CN108342408A - A kind of Genetic elements accurately controlling gene rearrangement and its recombinant plasmid and application - Google Patents
A kind of Genetic elements accurately controlling gene rearrangement and its recombinant plasmid and application Download PDFInfo
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Abstract
The present invention relates to biotechnology, a kind of Genetic elements accurately controlling gene rearrangement and its recombinant plasmid and application are specifically disclosed.Genetic elements of the present invention are sequentially spliced by pGAL1 promoters, Cre EBD antigen-4 fusion protein genes and terminator.The present invention collectively constitutes " switch " of gene rearrangement of the control based on SCRaMbLE by pGAL1 promoters, Cre EBD antigen-4 fusion protein genes and terminator, the expression of Cre can be controlled, the opening and closing for accurately controlling gene rearrangement avoid the problem that leakage expression.And it can be applied in the structure of subsequent recombination plasmid and restructuring yeast strains and in the screening of advantage phenotype yeast strain.
Description
Technical field
The present invention relates to biotechnology, more particularly to a kind of Genetic elements accurately controlling gene rearrangement
And its recombinant plasmid and application.
Background technology
In recent years, it as the ability of gene order-checking is constantly promoted, has been able to parse over indiscoverable universal
Existing genome rearrangement phenomenon.The genome mutation principal mode that the research of 20th century is found is single nucleotide polymorphism (SNP)
With heterochromatin polymorphism (HP).However, in recent years the study found that microorganism (such as Yersinia ruckeri, yeast), plant (such as
Green grass, rice), animal (such as lamprey, mouse), in human genome it is commonly found that genome rearrangement phenomenon, especially, base
Because a large amount of structure variations (SV) (including fragment deletion, repetition, lateral transfer, mutual transposition, inversion etc.) are comprising more in group
The higher frequency hereditary variation mode of information change, this pushes life concern to provide key evidence for genome by rearrangement.
There are universal relations between the rearrangement of natural gene group and character change, and study life concern for us provides with new function generation
Important source.
Natural gene group is reset and variation time span is larger, and function orthogenesis controllability is low.Pass through engineer's reality
Existing genome different scale is reset and variation can greatly make up the deficiency that natural gene is reset, and accelerates the discovery of new function.Change
The new approaches that synthetic gene group provides a kind of " from bottom to top, going to understand with creation " are learned, are for life-information understanding, function
It was found that brand-new strong tools.Using similar mechanism, is preset in chemically synthesized yeast chromosome body and " be based on LoxPsym
Point specificity recombination synthesis type genome rearrangement system (SCRaMbLE) ", the Design Fundamentals realize single-gene segment deletion,
It replicates, the genome rearrangement method of transposition.Such as in synthesis type synIXR chromosomes saccharomyces cerevisiae, synthesis type in 2014 in 2011
SynIII chromosomes saccharomyces cerevisiae and synthesis type synV chromosome saccharomyces cerevisiaes in 2017.
Wherein, synIXR chromosomes were synthesized in 2011 and studied works with synthesis synIII chromosomes this two in 2014
In work, researcher carries out the rearrangement work of chemical synthesis genome using pSCW11-Cre-EBD plasmids (pCRE1).
PSCW11 promoters are daughter cell (Daughter cell) specificity promoters, i.e., during yeast gemmation,
PSCW11 promoters are only expressed in daughter cell.Cre-EBD is melting for Cre recombinases and estrogen binding structural domain (EBD)
Hop protein.Theoretically, Cre-EBD missing estradiol (Estradiol) environment in by with the Hsp90 albumen in cytoplasm
In conjunction with and rest in cytoplasm.After estradiol is added in culture environment, the Cre- for generating and activating is combined with EBD structural domains
EBD compounds are disintegrated down from Hsp90 albumen, and enter nucleus under the guiding of Cre native signal peptides, act on loxp
Site, and then generate the rearrangement of chemical synthesis genome.
However in being reported at this two, even if researchers have found to cultivate synthesis in the culture medium for being added without estradiol
Type yeast also has a small amount of bacterial strain and SCRaMbLE occurs, and at the same time cell growth curve is slightly below control group and (is free of
PSCW11-Cre-EBD), show cell growth by minimal effect.Although the sites loxPsym are only inserted into nonessential gene 3 ' and are held
UTR region, but under normal conditions on synthesis type chromosome must gene and nonessential gene be dispersed in whole gene group
In so that the deletion that necessary gene can equally occur in rearrangement process, so as to cause cell death, growth rate declines.Therefore
The generation of comparison cell growth rate indirect proof rearrangement reaction can be passed through.In conclusion it is seen that pSCW11-Cre-EBD
There is leakage expression in plasmid, this is very unfavorable for control gene rearrangement.
Invention content
In view of this, the purpose of the present invention is to provide a kind of Genetic elements accurately controlling gene rearrangement so that described
Genetic elements can accurately control gene rearrangement in SCRaMbLE (gene rearrangement based on the sites LoxPsym), avoid the occurrence of
The problem of leakage expression；
Another object of the present invention is to provide a kind of recombinant plasmid containing said gene element and utilizes institute
State the control method of Genetic elements or its recombinant plasmid to the gene rearrangement based on SCRaMbLE.
Another object of the present invention is that providing a kind of utilization Genetic elements or its construction of recombinant plasmid has
The restructuring yeast strains of artificial control gene rearrangement function, to the screening operation conducive to follow-up bacterial strain；
Another object of the present invention is to provide a kind of utilizes above-mentioned restructuring yeast strains to carry out taking turns iteration genes more
The method of the screening advantage phenotype bacterial strain of rearrangement.
For achieving the above object, the present invention provides the following technical solutions：
A kind of Genetic elements accurately controlling gene rearrangement, by pGAL1 promoters, Cre-EBD antigen-4 fusion protein genes and
Terminator is sequentially spliced.
There are problems that leakage expression, the present invention pass through pGAL1 in controlling gene rearrangement for existing Genetic elements
Promoter, Cre-EBD antigen-4 fusion protein genes and terminator collectively constitute the element of gene rearrangement of the control based on SCRaMbLE,
The expression that Cre can be controlled accurately controls the opening and closing of gene rearrangement, avoids the problem that leakage expression.
In the present invention is embodied, the terminator selection tCYC1 terminators carry out the explanation of example.
To compare different promoters and different control modes to the active controls of Cre, in the specific embodiment of the invention
Build pCRE1 (pSCW11-Cre-EBD), pCRE2 (pZEO1-Cre-EBD), pCRE3 (pGAL1-Cre) and pCRE4 (pGal1-
Cre-EBD, the present invention) four kinds of Genetic elements " switch ", and respectively recombinant plasmid is constructed with pRS413；Above four kinds " are opened
Pass " plasmid and control plasmid pRS413 are converted respectively to No. 5 chromosome saccharomyces cerevisiaes of synthesis type and (are had the synthesis type of SCRaMbLE
Chromosome yeast strain), it is corresponding respectively to obtain recombinant bacterial strain pRS413-synV, pCRE1-synV, pCRE2-synV, pCRE3-
SynV and pCRE4-synV cultivates 32h in dextrose culture-medium, is spaced 2h sample detections OD600 and draws growth curve.Knot
Fruit shows, pCRE4-synV and pRS413-synV shows similar growth conditions, and pCRE1-synV, pCRE2-synV with
PCRE3-synV shows certain growth defect compared to pRS413-synV, this illustrates the control of independent EBD and GAL1
The control of promoter cannot completely inhibit the leakage activity of Cre, and pCRE4 combinations EBD is controlled and the control of GAL1 promoters
The Genetic elements that system is formed can be good at control leakage expression.
Meanwhile also never the expression intensity of isogeneous induction culture medium and Genetic elements aspect demonstrates the present invention to the present invention respectively
The Genetic elements accurately control effect on to gene rearrangement.Therefore, the present invention proposes the Genetic elements and is building
The recombinant plasmid or structure of control gene rearrangement have the application in the restructuring yeast strains of artificial control gene rearrangement function.
Wherein, the gene rearrangement is the gene rearrangement based on SCRaMbLE, that is, is based on LoxPsym locus specificities and recombinates
Synthesis type genome rearrangement system, it is general with SCRaMbLE be mostly synthesis type chromosome yeast strain, especially make
In brewer yeast (plan of saccharomyces cerevisiae synthetic gene group), such as synthesis type synIXR chromosomes saccharomyces cerevisiae in 2011,2014
Synthesis type synIII chromosomes saccharomyces cerevisiae and synthesis type synV chromosome saccharomyces cerevisiaes in 2017；In such yeast, one
The important design principle of item is in 3 ' one site loxPsym of end non-translational regions (UTR) insertion of nonessential gene.On chromosome
Necessary gene and nonessential gene be distributed immediately under normal conditions, such synthesis type chromosome will be numerous
The sites loxPsym interval is divided into different recomposition units, and the sites loxPsym are the full symmetric palindromic sequences of 34bp.In Cre
Under the action of recombinase, the sites any two loxPsym may all interact, and lead to the region meeting between two sites
The random missing and inversion that chromosome segment occurs, while the cell in duplication state can contaminate at random under recombination
Gene rearrangement is realized in the repetition of chromosome fragment.
According to the application of the Genetic elements, the present invention provides a kind of recombinant plasmids of control gene rearrangement, on basis
Contain Genetic elements of the present invention on plasmid.The Basic plasmid can be commercially available using any according to actual needs
It is commercialized plasmid, is such as commercialized plasmid pRS413 (plasmid map is shown in Fig. 1), and is according to the multiple cloning sites on commercialization plasmid
Genetic elements of the present invention are connected on Basic plasmid using digestion mode and form recombinant plasmid.
Similarly, the present invention also provides the recombinant plasmids has the artificial recombination for controlling gene rearrangement function in structure
Application in yeast strain, the gene rearrangement are similarly the gene rearrangement based on SCRaMbLE.
Specifically, a kind of restructuring yeast strains having artificial control gene rearrangement function, have institute of the present invention for conversion
The yeast strain for having SCRaMbLE of recombinant plasmid is stated, the method for transformation is referred to this field convenient technical process.It is logical
Cross build this kind of restructuring yeast strains the superior phenotype of yeast strain can be carried out screening and basic research.Meanwhile more into one
Preferably, which has knocked out YEL013W genes and/or YER042W genes to step, can be carried based on this operation
Its high carotenoid output.
In the specific embodiment of the invention, the present invention provides controls to utilize the weight of Genetic elements of the present invention structure
Group yeast strain gene rearrangement method, i.e., by restructuring yeast strains of the present invention in SGal-aa galas sugar culture-medium, contain
Gene rearrangement is opened in culture in the SC-aa dextrose culture-mediums of estradiol or SGal-aa gala sugar culture-mediums containing estradiol,
After the completion of rearrangement, thalline centrifuges and washes and be changed to conventional medium (SC-aa dextrose culture-mediums) culture, closes gene weight
Row.Wherein, aa in each culture medium indicates amino acid, and SC-aa indicates the culture medium for lacking the aa, lacking in aa according to
Entrained amino acid screening label is corresponding on recombinant plasmid, conveniently filters out the bacterial strain for whether importing properly recombinant plasmid；
By taking His labels as an example, above-mentioned each culture medium is just SGal-His galas sugar culture-medium and SC-His dextrose culture-mediums.
Synthesis type genome rearrangement is induced by opening, and closes induction, Growth and Differentiation and this period of screening and identification into
Capable, increase the type and quantity of rearrangement event really to the genome rearrangement process of the single of diploid, but can not
Ensure to obtain best rearranged result in primary reset.And nature biotechnology evolution is a very long and lasting process.For
Chromosome multiformity is rapidly and continuously generated on a large amount of cell colonys, the present invention screens according to the restructuring yeast strains
The concrete application of advantage phenotype yeast strain provides one kind and taking turns iteration gene rearrangement (Multiplex more on this basis
SCRaMbLE Iterative Cycling) yeast strain screening technique, i.e., screened by selecting each end cycle
Character improvement or the bacterial strain of promotion carry out new genome rearrangement reaction again.It is reacted by this iteration genome rearrangement,
The advantage phenotype of bacterial strain is constantly improved step by step.
In addition, the obtained diploid yeast bacterial strain with advantage phenotype can generate spore by meiosis, will sieve
The spore chosen mates with new synthesis type chromosome yeast, generates new diploid, then carries out more wheel iteration genomes
Rearrangement reaction.The a plurality of synthesis type chromosome of fusion that in this way can be gradually is intracellular at one, and expanding genome rearrangement can act on
Range, completely new target spot can be screened in a wider context.The flow diagram of above-mentioned two method is shown in Fig. 2.
Specifically, according to the first realization method of above-mentioned more wheel iteration genome rearrangements, the screening technique includes：
Step 1, the gene rearrangement for opening restructuring yeast strains of the present invention close gene rearrangement and to completing gene weight
The yeast strain of row is cultivated, and is then screened, is chosen to the yeast strain for completing gene rearrangement according to required phenotype
Has the yeast strain of superior phenotype；
Step 2, the yeast strain for having superior phenotype is opened in the way of step 1 gene rearrangement and continue into
Phenotype more preferably yeast strain is chosen in row screening；
The yeast strain for having superior phenotype described in the phenotype more preferably yeast strain replacement is repeated step by step 3
2。
According to second of realization method of above-mentioned more wheel iteration genome rearrangements, the screening technique includes：
Step 1, the gene rearrangement for opening restructuring yeast strains described in claim 9 close gene rearrangement and to completing base
Because the yeast strain of rearrangement is cultivated, then the yeast strain for completing gene rearrangement is screened according to required phenotype,
Choose the yeast strain for having superior phenotype；
Step 2, by the yeast strain for having superior phenotype by meiosis Haploid production spore, by tearing born of the same parents open
The haplospore (spore for inheriting superior phenotype) for filtering out color burn, by the haplospore of color burn and newly
The haploid yeast strain mating for having SCRaMbLE form new diploid yeast bacterial strain, then in the way of step 1
It opens gene rearrangement and continues to screen, choose phenotype more preferably yeast strain；
The yeast strain for having superior phenotype described in the phenotype more preferably yeast strain replacement is repeated step by step 3
2。
In above two screening technique, the yeast strain for having potential advantages phenotype often is different from setting out in color
Bacterial strain, this can will be provided with the saccharomycete of potential advantages phenotype by convenient visual method according to required phenotype type
Strain is picked out, but when color is deeper, is distinguished by being visually not easy, therefore the present invention is preferably using passing through bacterial strain color
When range estimation screening, by bacterial strain to be screened, the gradually heating culture within the scope of 30-37 DEG C, further screens bacterial strain similar in color.
In the range, as the raising of temperature, the very deep bacterial strain color of numerous colors can shoal, relative different is obvious, can be with
Pick out the relatively deeper bacterium of wherein color.Also, to restore to after bacterial strain optimum temperature, color can also be restored to normal,
Show that the advantage phenotype of bacterial strain will not be had an impact.
Phenotype type of the present invention can be any phenotype type for being conducive to actual production and research in practice, such as
High yield, drug resistance, high temperature resistant etc., and to having the bacterial strain of these advantage phenotypes, there is a set of screening technique of comparative maturity in this field.
By above technical scheme it is found that the present invention passes through pGAL1 promoters, Cre-EBD antigen-4 fusion protein genes and termination
Son collectively constitutes " switch " of gene rearrangement of the control based on SCRaMbLE, can control the expression of Cre, accurately control gene weight
The opening and closing of row avoid the problem that leakage expression.And it can be applied to subsequent recombination plasmid and restructuring yeast strains
In structure and in the screening of advantage phenotype yeast strain.
Description of the drawings
Fig. 1 show pRS413 plasmid maps；
Fig. 2 show the flow diagram of the yeast strain screening technique of more wheel iteration gene rearrangements of the present invention；
Fig. 3 show the structural schematic diagram of Genetic elements and pSCW11-Cre-EBD of the present invention；
Fig. 4 show the plasmid map of recombinant plasmid pCRE4 of the present invention；
Fig. 5 show the plasmid map of recombinant plasmid pCRE1；
Fig. 6 show the synthesis type synIII saccharomyces cerevisiaes and synthesis type synV ferment for converting pCRE1 and pCER4 respectively
Female plated growth result；Wherein, the dilution of tablet from left to right is followed successively by 10-1、10-2、10-3、10-4、10-5With 10-6；
Fig. 7 show the plasmid map of recombinant plasmid pCRE2；
Fig. 8 show the plasmid map of recombinant plasmid pCRE3；
Fig. 9 show the structure of Genetic elements of the present invention, pSCW11-Cre-EBD, pZEO1-Cre-EBD and pGAL1-Cre
Schematic diagram；
Figure 10 show the OD600 growth curves that conversion has the synthesis type synV saccharomyces cerevisiaes of different recombinant plasmids；
Figure 11, which show conversion, has the synthesis type synV saccharomyces cerevisiaes of recombinant plasmid pCRE4 of the present invention to be lured in difference
Lead under culture medium for when column diagram；Wherein, below column diagram table indicate successively dextrose culture-medium, gala sugar culture-medium with
And estradiol；
Figure 12 show the structural schematic diagram of pGAL1-Cre-EBD-GFP and pSCW11-Cre-EBD-GFP；
Figure 13 show the OD600 that conversion respectively has the synthesis type synV saccharomyces cerevisiaes of recombinant plasmid pCRE5 and pCRE6
Fluorescent intensity degree column diagram；Wherein, Initial indicates that original state, Switch on indicate open state, Switch off tables
Show closed state；Cylindricality A indicates that conversion has the fluorescence intensity of the synthesis type synV saccharomyces cerevisiaes of recombinant plasmid pCRE5, cylindricality B
Indicate that conversion has the fluorescence intensity of synthesis type synV saccharomyces cerevisiaes of recombinant plasmid pCRE6；
Figure 14 show the cell that conversion respectively has the synthesis type synV saccharomyces cerevisiaes of recombinant plasmid pCRE5 and pCRE6
GFP images；Wherein, Initial indicates that original state, Switch on indicate that open state, Switch off indicate to close shape
State；CRE5 indicates that conversion has the cell image of the synthesis type synV saccharomyces cerevisiaes of recombinant plasmid pCRE5, CRE6 to indicate that conversion has
The cell image of the synthesis type synV saccharomyces cerevisiaes of recombinant plasmid pCRE6；
Figure 15 show the synthesis type synV saccharomyces cerevisiaes using Genetic elements of the present invention structure after gene rearrangement
Carotenoid (lycopene+beta carotene) yield column diagram of the bacterial strain of screening；
Figure 16 show the 5 generation bacterial strains that the yeast strain screening technique of more wheel iteration gene rearrangements of the present invention filters out
Carotenoid (lycopene+beta carotene) yield column diagram；
Figure 17 show the color result for the bacterial strain cultivated under 30-37 DEG C of different temperatures；Wherein a figures indicate that a yeast turns
Change to be respectively placed at different temperature after being applied to multiple tablets and be cultivated；B figures indicate the yeast colony on a tablet
It reprints to be positioned at different temperature after multiple tablets and be cultivated；
Figure 18 show carotenoid (lycopene+beta carotene) the yield column for the saccharomyces cerevisiae for knocking out different genes
Shape figure；Wherein, table corresponds to the situation that each bacterial strain knocks out gene below column diagram.
Specific implementation mode
The invention discloses a kind of Genetic elements accurately controlling gene rearrangement and its recombinant plasmid and application, this field skills
Art personnel can use for reference present disclosure, be suitably modified technological parameter realization.In particular, it should be pointed out that all similar replacements and
Change apparent to those skilled in the art, they are considered as being included in the present invention.Gene of the present invention
Element and its recombinant plasmid and related application are described by preferred embodiment, and related personnel can obviously not depart from
Genetic elements described herein and its recombinant plasmid and related application are modified in the content of present invention, spirit and scope or suitably
It changes and combines, to realize and apply the technology of the present invention.
According to the technology of the present invention core, the Genetic elements by pGAL1 promoters, Cre-EBD antigen-4 fusion protein genes and
Terminator is sequentially spliced, i.e. pGAL1 promoters+Cre-EBD antigen-4 fusion protein genes+terminator, pGAL1 promoters and Cre-
EBD antigen-4 fusion protein genes have unique corresponding gene order in the art.
In verifying in use, involved gene order and culture medium prescription is as follows for embodiment：
PGAL1 sequences such as SEQ ID NO:Shown in 1, Cre-EBD antigen-4 fusion protein genes sequence is as such as SEQ ID NO:2 institutes
Show, tCYC1 sequences such as SEQ ID NO:Shown in 3, pZEO1 sequences are as such as SEQ ID NO:Shown in 4.
SC-His dextrose culture-mediums：Glucose 20g/L, YNB 6.7g/L, four scarce powder of amino acids 2g/L (- Ura ,-
His ,-Leu ,-Trp), Ura 20mg/L, Trp 20mg/L, Leu 100mg/L, pH are adjusted to 6.0；
SGal-His gala sugar culture-mediums：Galactolipin 20g/L, YNB 6.7g/L, four scarce powder of amino acids 2g/L (- Ura ,-
His ,-Leu ,-Trp), Ura 20mg/L, Trp 20mg/L, Leu 100mg/L, pH are adjusted to 6.0；
SC-His culture mediums：YNB 6.7g/L, four scarce powder of amino acids 2g/L (- Ura ,-His ,-Leu ,-Trp), Ura
20mg/L, Trp 20mg/L, Leu 100mg/L, pH are adjusted to 6.0；
With reference to embodiment, the present invention is further explained.
Embodiment 1：The shadow that Genetic elements of the present invention grow synthesis type yeast chromosomal with pSCW11-Cre-EBD
It rings
According to pGAL1 promoters, the gene order of Cre-EBD antigen-4 fusion protein genes and tCYC1 terminators, sequentially splice
Artificial synthesized Genetic elements of the present invention (schematic diagram is shown in Fig. 3), and be connected on pRS413 plasmids, obtain recombinant plasmid pCRE4
(pGal1-Cre-EBD), plasmid map is shown in Fig. 4；Simultaneously with according to pSCW11 promoters, Cre-EBD antigen-4 fusion protein genes and
The gene order of tCYC1 terminators is sequentially spliced artificial synthesized crt gene element (schematic diagram is shown in Fig. 3), and is connected to
On pRS413 plasmids, recombinant plasmid pCRE1 (pSCW11-Cre-EBD) is obtained, plasmid map is shown in Fig. 5；
It (is to have that pCRE1 and pCER4, which are converted respectively to synthesis type synIII yeast and synthesis type synV yeast,
The synthesis type chromosome saccharomyces cerevisiae of SCRaMbLE), dilute (10 by contact plate-1、10-2、10-3、10-4、10-5、10-6) comparison life
Long situation.As shown in fig. 6, compared with blank control (pRS413 plasmids), (pRS413 plasmids contain in SC-His culture mediums
His labels can screen the bacterial strain for whether being transferred to recombinant plasmid in SC-His culture mediums), it is 10 in diluted concentration-1~
10-2When, it can not observe difference since bacterium colony is intensive；And work as a concentration of 10-4~10-6When, pCRE1-synV and pCRE1-
The clump count of synIII dilution groups is significantly reduced compared to control group, pCRE4-synV and pCRE4-synIII dilutions group ground
Clump count does not have significant difference compared to control group.Experimental result shows that pCRE1 causes synV yeast and synIII yeast to go out
Existing growth defect, and pCRE4 is without result in this phenomenon.
This demonstrate the excessive Cre-EBD expressed due to pCRE1 not with Hsp90 in conjunction with and diffuse into nucleus, and
The transcription control of pGAL1 promoters and the location control of Cre-EBD effectively have adjusted Cre recombinases in pCRE4, demonstrate,prove
Bright pGAL1-Cre-EBD can accurately control the rearrangement of synthesis type genome.
Embodiment 2：Influence of the different genes element to synthesis type yeast chromosomal gene rearrangement
To compare different promoters and different control modes to the active controls of Cre, pCRE1 is built in the present embodiment
(pSCW11-Cre-EBD), pCRE2 (pZEO1-Cre-EBD, plasmid map are shown in Fig. 7), pCRE3 (pGAL1-Cre, plasmid map
See Fig. 8) and four kinds of " switch " plasmids (Fig. 9) of pCRE4 (pGal1-Cre-EBD), wherein pZEO1 is the weak of constitutive expression
Promoter, this allow for pSCW11 it is whether too strong and cause leakage expression carry out comparison setting；The EBD of pCRE3 engages domain
It is removed.Above four kinds of " switch " plasmids and control plasmid pRS413 are converted respectively to synthesis type synV saccharomyces cerevisiaes, and
Recombinant bacterial strain pRS413-synV, pCRE1-synV, pCRE2-synV, pCRE3-synV and pCRE4-synV are trained in glucose
It supports and cultivates 32h in base, be spaced 2h sample detections OD600 and draw growth curve.
As shown in Figure 10, pCRE4-synV and pRS413-synV show similar growth conditions, and pCRE1-synV,
PCRE2-synV and pCRE3-synV shows certain growth defect compared to pRS413-synV, this illustrates independent EBD's
Control and the control of GAL1 promoters cannot completely inhibit the leakage activity of Cre, and pCRE4 combinations EBD is controlled and
The Genetic elements that the control of GAL1 promoters is formed can be good at control leakage expression.
Embodiment 3：Genetic elements of the present invention are under different induction modes to synthesis type yeast chromosomal gene rearrangement
Influence
To assess the performance of Genetic elements of the present invention, pRS413-synV (control) and pCRE4-synV in embodiment 2 are existed
It is cultivated under 4 kinds of different conditions：(1) SC-His dextrose culture-mediums；(2) the SC-His dextrose culture-mediums with 1 μM of estradiol；
(3) SGal-His galas sugar culture-medium；(4) the SGal-His gala sugar culture-mediums of 1 μM of estradiol are added with.Remembered by microplate reader
Record growth curve, and according to formula calculate yeast for when (Doubling Time).
As shown in figure 11, when with SC-His dextrose culture-medium culture cells, between pCRE4-synV and control group
Dai Shiwu significant differences.When with 1 μM of estradiol is added in dextrose culture-medium, the Dai Shiyou of pCRE4-synV compared to the control group
Extended, this explanation pGAL1 promoter in glucose sugar culture-medium also has micro leakage expression.When with SGal-His galactolipins
When medium culture cell, pCRE4-synV significantly extends 8.3% when being compared with a control its generation.This illustrates GAL1 promoters
Expressed after being induced by galactolipin, and generate a large amount of Cre-EBD, wherein excessive part enter nucleus cause genome rearrangement it is anti-
It answers.When galactolipin and estradiol are added in culture medium simultaneously, pCRE4-synV yeast for when significantly extend to 10 hours,
It is 1.5 times of control group.It proves that Cre-EBD is largely transported into nucleus, violent synthetic gene group rearrangement reaction occurs.
Embodiment 4：The expression intensity of Genetic elements and pSCW11-Cre-EBD of the present invention compares
For the expression intensity of the two switches of research pCRE1 and pCRE4, Cre-EBD and GFP green fluorescent protein structures are utilized
Fusion protein is built, and is respectively placed under the control of pSCW11 and pGAL1 promoters, pCRE5 (pSCW11-Cre-EBD- have been obtained
) and pCRE6 (pGAL1-Cre-EBD-GFP-tCYC1) the two plasmids (Figure 12) GFP-tCYC1.
The inductive condition that the two are switched due to pCRE5 and pCRE6 is different, defines respectively here, for
PCRE5, " initial " (Initial) state are to be cultivated 8 hours in SC-His dextrose culture-mediums, " unlatching " (Switch on)
State is to add the SC-His dextrose culture-mediums culture 8 hours of 1 μM of estradiol, and " closings " (Switch off) state is to undergo
After " unlatching " state culture 8 hours, thalline centrifuges and washes after twice that be resuspended in culture 8 in SC-His dextrose culture-mediums small
When.
For pCRE6, " initial " state is to be cultivated in SC-His dextrose culture-mediums, and " unlatching " state is 1 μM of addition
The SGal-His gala sugar culture-mediums of estradiol, "Off" state are after undergoing the culture of " unlatching " state 8 hours, and thalline centrifuges simultaneously
It is resuspended in SC-His dextrose culture-mediums and cultivates 8 hours after washing twice.Measured respectively with microplate reader pCRE5-synV with
Fluorescence intensity/OD600s of the pCRE6-synV in respective " initial " " unlatching " and " closing ".
As shown in figure 13, Cre-EBD-s of the pCRE5-synV under " initial " state " unlatching " state and "Off" state
GFP expression quantity is respectively 10.9,16.3 and 13.4 (a.u.).And pCRE6-synV is in " initial " state, " unlatching " state and " pass
Closing " the Cre-EBD-GFP expression quantity under state is respectively 1.5,99.5 and 1.8 (a.u.).Compared with pCRE5, pCRE6 " is being opened
Open " there is under state higher expression intensity, with lower expression intensity under " initial " state and "Off" state.
As shown in figure 14, while with fluorescence microscope the cell of record pCRE5-synV and pCRE6-synV is shot respectively
GFP fluorescent images.It can be found that pCRE5-synV cells have low-level green fluorescence under three circumstances.pCRE6-synV
Green fluorescence is not observed under " initial " state and "Off" state, has significant green fluorescence under " unlatching " state.
These results indicate that Genetic elements of the present invention have higher ON/OFF ratios compared to pSCW11-Cre-EBD.This may be
Since pSCW11 is only expressed in daughter cell, pGAL1 is equivalent to constitutive promoter in the on state, theoretically all thin
Born of the same parents can express, therefore theoretically pGAL1-Cre-EBD can act on the ratio bigger that yeast cells generates genome rearrangement.
Embodiment 5：It is screened using the restructuring yeast strains for having artificial control gene rearrangement function that the present invention is built excellent
Good phenotype
To produce the synthesis type synV saccharomyces cerevisiaes of carotenoid as starting strain (number yJBH000, by whole
Tri- gene integrations of external source crtE, crtI and crtYB are closed to in synV yeast YEL063C/CAN1 locus, are constructed heterologous
Carotenogenesis approach), screening has the saccharomyces cerevisiae of High Yield of Carotenoid superior phenotype, and method is specific as follows：
The pCRE4 plasmids that previous embodiment is built are transformed into yJBH000 saccharomyces cerevisiaes, bacterium solution is applied to SC-His grapes
On sugared agar, cultivated 72 hours at 30 DEG C.
Single bacterium colony is seeded in 5mL SC-His and is incubated overnight, is washed twice with dd H2O within second day, with containing 1 μM
It is resuspended to OD600=1.0 in 2% galactolipin SGal-His culture mediums of estradiol (Sigma-Aldrich).It is induced at 30 DEG C
The Cre-EBD that bacterial strain makes in cell for 8 hours is expressed, and starts SCRaMbLE progress.
It takes 1mL cultures to centrifuge yeast cells, is washed 2 times with ddH2O, and be resuspended in the training of 1mLSC-His glucose
It supports in base.
The washed bacterium of previous step is adjusted to OD600=1, dilutes 10 with SC-His dextrose culture-mediums-3, 10-4With 10-5After be coated on SC-His dextrose agar plates, and bacterial strain, the carotenoid until observing yeast colony are incubated at 30 DEG C
Pigment has notable difference (72 hours to 120 hours).
Yeast colony is selected from this agar.Scribing line culture is carried out on SC-His agar glucoses, and SC-His is used in combination
Dextrose culture-medium is inoculated in 48 orifice plates, shake culture 48 hours at 30 DEG C.
The bacterial strain more darker than yJBH000 is selected to ferment.Three independent colony inoculations are taken to be cultivated to 5mL YPD
It is cultivated in base 24 hours, then with the YPD culture mediums (40g/L glucose) that OD600 is 0.1 inoculation and 40mL, at 30 DEG C,
Fermented and cultured 60 hours in 250rpm shaking tables.
2mL fermentation culture mediums are collected by centrifugation, 1mL acetone is used in combination to extract carotenoid from precipitation.Sample is analyzed by HPLC
Product.Mobile phase is by methanol-acetonitrile-dichloromethane (9：40：It 1v/v) forms, flow velocity 30mL/min.
It will eventually determine that the bacterial strain of high yield carries out full-length genome deep sequencing (2000 platforms of Illumina HiSeq).It tests
Demonstrate,prove the genome structure variation of carotenoid production.According to genomic sequence data, pass through the missing or mistake table in control cell
Up to the mutant of analysis structure variation.
Finishing screen select five plant heights production haploid strains, yJBH001, yJBH012, yJBH026, yJBH027 and
19.22mg/L, 21.76mg/L, 19.59mg/L, 19.15mg/L is respectively increased in yJBH029, carotenoid output,
19.38mg/L.As shown in figure 15, the carotenoid output of yJBH000 is 12.53mg/L, in contrast, genome rearrangement
The carotenoid output of yJBH001, yJBH012, yJBH026, yJBH027 and yJBH029 after evolution improves 1.53~
1.74 again.
Embodiment 6：The present invention takes turns the yeast strain screening technique of iteration gene rearrangement more
It is still that basic strain construction produces carotenoid with synV saccharomyces cerevisiaes with reference to the screening technique in embodiment 5
Bacterial strain (number yJBD000, by integrating tri- gene integrations of external source crtE, crtI and crtYB to synV yeast
In YEL063C/CAN1 locus, heterologous carotenoid route of synthesis is constructed) first round screening is carried out, obtain high yield class recklessly
The yJBD001 bacterial strains of radish element take turns out bacterium germination using it as second, continue to carry out genome rearrangement simultaneously according to the method for embodiment 5
Superior strain is screened, yJBD038 is obtained.With this recursion, third round obtains yJBD048, and fourth round obtains yJBD057, and the 5th takes turns
To yJBD069.As a result Analysis offermehtations (Figure 16), is compared and yJBD000, yJBD038, yJBD048, yJBD057 and yJBD069
Carotenoid production increases to 12.37mg/L (12.8x), 29.98mg/L (31.1x), 35.83mg/L (37.2x) and
37.39mg/L(38.8x).Productivity has larger growth in the first four period, the last one period only has production slightly
Power increases, this may be the upper limit due to having reached current system, and growth defect caused by high-level carotenoid accumulation is most
Caused by big ability.These results indicate that mostly wheel iteration genome rearrangement is a kind of effective screening technique, can constantly be enriched with
Superiority inheritance character, lasting increase genotypic diversity promote productivity.
Embodiment 7：The evolution bacterial strain of high-temperature cultivation assisting sifting advantage phenotype
A common problem can be encountered when carrying out high flux screening by dithering, i.e., when to be screened
When bacterial strain color is very deep, naked eyes are difficult to repartition out wherein darker bacterial strain.With yJBH000 saccharomyces cerevisiaes or
When yJBD000 saccharomyces cerevisiaes are that starting strain screens the evolution bacterial strain of high carotenoid output, relative to yJBH000 wine brewing ferment
The evolution bacterial strain color of female or yJBD000 saccharomyces cerevisiaes (being light yellow), potential High Yielding Heterosis phenotype shows as red, orange
Red, orange or yellow, color is deeper, and the possibility for representing high yield is bigger.
In existing research, Keaslin is in the work for striking searching target spot in library using carotenoid dithering yeast list
Solve the problems, such as this, selection significantly reduces out using YB/I/BTS1 the color of bacterium germination.But screen the mesh of advantage phenotype bacterial strain
Be exactly persistently exist for advantage phenotype, cannot permanently reduce color by changing gene and screen.Therefore the present invention provides
A kind of method by high-temperature cultivation assisting sifting advantage phenotype yeast strain
As shown in a in Figure 17, four SC-His grapes will be coated onto using the saccharomycete after 5 method genome rearrangement of embodiment
On sugared agar plate, and cultivated 3 days at 30 DEG C, 33 DEG C, 35 DEG C and 37 DEG C respectively；In Figure 13 in b, the same tablet is turned over respectively
It prints on four SC-His dextrose agar plates, is also respectively put into and is cultivated 3 days at 30 DEG C, 33 DEG C, 35 DEG C and 37 DEG C.
It is observed that with the raising of cultivation temperature, colony colour integrally shows downward trend, and class is produced at 37 DEG C
Carrotene yeast is nearly close to white.Show to cultivate the background color that can reduce flora at high temperature, it can be with by naked eyes
Color is not easy the potential advantages phenotype bacterial strain differentiated more deeply before accurate resolution.Saccharomyces cerevisiae optimum growth temperature is 30 DEG C, temperature
It is unfavorable to cell metabolism to spend height, therefore colony colour can shoal when higher than 30 degree of cultures, but this method of the invention is to allow
Yeast cells temporarily reduces yield, when being cultivated again at 30 degree, and can restore normal color.
Embodiment 8：Genetic knock-out experiment
To these three genes of YEL013W in the yJBH000 in embodiment 5, YEL014C (for compareing) and YER042W point
Knockout is not individually knocked out and combined, obtains yJBN009 (knocking out YEL013W genes), yJBN010 (knocks out YEL014W bases
Cause, yJBN006 (knock out YEL014C and YEL013W genes), and yJBN007 (knocking out YER042W genes) and yJBN008 (are knocked out
YEL014C, YEL013W and YER042W gene) five kinds of test strains.Then fermenting and producing as follows：
Activation culture 24 hours in inoculation to 5mL YPD culture mediums are taken, are then 0.1 inoculation and 40mL with OD600
YPD culture mediums (40g/L glucose), fermented and cultured 60 hours in 30 DEG C, 250rpm shaking tables.
2mL fermentation culture mediums are collected by centrifugation, 1mL acetone is used in combination to extract carotenoid from precipitation.Sample is analyzed by HPLC
Product.Mobile phase is by methanol-acetonitrile-dichloromethane (9：40：It 1v/v) forms, flow velocity 30mL/min.
The carotenoid total output of the result is shown in Figure 18, yJBN009, yJBN010, yJBN006, yJBN007 and yJBN008
It is respectively：10.97mg/L, 18.94mg/L, 9.63mg/L, 16.05mg/L, 12.73mg/L and 17.56mg/L.It does not knock out and appoints
The yJBH000 bacterial strains of what gene are compared, and the yield of yJBN009, yJBN006 and yJBN008 are obviously improved, to prove
The deletion of YEL013W target spots can promote the synthesis of carotenoid, in addition it can also be seen that the deletion of YER042W is in high yield class
The ratio of beta carrotene can be improved in carrotene yeast.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>University Of Tianjin
<120>A kind of Genetic elements accurately controlling gene rearrangement and its recombinant plasmid and application
<130> MP1728662
<160> 4
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<213>Artificial sequence (Artificial Sequence)
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cggattagaa gccgccgagc gggcgacagc cctccgacgg aagactctcc tccgtgcgtc 60
ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca ctgctccgaa 120
caataaagat tctacaatac tagcttttat ggttatgaag aggaaaaatt ggcagtaacc 180
tggccccaca aaccttcaaa ttaacgaatc aaattaacaa ccataggatg ataatgcgat 240
tagtttttta gccttatttc tggggtaatt aatcagcgaa gcgatgattt ttgatctatt 300
aacagatata taaatggaaa agctgcataa ccactttaac taatactttc aacattttca 360
gtttgtatta cttcttattc aaatgtcata aaagtatcaa caaaaaattg ttaatatacc 420
tctatacttt aacgtcaagg ag 442
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atgtccaatt tactgaccgt acaccaaaat ttgcctgcat taccggtcga tgcaacgagt 60
gatgaggttc gcaagaacct gatggacatg ttcagggatc gccaggcgtt ttctgagcat 120
acctggaaaa tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac 180
cggaaatggt ttcccgcaga acctgaagat gttcgcgatt atcttctata tcttcaggcg 240
cgcggtctgg cagtaaaaac tatccagcaa catttgggcc agctaaacat gcttcatcgt 300
cggtccgggc tgccacgacc aagtgacagc aatgctgttt cactggttat gcggcggatc 360
cgaaaagaaa acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact 420
gatttcgacc aggttcgttc actcatggaa aatagcgatc gctgccagga tatacgtaat 480
ctggcatttc tggggattgc ttataacacc ctgttacgta tagccgaaat tgccaggatc 540
agggttaaag atatctcacg tactgacggt gggagaatgt taatccatat tggcagaacg 600
aaaacgctgg ttagcaccgc aggtgtagag aaggcactta gcctgggggt aactaaactg 660
gtcgagcgat ggatttccgt ctctggtgta gctgatgatc cgaataacta cctgttttgc 720
cgggtcagaa aaaatggtgt tgccgcgcca tctgccacca gccagctatc aactcgcgcc 780
ctggaaggga tttttgaagc agctcatcga ctgatttacg gcgctgagga tgactctggt 840
cagaggtacc tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg agatatggcc 900
cgcgctggag tttcaatacc ggagatcatg caagctggtg gctggaccaa tgtaaatatt 960
gtcatgaact atatccgtaa cctggatagt gaaacagggg caatggtggt cctgctggaa 1020
gatggcgatc tcgagccatc cgctggagac atgagagctg ccaacctttg gccaagcccg 1080
ctcatgatca aacgctctaa ggagaacagc ctggccttgt ccctgacggc cgaccagatg 1140
gtcagtgcct tgttggatgc tgagcccccc atactctatt ccgagtatga tcctaccaga 1200
cccttcagtg aagcttcgat gatgggctta ctgaccaacc tggcagacag ggagctggtt 1260
cacatgatca actgggcgaa gagggtgcca ggctttgtgg atttgaccct ccatgatcag 1320
gtccaccttc tagaatgtgc ctggctagag atcctgatga ttggtctcgt ctggcgctcc 1380
atggaacacc cggggaagct cctgtttgct cctaacttgc tcctggacag gaatcaaggt 1440
aaatgtgtgg aaggcatggt ggagatcttc gacatgctgc tggttacatc atctcggttc 1500
cgcatgatga atctgcaggg agaggagttt gtgtgcctca aatctattat tttgcttaat 1560
tctggagtat acacatttct gtccagcacc ctgaagtctc tcgaggagaa ggaccatatc 1620
caccgagtcc tggacaagat cacagacact ttgatccacc tgatggccaa ggcaggcctg 1680
accctgcagc agcagcacca gcggctagcc cagctcctcc tcctcctctc ccacatcagg 1740
cacatgagta acgaaggcat ggagcatctg tacagcatga agtgcaagaa cgtggtaccc 1800
ctctatgacc tgctgctgga gatgctagac gcccaccgcc tacatgcgcc cactagtcgt 1860
ggaggggcat ccgtggagga gacggaccaa agccacttgg ccactgcggg ctctacttca 1920
tcgcattcct tgcaaaagta ttacatcacg ggggaggcag agggtttccc tgccacagtc 1980
tga 1983
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aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt 120
agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta 180
cgcatgtaac attatactga 200
<210> 4
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<213>Artificial sequence (Artificial Sequence)
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gttaaacgtg tggtttatgg gtgcaccagg gctttatcgt gttttatatc gatggcgatt 60
tgtgcctcca gtgtattttt gtatatccaa ttaaggtttc ttacctaatt ttatttttat 120
catctttagt taatgctggt ttgctctgtt tctgctgctt tctgtgcggt tctcctcttc 180
tcttgtttct tcgtgttgtc ccccatcgcc gatgggctta tatggcgtat atatatagag 240
cgagttttta cgtcgaagat catctcagtt tgcttgatag cctttctact ttattacttt 300
cgtttttaac ctcattatac tttagttttc tttgatcggt ttttttctct gtatacttaa 360
aagttcaaat caaagaaaca tacaaaacta cgtttatatc aattaata 408
Claims (18)
1. a kind of Genetic elements accurately controlling gene rearrangement, which is characterized in that by pGAL1 promoters, Cre-EBD fusion proteins
Gene and terminator are sequentially spliced.
2. Genetic elements according to claim 1, which is characterized in that the terminator is tCYC1 terminators.
3. Genetic elements described in claims 1 or 2 have artificial control in the recombinant plasmid or structure of structure control gene rearrangement
Application in the restructuring yeast strains of gene rearrangement function.
4. applying according to claim 3, which is characterized in that the gene rearrangement is the gene rearrangement based on SCRaMbLE.
5. a kind of recombinant plasmid of control gene rearrangement, which is characterized in that containing described in claims 1 or 2 on Basic plasmid
Genetic elements.
6. applying according to claim 5, which is characterized in that the Basic plasmid is commercialization plasmid pRS413.
7. the answering in the restructuring yeast strains that structure has artificial control gene rearrangement function of recombinant plasmid described in claim 5
With.
8. applying according to claim 7, which is characterized in that the gene rearrangement is the gene rearrangement based on SCRaMbLE.
9. a kind of restructuring yeast strains having artificial control gene rearrangement function, any one to convert the requirement 6-8 that has the right
The yeast strain for having SCRaMbLE of the item recombinant plasmid.
10. restructuring yeast strains according to claim 9, which is characterized in that knocked out YEL013W genes and/or YER042W
Gene.
11. application of the restructuring yeast strains of claim 9 or 10 in screening superior phenotype yeast strain.
12. a kind of method of the control restructuring yeast strains gene rearrangement of claim 9 or 10, which is characterized in that by right
It is required that 9 or 10 restructuring yeast strains are in SGal-aa galas sugar culture-medium, the SC-aa dextrose culture-mediums containing estradiol
Or gene rearrangement is opened in culture in the SGal-aa gala sugar culture-mediums containing estradiol, after the completion of rearrangement, thalline is centrifuged and is washed
And it is changed to conventional medium culture, close gene rearrangement.
13. a kind of yeast strain screening technique of more wheel iteration gene rearrangements, which is characterized in that including：
Step 1, the gene rearrangement for opening the restructuring yeast strains of claim 9 or 10 close gene rearrangement and to completing base
Because the yeast strain of rearrangement is cultivated, then the yeast strain for completing gene rearrangement is screened according to required phenotype,
Choose the yeast strain for having superior phenotype；
The yeast strain for having superior phenotype in the way of step 1 is opened gene rearrangement and continues to sieve by step 2
Phenotype more preferably yeast strain is chosen in choosing；
The yeast strain for having superior phenotype described in the phenotype more preferably yeast strain replacement is repeated step 2 by step 3.
14. according to screening technique described in claim 13, which is characterized in that screening described in step 1 is to be estimated by bacterial strain color
Screening.
15. according to screening technique described in claim 14, which is characterized in that the range estimation screening further includes that bacterial strain to be screened exists
Gradually heating culture, further screens bacterial strain similar in color within the scope of 30-37 DEG C.
16. a kind of yeast strain screening technique of more wheel iteration gene rearrangements, which is characterized in that including：
Step 1, the gene rearrangement for opening the restructuring yeast strains of claim 9 or 10 close gene rearrangement and to completing base
Because the yeast strain of rearrangement is cultivated, then the yeast strain for completing gene rearrangement is screened according to required phenotype,
Choose the yeast strain for having superior phenotype；
Step 2, by the yeast strain for having superior phenotype by meiosis Haploid production spore, by tearing born of the same parents' screening open
The haplospore for going out color burn, by the haplospore of color burn and the new haploid yeast bacterium for having SCRaMbLE
Strain mating forms new diploid yeast bacterial strain, and gene rearrangement is then opened in the way of step 1 and continues to screen, and selects
Take phenotype more preferably yeast strain；
The yeast strain for having superior phenotype described in the phenotype more preferably yeast strain replacement is repeated step 2 by step 3.
17. according to screening technique described in claim 16, which is characterized in that screening described in step 1 is to be estimated by bacterial strain color
Screening.
18. according to screening technique described in claim 17, which is characterized in that the range estimation screening further includes that bacterial strain to be screened exists
Gradually heating culture, further screens bacterial strain similar in color within the scope of 30-37 DEG C.
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| CN108342408B (en) | 2020-07-14 |
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