CN103571765A - Saccharomyces cerevisiae engineering bacteria with low-yielding ethyl carbamate, and building method and application of saccharomyces cerevisiae engineering bacteria - Google Patents

Abstract

The invention discloses saccharomyces cerevisiae engineering bacteria with low-yielding ethyl carbamate. A regulatory factor G1n3p in saccharomyces cerevisiae is transformed through engineering bacteria. The concrete strategy is that a phosphorylation site on a G1n3p nuclear localization sequence is subjected to mutation to form three phosphorylation sites on the G1n3p nuclear localization sequence; the three phosphorylation sites respectively are the 344th, 347th and 355th serine. In order to obtain a better technical effect, a nuclear localization regulatory region of combining G1n3p with an upstream regulatory factor can be further removed; the G1n3p of a regulatory sequence at the tail end of C is removed by truncated expression, wherein an expression sequence is 1-653. The EC yield of the engineering bacteria disclosed by the invention is reduced by 62% by detection of a yellow rice wine simulation system. In addition, the content of main ingredients is not significantly changed, and the fermentation characteristics and the growth characteristics of a bacterial strain are not affected after the content of other ingredients in a fermentation liquor after fermentation is detected.

Description

A kind of saccharomyces cerevisiae engineered yeast of low yield urethanum and construction process thereof and application

Technical field

The yeast metabolism engineering bacteria and the construction process thereof that the present invention relates to a kind of low yield urethanum, belong to microbiological genetic engineering field.

Background technology

China's traditional zymotic industry is with a long history, has a far reaching influence.Traditional zymotic product complexity is various, broad covered area, and as vinegar, soy sauce, fermented soya bean, fermented bean curd, white wine, yellow rice wine etc. have formed fairly large, and infiltration is in the every aspect of people's dietetic life.Especially enjoy rarity in wine, the yellow rice wine consumption market expanding day of the title of liquid cake, is popular in countries in the world gradually, by country, is classified as one of alcoholic drink of giving priority to and developing.Yet research is found, in fermenting process, the incomplete metabolism meeting of nitrogenous compound produces urethanum (Ethyl carbamate, be called for short EC) etc. amine (ammonia) the class material of harm health of people, had a strong impact on people ' s health, the export trade and the international image of China.

EC affects harmful amine (ammonia) the class material of a class the most widely in leavened food.20th century are early stage, it is found that EC anti-bacteria, plant tissue in vitro, and the growth of the cancer cells of mouse, so it is used as antitumour drug within for some time.But in research subsequently, EC is found to have potential carinogenicity.Result of study shows that the test animals such as mouse, rat, hamster, monkey all can be induced optimum or malignant tumour under EC stimulates.In 2007 Nian， international cancer research institutions, propose to list EC in 2A level carcinogenic substance, with formaldehyde in same rank.EC is only for scientific research at present.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of saccharomyces cerevisiae engineered yeast of low yield urethanum, by genetically engineered, regulatory factor Gln3p in yeast saccharomyces cerevisiae is transformed, specific strategy is that the phosphorylation site on Gln3p nuclear localization sequence is suddenlyd change.

Describedly sport 3 phosphorylation sites on Gln3p nuclear localization sequence, be respectively the Serine of the 344th, 347 and 355.

In order further to reduce EC output, on the basis suddenling change at the phosphorylation site on Gln3p nuclear localization sequence, the position of appraising and deciding that removal Gln3p combines with upstream regulatory factor regulates and controls a region, removes the Gln3p of C-terminal regulating and controlling sequence by shorten expression, and expressed sequence is 1-653.

Obtain the method for above-mentioned Yeast engineering bacteria, it is characterized in that concrete steps are as follows:

1) the GLN3 gene fragment of the removal C-terminal regulating and controlling sequence of clone's acquisition length 1959bp from Yeast genome; Be connected to T vector construction plasmid T-GLN3, and be converted in E.coli JM109 bacterial strain;

2) use sudden change test kit to suddenly change to the phosphorylation site of the GLN3 gene on plasmid T-GLN3 in step 1, the plasmid after sudden change is converted in E.coli JM109 bacterial strain to preservation strain;

3) to step 2) improved T-GLN3 plasmid, be cloned into plasmid pYX212, build plasmid pYX212-GLN3;

4) pYX212-GLN3 plasmid step 3) being obtained, is converted in S.cerevisiae CEN PK2, after bacterium colony PCR checking, and the engineering strain that obtains building.

Saccharomyces cerevisiae engineered yeast after transformation provided by the invention, urea utilizes ability significantly to promote.In yellow rice wine simulated system, to compare with wild strain, in engineering bacterium fermentation liquid, the content of urea and EC has declined respectively 70% and 62%, has realized and has reduced the object that in yellow rice wine, EC forms.

Accompanying drawing explanation

All primers in table 1 the present invention

Contents of Main Components detected result in table 2 genetic engineering bacterium and wild mushroom yellow rice wine simulated system

Fig. 1 increase GLN3 and GLN3 _1-653the electrophoresis detection figure of gene

Fig. 2 Gln3p nuclear localization sequence suddenlys change and appraises and decides a regulating and controlling sequence disappearance affects quantitative PCR detection result to urea metabolic gene

Fig. 3 genetic engineering bacterium and wild mushroom urea metabolic gene quantitative PCR detection result

Fig. 4 genetic engineering bacterium and wild mushroom shake flask fermentation urea utilize situation detected result

EC content detection result in Fig. 5 genetic engineering bacterium and wild mushroom yellow rice wine simulated system

Specific implementation method

Embodiment 1 impact of regulatory factor Gln3p nuclear localization sequence sudden change on bacterial strain urea Metabolic Gene Expression amount

Construction process is as follows:

1) with GLN3-F and GLN3-R (referring to table 1) primer, from the genome of yeast saccharomyces cerevisiae, increase and obtain obtaining the GLN3 gene fragment (Fig. 1) of length 2193bp.The GLN3 gene that pcr amplification is obtained, after glue reclaims purifying, is connected with pMD19-T Vector (Takara), and is converted in E.coli JM109.On amicillin resistance flat board, through bacterium colony PCR, select the positive colony T-GLN3 of conversion, deliver to the raw work sequence verification in Shanghai.By through the correct bacterial strain of sequence verification sequence, ℃ preservation of glycerine pipe-20;

2) after the bacterial strain of previous step preservation is activated, extract T-GLN3 plasmid.Take T-GLN3 plasmid as template, and the mutant primer in table 1 is primer, and according to the MutanBEST point mutation test kit specification sheets operation of precious biotech firm, by 344 on Gln3p, 347 and 355 Ser sites all sport L-Ala.By gained T-GLN3 _{s344A, S347A, S355A}plasmid is converted in E.coli JM109, and delivers to the raw work sequence verification in Shanghai.By through the correct bacterial strain of sequence verification sequence, ℃ preservation of glycerine pipe-20;

3) extract the T-GLN3 building _{s344A, S347A, S355A}with the pYX212 plasmid of preserving, after Nco I and Sac I double digestion, glue reclaims respectively.The GLN3 that recovery is obtained _{s344A, S347A, S355A}fragment is connected with linearizing pYX212 plasmid, and is converted in E.coli JM109.After amplification cultivation, extract the pYX212-GLN3 building _{1-653, S344A, S347A, S355A}plasmid is converted into S.cerevisiae CEN PK2 bacterial strain.Through yeast colony PCR screening, obtain after positive colony ℃ preservation of glycerine pipe-20.

In order to check the effect of Gln3p nuclear localization sequence sudden change, by the method for real-time quantitative PCR, at transcriptional level, detected the changing conditions of yeast saccharomyces cerevisiae urea metabolic gene (DUR1,2 and DUR3) expression amount.Experiment be take wild strain as contrast, and result as shown in Figure 2.Experimental result shows, in the situation that glutamine exists, expressing pYX212-GLN3 _{, S344A, S347A, S355A}in the bacterial strain of plasmid, DUR1,2 and the expression amount of DUR3 improved respectively 2.4 and 1.8 times.Explanation can improve the expression amount of urea metabolism related gene to the sudden change of Gln3p nuclear localization sequence.

Embodiment 2 regulatory factor Gln3p appraise and decide the impact of regulating and controlling sequence disappearance on the metabolism of bacterial strain urea

Construction process is as follows:

1) with GLN3-F ' and GLN3-R ' (referring to table 1) primer, from the genome of yeast saccharomyces cerevisiae, increase and obtain obtaining the GLN3 of length 1959bp _1-653gene fragment (Fig. 1).The GLN3 that pcr amplification is obtained _1-653gene, after glue reclaims purifying, is connected with pMD19-TVector (Takara), and is converted in E.coli JM109.On amicillin resistance flat board, through bacterium colony PCR, select the positive colony T-GLN3 of conversion _1-653, deliver to the raw work sequence verification in Shanghai.By through the correct bacterial strain of sequence verification sequence, ℃ preservation of glycerine pipe-20;

2) extract the T-GLN3 building _1-653with the pYX212 plasmid of preserving, after Nco I and Sac I double digestion, glue reclaims respectively.The GLN3 that recovery is obtained _1-653fragment is connected with linearizing pYX212 plasmid, and is converted in E.coli JM109.After amplification cultivation, extract the pYX212-GLN3 building _1-653plasmid is converted into S.cerevisiae CEN PK2 bacterial strain.Through yeast colony PCR screening, obtain after positive colony ℃ preservation of glycerine pipe-20.

In order to check Gln3p to appraise and decide the effect of regulating and controlling sequence disappearance, by the method for real-time quantitative PCR, at transcriptional level, detected the changing conditions of yeast saccharomyces cerevisiae urea metabolic gene (DUR1,2 and DUR3) expression amount.Experiment be take wild strain as contrast, and result as shown in Figure 2.Experimental result shows, in the situation that glutamine exists, expressing pYX212-GLN3 _1-653in the bacterial strain of plasmid, DUR1,2 and the expression amount of DUR3 improved respectively 3.3 and 2.6 times.Illustrate that the disappearance that Gln3p appraises and decides a regulating and controlling sequence also can improve the expression amount of urea metabolism related gene, and effect comparison Gln3p nuclear localization sequence suddenlys change and also will get well.

The structure of the more excellent low yield EC of embodiment 3 Yeast engineering bacteria

Gln3p nuclear localization sequence is being suddenlyd change and appraising and deciding a regulating and controlling sequence and all obtain on the basis of forward result, the Yeast engineering bacteria by two kinds of methods in conjunction with the more excellent low yield EC of structure.Construction process is as follows:

With the T-GLN3 building in embodiment 2 _1-653plasmid is template, and the mutant primer in table 1 is primer, according to the MutanBEST point mutation test kit specification sheets operation of precious biotech firm, by Gln3p _1-653on 344,347 and 355 Ser sites all sport L-Ala.By gained T-GLN3 _{1-653, S344A, S347A, S355A}plasmid is converted in E.coli JM109, and delivers to the raw work sequence verification in Shanghai.By through the correct bacterial strain of sequence verification sequence, ℃ preservation of glycerine pipe-20.

In order to check Gln3p to appraise and decide the effect of regulating and controlling sequence disappearance, by the method for real-time quantitative PCR, at transcriptional level, detected the changing conditions of yeast saccharomyces cerevisiae urea metabolic gene (DUR1,2 and DUR3) expression amount.Experiment be take wild strain as contrast, and result as shown in Figure 3.Experimental result shows, in the situation that glutamine exists, expressing pYX212-GLN3 _{1-653, S344A, S347A, s355A}in the bacterial strain of plasmid, DUR1,2 and the expression amount of DUR3 improved respectively 4.3 and 3.2 times.The significantly raising of urea Metabolism-Related Genes Expression amount, illustrates and really can remove the inhibition of NCR effect to urea Metabolic Gene Expression to the transformation of Gln3p, has confirmed that this method for the transformation of regulatory factor specificity is feasible.

Embodiment 4 genetic engineering bacteriums and the experiment of wild strain shake flask fermentation

In the shake flask fermentation of 48 hours, further detect the utilize situation of genetic engineering bacterium to urea, result is as shown in Figure 4.Experimental result shows to compare with wild strain through the genetic engineering bacterium of transformation, and urea utilization ratio significantly improves.Expressed pYX212-GLN3 _{1-653, S344A, S347A, S355A}the urea accumulation volume of plasmid bacterial strain has declined 54.5%.Illustrate by the transformation to Gln3p, at DUR1,2 and after the expression amount of DUR3 improves, bacterial strain is also improved to the utilization ratio of urea.Urea in yeast born of the same parents is degraded fast, therefore cannot accumulate.Genetic engineering bacterium and wild mushroom are done to biomass detection, and difference is not remarkable between the two.

Embodiment 5 genetic engineering bacteriums and the fermenting experiment of wild strain in yellow rice wine simulated system

Because the main object of this patent is to utilize the method for metabolic engineering to reduce the content of EC in yellow rice wine, therefore on the basis of quantitative PCR and shake flask fermentation checking, can engineering strain real attenuation in yellow rice wine simulated system that necessary utilization transformation obtains detects the generation of EC, to detect engineering strain, be applied in industrialized production.In the yellow rice wine simulated system of standard, carried out the shake flask fermentation of 25 days, every sampling in 5 days, once detected respectively the EC content in different samples, it is contrast that experiment still be take without the wild strain of transforming, and result as shown in Figure 5.The result that yellow rice wine simulated system detects is comparatively identical with the result of experiment before.Engineering strain EC generation in the fermenting process of yellow rice wine simulated system has been compared and has been declined 62% with the wild strain of not transforming.In addition, detecting the content of other compositions in fermented liquid and find after fermentation ends, all there is not significant change (table 2) in Contents of Main Components.Show that improved bacterial strain not only can make the content of EC in fermented liquid decline to a great extent, and can not impact the fermentation character of bacterial strain.These presentation of results the bacterial strain that obtains of method by metabolic engineering Gln3p have the potentiality of the actual production of being applied to.

Table 1

Table 2

Claims (4)

1. a saccharomyces cerevisiae engineered yeast for low yield urethanum, is characterized in that by genetically engineered, regulatory factor Gln3p in yeast saccharomyces cerevisiae being transformed, and specific strategy is that the phosphorylation site on Gln3p nuclear localization sequence is suddenlyd change.

2. saccharomyces cerevisiae engineered yeast as claimed in claim 1, sports 3 phosphorylation sites on Gln3p nuclear localization sequence described in it is characterized in that, is respectively the Serine of the 344th, 347 and 355.

3. saccharomyces cerevisiae engineered yeast according to claim 2, is characterized in that further removing that Gln3p combines with upstream regulatory factor appraises and decides a regulation and control region, position, removes the Gln3p of C-terminal regulating and controlling sequence by shorten expression, and expressed sequence is 1-653.

4. the method that obtains saccharomyces cerevisiae engineered yeast described in claim 3, is characterized in that concrete steps are as follows:

1) the GLN3 gene fragment of the removal C-terminal regulating and controlling sequence of clone's acquisition length 1959bp from Yeast genome; Be connected to T vector construction plasmid T-GLN3, and be converted in E.coli JM109 bacterial strain;

2) use sudden change test kit to suddenly change to the phosphorylation site of the GLN3 gene on plasmid T-GLN3 in step 1, the plasmid after sudden change is converted in E.coli JM109 bacterial strain to preservation strain;

3) to step 2) improved T-GLN3 plasmid, be cloned into plasmid pYX212, build plasmid pYX212-GLN3;

4) pYX212-GLN3 plasmid step 3) being obtained, is converted in S.cerevisiae CEN PK2, after bacterium colony PCR checking, and the engineering strain that obtains building.

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