energy successful expression goes out to recombinate astaxanthin albumen, synthesizes and accumulate 3S, the shrimp blueness of 3 ' S configurations using beta carotene as substrate
Element, expression quantity height, yield and the yield height of the transgenosis phaffiafhodozyma of acquisition, antiviral antibiotic ability is strong, and service life extends,
Be conducive to practical metaplasia to produce, there is good prospects for commercial application.
Above-mentioned integration is using electroporated；Electric shock condition is voltage 5KV/cm, and electric shock medium is to contain 0.11 μ L N, N- bis-
The STM buffer of methylacetamide and 0.03 μ L ethyl lactate.Vector plasmid containing foreign gene, can be under electric current stimulation
The rDNA of phaffiafhodozyma is integrated, and in the medium that shocks by electricity n,N-dimethylacetamide and ethyl lactate presence, on the one hand
The two can be replaced with the hydrogen of DNA insertion point intramolecular base centering, the extensibility at insertion point be improved, in purpose base
When because of insertion, the active force for moving and colliding between medium intermediate ion is slowed down, so that the copy number of foreign gene insertion increases, from
And the ability of thallus antiviral antibiotic is enhanced, so that service life of the transgenosis thallus under antibiotic environment extends, effectively expand
The scope of application for having increased transgenosis thallus is conducive to practical metaplasia and produces, on the other hand can reduce to the maximum extent to enzymatic activity
Damage, and the expression activity of exogenous enzymes is protected, it can especially promote hydroxylase to add hydroxyl base on No. 3 carbon of ketone group enzyme product
Group, to effectively convert astaxanthin for intermediate material, improves and avoids the accumulation of ketone group carotenoid by-product, simultaneously
Improve the yield of astaxanthin.
Plasmid concentration is that cell is resuspended in 0.75 μ g/ μ L；Above-mentioned resuspension cell is with the dithiothreitol (DTT) of 48mmol/L concentration
Red Producing Strain Phaffia rhodozyma beta carotene Mutant Cells handle, in logarithmic growth phase.Method in mid log phase
Husband's yeast cell growth is vigorous, and cell wall structure is loose compared with the cell wall structure of stationary phase cell, is more suitable as transgenosis
Acceptor material, dithiothreitol (DTT) can be acted on yeast cells with it, cell wall can be made to generate with-S-S- the key of loose protein
It is loose, be conducive to voltage and acted on yeast cell wall, form Micro-v oid, exogenous plasmid dna is promoted to enter cell.
The plasmid preparation step of expression vector containing beta carotene ketone group enzyme bkt and beta carotene hydroxylase crtZ
It is as follows: expression vector pPRcDNAbkt is connected on pMON carrier by pcr amplification product, then reuses PCR reaction,
And product is connected on pMON carrier, target fragment Pgpd+bkt+Tgpd is obtained, then uses the mono- enzyme of Hind III simultaneously
Cut target fragment Pgpd+bkt+Tgpd and carrier pPRcDNAcrtZ, then with T4DNA ligase be attached reaction to get
The plasmid of pPRcDNAcrtZ-bkt expression vector.
The preparation step of above-mentioned expression vector pPRcDNAbkt are as follows: expand beta carotene ketone group enzyme bkt gene using PCR
Increase to increase EcoRI and XhoI restriction enzyme site, PCR product after purification, is connected on carrier pMON, then by carrier pMON-
Bkt and carrier pPRcDNA through EcoRI and XhoI double digestion, reuses T4DNA ligase and is attached reaction, connection produces respectively
Object is transformed into genome of E.coli, the positive colony filtered out, as pPRcDNAbkt expression vector.
The preparation step of above-mentioned expression vector pPRcDNAcrtZ are as follows: beta carotene hydroxylase crtZ is connected to carrier
On pMON, and as template PCR amplifications, while carrier pPRcDNA is also subjected to PCR amplification, then with above-mentioned two PCR
Product is template, carries out PCR amplification again, obtains the crtZ to link together by over-lap PCR and promoter Pgpd, then
This PCR product is connected on pMON carrier, using NotI and XhoI double digestion PCR fragment, and meanwhile it is bis- using NotI and XhoI
Digestion carrier pPRcDNA, product are attached reaction using T4DNA ligase, and connection product is transformed into Escherichia coli base
Because of the positive colony in group, filtered out, as pPRcDNA crtZ expression vector.
PCR reaction system are as follows: 10 × Buffer, 5 μ L, MgCl23 μ L, 4 μ L of dNTP (25mM), 0.5 μ L of DNA profiling, Taq
Polymerase (5u/ μ L) 0.25 μ L, on draw 1 μ L, under draw 1 μ L, then be added sterile water be assigned to 50 μ L.Pcr amplification reaction journey
Sequence are as follows: 94 DEG C of initial denaturation 4min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 30 recycle, last 72 DEG C of ends
Extend 10min, is cooled to 4 DEG C of end.
Digestion system are as follows: 10 × enzyme cutting buffering liquid, 2 μ L, ddH216 μ L of O, DNA0.3 μ L, 0.6 μ L of restriction enzyme, so
Sterile water is added afterwards and is assigned to 20 μ L, system is then incubated into 1h at 20 DEG C, it is restricted interior when above-mentioned system is double digestion system
Enzyme cutting is added with the ratio of 1:1.
The coupled reaction system of T4DNA ligase are as follows: 10 × T4buffer, 2 μ L, ddH217 μ L of O, T4DNA ligase
0.5 μ L, 0.08 μ L of fatty diglycollic amide, 0.05 μ L of isobutyl acetate, segment Pgpd+bkt+Tgpd and carrier pPRcDNA
4 μ L of crtZ, distilled water add to 25ul；Above-mentioned Pgpd+bkt+Tgpd segment and the weight ratio of carrier pPRcDNA crtZ are
1.5:1 is used after incubating 2h at 23 DEG C after above-mentioned connection reaction mixes.Fatty diglycollic amide in coupled reaction system
It with the special presence of isobutyl acetate, is integrated on the viscous terminal residue of carrier surface, prevents to attract each other between carrier, avoid carrying
Body is formed from even causing active site occupied, can be reduced the usage amount of carrier, be increased the joint efficiency and yield of expression vector,
The stability of vector plasmid can be improved simultaneously, and then guarantees that transgenosis phaffiafhodozyma can succeed, high efficient expression goes out astaxanthin conjunction
At albumen, increase the yield and yield of astaxanthin.
Specific preparation process is as follows for the red phaffia rhodozyma of transgenosis:
Step 1: extracting the total serum IgE of red phaffia rhodozyma with TRizol, then carried out total serum IgE with Reverse Transcriptase kit inverse
It is transcribed into cDNA template, obtains the plasmid gene group of red phaffia rhodozyma, and as template, carries out the building of expression vector；
Step 2: expression vector of the building containing beta carotene ketone group enzyme bkt and beta carotene hydroxylase crtZ, it will
It after carrier is connect with pMON, is transformed into genome of E.coli, the positive colony filtered out, as pPRcDNAcrtZ-bkt
The plasmid of expression vector；
Step 3: after being activated to red phaffia rhodozyma with YEPD culture medium and expand culture, collecting and be in logarithmic growth phase
Thallus after, with containing 48mmol/L dithiothreitol (DTT) kaliumphosphate buffer be resuspended thallus, vibrate 10min after, be centrifuged 5min,
Liquid is discarded supernatant, after then using STM buffer washing thalline 3 times, thallus is placed in containing 0.11 μ L n,N-dimethylacetamide
In the STM buffer of 0.03 μ L ethyl lactate, then it is placed in save on ice and cell is resuspended to get phaffiafhodozyma competence；
Step 4: taking the plasmid of pPRcDNA crtZ-bkt expression vector, using sfiI single endonuclease digestion plasmid, keep plasmid linear
Change, the plasmid after linearisation is more convenient on the rDNA for being inserted into red phaffia rhodozyma；
Step 5: taking phaffiafhodozyma competence that cell is resuspended and contain expression vector pPRcDNA crtZ- with what is linearized
The Plasmid DNA of bkt mixes, and the plasmid concentration in mixed system is that cell is resuspended in 0.75 μ g/ μ L, is subsequently placed in the electric shock of ice pre-cooling
In equipment, voltage is set as 5KV/cm, after carrying out electroporated 1min, the YEPD culture medium of ice pre-cooling is added, after mixing, by bacterium
Suspension cultivates 2h at 22 DEG C, is then transferred on YEPD solid medium, and 60h is cultivated at 22 DEG C, and screening is collected and protected
Thallus is deposited to get the red phaffia rhodozyma of transgenosis.
Beta carotene ketone group enzyme source is in haematococcus pluvialis；Beta carotene hydroxylase derives from Ou Wenshi bacterium.It is red
Assimilation and hydroxylating betide the later period of carotenogenesis in phaffia rhodozyma chemical activators approach, are added outside two kinds
Source gene can form facilitation to assimilation and hydroxylating, resist Metopirone and piperonyl butoxide to chemical activators
Inhibiting effect increases yield to accumulate and be formed more astaxanthin products.
A kind of application of the preparation method of the red phaffia rhodozyma high-yield astaxanthin of above-mentioned transgenosis, the i.e. red Fife's ferment of the transgenosis
Mother produce astaxanthin is configured as 3S, 3'S configuration.
Embodiment 2:
A kind of red phaffia rhodozyma high yield 3S of transgenosis, the method for 3 ' S astaxanthins, including,
A kind of red phaffia rhodozyma of transgenosis is provided, is integrated in the red phaffia rhodozyma cell of the transgenosis containing beta carotene
The plasmid of ketone group enzyme and beta carotene hydroxylase expression vector；
A kind of culture medium, the red phaffia rhodozyma of the above-mentioned transgenosis of fermented and cultured are provided；And
3S, the astaxanthin of 3 ' S configurations are extracted from tunning.
It is 3.5:1 that total carbon source concentration, which is 15g/L, wherein glucose and sucrose weight ratio, in above-mentioned culture medium；Culture medium is total
Nitrogen source is peptone and (NH4)2SO4, the method fermented respectively using single nitrogen source；It further include 5.5g/L's in above-mentioned culture medium
Malt juice extract.
Above-mentioned fermentation culture conditions are as follows: temperature is 22 DEG C, aeration quantity 8.5L/h, intensity of illumination 700Lux.Control fermentation
Temperature is conducive to the growth metabolism and pigment accumulation of red phaffia rhodozyma, using the dextrose and saccharose collaboration in cellobiose as carbon
Source, it is the most advantageous to thalli growth, while during fermented and cultured, it keeps enough aeration quantitys most important, is because of ketone
Change the participation for requiring oxygen with hydroxylating, guarantees the astaxanthin for producing higher yield and fine quality with this.
The red phaffia rhodozyma high yield 3S of transgenosis, specific preparation process is as follows for 3 ' S astaxanthins:
Step 1: the red phaffia rhodozyma strain of transgenosis being seeded in YPD seed culture medium, temperature is 22 DEG C, revolving speed is
10h is cultivated under conditions of 300r/min and obtains level-one kind culture, then level-one kind culture is pressed to the inoculum concentration of 8.5% (V/V)
It is seeded in fermentation medium, culture 10h is standby to get second level kind under conditions of temperature is 22 DEG C, revolving speed is 300r/min
With the condition of culture of the step condition of culture and fermenting and producing is close, in bacterial cell culture, can improve and medium component
Contact and oxygen supply, breeding is than more uniform, and efficiency is also high, not will form mycoderm, will not form long mycelia institute shape
At bead, prepare for fermentation tank culture；
Step 2: second level kind is inoculated into fermentor by the inoculum concentration of 9% (V/V), cultivates 4d under conditions of 22 DEG C,
Then it is centrifuged 10min under conditions of 5000r/min and collects thallus, distilled water is cleaned to get yeast thallus, spare, the step
Sufficient nutriment and good survival and reproduction condition can be provided for strain, guarantee the quick breeding of strain, obtain saccharomycete
Body, while can induce the expression of foreign gene, increase its expression quantity, product is stablized, easy purification；
Step 3: under conditions of being protected from light, STET buffer being added into yeast thallus, under conditions of 1000r/min
Then magnetic agitation 20min is added the lysozyme of Fresh to its final concentration of 120 μ g/mL, stirs evenly and be placed on 35 DEG C
Water-bath in 30min, then be added cell pyrolysis liquid 10min is heated in boiling water bath, be rapidly cooled to room temperature, then exist
Be centrifuged 15min under conditions of 10000r/min, then supernatant be added in the isopropanol of pre-cooling of 0.5 times of volume, after mixing-
10min is settled under conditions of 6 DEG C, is then centrifuged 10min under conditions of 10000r/min, is discarded supernatant, is dissolved with sterile water
Precipitating is freeze-dried to get astaxanthin.
Total carotinoid yield obtained by the present embodiment is 194.49mg/L, and wherein 3S, the yield of 3'S configuration astaxanthin are
186.45mg/L。
Comparative example 1:
A kind of preparation method of the red phaffia rhodozyma of transgenosis, is by that will contain beta carotene ketone group enzyme bkt and β-Hu
The plasmid integration of radish element hydroxylase crtZ expression vector is into host cell, to obtain the red phaffia rhodozyma of transgenosis；It is above-mentioned
Integration use is electroporated, and electric shock medium is STM buffer, that is, is not added with n,N-dimethylacetamide and ethyl lactate.
This comparative example compares test on the basis of embodiment 1, consistent in other preparation steps and embodiment 1, system
Obtain the red phaffia rhodozyma of transgenosis.
The red phaffia rhodozyma of transgenosis obtained by this comparative example is taken, according to the high yield 3S in embodiment 2, the method for 3 ' S astaxanthins
It is cultivated and is extracted, gained total carotinoid yield is 167.68mg/L, and wherein 3S, the yield of 3'S configuration astaxanthin are
104.68mg/L, the compounds content with ketone group reach 43.23mg/L.Illustrate the DMAC N,N' dimethyl acetamide in electric shock medium
The expression activity that exogenous enzymes can be protected with ethyl lactate improves and avoids the accumulation of ketone group carotenoid by-product, improves
The yield of 3S, 3'S configuration astaxanthin.
Comparative example 2:
A kind of preparation method of the red phaffia rhodozyma of transgenosis, the wherein coupled reaction system of T4DNA ligase are as follows: 10 ×
T4buffer 2μL、ddH217 μ L of O, 0.5 μ L of T4DNA ligase, segment Pgpd+bkt+Tgpd and carrier pPRcDNAcrtZ 4
μ L, distilled water add to 25ul.
1) the Pgpd+bkt+Tgpd segment in coupled reaction system and the weight ratio of carrier pPRcDNAcrtZ are 1.5:
1。
2) the Pgpd+bkt+Tgpd segment in coupled reaction system and the weight ratio of carrier pPRcDNAcrtZ are 3.5:
1。
This comparative example compares test on the basis of embodiment 1, consistent in other preparation steps and embodiment 1, system
Obtain the red phaffia rhodozyma of transgenosis.
1) and 2) this comparative example gained red phaffia rhodozyma of transgenosis is taken respectively, according to the high yield 3S in embodiment 2,3 ' S shrimps
The method of green element is cultivated and is extracted, wherein 1) gained total carotinoid yield is 138.35mg/L, wherein 3S, and 3'S structure
The yield of type astaxanthin is 121.54mg/L；2) gained total carotinoid yield is 157.87mg/L, wherein 3S, 3'S configuration
The yield of astaxanthin is 135.27mg/L.
From the above results, when other conditions are identical, fatty diglycollic amide and acetic acid in coupled reaction system
The presence of isobutyl ester can increase the yield and stability of effective carrier, promote expression of the transgenic yeast to foreign gene, increase
The content of target product is added；In the case where not adding fatty diglycollic amide and isobutyl acetate, Pgpd+bkt+ is improved
Tgpd segment and the weight ratio of carrier pPRcDNAcrtZ are it is also possible that astaxanthin yield increases, but increases foreign gene
With the usage amount of carrier, production cost and energy consumption are in contrast increased, reduces economic benefit in actual production.
Comparative example 3:
A kind of preparation method of the red phaffia rhodozyma of transgenosis is not added with N, N- bis- wherein electric shock medium is STM buffer
Methylacetamide and ethyl lactate.Simultaneously T4DNA ligase coupled reaction system in be not added with fatty diglycollic amide and
The weight ratio of isobutyl acetate, Pgpd+bkt+Tgpd segment and carrier pPRcDNAcrtZ in coupled reaction system is 1.5:
1。
This comparative example compares test on the basis of embodiment 1, consistent in other preparation steps and embodiment 1, system
Obtain the red phaffia rhodozyma of transgenosis.
The red phaffia rhodozyma of transgenosis obtained by this comparative example is taken, according to the high yield 3S in embodiment 2, the method for 3 ' S astaxanthins
It is cultivated and is extracted, gained total carotinoid yield is 128.36mg/L, and wherein 3S, the yield of 3'S configuration astaxanthin are
73.54mg/L, the compounds content with ketone group reach 37.58mg/L.
Test example 1:
The animal body acute toxicity test of astaxanthin
According to the mouse test in GB15193.3-2003 " acute toxicity test " in maximum tolerated dose method: taking 15g real
It applies example 2 and extracts obtained astaxanthin sample constant volume in 100mL sterile pure water, be made into the sample solution that concentration is 0.15g/mL.
Before administration, the weight of every KM mouse is measured.Growth normal mouse 20 are randomly selected, is administered in a manner of stomach-filling, 2.0mL/
Only, 0.4mL/ times, interior point of 5 administration of 12h.Observation calculates its death rate after 7 days, 7 days and weighs.
Thais clavgeria Antihypertensive Peptides oxicity analysis: 20 mouse are no different paradoxical reaction, and also without death, weight is obviously increased, hair
It is normal, half lethal dose (LD50) > 15g/kg weight.It is classified according to acute toxicity, obtained astaxanthin in the present invention
Belong to practical innocuous substance.
Test example 2:
Application of the astaxanthin in perch cultivates
Test material: purchase average size is 3000 tail of perch seedling of 21.2 ± 5.3g/ tail as test fish, and test fish exists
It is temporarily supported after two weeks in 23 ± 1 DEG C of pond, starts to be tested.Test fish is equally divided into 3 groups, 1 group of feeding of test contains reality
2 gained Carotenoids Extractss of example (per kilogram feed addition 50mg) are applied, 2 groups of feedings of test contain 1 gained class of comparative example recklessly
Radish extract (per kilogram feed addition 50mg), control group fed equivalent blank feed are continuously fed 8 weeks, count nature
Death rate of the onset and survival rate, as a result as shown in table 1.
Application test result of 1 astaxanthin of table in perch cultivates
As seen from the above table, test group perch survival rate is significantly larger than control group, and average survival reaches 85% or more, shows
Feeding experiment group feed plays protective effect in enhancing perch immunity；And 1 group of test more excellent compared with 2 groups of results of test, is
Due to 3S in the Carotenoids Extractss of 1 group of addition of test, the content astaxanthin of 3'S configuration is higher, and more conducively animal body is inhaled
It receives and utilizes.
Test example 3:
Application of the astaxanthin in shrimp culture
Test material: test shrimp is averaged by identical 4500 tail of Chinese prawn shrimp seedling of purchase average size as test shrimp
It is divided into 3 groups, 1 group of feeding of test contains 2 gained Carotenoids Extractss of embodiment (per kilogram feed addition 30mg), test 2
Group feeding contains 1 gained Carotenoids Extractss of comparative example (per kilogram feed addition 30mg), control group fed equivalent blank
Feed is continuously fed 8 weeks, counts the natural occurrence death rate and survival rate, the results are shown in Table 2.
Application test result of 2 astaxanthin of table in shrimp culture
| Put number/tail in a suitable place to breed | Death toll/tail | Survival number/tail | Survival rate % | Relative immunity protective rate % | |
| Test 1 group | 1500 | 246 | 1254 | 83.6 | 41.8 |
| Test 2 groups | 1500 | 295 | 1205 | 80.3 | 30.3 |
| Control group | 1500 | 423 | 1077 | 71.8 | - |
As seen from the above table, test group prawn survival rate is significantly larger than control group, and average survival reaches 80% or more, shows
The disease incidence of Chinese prawn is remarkably decreased, and feeding experiment group feed rises on enhancing immunity of prawn and disease resistance ability
Positive effect is arrived；And 1 group of test more excellent compared with 2 groups of results of test, is the Carotenoids Extractss due to testing 1 group of addition
The content astaxanthin of middle 3S, 3'S configuration is higher, and more conducively animal body is absorbed and utilized.
The prior art of routine techniques dawn known to those skilled in the art in above-described embodiment, therefore herein no longer in detail
It repeats.
The above embodiments are only used to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this field
Member can also make a variety of changes and modification without departing from the spirit and scope of the present invention.Therefore, all equivalent
Technical solution also belong to scope of the invention, scope of patent protection of the invention should be defined by the claims.
Claims (10)
1. a kind of preparation method of red phaffia rhodozyma high yield 3S, the 3'S astaxanthin of transgenosis, it is characterised in that: including,
A kind of red phaffia rhodozyma of transgenosis is provided, is integrated in the red phaffia rhodozyma cell of transgenosis containing beta-carotenone
The plasmid of base enzyme and beta carotene hydroxylase expression vector；
A kind of culture medium, the red phaffia rhodozyma of transgenosis described in fermented and cultured are provided；And
3S, the astaxanthin of 3'S configuration are extracted from tunning.
2. a kind of preparation method of red phaffia rhodozyma high yield 3S, the 3'S astaxanthin of transgenosis according to claim 1, special
Sign is: the red phaffia rhodozyma of transgenosis is by that will contain beta carotene ketone group enzyme bkt and beta carotene hydroxylase
The plasmid integration of crtZ expression vector is into host cell, to obtain beta carotene ketone group enzyme bkt and beta carotene hydroxyl
Change enzyme crtZ in the host cell inner expression, and is able to produce 3S, the transgenic yeast of 3'S astaxanthin；The host cell
For red Producing Strain Phaffia rhodozyma beta carotene mutant strain.
3. a kind of preparation method of red phaffia rhodozyma high yield 3S, the 3'S astaxanthin of transgenosis according to claim 2, special
Sign is: the integration is using electroporated；The electric shock condition is 5 ~ 6KV/cm of voltage, electric shock medium be containing 0.08 ~
The STM buffer of 0.12 μ L DMAC N,N' dimethyl acetamide and 0.02 ~ 0.05 μ L ethyl lactate.
4. a kind of preparation method of red phaffia rhodozyma high yield 3S, the 3'S astaxanthin of transgenosis according to claim 2, special
Sign is: the plasmid concentration is that cell is resuspended in 0.65 ~ 0.8 μ g/ μ L；The resuspension cell is with 45 ~ 50mmol/L concentration
Red phaffia rhodozyma cell that dithiothreitol (DTT) is handled, in logarithmic growth phase.
5. a kind of preparation method of red phaffia rhodozyma high yield 3S, the 3'S astaxanthin of transgenosis according to claim 2, special
Sign is: the plasmid containing beta carotene ketone group enzyme bkt and beta carotene hydroxylase crtZ expression vector by with
The preparation of lower section method: expression vector pPRcDNA bkt is connected on pMON carrier by pcr amplification product, is then reused
PCR reaction, and product is connected on pMON carrier, target fragment Pgpd+bkt+Tgpd is obtained, then uses Hind simultaneously
III single endonuclease digestion target fragment Pgpd+bkt+Tgpd and carrier pPRcDNA crtZ, then reaction is attached with T4DNA ligase
To obtain the final product.
6. a kind of preparation method of red phaffia rhodozyma high yield 3S, the 3'S astaxanthin of transgenosis according to claim 5, special
Sign is: the coupled reaction system of the T4DNA ligase are as follows: 10 × T4 buffer, 2 μ L, 17 ddH2O μ L, T4DNA connection
0.5 μ L of enzyme, 0.05 ~ 0.1 μ L of fatty diglycollic amide, 0.03 ~ 0.06 μ L of isobutyl acetate, segment Pgpd+bkt+Tgpd and
4 μ L of carrier pPRcDNA crtZ, distilled water add to 25ul；The Pgpd+bkt+Tgpd segment and carrier pPRcDNA crtZ's
Weight ratio is 1.5 ~ 2.5:1.
7. a kind of preparation method of red phaffia rhodozyma high yield 3S, the 3'S astaxanthin of transgenosis according to claim 1, special
Sign is: beta carotene ketone group enzyme source is in haematococcus pluvialis；The beta carotene hydroxylase derives from Ou Wenshi bacterium.
8. a kind of preparation method of red phaffia rhodozyma high yield 3S, the 3'S astaxanthin of transgenosis according to claim 1, special
Sign is: the total carbon source concentration of culture medium is that 15 ~ 20g/L, wherein glucose and sucrose weight ratio are 3 ~ 4:1；The culture
The total nitrogen source of base is peptone and (NH4)2SO4, the method fermented respectively using single nitrogen source；It further include 4 ~ 6g/ in the culture medium
The malt juice extract of L.
9. a kind of preparation method of red phaffia rhodozyma high yield 3S, the 3'S astaxanthin of transgenosis according to claim 1, special
Sign is: the fermentation culture conditions are as follows: temperature is 20 ~ 22 DEG C, 6 ~ 10L/h of aeration quantity, and intensity of illumination is 500 ~ 800Lux.
10. a kind of such as preparation method of the red phaffia rhodozyma high-yield astaxanthin of the described in any item transgenosis of claim 1 ~ 9 is answered
With, it is characterised in that: the astaxanthin is configured as 3S, 3'S configuration.
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| CN112430637A (en) * | 2019-08-26 | 2021-03-02 | 中国科学院青岛生物能源与过程研究所 | Method for improving yield of phaffia rhodozyma astaxanthin |
| CN114437950A (en) * | 2021-12-24 | 2022-05-06 | 青岛尚德生物技术有限公司 | Phaffia rhodozyma GOY1 for improving aquatic animal body color and preparation method and application of culture of phaffia rhodozyma GOY1 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112430637A (en) * | 2019-08-26 | 2021-03-02 | 中国科学院青岛生物能源与过程研究所 | Method for improving yield of phaffia rhodozyma astaxanthin |
| CN112430637B (en) * | 2019-08-26 | 2022-07-26 | 中国科学院青岛生物能源与过程研究所 | Method for improving yield of phaffia rhodozyma astaxanthin |
| CN114437950A (en) * | 2021-12-24 | 2022-05-06 | 青岛尚德生物技术有限公司 | Phaffia rhodozyma GOY1 for improving aquatic animal body color and preparation method and application of culture of phaffia rhodozyma GOY1 |
| CN114437950B (en) * | 2021-12-24 | 2023-11-24 | 青岛尚德生物技术有限公司 | Phaffia rhodozyma GOY1 for improving body color of aquatic animals and preparation method and application of culture of Phaffia rhodozyma GOY |
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