CN105368867B - A method of transformation transport protein Bap2p promotes saccharomyces cerevisiae to utilize branched-chain amino acid - Google Patents
A method of transformation transport protein Bap2p promotes saccharomyces cerevisiae to utilize branched-chain amino acid Download PDFInfo
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- CN105368867B CN105368867B CN201510847226.XA CN201510847226A CN105368867B CN 105368867 B CN105368867 B CN 105368867B CN 201510847226 A CN201510847226 A CN 201510847226A CN 105368867 B CN105368867 B CN 105368867B
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Abstract
The invention discloses a kind of methods that transformation transport protein Bap2p promotes saccharomyces cerevisiae to utilize branched-chain amino acid, belong to microorganism hereditary and molecular biology field.The present invention eliminates the ubiquitination site of Bap2p, to release the regulation of ubiquitination suffered by Bap2p.The ubiquitination regulation that saccharomyces cerevisiae is subject to is weakened, its utilization to branched-chain amino acid is improved.
Description
Technical field
The present invention relates to a kind of methods that transformation transport protein Bap2p promotes saccharomyces cerevisiae to utilize branched-chain amino acid, belong to
Microorganism hereditary and molecular biology field.
Background technique
Transport protein Bap2p is a kind of branched-chain amino acid specificity permease on cell membrane, the amino acid of transhipment
Including leucine (Leu), isoleucine (Ile) and valine (Val), there is very high affinity for Leu.Ubiquitination is egg
One of the major way of white matter posttranslational modification will enter Ubiquitin-Proteasome Pathway by the protein of ubiquitin tag, and go forward side by side one
Step is degraded by the protease in vacuole.Bap2p is regulated and controled by ubiquitination, i.e. the ubiquitination site (lysine) of Bap2p is general
Element is identified and is marked, and is finally degraded by protease.Therefore, the ubiquitination for releasing or mitigating Bap2p, to reduce
It is degraded, it will help its being stabilized on cell membrane simultaneously plays amino acid transport function.
The branched-chain amino acids such as leucine, isoleucine and valine are the non-preference type nitrogen source of saccharomyces cerevisiae, when in culture medium
There are when preference type nitrogen source (glutamine, asparagine, glutamic acid etc.), saccharomyces cerevisiae preferentially utilizes preference type nitrogen source；Only
When preference type nitrogen source exhausts, non-preference type nitrogen source is just started with.This preferential side using preference type nitrogen source of saccharomyces cerevisiae
Formula can be brought many unfavorable as a result, being unfavorable for making full use of nitrogen source such as (1), (2) unwanted metabolic products (such as carbamic acid
Ethyl ester) accumulation etc..Therefore, weaken the ubiquitination regulation that branched-chain amino acid transport protein Bap2p is subject to, it can be in cell
It plays stably on film function, helps to improve the utilization of branched-chain amino acid, to facilitate making full use of for nitrogen source, and not comprehensively
It will cause the accumulation of the noxious products such as urethanes.
Summary of the invention
The problem to be solved in the present invention is to provide a kind of transformation transport protein Bap2p, and saccharomyces cerevisiae to be promoted to utilize branched-amino
The method of acid.
The transformation transport protein Bap2p is the ubiquitination site for eliminating Bap2p, to release suffered by Bap2p
Ubiquitination regulation.
The ubiquitination site of the elimination Bap2p, is that its the 12nd and/or 13 and/or 38 and/or 69 lysine is dashed forward
Become arginine.
In one embodiment of the invention, the nucleotide sequence such as SEQ ID of the transport protein Bap2p is encoded
Shown in NO.1.
In one embodiment of the invention, it is described by lysine mutation be arginine, be different mutational site
Combination.
In one embodiment of the invention, it is described by lysine mutation be arginine, be by the 12nd, 13,38 and
69 lysines all sport arginine, obtain mutant Bap2pK12,13,38,69R。
The branched-chain amino acid refers to leucine, isoleucine and valine.
The present invention has carried out genetic modification to saccharomyces cerevisiae branched-chain amino acid transport protein, weakens its ubiquitination being subject to
Regulation, improves its utilization to branched-chain amino acid.
Detailed description of the invention
The combination of Fig. 1 rite-directed mutagenesis
Fig. 2 Bap2p series mutants branched-chain amino acid utilization power
Specific embodiment
Materials and methods
Wine brewing yeast strain used in following embodiments is S.cerevisiae CEN.PK2-1D- Δ ubi4 (MAT α ura3-
52；trp1-289；leu2-3,112；his3Δ1；Δubi4::LEU2；MAL2-8C；SUC2) monoploid type strain, wine brewing
Yeast CEN.PK2-1D- Δ ubi4 is by saccharomyces cerevisiae CEN.PK2-1D (MAT α ura3-52；trp1-289；leu2-3,112；
his3Δ1；MAL2-8C；SUC2 gene UBI4 transformation) is knocked out, saccharomyces cerevisiae CEN.PK2-1D is purchased from EUROSCARF
(Frankfurt, Germany), other operations are conventional molecular biological operation.
Embodiment 1
YNB fluid nutrient medium: 1.74g/L Yeast Nitrogen Base without Amino Acids and
Ammonium Sulfate, 20g/L D-glucose, 5g/L (NH4)2SO4.The double deficiency culture medium (DM- of leucine, uracil
leu-, ura-): 50 μ g/mL histidines, 50 μ g/mL tryptophans are added in YNB culture medium.Solid medium is the training of corresponding liquid
It supports and 20g/L agar powder is added in base.
Using Saccharomyces Cerevisiae in S .cerevisiae CEN.PK2-1D- Δ ubi4 genomic DNA as template, primer pair is used
BAP2-F/BAP2-R (table 1) amplification obtains gene BAP2 (SEQ ID NO.1).BAP2 is sequenced through picking single colonie and Sanger
After verifying is correct, carrier pUbDetec16 (SEQ ID NO.2) is connected to by restriction enzyme site NotI and SmaI, is obtained
Recombinant expression carrier pUbDetec16-BAP2.Recombinant expression carrier is carried out using primer pair pUbDetec16-ver-F/R (table 1)
Verifying.It selects the correct recombinant plasmid of sequencing and converts S.cerevisiae CEN.PK2-1D- Δ using lithium acetate transformation method
Ubi4 is coated with DM-leu-, ura-Solid medium.In 30 DEG C of incubator culture 3-4d, picking individual colonies carry out bacterium colony PCR verifying
After be inoculated with corresponding fluid nutrient medium.It is to be grown to logarithmic phase transfer in order to follow-up test.
The lithium acetate transformation method of saccharomyces cerevisiae: by S.cerevisiae CEN.PK2-1D- Δ ubi4 in YPD culture medium
It 30 DEG C, after 200rpm overnight incubation, is forwarded in fresh 40mL YPD culture medium, and make final concentration of 106cell·mL-1。
30 DEG C, 200rpm cultivates about 6h, is 1.2-1.5 × 10 to thalli growth to concentration7cell·mL-1(OD600=1.2-1.5).It receives
Collect thallus and collect whole cells in 4 DEG C of centrifugation 5min of 4000rpm, with the sterile water washing cell of the pre-cooling of 1 times of volume.4000rpm
4 DEG C of centrifugation 5min collect cell, with the sterile water washing cell of the pre-cooling of 1/2 volume.4 DEG C of centrifugation 5min of 4000rpm collect thin
Born of the same parents are added 4mL conversion fluid and are uniformly mixed cell and conversion fluid with liquid-transfering gun, are incubated at room temperature 30min.4 DEG C of 4000rpm from
Heart 5min collects cell, with 1mL 1molL-1Sorbierite suspension cell is simultaneously transferred in 1.5mL centrifuge tube.Room temperature 13000rpm
It is centrifuged 30s, abandons supernatant, repeats the step twice.With 100 μ L 1molL-1Sorbierite suspension cell makes final concentration of
1010cell·mL-1.The competent cell and addition 5 μ L (100 μ g) plasmid or linear DNA for taking 40 μ L to prepare, are transferred to after mixing
In the 0.2cm electricity revolving cup being pre-chilled on ice, it is incubated for 5min on ice.Electric revolving cup is put into electroporation, parameter setting 1.5kV, 25 μ
1mL1molL is added after electric shock in F immediately-1Sorbierite.After mixing (gently pressure-vaccum not generate bubble) with liquid-transfering gun,
Obtained electric shock mixture is transferred in 1.5mL centrifuge tube, 30 DEG C of stationary incubation 1h.It takes 0.2mL to be coated with corresponding nutrition to lack
Swaged plate observes result after being placed in 30 DEG C of incubator 3-4d.
1 pUbDetec16-BAP2 expression vector establishment of table and verifying the primer
The rite-directed mutagenesis in ubiquitination site: using the method for rite-directed mutagenesis, corresponding ubiquitination site (lysine) is prominent
Become arginine.Rite-directed mutagenesis is carried out using the method for fast enzyme cutting DpnI digestion template.Firstly, with recombinant plasmid
PUbDetec16-BAP2 is template, expands complete recombinant plasmid using 2 × Super pfu Mix archaeal dna polymerase.PCR is produced
Object adds DpnI in 37 DEG C after column recycles, and reacts 1h, since the plasmid template of thallus itself can be methylated modification, and expands
Plasmid will not then be methylated.Primary template plasmid can be eliminated after digesting by DpnI.By the mixing after endonuclease reaction
For liquid in 80 DEG C of PCR instrument, 5min can Transformed E .coli JM109 after inactivating enzyme.Transformation system coating contains appropriate amounts of ammonia benzyl mould
The LB plate of element, stands overnight culture in 37 DEG C of incubators.Random picking single colonie carries out bacterium colony PCR verifying, verifies primer
Using pUbDetec16-ver-F/R.Continue on for next round mutation through the correct monoclonal of Sanger sequence verification, until complete
It is mutated at quadruple, the site sequence such as Fig. 1 of rite-directed mutagenesis.
2 pUbDetec16 series expression vector rite-directed mutagenesis the primer of table
Eliminate the influence to branched-amino acid metabolic in ubiquitination site: respectively by the CEN.PK2-1D- Δ ubi4 of activation
[pUbDetec16]、CEN.PK2-1D-Δubi4[pUbDetec16-Bap2p]、CEN.PK2-1D-Δubi4
[pUbDetec16-Bap2pK12,13R]、CEN.PK2-1D-Δubi4[pUbDetec16-Bap2pK12,13,38R]、CEN.PK2-1D-
Δubi4[pUbDetec16-Bap2pK12,13,38,69R] inoculation YNB fluid nutrient medium (not adding ammonium salt), and add in the medium
Add each 10mM of Leu, Ile, Val.In 30 DEG C, 200rpm cultivation cycle 48h, respectively in 0h and 48h sample detection branched-chain amino acid
Metabolic condition.
From result (Fig. 2) as can be seen that relative to negative control CEN.PK2-1D- Δ ubi4 [pUbDetec16], engineering
Bacterial strain to the utilization rates of 3 kinds of branched-chain amino acids as the increase of ubiquitination site mutation quantity is continuously increased, and quadruple mutant
CEN.PK2-1D-Δubi4[pUbDetec16-Bap2pK12,13,38,69R] to the utilization rate highest of branched-chain amino acid, it respectively reaches
30.1 ± 1.5% (Val), 41.5 ± 1.8% (Ile) and 30.8 ± 0.8% (Leu), are respectively increased relative to control group
88.1%, 48.2% and 47.4%.Should the result shows that, the transformation in Bap2p ubiquitination site is for improving engineering strain branch
The utilization of chain amino acid achieves significant effect.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (4)
1. a kind of method for promoting saccharomyces cerevisiae to utilize branched-chain amino acid, which is characterized in that be to eliminate saccharomyces cerevisiae transport protein
The ubiquitination site of Bap2p, to release the regulation of ubiquitination suffered by Bap2p；The ubiquitination position of the elimination Bap2p
Point is that the 12nd, 13,38 and 69 lysine of saccharomyces cerevisiae transport protein Bap2p is all sported arginine；
The nucleotide sequence of the transport protein Bap2p is encoded as shown in SEQ ID NO.1；
The saccharomyces cerevisiae is S.cerevisiae CEN.PK2-1D- Δ ubi4 (MAT α ura3-52；trp1-289；leu2-3,
112；his3Δ1；Δubi4::LEU2；MAL2-8C；SUC2)；
The saccharomyces cerevisiae CEN.PK2-1D- Δ ubi4 is by saccharomyces cerevisiae CEN.PK2-1D (MAT α ura3-52；trp1-289；
leu2-3,112；his3Δ1；MAL2-8C；SUC2) knock out what gene UBI4 was obtained.
2. the method according to claim 1, wherein the branched-chain amino acid refers to leucine, different bright ammonia
Acid and valine.
3. a kind of saccharomyces cerevisiae improved using branched-chain amino acid ability, which is characterized in that the transport protein of the saccharomyces cerevisiae
The ubiquitination site of Bap2p is eliminated, to release the regulation of ubiquitination suffered by Bap2p；The ubiquitin of the elimination Bap2p
Change site, is that the 12nd, 13,38 and 69 lysine of saccharomyces cerevisiae transport protein Bap2p is all sported into arginine；
The nucleotide sequence of the transport protein Bap2p is encoded as shown in SEQ ID NO.1；
The saccharomyces cerevisiae is S.cerevisiae CEN.PK2-1D- Δ ubi4 (MAT α ura3-52；trp1-289；leu2-3,
112；his3Δ1；Δubi4::LEU2；MAL2-8C；SUC2)；
The saccharomyces cerevisiae CEN.PK2-1D- Δ ubi4 is by saccharomyces cerevisiae CEN.PK2-1D (MAT α ura3-52；trp1-289；
leu2-3,112；his3Δ1；MAL2-8C；SUC2) knock out what gene UBI4 was obtained.
4. application of the saccharomyces cerevisiae as claimed in claim 3 in rice wine production.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1973039A (en) * | 2004-06-22 | 2007-05-30 | 三得利株式会社 | Stabilized proline transporter |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1973039A (en) * | 2004-06-22 | 2007-05-30 | 三得利株式会社 | Stabilized proline transporter |
Non-Patent Citations (3)
| Title |
|---|
| The N-Terminal Domain of the Yeast Permease Bap2p Plays a Role in Its Degradation;Fumihiko Omura et al.;《Biochemical and Biophysical Research Communications》;20011012;第287卷;摘要，第1045页左栏第1段至第1049页右栏第2段，图1 |
| 氮代谢调控相关蛋白质泛素化修饰筛选;吕永坤 等;《Journal of Food Science and Biotechnology》;20140615;第33卷(第6期);第576-581页 |
| 酿酒酵母转运蛋白Agp1p 泛素化对氮源利用的影响;李应宇 等;《微生物学报》;20150206;第55卷(第5期);第570-578页 |
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