CN105274133A - Method for reducing saccharomyces cerevisiae urea accumulation by modifying urea metabolism regulation approach - Google Patents
Method for reducing saccharomyces cerevisiae urea accumulation by modifying urea metabolism regulation approach Download PDFInfo
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- CN105274133A CN105274133A CN201510816366.0A CN201510816366A CN105274133A CN 105274133 A CN105274133 A CN 105274133A CN 201510816366 A CN201510816366 A CN 201510816366A CN 105274133 A CN105274133 A CN 105274133A
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- dal81p
- yeast
- urea
- dal82p
- saccharomyces cerevisiae
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Abstract
The invention discloses a method for reducing saccharomyces cerevisiae urea accumulation by modifying a urea metabolism regulation approach and belongs to the field of microbial genetics and molecular biology. According to the method, related activating factors of a urea metabolic pathway are modified, and particularly, on the basis of eliminating ubiquitination regulatory sites of Dal81p, Dal81p<K69A>, <K918A> and Dal82p are subjected to overexpression. After the related activating factors of the saccharomyces cerevisiae urea metabolic pathway are subjected to genetic modification according to the method, urea accumulation during saccharomyces cerevisiae fermentation can be reduced fundamentally, and urea accumulation quantity is reduced by 55.7%.
Description
Technical field
The present invention relates to a kind of method transforming the accumulation of urea metabolism regulatory pathway reduction yeast saccharomyces cerevisiae urea, belong to microorganism hereditary and biology field.
Background technology
Urethanum is considered to a kind of material people being had to potential carinogenicity, and is divided into the carcinogenic compound of 2B level in 1974 by international cancer research institution (IARC).After this, researchist finds that again urethanum also directly can bring out the liver cancer of people, impels IARC, in 2007, the carcinogenic grade of urethanum has been risen to 2A level (at the same level with formaldehyde) by 2B level further.And the urethanum in yellow rice wine, become harm consumer health and the important hidden danger affecting its market competitiveness.
In pure mellow wine and yellow rice wine, the urea produced by yeast saccharomyces cerevisiae metabolism is all the topmost precursor substance of urethanum.During the fermentation, the urea in yeast cell produces primarily of arginine metabolism.The activity of the power of yeast saccharomyces cerevisiae urea metabolism and two specific incitant Dal81p and Dal82p is closely related.
Dal81p and Dal82p is the important incitants of two of urea metabolism in yeast saccharomyces cerevisiae, and the promotor of urea metabolism gene (DUR1,2 and DUR3) has their land.Lack the activation of Dal81p and Dal82p, DUR1,2 and the expression amount of DUR3 will decline significantly.In order to strengthen the urea metabolism ability of bacterial strain, the strategy of Dal81p and Dal82p overexpression can be taked.
Ubiquitination regulation and control are a kind of modes that yeast saccharomyces cerevisiae controls non-preference type Metabolism of nitrogen source in born of the same parents.In preference type nitrogenous source environment, the permease of non-preference type nitrogenous source or incitant by ubiquitin tag, and can be transported to degraded in vacuole.Therefore, by carrying out rite-directed mutagenesis to the ubiquitination site on permease or incitant, the retention time of these albumen in born of the same parents can be extended, improving the metabolic rate of non-preference type nitrogenous source.
Summary of the invention
The present invention, by the transformation urea metabolism approach associated activation factor, reduces urea accumulation in fermentation by saccharomyces cerevisiae process, by this genetic modification, fundamentally can reduce the accumulation of urea in fermentation by saccharomyces cerevisiae process.
First object of the present invention is to provide a kind of method reducing the accumulation of yeast urea, and described method transforms urea metabolism approach regulatory factor Dal81p and Dal82p.
Overexpression regulatory factor Dal82p and the Dal81p eliminating ubiquitination regulatory site while that described transformation being.
In one embodiment of the invention, the ubiquitination regulatory site of described Dal81p refer to its 69th and 918 Methionins (K).
In one embodiment of the invention, the Dal81p eliminating ubiquitination regulatory site described in refers to aminoacid sequence to be that the amino acid mutation of the 69th, 918 of the Dal81p of SEQIDNO.1 becomes L-Ala.
In one embodiment of the invention, the aminoacid sequence of described Dal82p is the sequence shown in SEQIDNO.2.
In one embodiment of the invention, described overexpression, refers to Dal81p
k69A, K918Asuccessively be connected on expression vector pY26-GPD-TEF with Dal82p, construction of expression vector pY26-Dal81p
k69A, K918A-Dal82p, and be transformed in yeast and express.
In one embodiment of the invention, the nucleotide sequence of described expression vector pY26-GPD-TEF is the sequence shown in SEQIDNO.11.
In one embodiment of the invention, described method specifically: be that the amino acid mutation of the 69th, 918 of the Dal81p of SEQIDNO.1 becomes L-Ala to obtain Dal81p by aminoacid sequence
k69A, K918A, then by Dal82pDal81p
k69A, K918Asuccessively be connected on expression vector pY26-GPD-TEF with the Dal82p that aminoacid sequence is SEQIDNO.2, build and obtain recombinant vectors pY26-Dal81p
k69A, K918A-Dal82p, and recombinant vectors is transformed in host yeast expresses.
In one embodiment of the invention, described host yeast is yeast saccharomyces cerevisiae.
In one embodiment of the invention, described host yeast is yeast saccharomyces cerevisiae CEN.PK2-1C (MATa; Ura3-52; Trp1-289; Leu2-3_112; His3 Δ 1; MAL2-8
c; SUC2) monoploid type strain.
Second object of the present invention is to provide the yeast that a kind of urea accumulation ability reduces, described yeast process LAN regulatory factor Dal82p and the Dal81p eliminating ubiquitination site.
In one embodiment of the invention, the construction process of described yeast is: aminoacid sequence is that the amino acid mutation of the 69th, 918 of the Dal81p of SEQIDNO.1 becomes L-Ala to obtain Dal81p by (1)
k69A, K918A; (2) by Dal82pDal81p
k69A, k918Asuccessively be connected on expression vector pY26-GPD-TEF with the Dal82p that aminoacid sequence is SEQIDNO.2, build and obtain recombinant vectors pY26-Dal81p
k69A, K918A-Dal82p; (3) recombinant vectors is transformed in host yeast expresses.
In one embodiment of the invention, described host yeast is yeast saccharomyces cerevisiae, can be yeast saccharomyces cerevisiae CEN.PK2-1C.
The present invention's also application of claimed described yeast in food.
Described urea accumulation reduces, and refers to engineering strain relative to wild strain, the accumulation of urea in preference type nitrogen repression substratum.Preference type nitrogen repression substratum refers to and with the addition of yeast saccharomyces cerevisiae preference type nitrogenous source in YNB substratum.
Usefulness of the present invention: the present invention has carried out genetic modification to the yeast saccharomyces cerevisiae urea metabolism approach associated activation factor, and by the transformation to 2 incitants, urea accumulation volume decreases 55.7% respectively.By this genetic modification to yeast saccharomyces cerevisiae, can the fundamentally accumulation of urea in less fermentation by saccharomyces cerevisiae process.
Accompanying drawing explanation
Fig. 1: Metabolically engineered Dal81p and Dal82p is on the impact of bacterial strain urea utilization power.
Embodiment
The detection method reference KnorstMT of urea, NeubertR, WohlrabW.Analyticalmethodsformeasuringureainpharmaceutic alformulations.JournalofPharmaceuticalandBiomedicalAnaly sis15,1627-1632 (1997).
Materials and methods
The present invention's Wine brewing yeast strain used is CEN.PK2-1C (MATa; Ura3-52; Trp1-289; Leu2-3_112; His3 Δ 1; MAL2-8
c; SUC2) monoploid type strain, other operations are conventional molecular biological operation.
Colibacillary cultivation uses LB substratum, and the activation of yeast saccharomyces cerevisiae uses YPD substratum, and yeast saccharomyces cerevisiae urea utilization power uses preference type nitrogen repression substratum.The formula of these substratum is as follows.
LB substratum: 10gL
-1peptone, 5gL
-1yeast extract, 10gL
-1naCl, pH7.4.During screening E.coliJM109 transformant, in substratum, add penbritin 100 μ gmL
-1;
YPD substratum: 20gL
-1peptone, 10gL
-1yeast extract, 20gL
-1glucose;
Yeast conversion screening culture medium: 1.7gL
-1yNB synthetic medium (without amino acid and ammonium sulfate), 20gL
-1glucose, 10mmolL
-1ammonium sulfate, 40mgL
-1histidine, 40mgL
-1tryptophane, 40mgL
-1leucine;
Preference type nitrogen repression substratum: 1.7gL
-1yNB synthetic medium (without amino acid and ammonium sulfate), 20gL
-1glucose, 10mmolL
-1glutamine, 10mmolL
-1(urea or other single non-preference type nitrogenous sources).When cultivating different auxotroph engineering strains, the necessary amino acid of strain growth or uridylic (40mgL need be added
-1).
Embodiment 1
The elimination in Dal81p ubiquitination site: Methionin (K) site mutation using in Dal81p sequence that aminoacid sequence is SEQIDNO.1 by MutanBESTkit site-directed mutagenesis kit the 69th and the 918th is L-Ala (A), usedly quotes in table 1.
Ubiquitination site rite-directed mutagenesis primer on table 1Dal81p
Variants is connected to cloning vector pMD-19TSimple, transformation of E. coli JM109.Picking positive transformant, through the overexpression for next step that Sanger sequence verification is correct.
Dal81p
k69A, K918Awith the structure of Dal82p expression vector: design primer amplification obtain DAL81
k69A, K918A(forward primer adds SmaI or NotI restriction enzyme digestion sites with aminoacid sequence such as the DAL82 gene of SEQIDNO.2; Reverse primer adds HindIII or BglII restriction enzyme digestion sites) (see table 2).By the mode that enzyme is cut-connected, by DAL81
k69A, k918Abe taken up in order of priority subclone on expression vector pY26-GPD-TEF with DAL82, build recombinant vectors pY26-Dal81p
k69A, k918A-Dal82p.
Table 2 increases DAL81
k69A, K918Awith the primer of DAL82 gene
By recombinant vectors transformed saccharomyces cerevisiae CEN.PK2-1C, picking positive transformant in yeast conversion screening culture medium, for the further research of urea utilization power.
By engineering strain and control strain (bacterial strain without transformation), cultivate after 24h activation through 30 DEG C of shaking tables (200r/min), be seeded in preference type nitrogen repression substratum, under the condition of 30 DEG C of shaking tables (200r/min), cultivate 48h, detect the utilization power of urea in substratum therebetween every 8h.
To Saccharomyces cerevisiae gene engineering bacteria strain CEN.PK2-1C (pY26-Dal81p
k69A, K918A-Dal82p) and control strain under real fermentation condition, urea utilization power detects.In the preference type nitrogen repression substratum that with the addition of urea, have detected the changing conditions of urea content in 48h fermenting process, experimental result as shown in Figure 1.Result shows, the urea content detected after 48h have dropped 55.7% respectively than control strain.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. reduce a method for yeast urea accumulation, it is characterized in that, described method transforms urea metabolism approach regulatory factor Dal81p and Dal82p.
2. method according to claim 1, is characterized in that, overexpression regulatory factor Dal82p and the Dal81p eliminating ubiquitination regulatory site while that described transformation being.
3. method according to claim 2, is characterized in that, described in eliminate ubiquitination regulatory site Dal81p refer to aminoacid sequence to be that the amino acid mutation of the 69th, 918 of the Dal81p of SEQIDNO.1 becomes L-Ala.
4. method according to claim 1, is characterized in that, the aminoacid sequence of described Dal82p is the sequence shown in SEQIDNO.2.
5. method according to claim 1, is characterized in that, described method specifically: be that the amino acid mutation of the 69th, 918 of the Dal81p of SEQIDNO.1 becomes L-Ala to obtain Dal81p by aminoacid sequence
k69A, K918A, then by Dal82pDal81p
k69A, K918Asuccessively be connected on expression vector pY26-GPD-TEF with the Dal82p that aminoacid sequence is SEQIDNO.2, build and obtain recombinant vectors pY26-Dal81p
k69A, K918A-Dal82p, and recombinant vectors is transformed in yeast expresses.
6., according to the arbitrary described method of claim 1-5, it is characterized in that, described yeast is yeast saccharomyces cerevisiae.
7. a yeast for urea accumulation ability reduction, is characterized in that, described yeast process LAN regulatory factor Dal82p and the Dal81p eliminating ubiquitination site.
8. yeast according to claim 7, is characterized in that, the construction process of described yeast is: aminoacid sequence is that the amino acid mutation of the 69th, 918 of the Dal81p of SEQIDNO.1 becomes L-Ala to obtain Dal81p by (1)
k69A, K918A; (2) by Dal82pDal81p
k69A, K918Asuccessively be connected on expression vector pY26-GPD-TEF with the Dal82p that aminoacid sequence is SEQIDNO.2, build and obtain recombinant vectors pY26-Dal81p
k69A, K918A-Dal82p; (3) recombinant vectors is transformed in yeast expresses.
9. the yeast according to claim 7 or 8, is characterized in that, described yeast is yeast saccharomyces cerevisiae.
10. the application of yeast described in claim 7 in food.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107362A (en) * | 2021-11-18 | 2022-03-01 | 江南大学 | Application of chromatin regulation factor Ahc1p in reducing saccharomyces cerevisiae urea accumulation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101550401A (en) * | 2009-01-19 | 2009-10-07 | 中国食品发酵工业研究院 | Method for screening yellow wine yeast of high-yield urea |
| CN103571765A (en) * | 2013-11-05 | 2014-02-12 | 江南大学 | Saccharomyces cerevisiae engineering bacteria with low-yielding ethyl carbamate, and building method and application of saccharomyces cerevisiae engineering bacteria |
-
2015
- 2015-11-20 CN CN201510816366.0A patent/CN105274133A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101550401A (en) * | 2009-01-19 | 2009-10-07 | 中国食品发酵工业研究院 | Method for screening yellow wine yeast of high-yield urea |
| CN103571765A (en) * | 2013-11-05 | 2014-02-12 | 江南大学 | Saccharomyces cerevisiae engineering bacteria with low-yielding ethyl carbamate, and building method and application of saccharomyces cerevisiae engineering bacteria |
Non-Patent Citations (3)
| Title |
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| CHURCHER C等: ""Dal81p [Saccharomyces cerevisiae S288c]"", 《GENBANK DATABASE》 * |
| PHILIPPSEN P等: ""Dal82p [Saccharomyces cerevisiae S288c]"", 《GENBANK DATABASE》 * |
| 赵鑫锐: ""代谢工程改造酿酒酵母降低黄酒中的氨基甲酸乙酯"", 《中国博士学位论文全文数据库（工程科技Ⅰ辑）电子期刊》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107362A (en) * | 2021-11-18 | 2022-03-01 | 江南大学 | Application of chromatin regulation factor Ahc1p in reducing saccharomyces cerevisiae urea accumulation |
| CN114107362B (en) * | 2021-11-18 | 2023-07-25 | 江南大学 | Application of chromatin regulating factor Ahc1p in reducing urea accumulation of saccharomyces cerevisiae |
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