CN104232495B - Knock out the transformation saccharomyces cerevisiae C5D P and its function of CIT2 genes - Google Patents
Knock out the transformation saccharomyces cerevisiae C5D P and its function of CIT2 genes Download PDFInfo
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- CN104232495B CN104232495B CN201310224677.9A CN201310224677A CN104232495B CN 104232495 B CN104232495 B CN 104232495B CN 201310224677 A CN201310224677 A CN 201310224677A CN 104232495 B CN104232495 B CN 104232495B
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- cit2
- xylose
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The present invention relates to a kind of xylose utilization rate engineering strain high.Present invention discover that CIT2 genes are related to the negative regulation that xylose is transported in saccharomyces cerevisiae C5D P, by knocking out CIT2 genes in C5D P, the transformation bacterium for obtaining can improve xylose utilization.It is verified by experiments, the transformation bacterium also has growth rate high, the original glucose utilization rate of bacterium of setting out is not influenceed, the advantages of ethanol production is significantly increased than starting strain.
Description
Technical field：
The present invention relates to a kind of xylose utilization rate engineering strain high, and in particular in knockout saccharomyces cerevisiae C5D-P
The engineering strain that CIT2 genes are obtained.
Background technology：
C5D-P is the engineered Saccharonayces yeast strain for being transferred to Piromyces sp.njau1 strain X I genes, by nature
The metabolic pathway of middle xylose introduces saccharomyces cerevisiae and obtains activity expression（Chinese patent application 201110376729.5）.
C5D-P can be while glucose fermentation and xylose.And the strain of most of an industrial strain of S.cerevisiae is unable to fermenting xylose, reason be it from
Body lacks xylose to the metabolic pathway of xylulose.
But the xylose utilization rate of saccharomyces cerevisiae recombinant bacterial strain C5D-P can not still reach industrialization production requirements.Research shows
The transdermal delivery of xylose is one of major obstacle that engineering bacteria utilizes xylose（Wang Y., 2004）, this is because saccharomyces cerevisiae lacks
Few single-minded xylose transport albumen, and GLUT is very low for the binding ability of xylose.
Therefore, the research for its regulation and control xylose utilization mechanism of the transdermal delivery GAP-associated protein GAP of xylose, transformation wine brewing are carried out
Yeast C5D-P, improving its xylose transport capacity has highly important theoretical and application value.
The content of the invention：
It is an object of the invention to pass through to transform saccharomyces cerevisiae C5D-P, its xylose transport capacity is improved, obtain a kind of new
It is capable of the engineering strain of efficient common fermentation glucose and xylose.
The present invention puts it briefly has carried out following work：
1st, it has been investigated that the negative regulation that CIT2 genes are very possible to xylose is transported in C5D-P is related
Inventor to being transferred to the Engineering Yeast bacterial strain C5D-P of Piromyces sp.njau1 strain X I genes, with grape
Sugar and xylose（2:1, w/w）For mixed carbon source is cultivated, the feelings that xylose is started with are finished in chromatogram monitoring glucose consumption
Under condition, gene expression spectrum analysis are carried out by control of recipient bacterium, result retrieval obtains 29 albumen for having notable difference to express, its
Middle transdermal delivery GAP-associated protein GAP CIT2 expression is substantially lowered.Very possible and xylose transport the negative regulation phase of this explanation CIT2 gene
Close.
Close day CIT2 genes and belong to DUP240 families, but study less.On the basis of C5D-P utilizes xylose, by knocking out
The method of CIT2 genes can identify that it utilizes the function of xylose mechanism to saccharomyces cerevisiae.
2nd, by knocking out CIT2 genes in saccharomyces cerevisiae C5D-P, it was confirmed that above-mentioned discovery, it is improved the base of xylose utilization
Because of engineered strain C5D-P-CIT2 Δs
It is specific as follows：
1）With plasmid pUG6 as template, Kan of the two ends comprising loxp sites is cloned with designed 60bp primersrGene,
Its size is 1808bp, and glue reclaim PCR knocks out component fragment, connects carrier T, sequencing；
2）Turn saccharomyces cerevisiae C5D-P with the knockout modularization of CIT2 genes, screening rotates into the bacterial strain of G418 resistance fragments；
3）Change turns pSH65 in G418 resistant strains obtained as above, with YPG fluid nutrient medium induced expression pSH65 matter
Cre gene expressions are by Kan in saccharomyces cerevisiae genome on grainrGene excision, finally gives the engineering bacteria for knocking out CIT2 genes
C5D-P-CIT2Δ。
CIT2 sequences such as SEQ ID NO:Shown in 1；
Knock out the CIT2 Δs sequence such as SEQ ID NO of CIT2 genes:Shown in 2.
By comparing transformation bacterial strain（C5D-P-CIT2Δ）With starting strain（C5D-P）Four aspect below, it was demonstrated that this hair
Bright beneficial effect：
1st, C5D-P-CIT2 Δs compare with C5D-P xylose utilizations
As a result：48h recombinant bacterial strain C5D-P-CIT2 Δ xylose utilization rates reach 37.76%%；And starting strain C5D-P xyloses
Utilization rate only reaches 19.19%.
Conclusion：CIT2 genes are to influence it to utilize one of factor of xylose in saccharomyces cerevisiae, and the knockout of CIT2 genes is favourable
In the raising of its xylose utilization rate.
2nd, C5D-P-CIT2 Δs compare with C5D-P growth rates
As a result：Bacterium C5D-P-CIT2 Δs OD is transformed after culture 48h600Value improves 9.33% than C5D-P
Conclusion：Further prove that transformation bacterium C5D-P-CIT2 Δs can preferably utilize xylose after glucose utilization is finished
As carbon source.
3rd, C5D-P-CIT2 Δs compare with C5D-P glucose utilizations
As a result：C5D-P-CIT2 Δs and C5D-P have equally utilized glucose completely；
Conclusion：Transformation bacterial strain does not disturb the utilization to glucose
4th, C5D-P-CIT2 Δs compare with C5D-P ethanol productions
As a result：The transformation bacterium C5D-P-CIT2 Δ ratios bacterium C5D-P that sets out is significantly increased, and 48h ethanol productions improve
8.91%；
Conclusion：Further prove that transformation bacterium C5D-P-CIT2 Δs can preferably utilize xylose after glucose utilization is finished
As carbon source.
Brief description of the drawings：
Fig. 1 is that C5D-P-CIT2 Δs compare with C5D-P growth rates；Abscissa is incubation time, and ordinate is that thalline is close
Degree OD600Value；
Fig. 2 is that C5D-P-CIT2 Δs compare with C5D-P glucose utilizations；Abscissa is incubation time, and ordinate is grape
Sugared concentration；
Fig. 3 is that C5D-P-CIT2 Δs compare with C5D-P xylose utilizations；Abscissa is incubation time, and ordinate is that xylose is dense
Degree；
Fig. 4 is that C5D-P-CIT2 Δs compare with C5D-P ethanol productions.Abscissa is incubation time, and ordinate is that ethanol is dense
Degree.
Specific embodiment：
The plasmid lifted in following examples, bacterial strain are only intended to be described in further detail the present invention, not to this
The substance of invention is any limitation as.
The experimental methods of molecular biology for illustrating, equal reference are not made in following examples《Molecular Cloning:A Laboratory guide》
（The third edition）J. specific method listed in the book of Pehanorm Brooker one is carried out, or is carried out according to kit and product description.
Test material and reagent
1st, bacterial strain and carrier：Plasmid pUG6, pSH65 are purchased from Invitrogen companies, Wine brewing yeast strain C5D-P（Table
Type is：MatA ura3-52）Preserved by place laboratory.
2nd, enzyme and other biochemical reagents：Ligase is purchased from NEB companies, and other reagents be all domestic as illustrated
Reagent（Can be commercially available from common biochemical Reagent Company）.
3rd, culture medium：
（1）Escherichia coli culture medium LB（1% peptone, 0.5% yeast extract, l%NaCl, pH7.0）.
（2）Yeast culture medium YPD（1% yeast extract, 2% peptone, 2% glucose, flat board add 2% agar）
（3）Selective agar medium SC（0.67%YNB, 2% glucose, flat board add 2% agar）
The knockout of CIT2 genes in the saccharomyces cerevisiae C5D-P of embodiment 1
According to known CIT2 genes and the nucleotide sequence of plasmid pUG6, primer is designed：Design direction is to need amplification
Direction, primer length is 60bp, and annealing temperature is at 60-65 DEG C.And they are named as strike P25354-Pug6F, CIT2 upstream
The upper F of F, CIT2（Upstream specific primer）, strike upper R, CIT2 downstream R of P25354-Pug6R, CIT2（Downstream specific primer）See
Table 1.
The CIT2 gene knockouts of table 1 and checking primer
Obtain knocking out component by PCR（1808bp）, amplification obtain product glue reclaim after send Hua Da gene sequencing, by than
To analysis sequencing sequence, it was demonstrated that the sequence of acquisition is consistent with Theoretical Design primer sequence.
Embodiment 2 knocks out modularization and turns saccharomyces cerevisiae C5D-P
(1) saccharomyces cerevisiae C5D-P is activated, adds 1ml sterile deionized waters to take 100 μ after cleaning twice with sterile deionized water
L is standby；
(2) 50 μ l of salmon essence are put into boiling water that to boil 10min standby；
(3) PEG3350 takes 240 μ l and LiAC36 μ l addition above saccharomyces cerevisiaes with the outstanding mixing of salmon essence and knockout component whirlpool
After be put into 30min in 30 DEG C of water-baths, reenter in 42 DEG C of 45min；
(4) 1mlYPD cultures are added based on 30 DEG C of 120rpm cultures 1h；
(5) take 20 μ l and be applied to 48h or so observations on the YPD flat boards containing G418 antibiotic.
The engineered strain of embodiment more than 3 turns pSH65 and KanrThe excision of gene（Resistance screening mark in knockout component
Removal）
(1) activation rotates into the saccharomyces cerevisiae C5D-P for knocking out component, and 1ml sterilizings are added after cleaning twice with sterile deionized water
It is standby that deionized water takes 100 μ l；
(2) 50 μ l of salmon essence are put into boiling water that to boil 10min standby；
(3) PEG3350 takes 240 μ l and LiAC36 μ l addition above saccharomyces cerevisiaes with the outstanding mixing of salmon essence and knockout component whirlpool
After be put into 30min in 30 DEG C of water-baths, reenter in 42 DEG C of 45min；
(4) 1mlYPD cultures are added based on 30 DEG C of 120rpm cultures 1h；
(5) take 20 μ l and be applied to 48h or so observations on the YPD flat boards containing antibiotic phleomycin.
(6) the bacterial strain YPG medium culture 4-7h of pSH65 will be rotated into, and Cre gene expressions, cut on inducing plasmid pSH65
Except Kan on genomerGene, removes resistance；
(7) passed on YPD culture mediums and lose plasmid pSH65 in saccharomyces cerevisiae, obtain the C5D-P-CIT2 Δs of CIT2 missings.
The measure of the engineered strain Physiology and biochemistry of embodiment 4
1st, experimental subjects
Experimental strain：Missing CIT2 genes and the saccharomyces cerevisiae C5D-P containing pYES-AXI；
Control strain：Saccharomyces cerevisiae C5D-P containing pYES-AXI
2nd, experimental technique
With the mixture of glucose/xylose（The initial concentration of respectively 20.0g/L and respectively 40g/L）It is sole carbon
The weighing apparatus culture growth in source, 30 DEG C of 180rpm shake training 71h, and interval 3h to 8h is sampled once, high performance liquid chromatography（HPLC）Survey
Determine supernatant glucose and xylose content, compare sugar utilization, growth rate and alcohol yied.
Experiment above is repeated 3 times the above.
3rd, result
1）Growth rate
The transformation bacterium C5D-P-CIT2 Δ ratios bacterium C5D-P that sets out improves 9.33% in 48h；
2）Glucose and xylose utilization rate
Cultivated by 48h, the glucose of recombinant bacterial strain C5D-P-CIT2 Δ culture mediums and the grape of control strain culture medium
Sugar has almost been utilized completely；Show that recombinant bacterial strain does not influence the utilization of glucose.
3）Xylose utilization rate
Cultivated by 48h, the xylose utilization rate of recombinant bacterial strain C5D-P-CIT2 Δ culture mediums reaches 37.76%；And compare bacterium
The xylose utilization rate of strain culture medium only reaches 19.19%；
4）Ethanol production
The transformation bacterium C5D-P-CIT2 Δ ratios bacterium C5D-P that sets out improves 8.91% in 48h.
It is all of above the results detailed in accompanying drawing 1~4.
4th, conclusion
1）Transformation bacteria growing speed is high；
2）Transformation bacterium does not influence the original glucose utilization rate of bacterium of setting out；
3）Transformation bacterium xylose utilization rate is significantly increased than starting strain；
4）Transformation bacterium ethanol production is significantly increased than starting strain；
5）Prove present invention discover that saccharomyces cerevisiae in CIT2 genes be influence its utilize one of factor of xylose, CIT2 bases
The knockout of cause is conducive to the raising of its xylose utilization rate.
Claims (3)
1. a kind of engineering strain, is characterized in contain SEQ ID NO in saccharomyces cerevisiae C5D-P:Nucleotide sequence shown in 2；
The SEQ ID NO:The construction method of nucleotide sequence shown in 2 is：Build SEQ ID NO:The clpp gene of sequence shown in 1
Except component, the knockout component is transferred to yeast C5D-P, knocks out SEQ ID NO:The gene of sequence shown in 1, removes resistance mark
Note, finally gives SEQ ID NO:Nucleotide sequence shown in 2.
2. application of the bacterial strain described in claim 1 in glucose fermentation and xylose.
3. application of the bacterial strain described in claim 1 in alcohol production.
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