CN107641605A - A kind of method of rapid Optimum yeast cells factory - Google Patents
A kind of method of rapid Optimum yeast cells factory Download PDFInfo
- Publication number
- CN107641605A CN107641605A CN201710772087.8A CN201710772087A CN107641605A CN 107641605 A CN107641605 A CN 107641605A CN 201710772087 A CN201710772087 A CN 201710772087A CN 107641605 A CN107641605 A CN 107641605A
- Authority
- CN
- China
- Prior art keywords
- yeast
- gene
- strain
- label
- diploid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to biological technical field, specifically discloses a kind of method of rapid Optimum yeast cells factory.The present invention can be by the characteristics of the forming diploid that mate between utilizing monoploid saccharomyces cerevisiae, and the specific recombinant technique of improved Cre LoxP systems, substantially increase the efficiency of genome rearrangement, based on the yeast strain library reset by the genome of substantial amounts, quickly and easily aimed strain is optimized and filters out superior strain, it is more universal relative to the optimisation technique of some gene in existing transformation metabolic pathway, the inventive method, it is more efficient and cost is lower.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of side of rapid Optimum yeast cells factory
Method.
Background technology
As health is more focused in economic growth and social progress and the further raising of medical level, people,
Safe and efficient especially to perfect in workmanship, quality-high and inexpensive functional nutrient chemicals, the market demand of cosmetics further expand
Greatly.Global resources shortage, environmental pollution background under, traditional chemical industry because of the problems such as energy consumption is big, and pollutant emission is serious,
Increasingly it is not suitable with the requirement of sustainable development.
With the popularization of DNA sequencing technology, the full-length genome information of more and more species is revealed, in addition, transcript profile, egg
The related omics technologies such as white matter group, metabolism group development cause scientists to various complicated metabolic pathways in biological cell with
And fine regulatory mechanism has more deep cognition.Such as the necessary Genetic elements of saccharomyces cerevisiae production beta carotene are
GGPP synthase genes crtE, phytoene synthase gene crtB, Phytoene dehydrogenase gene crtI and kind
Lycopene cyclase gene crtY；The necessary Genetic elements of saccharomyces cerevisiae production lycopene are GGPP synthase genes crtE, eight
Hydrogen lycopene synthase gene crtB and Phytoene dehydrogenase gene crtI；Saccharomyces cerevisiae production violacein
Must Genetic elements be vioA, vioB, vioC, vioD and vioE.
The further investigation of synthetic biology accelerates the exploitation of green non-pollution organic chemicals cell factory.In industrial bacterium
In the improvement of strain, it is already possible to reduce cost by groping the measures such as fermentation condition, optimization cell metabolism path, improve and produce
Amount.
In microbiological industry, although the chemical attack of production is different, the requirement to industrial strain is also different,
Commercial object is all the bacterial strain that relative high yield is obtained under equal working condition.In long term production, to industrial strain
It is little by changing the external environment raising yield rising space merely, it is necessary to through groping to optimal fermentation condition to bacterial strain certainly
Body carries out essence improvement, that is, transforms the gene of bacterial strain.Traditional genetic engineering, often through some base changed in metabolic pathway
The expression of cause, or yield is improved by the optimization of regulatory mechanism, but have that cost is high and the R＆D cycle long, resource consumption
The shortcomings that big.
So that beta carotene produces as an example, beta carotene (C40H56) it is one of carotenoid, it is the precursor of vitamin A,
Also referred to as provitamin A, it is crocus fat-soluble compound.Beta carotene is widely present in plant, algae and fungi, but dynamic
Beta carotene can not be synthesized in thing and human body, it is necessary to absorb from the external world.Beta carotene is to safeguard that health is indispensable
Nutrient, the yctalopia caused by vitamin A deficiency can be prevented；As a kind of high-efficiency antioxidant agent, have anti-aging,
Antitumor, radioresistance and other effects；As food additives, beta carotene plays pigment and nutrition fortifier.In recent years,
With more health cares of beta carotene and the discovery of medicinal function, beta carotene has increasingly obtained the attention of people.
Much more industrial to use the three mould producing beta-carotene by fermentation of spore cloth Laplace, because it has, biomass is relatively large, unit
The characteristics of thalline pigment production is high.But by contrast, the genetic background of S. cervisiae is apparent, growth cycle is shorter and is more easy to
Culture, while the artificial synthesized of genome sequencing and chromosome dyad has been completed, utilize saccharomyces cerevisiae structure metabolic pathway
Beta carotene is produced, more there is researching value and exploitation future.
At present, on the basis of fully being parsed in herxheimer-liked reaction path, the country is had been carried out in wine brewing ferment
Beta carotene route of synthesis is constructed in female bacterium and carries out effective expression, however, disclosing being made wine using restructuring for report so far
Very big gap still be present compared with recombination bacillus coli in the yield of yeast synthesis beta carotene.Before 2013 report
The yield highest for synthesizing beta carotene in saccharomyces cerevisiae only has 6.29mg/gDCW.2013, Reyes et al. was made with hydrogen peroxide
For screening pressure, by microevolution by 3 times of the output increased of beta carotene, reach 18mg/gDCW.At the same time, Zhejiang is big
Learn and 11mg/gDCW (7.41mg/gDCW beta carotenes) is realized in saccharomyces cerevisiae using inducible expression in Hong Wei seminars
Total carotinoid yield；Then, the seminar incites somebody to action by lowering the series of optimum means such as ERG9, and by fermentation optimization
The output increased of total carrotene is to 1156mg/L (20.79mg/gDCW), but the purity of wherein beta carotene is relatively low, only accounts for total
The 30% of yield.It would therefore be highly desirable to a kind of method of the yeast cells of rapid Optimum production of chemicals is developed, so as to obtain height
The recombinant Saccharomyces cerevisiae bacterial strain of a kind of chemicals such as purity, high yield beta carotene, lycopene, violacein.
The content of the invention
In view of this, it is an object of the invention to provide a kind of method of rapid Optimum yeast cells factory so that described
Method can easy, fast and efficiently improve the ferment of a kind of chemicals such as production beta carotene, lycopene, violacein
Female yield.
For achieving the above object, the present invention provides following technical scheme：
A kind of method of rapid Optimum yeast cells factory, including：
Step 1, structure are in 3 ' ends of at least two dispensable genes or at least two complete function elements 3 ' end insertion SEQ
ID NO:The haploid yeast to be mated of nucleotide sequence shown in 1；
Step 2, the haploid yeast haploid yeast to be mated with described in step 1 of production of chemicals for needing to optimize entered
Row mating, forms diploid yeast bacterial strain；
Step 3, the diploid yeast bacterium will be transformed into the plasmid that can express Cre recombinases for screening label
Strain, then cultivated corresponding to the screening label on culture medium, while adding the expression progress that estrogen opens Cre recombinases
Gene rearrangement, the expression that estrogen closes Cre recombinases is removed after the completion of culture, obtain the diploid yeast bacterium of genome recombination
Strain library；
Step 4, by the diploid yeast bacterial strain dilution spread that step 3 is obtained described in step 3 screen label corresponding to
Cultivated on culture medium, compared with diploid yeast bacterial strain described in step 2, pick out that bacterium colony is larger and color distortion obvious two
Then times body yeast strain passes through shake flask fermentation and HPLC determines chemicals yield, yield highest as potential superior strain
Yeast is the yeast after optimization.
Cre/loxP restructuring enzyme system is usually used in gene targeting, and LoxP sites therein derive from P1 bacteriophages, are by two
The 8bp sequences of individual 13bp inverted repeats and midfeather collectively constitute, and 8bp intervening sequence determines LoxP direction,
13bp inverted repeats is the binding domain of Cre enzymes.Restructuring between two LoxP sites of Cre recombinase-mediateds is one dynamic
State, reversible process, when two loxP directions are consistent, the sequence among two loxP can only delete；When two
When loxP is in opposite direction, the sequence among two loxP can only invert, when the loxP between two DNA is reset
Two DNA can be caused to be shifted over.Present invention adjustment LoxP site 8bp intervening sequence ATGTATGC is ATGTACAT, between making
The directionality every sequence elimination, does not have a directive loxP sites (SEQ ID NO:Nucleotide sequence shown in 1), delete, overturn, move
Position, replicate etc. situation need not be confined to before limitation, have probability, can greatly increase reset combination diversity.Borrow
The characteristic is helped, using, by the characteristics of the forming diploid that mate, weight occurs for the genome for obtaining substantial amounts between haploid yeast
The yeast strain library of row, potential Producing Strain is just sifted out as standard using bacterium colony size and color distortion with starting strain contrast
Strain, the superior strain after optimization is then specifically filtered out by shake flask fermentation and HPLC.
In order to increase the diversity for resetting combination, SEQ ID NO:Nucleotide sequence shown in 1 inserts The more the better, as
It is preferred that step 1 holds insertion SEQ ID NO for structure at 3 ' ends of each dispensable gene or each complete function element 3 ':1 institute
Show the haploid yeast to be mated of nucleotide sequence.SEQ ID NO:The insertion of nucleotide sequence shown in 1 can be according to yeast homologous
The mechanism of restructuring, upstream and downstream homologous sequence is set at the sequence both ends, is inserted into predetermined site.For haploid yeast to be mated
For fully synthetic type or the situation of semi-synthetic chromosome, the sequence can be inserted into directly by DNA de novo formation mode pre-
Fixed insertion point.
Screening label mentioned in the present invention, which may be selected from being frequently used in microorganism field screening objective microbe, to be adopted
Screening label, as auxotrophy label because and resistance label.The auxotrophy label is generally amino acid defective labels
Such as URA, LEU and HIS；The resistance label is selected generally from KanMX, NAT and Hyg.
Label culture medium is screened according to corresponding to screening label is settable, such as using URA screening labels, then corresponding screening
Label culture medium be lack URA culture mediums (SC-URA), use KanMX screening label then it is corresponding screening label culture medium for containing
G418 culture medium.
Culture corresponding to label is being screened described in step 3 in the diploid yeast bacterial strain dilution spread for being obtained step 3
Before being cultivated on base, the present invention controls the opening and closing of the expression of Cre recombinases by the addition and removing of estrogen, female
The concentration of hormone is preferably 5mM, cleaning yeast strain cell directly can be resuspended using sterilized water when removing estrogen.In order to enter one
Step ensures that Cre recombinases have stopped expressing before potential Screening of strain with high productivity, i.e., genome is in stable state and (still provides for weight
The genome of row is unfavorable for subsequently screening), the present invention completes and removed after estrogen closes the expression of Cre recombinases to go back in culture
Following operation can be increased：
The diploid yeast bacterial strain of genome rearrangement will be passed through on the culture medium without screening pressure, such as YPD culture mediums
Cellar culture (generally 24h) is carried out, then reprint corresponding screening label culture medium again, collecting can not normal growth
Diploid strains, obtain the diploid yeast strain library of genome recombination.In aforesaid operations, because YPD etc. is without screening pressure
The culture medium of power does not have screening pressure to the plasmid of the frame of expression of enzymes containing Cre, can not be right in diploid yeast breeds fission process
Deng be distributed to that two sons are intracellular, without assigning to the filial generation yeast of plasmid due to no plasmid, growth and breeding can faster,
Its offspring does not contain plasmid yet, and a large amount of yeast without plasmid are had as incubation time extends, in cell colony and are existed.This
Class yeast on corresponding screening label culture medium can not normal growth, therefore can be properly separated by screening label culture medium
Go out to lose the diploid yeast of the plasmid of the expression cassette of recombinase containing Cre.
The haploid yeast of production of chemicals mentioned by the present invention is production beta carotene, lycopene or purple bar
The haploid yeast of rhzomorph.These haploid yeasts can had been built up with the list times for producing these chemicals abilities
Body yeast or the interim built-up monoploid ferment of indispensable gene that production of chemicals is imported using any yeast strain
It is female.Wherein, the haploid yeast of the production beta carotene includes crtE, crtB, crtI and crtY gene or by optimization
CrtE, crtB, crtI and crtY gene afterwards, the haploid yeast for producing lycopene include crtE, crtB and crtI
Gene or crtE, crtB and crtI gene after optimization, produce violacein haploid yeast include vioA,
VioB, vioC, vioD and vioE gene or vioA, vioB, vioC, vioD and vioE gene after optimization.It is above-mentioned
The optimization of gene can be any Optimized Measures for being beneficial to expression carried out according to yeast, such as known inclined according to codon
The optimization that good type is carried out.
Haploid yeast needs to be mated of the present invention mate with the haploid yeast of production of chemicals to be optimized
Type is different, it is ensured that mating can occur and form diploid yeast, be typically chosen the yeast of affiliation relatively and carry out, simultaneously
Also the haploid yeast of fully synthetic type or semi-synthetic chromosome is included, yeast of the present invention may be selected to be saccharomyces cerevisiae.
SynIII and No. synV artificial saccharomyces cerevisiae (No. III and No. V dyeing of saccharomyces cerevisiae is employed in specific embodiment party mode of the present invention
Body is artificial synthesized) the inventive method is described in detail.
The visible bacterial strain of shake flask fermentation of the present invention is carried out according to common fermentation processes, such as is seeded to YPD culture mediums
In, cultivated 2-3 days on 30 DEG C of shaking table.In the specific embodiment of the invention, the present invention can produce beta carotene at one plant
Bacterial strain on the basis of, optimize by the inventive method, obtain the recombinant Saccharomyces cerevisiae that one plant of beta carotene yield lifts 7.6 times
Bacterial strain.
From above technical scheme, the present invention utilizes the spy that can form diploid between monoploid saccharomyces cerevisiae by mating
Point, and the specific recombinant technique of improved Cre-LoxP systems, the efficiency of genome rearrangement is substantially increased, it is huge with quantity
Based on the yeast strain library that big genome is reset, quickly and easily aimed strain is optimized and filters out Producing Strain
Strain, relative to the optimisation technique of some gene in existing transformation metabolic pathway, the inventive method is more universal, and cost is more
It is low, it is more efficient.
Brief description of the drawings
The S. cervisiae that Fig. 1 show production beta carotene to be optimized carries out gained after locus specificity restructuring
The colony colour contrast difference of diploid yeast strain library different genotype；
Fig. 2 show the beta carotene yield comparison column diagram of potential superior strain and starting strain, wherein control is not
By the diploid strains yYW0243 that sets out of genome recombination, remaining is the diploid by genome recombination of different numberings
Bacterial strain.
Embodiment
The invention discloses a kind of method of rapid Optimum yeast cells factory, those skilled in the art can use for reference herein
Content, it is suitably modified technological parameter realization.In particular, all similar replacements and change are to people in the art
It is it will be apparent that they are considered as being included in the present invention for member.The method of the invention has passed through preferred embodiment
Be described, related personnel substantially can not depart from present invention, in spirit and scope to promoter described herein and bacterium
Strain is modified or suitably changed with combining, to realize and using the technology of the present invention.
Starting strain employed in the specific embodiment of the invention is to contain artificial synthesized type III chromosomes respectively
The saccharomyces cerevisiae of the synthesis type chromosome of (being abbreviated as synIII) and artificial synthesized type V chromosomes (being abbreviated as synV).Synthesis
Type chromosome is that SEQ ID NO will be carried by the way of de novo formation:The original dye of insertion of nucleotide sequence scale shown in 1
' end, the mode of de novo formation refers to existing synthesis technique to colour solid functional sequence (i.e. function element) 3, of the present invention
For bacterial strain merely to technical scheme is better described, it is not the technical characteristic for limiting the inventive method, according to
The difference being actually needed, selected yeast strain are also corresponding different.
With reference to embodiment, the present invention is expanded on further.
Embodiment 1：Produce the structure of beta carotene recombinant Saccharomyces cerevisiae bacterial strain
Using the yeast strain for carrying synV chromosomes as starting strain, inquired about first from GeneBank and get difference
Four kinds of genes for being used to synthesize beta carotene in source：GGPP synthase genes crtE, phytoene synthase gene
CrtB, Phytoene dehydrogenase gene crtI and lycopene cyclase gene crtY, meanwhile, for 4 kinds of genes are connected
Stable expression is incorporated into S. cerevisiae chromosomal after connecing, using yeast entogenous packaging strategy.By the above genetic fragment of acquisition
40bp or so homology arm, while gene design and the saccharomyces cerevisiae synV chromosome CAN sites of the left and right sides are designed between any two
Homologous sequence, length about 40bp.Said gene evades by saccharomyces cerevisiae codon optimization and suitably conventional Restriction Enzyme
Enzyme site, obtained after appropriate homology arm by artificial synthesized in addition.Obtained gene is imported into synV wine brewing ferment by converting
In mother, genetic recombination is carried out with integrating, and the phenotype that colony colour is in yellow is obtained by SC plate screenings.Choose phenotype stabilization
Bacterium colony, verified by bacterium colony PCR, carry out the confirmation of gene insertion site, be yYW0245 by correct Strain Designation.
Embodiment 2：Produce beta carotene recombinant Saccharomyces cerevisiae bacterial strain yYW245 optimization
1st, the structure of diploid Wine brewing yeast strain
By saccharomyces cerevisiae yYW0245 (MAT a), the artificial saccharomyces cerevisiae yYW0233 with carrying synIII chromosomes
(MAT α) is hybridized, each end of complete function element 3 ' insertion SEQ ID NO wherein on synIII chromosomes:Core shown in 1
Nucleotide sequence (will carry SEQ ID NO by the way of de novo formation:The original dye of insertion of nucleotide sequence scale shown in 1
Colour solid function element 3 ' end), the structure of diploid Wine brewing yeast strain comprises the following steps that：
Two Yeasts plate streakings activate after, while be inoculated into 5mlYPD (peptone 20g/L, dusty yeast 10g/L,
One glucose monohydrate 22g/L, agar powder 20g/L, pH 6.1) in fluid nutrient medium, after 30 DEG C of culture 4-6h, bacterium solution is scoring to
On YPD flat boards, using yeast micromanipulation instrument, manual picking saccharomyces cerevisiae diploid cell, or tonneau are thin with two primary yeasts
Auxotrophic complementation after born of the same parents' hybridization, after 30 DEG C are cultivated 2-3d, coating SC deficiency Screening of Media goes out positive hybrid cell.
Treat that the cell that microscope is chosen or flat screen is selected grows single bacterium colony at 30 DEG C, the diploid cell is verified with bacterium colony PCR methods
Mating type, if there are two kinds of mating types of MAT a and MAT α simultaneously, and synIII＆synV PCRtag exists, then proves hybridization
Success, pick out phenotype stabilization one plant, is yYW0243 by correct Strain Designation.
2nd, the heterogenous expression of Cre recombinases, establish and open site-specific recombination system
External structure carries the plasmid (Cre/EBD fusion expression plasmids) containing Cre recombinase expression cassettes of His labels,
After being expanded in Escherichia coli TOP10, plasmid is extracted, plasmid is transformed into diploid yeast bacterium yYW0243, passes through SC-His
The training of (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, lacking His kilnitamin powder 2g/L, 2% agar powder)
Support base screening positive transformant, by the line of correct transformant it is point pure after, be inoculated into the test tube of 5ml SC-His fluid nutrient mediums
Be incubated overnight, after be transferred in the new test tube of same medium, set up control group and experimental group, experimental group adds final concentration of
5mM estradiol (is dissolved in absolute ethyl alcohol), and control group only adds the absolute ethyl alcohol of same volume, 30 DEG C of more than shaking table culture 4h, so
Afterwards with sterile ddH2O is cleaned, and experimental group is the diploid yeast strain library of genome recombination after cleaning, and control group is gene
The diploid yeast strain library that group does not recombinate, then by the bacterial strain dilution spread SC-His flat boards in two libraries, is placed in 30 DEG C
2-3d, observation experiment group and control group colonial morphology difference are cultivated in incubator, sees Fig. 1.In Fig. 1, the bacterium colony of control group is yellow
The bacterium colony of color, the bacterium colony of other colors is experimental group bacterium colony.
3rd, the screening of potential superior strain
Comparative experiments group and starting strain yYW0243 (control group) morphological differences, the choosing colony from experimental group bacterium colony
The significantly potential superior strain of larger, color distortion.
Following steps can be also carried out after screening candidate's superior strain：
By inoculation in 5ml liquid YPD, after 30 DEG C of shaking table culture 2d, dilution spread YPD flat boards, single bacterium to be grown
Fall behind, by its photocopy on SC-His flat board, what can not be grown on SC-His flat board but can be grown on YPD is to lose
Fall the bacterial strain of the plasmid containing Cre recombinase expression cassettes.
4th, HPLC quantitatively detects beta carotene yield
The single bacterium colony for the potential superior strain that will be singled out is inoculated into the shaking flask for filling 50ml liquid YPD, 30 DEG C of shaking tables
After cultivating 2-3d, 10ml centrifuge tubes are taken, add 10ml bacterium solutions, 4000g centrifugations 5min, rear washing.80 DEG C of drying of cell after washing
(the 10ml centrifuge tubes for first weighing sky in advance) is weighed after to constant weight, records dry cell weight.2ml centrifuge tubes are taken, add 1ml bacterium solutions,
4000g centrifuges 5min, collects cell, is resuspended with 1ml 3M HCl, ice bath 3min after boiling water bath 3min, rear washing, 4000g centrifugations
3min, supernatant is abandoned, after adding 2ml acetone resuspension cell, vortex oscillation 5min, by acetone extract 4000g centrifugation 3min centrifugations
Afterwards, after using 0.2 μm of membrane filtration, it is added in sample bottle.By the mark product of beta carotene respectively with acetone gradient dilution to 5mg/
L, 10mg/l, 20mg/l, 30mg/l, 40mg/l, 50mg/l.The setting of HPLC location parameters, set wavelength as：450nm (tomatoes
Red pigment), 470nm (beta carotene)；Chromatographic column：C18 posts；Mobile phase：Acetonitrile：Methanol：Dichloromethane=(volume ratio 18：80：
2)；Flow velocity：1ml/min；Column temperature：40℃.
The beta carotene yield of starting strain yYW0243 and potential superior strain is detected, as bacterial strain secondary screening side
Method.HPLC data are handled, concentration-peak area standard curve of beta carotene mark product is made, simulates regression equation,
According to sample peak area, the concentration of sample beta carotene is calculated.Using beta carotene mark product as control, put down by setting three groups
Row measurement experiment, collects the HPLC data of the product, and makees Fig. 2, as a result shows, 3,5,7,9, No. 12 bacterial strains are than control bacterium
Beta carotene yield is high, and 4,6, No. 11 bacterium beta carotene yield are relatively low, using starting strain yYW0243 as control, wherein No. 3 bacterium
Yield is its 7.6 times, and No. 5 bacterium yield are its 5.12 times, and No. 7 bacterium yield are its 1.36 times, and No. 9 bacterium yield are its 1.72 times, 12
Number bacterium yield is its 1.17 times.In summary, using the method for rapid Optimum of the present invention, so that beta carotene produces bacterial strain as an example,
Optimal screening has obtained the yeast strain of two plants of relative high yields.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Sequence table
<110>University Of Tianjin
<120>A kind of method of rapid Optimum yeast cells factory
<130> MP1701571
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 34
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ataacttcgt ataatgtaca ttatacgaag ttat 34
Claims (10)
- A kind of 1. method of rapid Optimum yeast cells factory, it is characterised in that including：Step 1, structure are in 3 ' ends of at least two dispensable genes or at least two complete function elements 3 ' end insertion SEQ ID NO:The haploid yeast to be mated of nucleotide sequence shown in 1；Step 2, the haploid yeast haploid yeast to be mated with described in step 1 of production of chemicals for needing to optimize handed over Match somebody with somebody, form diploid yeast bacterial strain；Step 3, the diploid yeast bacterial strain will be transformed into the plasmid that can express Cre recombinases for screening label, so Cultivated afterwards corresponding to the screening label on culture medium, while adding the expression progress gene that estrogen opens Cre recombinases Reset, the expression that estrogen closes Cre recombinases is removed after the completion of culture, obtain the diploid yeast bacterial strain text of genome recombination Storehouse；Step 4, by the diploid yeast bacterial strain dilution spread that step 3 is obtained described in step 3 screen label corresponding to culture Cultivated on base, compared with diploid yeast bacterial strain described in step 2, pick out that bacterium colony is larger and the obvious diploid of color distortion Then yeast strain determines chemicals yield, yield highest yeast as potential superior strain by shake flask fermentation and HPLC For the yeast after optimization.
- 2. method according to claim 1, it is characterised in that step 1 is structure at the 3 ' ends or every of each dispensable gene The individual end of complete function element 3 ' insertion SEQ ID NO:The haploid yeast to be mated of nucleotide sequence shown in 1.
- 3. method according to claim 1, it is characterised in that the screening label is auxotrophy label or resistance label.
- 4. method according to claim 3, it is characterised in that the auxotrophy label is amino acid defective labels.
- 5. method according to claim 4, it is characterised in that the amino acid defective labels are selected from URA, LEU and HIS.
- 6. method according to claim 3, it is characterised in that the resistance label is selected from KanMX, NAT and Hyg.
- 7. method according to claim 1, it is characterised in that the concentration of the estrogen is 5mM.
- 8. method according to claim 1, it is characterised in that the haploid yeast of the production of chemicals is production β-Hu Luo Bu Su, lycopene or violacein haploid yeast.
- 9. method according to claim 8, it is characterised in that it is described production beta carotene haploid yeast include crtE, CrtB, crtI and crtY gene or crtE, crtB, crtI and crtY gene after optimization, produce lycopene Haploid yeast includes crtE, crtB and crtI gene or crtE, crtB and crtI gene after optimization, and production is purple The haploid yeast of color bacillin include vioA, vioB, vioC, vioD and vioE gene or vioA after optimization, VioB, vioC, vioD and vioE gene.
- 10. method according to claim 1, it is characterised in that the yeast is saccharomyces cerevisiae.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710772087.8A CN107641605A (en) | 2017-08-31 | 2017-08-31 | A kind of method of rapid Optimum yeast cells factory |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710772087.8A CN107641605A (en) | 2017-08-31 | 2017-08-31 | A kind of method of rapid Optimum yeast cells factory |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107641605A true CN107641605A (en) | 2018-01-30 |
Family
ID=61110197
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710772087.8A Pending CN107641605A (en) | 2017-08-31 | 2017-08-31 | A kind of method of rapid Optimum yeast cells factory |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107641605A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913613A (en) * | 2018-07-12 | 2018-11-30 | 天津大学 | A kind of bacterial strain and its application in the yield for improving microbial secondary metabolite |
| CN108913611A (en) * | 2018-07-11 | 2018-11-30 | 天津大学 | Genetic elements and its application in S. cerevisiae chromosomal number, copy number, structure variation |
| CN108913612A (en) * | 2018-07-12 | 2018-11-30 | 天津大学 | A kind of bacterial strain and its application in the yield for improving microbial secondary metabolite |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087407A (en) * | 2015-08-20 | 2015-11-25 | 天津大学 | Saccharomyces cerevisiae engineering strain as well as preparation method, application and fermentation culture method thereof |
| CN105483034A (en) * | 2016-02-23 | 2016-04-13 | 天津大学 | Saccharomyces cerevisiae mating type conversion method |
| CN105602934A (en) * | 2016-02-17 | 2016-05-25 | 天津大学 | Transfer method for saccharomyces cerevisiae chromosomes |
| CN106282224A (en) * | 2015-06-26 | 2017-01-04 | 深圳华大基因研究院 | Method and the eukaryotic cell of artificial chromosome is built in target cell |
-
2017
- 2017-08-31 CN CN201710772087.8A patent/CN107641605A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282224A (en) * | 2015-06-26 | 2017-01-04 | 深圳华大基因研究院 | Method and the eukaryotic cell of artificial chromosome is built in target cell |
| CN105087407A (en) * | 2015-08-20 | 2015-11-25 | 天津大学 | Saccharomyces cerevisiae engineering strain as well as preparation method, application and fermentation culture method thereof |
| CN105602934A (en) * | 2016-02-17 | 2016-05-25 | 天津大学 | Transfer method for saccharomyces cerevisiae chromosomes |
| CN105483034A (en) * | 2016-02-23 | 2016-04-13 | 天津大学 | Saccharomyces cerevisiae mating type conversion method |
Non-Patent Citations (3)
| Title |
|---|
| QIUHUI LIN等: "Robust orthogonal recombination system for versatile genomic elements rearrangement in yeast Saccharomyces Cerevisiae", 《SCIENTIFIC REPORTS》 * |
| RONALD H.HOESS等: "The role of the loxP spacer region in P1 site-specific recombination", 《NUCLEIC ACIDS RESEARCH》 * |
| 谢泽雄等: "酵母基因组工程", 《中国科学: 生命科》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913611A (en) * | 2018-07-11 | 2018-11-30 | 天津大学 | Genetic elements and its application in S. cerevisiae chromosomal number, copy number, structure variation |
| CN108913611B (en) * | 2018-07-11 | 2022-11-11 | 天津大学 | Gene element and application thereof in saccharomyces cerevisiae chromosome number, copy number and structural variation |
| CN108913613A (en) * | 2018-07-12 | 2018-11-30 | 天津大学 | A kind of bacterial strain and its application in the yield for improving microbial secondary metabolite |
| CN108913612A (en) * | 2018-07-12 | 2018-11-30 | 天津大学 | A kind of bacterial strain and its application in the yield for improving microbial secondary metabolite |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104962488B (en) | A kind of restructuring yeast strains and its construction method and application | |
| CN105420134B (en) | A kind of restructuring yeast strains and its construction method and application | |
| CN105087406B (en) | A kind of restructuring yeast strains and its construction method and application | |
| CN106566779B (en) | A kind of restructuring yeast strains and its construction method and application | |
| CN105779319B (en) | A kind of restructuring yeast strains and its construction method and application | |
| Yamada et al. | Gene copy number and polyploidy on products formation in yeast | |
| CN107641605A (en) | A kind of method of rapid Optimum yeast cells factory | |
| CN105039183B (en) | A kind of angstrom moral bacterium FS110 protoplasts and preparation method thereof and method for transformation | |
| Pollmann et al. | Engineering of the carotenoid pathway in Xanthophyllomyces dendrorhous leading to the synthesis of zeaxanthin | |
| CN109971664B (en) | Bacterial strain for high yield of astaxanthin and application thereof | |
| CN102972205A (en) | Liquefying method and use method of solid strains of edible fungi and medicinal fungi | |
| CN109943493B (en) | Mutant strain for realizing diversity of general enzyme catalytic functions and construction method thereof | |
| CN109988722A (en) | A kind of method of recombinant Saccharomyces cerevisiae bacterial strain and its application and production tyrosol and/or rhodioside | |
| den Haan et al. | Heterologous production of cellulose-and starch-degrading hydrolases to expand Saccharomyces cerevisiae substrate utilization: lessons learnt | |
| CN104805027B (en) | One kind restructuring Ye Shi solution fat yeast strains and its construction method and application | |
| CN108373980A (en) | A kind of S. cervisiae, its construction method and its application in fermentation prepares lycopene | |
| Watcharawipas et al. | Red yeasts and their carotenogenic enzymes for microbial carotenoid production | |
| CN106893684A (en) | A kind of restructuring yeast strains and its application | |
| KR100701319B1 (en) | Escherichia coli capable of producing lycopene with enhanced productivity and method for producing lycopene using the same | |
| CN107574128A (en) | A kind of method of external rapid Optimum bacterial strain metabolic pathway | |
| CN106893726B (en) | Promoter and recombinant yeast strain | |
| WO2018233531A1 (en) | Microorganism and use thereof | |
| CN107418968A (en) | A kind of method that astaxanthin is efficiently produced using Nicotiana tabacum plastid | |
| CN106754448A (en) | A kind of restructuring yeast strains and its application | |
| KR101034822B1 (en) | Method for genetic transformation of haematococcus pluvialis, strain and protein formed using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180130 |
|
| RJ01 | Rejection of invention patent application after publication |