CN101302535B - Construction method of plasmid expressing xylulokinase gene - Google Patents
Construction method of plasmid expressing xylulokinase gene Download PDFInfo
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- CN101302535B CN101302535B CN200810064831XA CN200810064831A CN101302535B CN 101302535 B CN101302535 B CN 101302535B CN 200810064831X A CN200810064831X A CN 200810064831XA CN 200810064831 A CN200810064831 A CN 200810064831A CN 101302535 B CN101302535 B CN 101302535B
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Abstract
The invention discloses a construction method for a plasmid used to express a xylulokinase gene, which relates to the construction method for a plasmid vector. The construction method solves the problems that: the prior saccharomyces cerevisiae altered by metabolic engineering has low xylose utilization rate, and a fermentation byproduct xylitol has high output. The construction method is as follows: a KanR gene, an XKS1 gene, an ADH1 terminator fragment, and a 2.2 kb rDNA fragment in the saccharomyces cerevisiae are cloned; then gene fragments are reclaimed and purified, and then are connected with a pGEM Easy vector; then a plasmid pR, a plasmid pRK and a plasmid pRKT are orderly constructed; finally the 2.2 kb rDNA fragment is added into the plasmid pRKT. The vector constructed by the method can perform high copy integration of the XKS1 gene to a chromosome genome of the saccharomyces cerevisiae, realize the stable overexpression of the XKS1, dredge metabolic flux that the saccharomyces cerevisiae utilizes xylose to produce alcohol, improve the utilization rate of the xylose, and reduce the output of the byproduct xylitol.
Description
Technical field
The present invention relates to a kind of construction process of plasmid vector.
Background technology
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) does not possess the xylose metabolism ability, therefore can't produce ethanol by fermenting plant fiber hydrolyzed solution.Adopt the metabolic engineering means in yeast saccharomyces cerevisiae, artificially to set up the xylose metabolism approach at present---Xylose reductase-xylitol dehydrogenase (XR-XDH) approach or xylose isomerase (XI) approach.But the xylose utilization rate of the yeast saccharomyces cerevisiae behind metabolic engineering is still very low, and fermentation byproduct Xylitol generation height, causes the ethanol yield to be difficult to improve.
Summary of the invention
The objective of the invention is that at present the yeast saccharomyces cerevisiae xylose utilization rate behind metabolic engineering is still very low in order to solve, and the high problem of fermentation byproduct Xylitol generation, and a kind of construction process of expressing the plasmid of xylulokinase gene that provides.
The construction process of expressing the plasmid of xylulokinase gene carries out according to the following steps: one, KanR gene among the cloned plasmids pKT0150; Two, clone's yeast saccharomyces cerevisiae XKS1 gene; Three, ADH1 terminator fragment among the cloned plasmids pKT0150; Four, 2.2kb rDNA fragment in clone's yeast saccharomyces cerevisiae; The gene fragment of five, recovery, purification step one to four preparation, and then under 16 ℃ condition, be connected 10～12h with pGEMT Easy carrier respectively, obtain pGMT-KanR, pGMT-XKS1, pGMT-ADH1T and pGMT-rDNA; Six, making up plasmid pR, is the gene KanR that skeleton is introduced resistance marker with yeast saccharomyces cerevisiae integrative vector p406ADH1; Seven, make up plasmid pRK, the XKS1 gene is added plasmid pR; Eight, make up plasmid pRKT, ADH1 terminator fragment is added plasmid pRK; Nine, 2.2kb rDNA fragment is added plasmid pRKT, promptly obtain expressing the plasmid of xylulokinase gene; Wherein amplification upstream primer sequence is 5 '-TTCCATATGTCTGTTTAGCTTGCCTC-3 ' in the step 1, and the downstream primer sequence is 5 '-CTTGACGTCTATCATCGATGAATTCGA-3 '; Amplification upstream primer sequence is 5 '-CGGACTAGTCTCAATCTTCAGCAAGCGAC-3 ' in the step 2, and the downstream primer sequence is 5 '-CGCGTCGACAACGGGGAACAAAATGATG-3 '; Amplification upstream primer sequence is 5 '-CGCGTCGACATTTGTTACTGCTGCTGGTATT-3 ' in the step 3, and the downstream primer sequence is 5 '-CTCGGCCGCCCTGTTATCCCTAGCGG-3 '; Amplification upstream primer sequence is 5 '-TTCCATATGGGAACCTCTAATCATTCGCT-3 ' in the step 4, and the downstream primer sequence is 5 '-TCTCGGCCGAACGAACGAGACCTTAACCT-3 '.
(xylulokinase XK) can catalysis xylulose phosphate formation X 5P, is one of rate-limiting step of xylose metabolism, is in the node location that the xylose metabolism thing enters phosphopentose pathway (PPP) for xylulokinase.Though yeast saccharomyces cerevisiae self has the metabolic downstream of xylulose enzyme system, but limited xylose metabolism owing to its activity is low and flowed to phosphopentose pathway (PPP), thereby cause the xylose utilization rate of the yeast saccharomyces cerevisiae behind metabolic engineering still lower, fermentation byproduct Xylitol generation height, the ethanol yield is still lower.
Yeast saccharomyces cerevisiae endogenous overexpression xylulokinase gene XKS1 can quicken xylose metabolism stream, improves the xylose utilization rate and the ethanol yield of yeast saccharomyces cerevisiae.The inventive method has made up a kind of multi-copy integration expression vector xylulokinase gene XKS1, that be suitable for the yeast saccharomyces cerevisiae industrial strain that carries, this carrier can be incorporated into the high copy of overexpression xylulokinase gene XKS1 on the chromogene group of yeast saccharomyces cerevisiae, thereby realized the excess stably express of xylulokinase XKS1, having dredged yeast saccharomyces cerevisiae utilizes the xylose metabolism of wood sugar producing and ethanol to flow, improve the utilization ratio of wood sugar, reduced the output of by product Xylitol.The plasmid that the inventive method is made up changes in the yeast saccharomyces cerevisiae, the contrast fermentation test proves that the ethanol yield of the yeast saccharomyces cerevisiae that changes the inventive method structure plasmid over to is than high more than 30% through the ethanol yield of the yeast saccharomyces cerevisiae behind the metabolic engineering, illustrating that plasmid that the inventive method makes up changes over to can eliminate wood sugar behind the yeast saccharomyces cerevisiae and transform restriction on the alcohol metabolism approach, has improved the activity of downstream enzyme system.
Embodiment
Embodiment one: the construction process that present embodiment is expressed the plasmid of xylulokinase gene carries out according to the following steps: one, KanR gene among the cloned plasmids pKT0150; Two, clone's yeast saccharomyces cerevisiae XKS1 gene; Three, ADH1 terminator fragment among the cloned plasmids pKT0150; Four, 2.2kbrDNA fragment in clone's yeast saccharomyces cerevisiae; The gene fragment of five, recovery, purification step one to four preparation, and then under 16 ℃ condition, be connected 10～12h with the pGEMTEasy carrier respectively, obtain pGMT-KanR, pGMT-XKS1, pGMT-ADH1T and pGMT-rDNA; Six, making up plasmid pR, is the gene KanR that skeleton is introduced resistance marker with yeast saccharomyces cerevisiae integrative vector p406ADH1; Seven, make up plasmid pRK, the XKS1 gene is added plasmid pR; Eight, make up plasmid pRKT, ADH1 terminator fragment is added plasmid pRK; Nine, 2.2kb rDNA fragment is added plasmid pRKT, promptly obtain expressing the plasmid of xylulokinase gene; Wherein amplification upstream primer sequence is 5 '-TTC in the step 1
CATATGTCTGTTTAGCTTGCCTC-3 ', the downstream primer sequence is 5 '-CTT
GACGTCTATCATCGATGAATTCGA-3 '; Amplification upstream primer sequence is 5 '-CGG in the step 2
ACTAGTCTCAATCTTCAGCAAGCGAC-3 ', the downstream primer sequence is 5 '-CGC
GTCGACAACGGGGAACAAAATGATG-3 '; Amplification upstream primer sequence is 5 '-CGC in the step 3
GTCGACATTTGTTACTGCTGCTGGTATT-3 ', the downstream primer sequence is 5 '-TCT
CGGCCGCCCTGTTATCCCTAGCGG-3 '; Amplification upstream primer sequence is 5 '-TTC in the step 4
CATATGGGAACCTCTAATCATTCGCT-3 ', the downstream primer sequence is 5 '-TCT
CGGCCGAACGAACGAGACCTTAACCT-3 '.
The DNA glue that the present embodiment step 5 is produced with Shanghai China Shun biotechnology company limited reclaims test kit (Gel Extraction Mini Kit) and reclaims.Four groups of linked systems in the step 5 are 10 μ L, connect the composition of damping fluid, 1 μ LT4DNA ligase enzyme and surplus double distilled water by the gene fragment of 1 μ L purifying, pGMT-easy, 1 μ L, the 10 * T4DNA of 1 μ L.
Connection product pGMT-KanR, pGMT-XKS1, pGMT-ADH1T and the pGMT-rDNA that the present embodiment step 5 obtains can preserve amplification by Transformed E .coli DH5 α.
The recognition site (underlining part) that adds Nde I in the present embodiment step 1 upstream primer, the recognition site (underlining part) of adding AatII in the step 1 downstream primer; The recognition site (underlining part) that adds Spe I in the step 2 upstream primer, the recognition site (underlining part) of adding Sal I in the step 2 downstream primer; The recognition site (underlining part) that adds Sal I in the step 3 upstream primer, the recognition site (underlining part) of adding Eag I in the step 3 downstream primer; The recognition site (underlining part) that adds Nde I in the step 4 upstream primer, the recognition site (underlining part) of adding EagI in the step 4 downstream primer.The rDNA fragment of present embodiment step 4 clone's 2.2kb is used to make up plasmid homologous recombination site, wherein contains plasmid and is used for the linear Hpa I single endonuclease digestion site that transforms.
Present embodiment plasmid pKT0150 is available from Addgene company, and genes of brewing yeast group DNA utilizes the fungal gene group DNA extraction test kit of day clean husky company to obtain.The medicine that uses in the present embodiment, reagent, enzyme, competent cell and plasmid etc. are all buied easily, if no particular requirement then concentration be that product marks concentration.
Present embodiment step 5 to nine is used operational manual referring to test kit.
The plasmid that each step of present embodiment step 5 to nine obtains can be transformed into after the PCR checking and preserve among the E.coliDH5 α and amplification.
Embodiment two: the difference of present embodiment and embodiment one is: the resistance marker in the step 6 is G418 (Geneticin).Other step and parameter are identical with embodiment one.
Embodiment three: the difference of present embodiment and embodiment one is: the PCR reaction system is 10 μ L in the step 1, is that the dNTP of 2.5mmol/L, the upstream primer that 1 μ L concentration is 1pmol/ μ L, downstream primer, the 0.8 μ L concentration that 1 μ L concentration is 1pmol/ μ L are the MgCl of 25mmol/L by 1 μ L, 10 * PCR buffer, 0.8 μ L concentration
2, 2 μ L plasmid pKT0150,0.2 μ L concentration are the Taq enzyme of 5U/mL and the composition of surplus double distilled water; The PCR reaction conditions: pre-94 ℃ of 2min of sex change, 94 ℃ of 1min of sex change, the 45 ℃ of 1min that anneal extend 72 ℃ of 2min, and 72 ℃ of 7min are extended in totally 30 circulations.Other step and parameter are identical with embodiment one.
Do not contain Mg among the 10 * PCR buffer that uses in the present embodiment
2+
Embodiment four: the difference of present embodiment and embodiment one is: the PCR reaction system is 10 μ L in the step 2, is that the dNTP of 2.5mmol/L, the upstream primer that 1 μ L concentration is 1pmol/ μ L, downstream primer, the 0.8 μ L concentration that 1 μ L concentration is 1pmol/ μ L are the MgCl of 25mmol/L by 1 μ L, 10 * PCR buffer, 0.8 μ L concentration
2, 2 μ L genes of brewing yeast group DNA, 0.2 μ L concentration are the Taq enzyme of 5U/mL and the composition of surplus double distilled water; The PCR reaction conditions: pre-94 ℃ of 5min of sex change, 94 ℃ of 1min of sex change, the 55 ℃ of 1min that anneal extend 72 ℃ of 2min, and 72 ℃ of 10min are extended in totally 30 circulations.Other step and parameter are identical with embodiment one.
Do not contain Mg among the 10 * PCR buffer that uses in the present embodiment
2+
Embodiment five: the difference of present embodiment and embodiment one is: the PCR reaction system is 10 μ L in the step 3, is that the dNTP of 2.5mmol/L, the upstream primer that 1 μ L concentration is 1pmol/ μ L, downstream primer, the 0.8 μ L concentration that 1 μ L concentration is 1pmol/ μ L are the MgCl of 25mmol/L by 1 μ L, 10 * PCR buffer, 0.8 μ L concentration
2, 2 μ L plasmid pKT0150,0.2 μ L concentration are the Taq enzyme of 5U/mL and the composition of surplus double distilled water; The PCR reaction conditions: pre-94 ℃ of 2min of sex change, 94 ℃ of 1min of sex change, the 50 ℃ of 1min that anneal extend 72 ℃ of 2min, and 72 ℃ of 7min are extended in totally 30 circulations.Other step and parameter are identical with embodiment one.
Do not contain Mg among the 10 * PCR buffer that uses in the present embodiment
2+
Embodiment six: the difference of present embodiment and embodiment one is: the PCR reaction system is 10 μ L in the step 4, is that the dNTP of 2.5mmol/L, the upstream primer that 1 μ L concentration is 1pmol/ μ L, downstream primer, the 0.8 μ L concentration that 1 μ L concentration is 1pmol/ μ L are the MgCl of 25mmol/L by 1 μ L, 10 * PCR buffer, 0.8 μ L concentration
2, 2 μ L genes of brewing yeast group DNA, 0.2 μ L concentration are the Taq enzyme of 5U/mL and the composition of surplus double distilled water; The PCR reaction conditions: pre-94 ℃ of 2min of sex change, 94 ℃ of 1min of sex change, the 53 ℃ of 1min that anneal extend 72 ℃ of 2.5min, and 72 ℃ of 7min are extended in totally 30 circulations.Other step and parameter are identical with embodiment one.
Do not contain Mg among the 10 * PCR buffer that uses in the present embodiment
2+
Embodiment seven: the difference of present embodiment and embodiment one is: with restriction enzyme Nde I and Aat II p406ADH1 and pGMT-KanR are carried out double digestion respectively in the step 6, connect with the T4DNA ligase enzyme then, obtain plasmid pR.Other step and parameter are identical with embodiment one.
The present embodiment step 6 uses operational manual to operate referring to test kit.
Embodiment eight: the difference of present embodiment and embodiment one is: with restriction enzyme Spe I and Sal I plasmid pR and pGMT-XKS1 are carried out double digestion respectively in the step 7, connect with the T4DNA ligase enzyme then, obtain plasmid pRK.Other step and parameter are identical with embodiment one.
The present embodiment step 7 uses operational manual to operate referring to test kit.
Embodiment nine: the difference of present embodiment and embodiment one is: with restriction enzyme Sal I and Eag I plasmid pRK and pGMT-ADH1T are carried out double digestion respectively in the step 8, connect with the T4DNA ligase enzyme then, obtain plasmid pRKT.Other step and parameter are identical with embodiment one.
The present embodiment step 8 uses operational manual to operate referring to test kit.
Embodiment ten: the difference of present embodiment and embodiment one is: with restriction enzyme Nde I, Eag I plasmid pRKT and pGMT-rDNA are carried out double digestion respectively in the step 9, connect with the T4DNA ligase enzyme then, promptly obtain expressing the plasmid of xylulokinase gene.Other step and parameter are identical with embodiment one.
The present embodiment step 9 uses operational manual to operate referring to test kit.
Embodiment 11: the difference of present embodiment and embodiment one is: resistance marker is G418 in the step 5; The PCR reaction system is 10 μ L in the step 1, is that the dNTP of 2.5mmol/L, the upstream primer that 1 μ L concentration is 1pmol/ μ L, downstream primer, the 0.8 μ L concentration that 1 μ L concentration is 1pmol/ μ L are the MgCl of 25mmol/L by 1 μ L, 10 * PCR buffer, 0.8 μ L concentration
2, 2 μ L plasmid pKT0150,0.2 μ L concentration are the Taq enzyme of 5U/mL and the composition of surplus double distilled water; The PCR reaction conditions: pre-94 ℃ of 2min of sex change, 94 ℃ of 1min of sex change, the 45 ℃ of 1min that anneal extend 72 ℃ of 2min, and 72 ℃ of 7min are extended in totally 30 circulations; The PCR reaction system is 10 μ L in the step 2, is that the dNTP of 2.5mmol/L, the upstream primer that 1 μ L concentration is 1pmol/ μ L, downstream primer, the 0.8 μ L concentration that 1 μ L concentration is 1pmol/ μ L are the MgCl of 25mmol/L by 1 μ L, 10 * PCRbuffer, 0.8 μ L concentration
2, 2 μ L genes of brewing yeast group DNA, 0.2 μ L concentration are the Taq enzyme of 5U/mL and the composition of surplus double distilled water; The PCR reaction conditions: pre-94 ℃ of 5min of sex change, 94 ℃ of 1min of sex change, the 55 ℃ of 1min that anneal extend 72 ℃ of 2min, and 72 ℃ of 10min are extended in totally 30 circulations; The PCR reaction system is 10 μ L in the step 3, is that the dNTP of 2.5mmol/L, the upstream primer that 1 μ L concentration is 1pmol/ μ L, downstream primer, the 0.8 μ L concentration that 1 μ L concentration is 1pmol/ μ L are the MgCl of 25mmol/L by 1 μ L, 10 * PCR buffer, 0.8 μ L concentration
2, 2 μ L plasmid pKT0150,0.2 μ L concentration are the Taq enzyme of 5U/mL and the composition of surplus double distilled water; The PCR reaction conditions: pre-94 ℃ of 2min of sex change, 94 ℃ of 1min of sex change, the 50 ℃ of 1min that anneal extend 72 ℃ of 2min, and 72 ℃ of 7min are extended in totally 30 circulations; The PCR reaction system is 10 μ L in the step 4, is that the dNTP of 2.5mmol/L, the upstream primer that 1 μ L concentration is 1pmol/ μ L, downstream primer, the 0.8 μ L concentration that 1 μ L concentration is 1pmol/ μ L are the MgCl of 25mmol/L by 1 μ L, 10 * PCR buffer, 0.8 μ L concentration
2, 2 μ L genes of brewing yeast group DNA, 0.2 μ L concentration are the Taq enzyme of 5U/mL and the composition of surplus double distilled water; The PCR reaction conditions: pre-94 ℃ of 2min of sex change, 94 ℃ of 1min of sex change, the 53 ℃ of 1min that anneal extend 72 ℃ of 2.5min, and 72 ℃ of 7min are extended in totally 30 circulations; With restriction enzyme Nde I and Aat II p406ADH1 and pGMT-KanR are carried out double digestion respectively in the step 6, connect with the T4DNA ligase enzyme then, obtain plasmid pR; With restriction enzyme Spe I and SalI plasmid pR and pGMT-XKS 1 are carried out double digestion respectively in the step 7, connect with the T4DNA ligase enzyme then, obtain plasmid pRK; With restriction enzyme Sal I and Eag I plasmid pRK and pGMT-ADH1T are carried out double digestion respectively in the step 8, connect with the T4DNA ligase enzyme then, obtain plasmid pRKT; With restriction enzyme Nde I, Eag I plasmid pRKT and pGMT-rDNA are carried out double digestion respectively in the step 9, connect with the T4DNA ligase enzyme then, promptly obtain expressing the plasmid of xylulokinase gene.Other step and parameter are identical with embodiment one.
Do not contain Mg among the 10 * PCR buffer that uses in the present embodiment
2+
The S. cervisiae streak inoculation on the YPD of different G418 concentration flat board, is cultivated 72h under 30 ℃ of conditions, filter out the transformant of high copy.Then with activatory yeast saccharomyces cerevisiae W5 picking list colony inoculation in the YPD liquid nutrient medium, under 30 ℃, 200r/min condition, cultivate 48h, get 100mL again and cultivate bacterium liquid centrifugal 5min under the 3500r/min condition, add 40mL 1 * TE after the abandoning supernatant and clean thalline, and then under the 3500r/min condition centrifugal 5min, abandon supernatant liquor and add 2mL1 * LiAc/0.5 * TE, room temperature is placed 10min, makes the yeast competent cell.
The plasmid of the expression xylulokinase gene of the linearizing present embodiment structure of Hpa I will be used, 10.7 μ L fish salmon sperm DNA, 100 μ L yeast HDY-01 competent cells and 700 μ L1 * LiAc/40PEG-4000/1 * TE mixes, under 30 ℃ of conditions, react 30min, add 88 μ L DMSO stoste uniform mixing afterwards, place 40 ℃ of water-bath heat shock 7min, the centrifugal 10s of 13000r/min then, abandoning supernatant liquor adds 1mL1 * TE again and cleans 2 times, the centrifugal 10s of 13000r/min, add 50～100 μ L, 1 * TE again after abandoning supernatant liquor, coat on the YPD plate culture medium that contains G418, under 30 ℃ of conditions, cultivate 48h, obtain high copy transformant W-XK.
Height is copied middle 60 generations of shake-flask culture of YEPD liquid nutrient medium (under the non-selective pressure) (every 24h was 10 generations) of transformant W-XK inoculation 40mL gradient dilution, every 10 from generation to generation, the plate count colony number carries out stability and measures calculating by formula, the result shows that the stability after 60 generations is 99%, show under non-selective pressure, the exogenous genetic fragment that is integrated on the yeast saccharomyces cerevisiae karyomit(e) has good genetic stability, can be suitable for industrial extensive environment.[plasmid stability calculation formula: stability (%)=(the colony number ÷ total count that has integrated plasmid) * 100%]
Transformant W-XK is cultured to logarithmic phase in selective pressure substratum (the YEPD substratum that contains G418), centrifugal collection thalline is also used 100mmol/L phosphoric acid buffer washing 2 times, be dissolved in lysis buffer (the 100mmol/L phosphoric acid buffer of new preparation then, 0.5mmol/LEDTA, 0.5mmol/L DTT, 1mmol/L PMSF, pH7.0) in, bacteria suspension adds granulated glass sphere (diameter is 0.5mm), handles 30～50s under cytoclasis instrument top speed, at ice bath 5min, the broken repetition 3～5 times, centrifugal 10min under the 10000g condition collects supernatant again, is crude enzyme liquid and is used for the enzyme assay analysis.(the thick liquid eggs white matter of enzyme Determination on content is carried out according to protein content determination test kit operation instructions.)
Present embodiment adopts continuous monitoring method to measure the xylulokinase enzyme and lives, and selects the linear reaction period to calculate enzyme activity, and measuring principle is as follows:
After above linked reaction reached balance, the speed of xylulokinase (XK) conversion of substrate D-xylulose equated with the minimizing speed of NADH in the reaction system, can calculate the activity of xylulokinase (XK) by the minimizing speed of measuring NADH.
Xylulokinase enzyme unit definition alive: under given experiment condition, the enzyme amount of per minute catalyzed conversion 1 μ molNADH is an enzyme unit (being 1U) alive.
In the formula:
6.22 * 10
3: the molar extinction coefficient (1mol of NADH
-1Cm
-1);
OD
340 1: t
1Testing sample is in the photoabsorption of 340nm wavelength constantly;
OD
340 2: t
2Testing sample is in the photoabsorption of 340nm wavelength constantly;
The second number (s) that 60:1min is suitable;
t
1, t
2: the time point of being got in the enzyme activity determination process (s);
C
Prouin: the protein concentration of crude enzyme liquid (mg/mL);
0.1: the volume of the crude enzyme liquid that adds in the reaction system (mL);
10: the multiple of crude enzyme liquid dilution.
Press in the table 1 xylulokinase enzyme activity determination reaction system in the data preparation crude enzyme liquid, volume is 0.9mL.
Table 1
Xylulokinase enzyme activity determination reaction system is balance 15min at ambient temperature, adds the crude enzyme liquid 0.1mL of 10 times of dilutions, and vortex oscillation device vibration mixing is a blank with distilled water, measures OD
340, and use automatic recording spectrophotometer record sample OD
340Over time.Choose the linear reaction period, calculate xylulokinase activity in the crude enzyme liquid.The enzyme activity determination result shows, the xylulokinase vigor of the yeast saccharomyces cerevisiae that plasmid the transformed reorganization bacterium W-XK of the expression xylulokinase gene that the present embodiment method makes up significantly improves than starting strain W5, the enzyme activity of starting strain W5 is 0.31U/mg, the enzyme activity of recombinant bacterial strain W-XK is 2.8U/mg, enzyme activity has improved 9 times, shows the high efficiency stable expression of having realized xylulokinase gene behind the plasmid vector transformed saccharomyces cerevisiae that the present embodiment method makes up.
Sequence table
＜110〉Heilongjiang University
＜120〉a kind of construction process of expressing the plasmid of xylulokinase gene
<160>8
<210>1
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the nucleotide sequence of plasmid pKT0150 and the pcr amplification upstream primer of the relative position design of KanR gene in plasmid.
<400>1
ttccatatgt?ctgtttagct?tgcctc?26
<210>2
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the nucleotide sequence of plasmid pKT0150 and the pcr amplification downstream primer of the relative position design of KanR gene in plasmid.
<400>2
cttgacgtct?atcatcgatg?aattcga?27
<210>3
<211>29
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the pcr amplification upstream primer of the gene order of yeast saccharomyces cerevisiae XKS1 design.
<400>3
cggactagtc?tcaatcttca?gcaagcgac?29
<210>4
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the pcr amplification downstream primer of the gene order of yeast saccharomyces cerevisiae XKS1 design.
<400>4
cgcgtcgaca?acggggaaca?aaatgatg?28
<210>5
<211>31
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the nucleotide sequence of plasmid pKT0150 and the pcr amplification upstream primer of the relative position design of ADH1 terminator fragment in plasmid.
<400>5
cgcgtcgaca?tttgttactg?ctgctggtat?t?31
<210>6
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the nucleotide sequence of plasmid pKT0150 and the pcr amplification downstream primer of the relative position design of ADH1 terminator fragment in plasmid.
<400>6
ctcggccgcc?ctgttatccc?tagcgg?26
<210>7
<211>29
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the segmental upstream primer of rDNA of the amplification 2.2kb of the gene order of yeast saccharomyces cerevisiae rDNA design.
<400>7
ttccatatgg?gaacctctaa?tcattcgct?29
<210>8
<211>29
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the segmental downstream primer of rDNA of the amplification 2.2kb of the gene order of yeast saccharomyces cerevisiae rDNA design.
<400>8
tctcggccga?acgaacgaga?ccttaacct?29
Claims (6)
1. construction process of expressing the plasmid of xylulokinase gene, the construction process that it is characterized in that expressing the plasmid of xylulokinase gene carries out according to the following steps: one, KanR gene among the cloned plasmids pKT0150; Two, clone's yeast saccharomyces cerevisiae XKS1 gene; Three, ADH1 terminator fragment among the cloned plasmids pKT0150; Four, 2.2kb rDNA fragment in clone's yeast saccharomyces cerevisiae; The gene fragment of five, recovery, purification step one to four preparation, and then under 16 ℃ condition, be connected 10～12h with pGEMT Easy carrier respectively, obtain pGMT-KanR, pGMT-XKS1, pGMT-ADH1T and pGMT-rDNA; Six, making up plasmid pR, is the gene KanR that skeleton is introduced resistance marker with yeast saccharomyces cerevisiae integrative vector p406ADH1; Seven, make up plasmid pRK, the XKS1 gene is added plasmid pR; Eight, make up plasmid pRKT, ADH1 terminator fragment is added plasmid pRK; Nine, 2.2kb rDNA fragment is added plasmid pRKT, promptly obtain expressing the plasmid of xylulokinase gene; Wherein amplification upstream primer sequence is 5 '-TTCCATATGTCTGTTTAGCTTGCCTC-3 ' in the step 1, and the downstream primer sequence is 5 '-CTTGACGTCTATCATCGATGAATTCGA-3 '; Amplification upstream primer sequence is 5 '-CGGACTAGTCTCAATCTTCAGCAAGCGAC-3 ' in the step 2, and the downstream primer sequence is 5 '-CGCGTCGACAACGGGGAACAAAATGATG-3 '; Amplification upstream primer sequence is 5 '-CGCGTCGACATTTGTTACTGCTGCTGGTATT-3 ' in the step 3, and the downstream primer sequence is 5 '-CTCGGCCGCCCTGTTATCCCTAGCGG-3 '; Amplification upstream primer sequence is 5 '-TTCCATATGGGAACCTCTAATCATTCGCT-3 ' in the step 4, the downstream primer sequence is 5 '-TCTCGGCCGAACGAACGAGACCTTAACCT-3 ', with restriction enzyme Nde I and Aat II p406ADH1 and pGMT-KanR are carried out double digestion respectively in the step 6, connect with the T4DNA ligase enzyme then, obtain plasmid pR, with restriction enzyme Spe I and SalI plasmid pR and pGMT-XKS1 are carried out double digestion respectively in the step 7, connect with the T4DNA ligase enzyme then, obtain plasmid pRK, with restriction enzyme Sal I and Eag I plasmid pRK and pGMT-ADH1T are carried out double digestion respectively in the step 8, connect with the T4 dna ligase then, obtain plasmid pRKT, use restriction enzyme Nde I in the step 9, Eag I carries out double digestion respectively to plasmid pRKT and pGMT-rDNA, connect with the T4DNA ligase enzyme then, promptly obtain expressing the plasmid of xylulokinase gene.
2. a kind of construction process of expressing the plasmid of xylulokinase gene according to claim 1 is characterized in that the resistance marker in the step 6 is G418.
3. a kind of construction process of expressing the plasmid of xylulokinase gene according to claim 1, it is characterized in that the PCR reaction system is 10 μ L in the step 1, is that the dNTP of 2.5mmol/L, the upstream primer that 1 μ L concentration is 1pmol/ μ L, downstream primer, the 0.8 μ L concentration that 1 μ L concentration is 1pmol/ μ L are the MgCl of 25mmol/L by 1 μ L10 * PCR buffer, 0.8 μ L concentration
2, 2 μ L plasmid pKT0150,0.2 μ L concentration are that the Taq enzyme of 5U/mL and the double distilled water of surplus are formed; The PCR reaction conditions: pre-94 ℃ of 2min of sex change, 94 ℃ of 1min of sex change, the 45 ℃ of 1min that anneal extend 72 ℃ of 2min, and 72 ℃ of 7min are extended in totally 30 circulations.
4. a kind of construction process of expressing the plasmid of xylulokinase gene according to claim 1, it is characterized in that the PCR reaction system is 10 μ L in the step 2, is that the dNTP of 2.5mmol/L, the upstream primer that 1 μ L concentration is 1pmol/ μ L, downstream primer, the 0.8 μ L concentration that 1 μ L concentration is 1pmol/ μ L are the MgCl of 25mmol/L by 1 μ L10 * PCR buffer, 0.8 μ L concentration
2, 2 μ L genes of brewing yeast group DNA, 0.2 μ L concentration are that the Taq enzyme of 5U/mL and the double distilled water of surplus are formed; The PCR reaction conditions: pre-94 ℃ of 5min of sex change, 94 ℃ of 1min of sex change, the 55 ℃ of 1min that anneal extend 72 ℃ of 2min, and 72 ℃ of 10min are extended in totally 30 circulations.
5. a kind of construction process of expressing the plasmid of xylulokinase gene according to claim 1, it is characterized in that the PCR reaction system is 10 μ L in the step 3, is that the dNTP of 2.5mmol/L, the upstream primer that 1 μ L concentration is 1pmol/ μ L, downstream primer, the 0.8 μ L concentration that 1 μ L concentration is 1pmol/ μ L are the MgCl of 25mmol/L by 1 μ L, 10 * PCR buffer, 0.8 μ L concentration
2, 2 μ L plasmid pKT0150,0.2 μ L concentration are that the Taq enzyme of 5U/mL and the double distilled water of surplus are formed; The PCR reaction conditions: pre-94 ℃ of 2min of sex change, 94 ℃ of 1min of sex change, the 50 ℃ of 1min that anneal extend 72 ℃ of 2min, and 72 ℃ of 7min are extended in totally 30 circulations.
6. a kind of construction process of expressing the plasmid of xylulokinase gene according to claim 1, it is characterized in that the PCR reaction system is 10 μ L in the step 4, is that the dNTP of 2.5mmol/L, the upstream primer that 1 μ L concentration is 1pmol/ μ L, downstream primer, the 0.8 μ L concentration that 1 μ L concentration is 1pmol/ μ L are the MgCl of 25mmol/L by 1 μ L, 10 * PCR buffer, 0.8 μ L concentration
2, 2 μ L genes of brewing yeast group DNA, 0.2 μ L concentration are that the Taq enzyme of 5U/mL and the double distilled water of surplus are formed; The PCR reaction conditions: pre-94 ℃ of 2min of sex change, 94 ℃ of 1min of sex change, the 53 ℃ of 1min that anneal extend 72 ℃ of 2.5min, and 72 ℃ of 7min are extended in totally 30 circulations.
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| CN1141057A (en) * | 1993-11-08 | 1997-01-22 | 普渡研究基金会 | Recombinant yeasts for effective fermentation of glucose and xylose |
| CN1225125A (en) * | 1996-05-06 | 1999-08-04 | 普渡研究基金会 | Stable recombinant yeasts for fermenting xylose to ethanol |
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| CN1141057A (en) * | 1993-11-08 | 1997-01-22 | 普渡研究基金会 | Recombinant yeasts for effective fermentation of glucose and xylose |
| CN1225125A (en) * | 1996-05-06 | 1999-08-04 | 普渡研究基金会 | Stable recombinant yeasts for fermenting xylose to ethanol |
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| 代谢木糖和葡萄糖的重组酿酒酵母的构建;刘继开等;可再生能源;20070228;25(1);13-16 * |
| 刘继开等.代谢木糖和葡萄糖的重组酿酒酵母的构建.可再生能源.2007,25(1),13-16. |
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