CN102719481B - Method for simultaneously transferring multiple genes into microbial genome - Google Patents
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- CN102719481B CN102719481B CN201210196996.9A CN201210196996A CN102719481B CN 102719481 B CN102719481 B CN 102719481B CN 201210196996 A CN201210196996 A CN 201210196996A CN 102719481 B CN102719481 B CN 102719481B
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Abstract
The invention discloses a method for simultaneously transferring multiple genes into microbial genome. The invention provides a method for preparing recombinant microorganism expressing multiple exogenous genes; the method comprises the following steps of: transferring expression cassettes of all genes into the host microorganism to obtain the recombinant microorganism combining multiple exogenous genes into the genome and expressing the multiple exogenous genes, wherein the 5' end of the expression cassette of the first gene has a homologous arm A, the 3' end of the expression cassette of the last gene has a homologous arm B, and the 3' tail end of the expression cassette of each gene and the 5' tail end of the expression cassette of the next gene have the same homologous arm; and the homologous arm A and the homologous arm B can be subjected to homologous recombination with the genome of the host microorganism. Through the method, multiple genes contained in a target metabolic pathway can be transferred into the host strain at one step and arranged according to a set sequence, the required engineering strain is directly obtained, and the troubles caused by multiple times of transformation and vector construction are avoided.
Description
Technical field
The present invention relates to a kind of method that a plurality of genes is imported simultaneously to microbial genome.
Background technology
The energy, population and environmental problem are current subject matter, if abundant renewable resources is changed into bioenergy, have not only solved the problem of energy and environment, also saved grain simultaneously, have solved the food problem being brought by populous.
At present genetic engineering modified having become improved Research for Industrial Microbial Germ level, promotes the core technology of biotechnology industry development.A genetic engineering modified important directions is the expression amount that imports new gene or improve original gene in Host Strains, can realize by the expression vector that contains goal gene fragment is transformed to Host Strains, thereby also can on recombinant vectors, transform Host Strains restructuring and realize to genome by goal gene fragment is inserted into, wherein the latter be due to more stable in bacterial classification goes down to posterity process and obtain application more preferably in industrial production.
Take yeast saccharomyces cerevisiae as example, and it has stronger resistance, is to produce the comparatively desirable production bacterial strain of alcohol, in the existing extensive application of the industrial circles such as food, fermentation.Along with in recent years, to the improving constantly of the requirements such as yeast saccharomyces cerevisiae physiological biochemical property and fermentation level, more and more need to carry out polygene operation.The probability that yeast saccharomyces cerevisiae homologous recombination occurs is very high, linear DNA fragment imports in brewing yeast cell can be easy to occur at homologous sequence place homologous recombination, and this expression, gene that makes to be easy to realize gene in yeast saccharomyces cerevisiae interrupts, gene substitution etc.But, when needed expressing gene is too much, there is following limitation: vector construction is more difficult on the one hand, needs repeatedly to connect conversion simultaneously; If carrier is excessive on the other hand, transformation ratio is more difficult, is not easy to obtain recombinant bacterial strain; If by integrative vector, the copy number of gene is often also lower, is difficult for realizing the high efficient expression of foreign gene.
Summary of the invention
The object of this invention is to provide a kind of method that a plurality of genes is imported simultaneously to microbial genome.
The invention provides a kind of prepare express a plurality of foreign genes recombinant microorganism method, comprise the steps: the expression cassette of each gene to import together host microorganism, obtain the recombinant microorganism that described a plurality of exogenous origin gene integrators is entered to genome and express described a plurality of foreign genes;
When described a plurality of foreign genes are two foreign genes, 5 ' end of the expression cassette of first gene has homology bracelet, 3 ' end of the expression cassette of second gene has homology arm second, and 3 ' end of the expression cassette of first gene has identical homology arm with 5 ' end of the expression cassette of second gene;
When described a plurality of foreign genes are three foreign genes, 5 ' end of the expression cassette of first gene has described homology bracelet, 3 ' end of the expression cassette of the 3rd gene has described homology arm second, 5 ' end of the expression cassette of second gene has identical homology arm with 3 ' end of the expression cassette of first gene, and 3 ' end of the expression cassette of second gene has identical homology arm with 5 ' end of the expression cassette of the 3rd gene;
When described a plurality of foreign genes are more than four during foreign gene, 5 ' end of the expression cassette of first gene has described homology bracelet, 3 ' end of the expression cassette of last gene has described homology arm second, and 5 ' end of 3 ' end of the expression cassette of each gene and the expression cassette of next gene has identical homology arm;
Described homology bracelet and described homology arm second can with the genome generation homologous recombination of described host microorganism.
The expression cassette of each gene includes promotor, described gene and terminator.
Described host microorganism can be prokaryotic micro-organisms or the eukaryotic microorganisms with high efficiency gene recombination ability, specifically can be yeast, is preferably yeast saccharomyces cerevisiae.
The site of described homologous recombination can be any site of described host microorganism chromosomal DNA, is preferably 18srDNA or the 26srDNA of described host microorganism.
More than described in each, the length of homology arm is 50bp.
Described homology bracelet specifically can be if the sequence 1 of sequence table be from as shown in the 1st to 766 Nucleotide of 5 ' end.
Described homology arm second specifically can be if the sequence 2 of sequence table be from as shown in the 2425th to 3148 Nucleotide of 5 ' end.
Described homology bracelet specifically can be if the sequence 7 of sequence table be from as shown in the 1st to 1180 Nucleotide of 5 ' end.
Described homology arm second specifically can be if the sequence 8 of sequence table be from as shown in the 1165th to 2281 Nucleotide of 5 ' end.
When described a plurality of foreign genes are two foreign genes: the expression cassette of first gene is from 5 ' end to 3 ' end is followed successively by following element: the sequence 7 of sequence table from the sequence 7 of the homology bracelet shown in the 1st to 1180 Nucleotide of 5 ' end, sequence table from the sequence 7 of the PGK promotor shown in the 1181st to 1778 Nucleotide of 5 ' end, first gene, sequence table from the AOX terminator shown in the 3120th to 3460 Nucleotide of 5 ' end; The expression cassette of second gene is from 5 ' end to 3 ' end is followed successively by following element: the sequence 8 of sequence table from the sequence 8 of the AOX terminator shown in the 1st to 341 Nucleotide of 5 ' end, sequence table from the sequence 8 of the trp promoter shown in the 342nd to 443 Nucleotide of 5 ' end, second gene, sequence table from the sequence 8 of the tryptophane terminator shown in the 1119th to 1164 Nucleotide of 5 ' end, sequence table from the homology arm second shown in the 1165th to 2281 Nucleotide of 5 ' end.
When described a plurality of foreign genes are three foreign genes: the expression cassette of first gene is from 5 ' end to 3 ' end is followed successively by following element: the sequence 1 of sequence table from the sequence 1 of the homology bracelet shown in the 1st to 766 Nucleotide of 5 ' end, sequence table from 5 ' end the 767th sequence 1 to the GPD promotor shown in 1444, first gene, sequence table from the CYC terminator shown in the 2759th to 3047 Nucleotide of 5 ' end; The expression cassette of second gene is from 5 ' end to 3 ' end is followed successively by following element: the sequence 2 of sequence table from the sequence 2 of the CYC terminator shown in the 1st to 289 Nucleotide of 5 ' end, sequence table, from the 290th to 887 Nucleotide of 5 ' end, be the sequence 2 of PGK promotor, second gene, sequence table from the sequence 2 of the ADH1 terminator shown in the 2691st to 3236 Nucleotide of 5 ' end, sequence table from the ADH1 promotor shown in the 3237th to 4643 Nucleotide of 5 ' end; The expression cassette of the 3rd gene is from 5 ' end to 3 ' end is followed successively by following element: the sequence 3 of sequence table from the sequence 3 of the ADH1 promotor shown in the 1st to 1407 Nucleotide of 5 ' end, the 3rd gene, sequence table from the sequence 3 of the Histidine terminator shown in the 2224th to 2424 Nucleotide of 5 ' end, sequence table from the homology arm second shown in the 2425th to 3148 Nucleotide of 5 ' end.
Described a plurality of foreign gene can be xylose isomerase gene, xylulokinase gene and G418 gene; Described xylose isomerase is as shown in the sequence 4 of sequence table; Described xylulokinase is as shown in the sequence 5 of sequence table; Described G418 is as shown in the sequence 6 of sequence table.Described xylose isomerase gene specifically can be if the sequence 1 of sequence table be from as shown in the 1445th to 2758 Nucleotide of 5 ' end.Described xylulokinase gene specifically can be if the sequence 2 of sequence table be from as shown in the 888th to 2690 Nucleotide of 5 ' end.Described G418 gene specifically can be if the sequence 3 of sequence table be from as shown in the 1408th to 2223 Nucleotide of 5 ' end.The expression cassette of described xylose isomerase gene is preferably as shown in the sequence 1 of sequence table; The expression cassette of described xylulokinase gene is preferably as shown in the sequence 2 of sequence table; The expression cassette of described G418 gene is preferably as shown in the sequence 3 of sequence table.Described host microorganism specifically can be yeast saccharomyces cerevisiae w303-1B.The microorganism called after recombinant microorganism first that adopts this section of scheme to obtain.
Described a plurality of foreign gene can be nadh oxidase gene and tryptophane gene; Described nadh oxidase gene is as shown in the sequence 9 of sequence table; Protein shown in the sequence 10 of described tryptophane gene coded sequence table.Described nadh oxidase gene specifically can be if the sequence 7 of sequence table be from as shown in the 1779th to 3119 Nucleotide of 5 ' end.Described tryptophane gene specifically can be if the sequence 8 of sequence table be from as shown in the 443rd to 1118 Nucleotide of 5 ' end.The expression cassette of described nadh oxidase gene is preferably as shown in the sequence 7 of sequence table; The expression cassette of described tryptophane gene is preferably as shown in the sequence 8 of sequence table.Described host microorganism specifically can be recombinant microorganism first.
The recombinant microorganism that arbitrary described method obtains all belongs to protection scope of the present invention.
The invention provides in microbe a plurality of gene one steps are incorporated into the method on genome, the method is utilized the principle of homologous recombination, needed a plurality of expression casettes are imported in host cell simultaneously, by the generation of self homologous recombination in body, realize a plurality of genes are integrated according to definite sequence one one-step site.By the method, contained a plurality of gene one steps of target pathways metabolism can be imported in host strain, and arrange by the order setting, directly obtain needed engineering strain, avoid the trouble repeatedly transforming and vector construction brings.
Accompanying drawing explanation
Fig. 1 is for expressing the result of the functional evaluation of xylose metabolism approach recombinant bacterial strain.
Fig. 2 is for expressing the result of nadh oxidase recombinant bacterial strain Function Identification.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.
Embodiment 1, in yeast saccharomyces cerevisiae, build xylose isomerase pathways metabolism
The present embodiment is expressed xylose isomerase gene (XI gene) and xylulokinase gene (XK gene) in yeast saccharomyces cerevisiae, simultaneously expression screening marker gene (G418 gene); For increasing copy number, the 18srDNA integration site that the genomic integration site that the present embodiment is selected is multiple copied.
One, the acquisition of xylose isomerase gene expression cassette
Synthetic xylose isomerase gene expression cassette (claims again C1 fragment, 3047bp), it is the double chain DNA molecule (in sequence 1,767 to 1444 Nucleotide of homologous sequence first, that are yeast saccharomyces cerevisiae 18srDNA from the 1st to 766 Nucleotide of 5 ' end are that GPD promotor, the 1445th to 2758 Nucleotide are that XI gene, the 2759th to 3047 Nucleotide are CYC terminator) shown in the sequence 1 of sequence table.Wherein, the protein shown in the sequence 4 of XI gene coded sequence table.
Two, the acquisition of xylulokinase gene expression cassette
Synthetic xylulokinase gene expression cassette (claims again C2 fragment, 4643bp), i.e. double chain DNA molecule shown in the sequence 2 of sequence table (sequence 2 is that CYC terminator, the 290th to 887 Nucleotide are that PGK promotor, the 888th to 2690 Nucleotide are that XK gene, the 2691st to 3236 Nucleotide are that ADH1 terminator, the 3237th to 4643 Nucleotide are ADH1 promotor from the 1st to 289 Nucleotide of 5 ' end).Wherein, the protein shown in the sequence 5 of XK gene coded sequence table.
Three, the acquisition of selection markers expression casette
Synthetic selection markers expression casette (claims again C3 fragment, 3148bp), it is the double chain DNA molecule (in sequence 3, being that ADH1 promotor, the 1408th to 2223 Nucleotide are that G418 gene, the 2224th to 2424 Nucleotide are that Histidine terminator, the 2425th to 3148 Nucleotide are the homologous sequence second of yeast saccharomyces cerevisiae 18srDNA from the 1st to 1407 Nucleotide of 5 ' end) shown in the sequence 3 of sequence table.Wherein, the protein shown in the sequence 6 of G418 gene coded sequence table.
Four, the comprehensive analysis of three expression cassettes
In above three expression cassettes, CYC terminator in xylose isomerase gene expression cassette can with xylulokinase gene expression cassette in CYC terminator generation homologous recombination, the ADH1 promotor in xylulokinase gene expression cassette can with selection markers expression casette in ADH1 promotor generation homologous recombination.The homologous sequence first of 18srDNA is interbody fusion site homology arm first half section, and the homologous sequence second of 18srDNA is the interbody fusion site homology arm second half section.
Five, the acquisition of recombinant bacterium
Yeast saccharomyces cerevisiae (w303-1B; Wild-type yeast saccharomyces cerevisiae): purchased from European yeast saccharomyces cerevisiae bacterial classification storehouse, the bacterial strain BMA64-1A of EUROpean Saccharomyces Cerevisiae numbering 20000A, network address is
http:// web.uni-frankfurt.de/fb15/mikro/euroscarf/data/w303.html).
By equimolar C1 fragment, C2 fragment and the C3 fragment transformed saccharomyces cerevisiae competent cell that shocks by electricity simultaneously, then containing 30 ℃ of inversions on the YPD solid medium of 200 μ g/ml G418, cultivating (within 3-4 days, can observe bacterium colony occurs), adopt gradient dilution method carrying out purifying cultivation containing on the YPD solid medium of 200 μ g/ml G418, obtain altogether the bacterial strain (restructuring fungus beetle) of 10 strain pure cultures.
Six, the Function Identification of recombinant bacterium
The substratum that the wood sugar of take is sole carbon source (pH 5.5): solvent is water, and solute and concentration thereof are as follows: yeast nitrogen base 0.67g/100mL, 2g/100mL wood sugar, 10mg/100ml uridylic, 10mg/100mL Histidine, 10mg/100mL tryptophane, 10mg/100mL leucine.
The restructuring fungus beetle obtaining from step 5, get at random 3 strains tests as follows respectively together with wild-type yeast saccharomyces cerevisiae:
Inoculation, to take the substratum that wood sugar is sole carbon source, is made to its initial OD
600=0.2, then 30 ℃, 220rpm(vibration radius 3cm) shaking culture 120 hours.Respectively within 25 hours, 50 hours, 75 hours, 100 hours, 120 hours and 145 hours, sampling and detect OD
600value, the results are shown in Figure 1(tri-strain restructuring fungus beetles, and every strain bacterium carries out revision test three times, averages; Wild-type yeast saccharomyces cerevisiae carries out revision test three times, averages).
Result shows, in wood sugar substratum, and wild-type Wine brewing yeast strain poor growth, recombinant bacterium can Fast Growth.Be by importing xylose isomerase gene, xylulokinase gene and selection markers gene, successfully to build and screen the yeast saccharomyces cerevisiae that has obtained having xylose isomerase pathways metabolism in the present embodiment, and two genes in this pathways metabolism have all obtained high efficient expression.
Embodiment 2, in yeast saccharomyces cerevisiae, express nadh oxidase gene
In the recombinant bacterium that the present embodiment obtains at embodiment 1, express nadh oxidase gene (noxE gene), to reduce the generation of Xylitol in wood-sugar fermentation; In order to increase copy number, in present case selection yeast saccharomyces cerevisiae, 26srDNA is as gene integration site.
One, the structure of nadh oxidase expression casette
Synthetic nadh oxidase expression casette (claims again C4 fragment, 3460bp), i.e. double chain DNA molecule shown in the sequence 7 of sequence table (1181 to 1778 Nucleotide of homologous sequence first, that sequence 7 is yeast saccharomyces cerevisiae 26srDNA from the 1st to 1180 Nucleotide of 5 ' end are that PGK promotor, the 1779th to 3119 Nucleotide are that noxE gene, the 3120th to 3460 Nucleotide are AOX terminator).Wherein, the protein shown in the sequence 9 of noxE gene coded sequence table.
Two, the structure of selection markers expression casette (trp-)
Synthetic selection markers expression casette (claims again C5 fragment, 2281bp), i.e. double chain DNA molecule shown in the sequence 8 of sequence table (sequence 8 is that AOX terminator, the 342nd to 443 Nucleotide are that trp promoter, the 444th to 1118 Nucleotide are that tryptophane gene, the 1119th to 1164 Nucleotide are that tryptophane terminator, the 1165th to 2281 Nucleotide are the homologous sequence second of yeast saccharomyces cerevisiae 26srDNA from the 1st to 341 Nucleotide of 5 ' end).Wherein, the protein shown in the sequence 10 of tryptophane gene coded sequence table.
Three, the comprehensive analysis of two expression cassettes
In above two each and every one expression cassettes, the AOX terminator in nadh oxidase expression casette can with selection markers expression casette in AOX terminator generation homologous recombination.The homologous sequence first of 26srDNA is interbody fusion site homology arm first half section, and the homologous sequence second of 26srDNA is the interbody fusion site homology arm second half section.
Four, the acquisition of recombinant bacterium
Auxotroph is selected dull and stereotyped (pH5.5): solvent is water, and solute and concentration thereof are as follows: yeast nitrogen base 0.67g/100mL, 2g/100mL glucose, 10mg/100mL uridylic, 10mg/100mL Histidine, 10mg/100mL leucine.
Equimolar C4 fragment, C5 fragment are shocked by electricity simultaneously and transform the competent cell of the recombinant bacterium that embodiment 1 obtains, then in auxotroph, select 30 ℃ of inversions on flat board to cultivate (within 3-4 days, can observe bacterium colony occurs), adopt gradient dilution method to select to carry out purifying cultivation on flat board in auxotroph, obtain altogether the bacterial strain (recombinant bacterium second) of 20 strain pure cultures.
Five, the evaluation of the function of recombinant bacterium
Wood-sugar fermentation substratum (YPX substratum, pH5.5): solvent is water, and solute and concentration thereof are as follows: yeast powder 10g/L, peptone 20g/L, wood sugar 45g/L.
The recombinant bacterium second obtaining from step 4, get at random together with the restructuring fungus beetle that 3 strain bacterium obtain with embodiment 1, test as follows respectively:
(1) by inoculation to YPD substratum, 30 ℃, 220rpm(vibration radius 3cm) shaking culture is to OD
600=8, bacterial strain reaches logarithmic phase, and centrifugal (4500rpm, 10min) collects thalline.
(2) thalline of step (1) being collected is seeded to wood-sugar fermentation substratum, and making its starting point concentration is OD
600=8, then 30 ℃ of static cultivations.Respectively within 0 hour, 7 hours, 23 hours, 31 hours and 48 hours and 57 hours, sampling and detect Xylitol concentration.
Detect the method for Xylitol concentration: by the centrifugal 2min of fermented liquid 13000rpm normal temperature, after 0.22 μ m membrane filtration, get filtrate and carry out HPLC; HPLC adopts Bio-rad HPX-87H pillar, and moving phase is 0.5mM aqueous sulfuric acid, and flow rate of mobile phase is 0.55mL/min; Xylitol standard substance (sigma company, article No. X3375) appearance time 13.3min, the peak area of 10g/L Xylitol standard substance is 2486387, so the function of Xylitol concentration and peak area is Y=X/2486387*10(Y, represents Xylitol concentration, and unit is g/L; X represents peak area).
The results are shown in Figure 2.Result shows, recombinant bacterium NADH coenzyme that embodiment 1 obtains is excessive causes a large amount of Xylitols accumulation, and the recombinant bacterium that the step 4 of embodiment 2 obtains is under the help of nadh oxidase, has reduced the accumulation to Xylitol.Be by importing nadh oxidase gene and selection markers gene, successfully to build and screen the yeast saccharomyces cerevisiae that has obtained high efficient expression nadh oxidase gene in the present embodiment.
Claims (3)
1. a method of preparing the recombinant microorganism of expressing a plurality of foreign genes, comprise the steps: the expression cassette of each gene to import together host microorganism, obtain the recombinant microorganism that described a plurality of exogenous origin gene integrators is entered to genome and express described a plurality of foreign genes; Described host microorganism is yeast saccharomyces cerevisiae;
When described a plurality of foreign genes are three foreign genes, 5 ' end of the expression cassette of first gene has described homology bracelet, 3 ' end of the expression cassette of the 3rd gene has described homology arm second, 5 ' end of the expression cassette of second gene has identical homology arm with 3 ' end of the expression cassette of first gene, and 3 ' end of the expression cassette of second gene has identical homology arm with 5 ' end of the expression cassette of the 3rd gene; Described a plurality of foreign gene is xylose isomerase gene, xylulokinase gene and G418 gene; Described xylose isomerase is as shown in the sequence 4 of sequence table; Described xylulokinase is as shown in the sequence 5 of sequence table; Protein shown in the sequence 6 of described G418 gene coded sequence table; Described xylose isomerase gene is if the sequence 1 of sequence table is from as shown in the 1445th to 2758 Nucleotide of 5 ' end; Described xylulokinase gene is if the sequence 2 of sequence table is from as shown in the 888th to 2690 Nucleotide of 5 ' end; Described G418 gene is if the sequence 3 of sequence table is from as shown in the 1408th to 2223 Nucleotide of 5 ' end;
When described a plurality of foreign genes are three foreign genes: the expression cassette of first gene is from 5 ' end to 3 ' end is followed successively by following element: the sequence 1 of sequence table from the sequence 1 of the homology bracelet shown in the 1st to 766 Nucleotide of 5 ' end, sequence table from 5 ' end the 767th sequence 1 to the GPD promotor shown in 1444, first gene, sequence table from the CYC terminator shown in the 2759th to 3047 Nucleotide of 5 ' end; The expression cassette of second gene is from 5 ' end to 3 ' end is followed successively by following element: the sequence 2 of sequence table from the sequence 2 of the CYC terminator shown in the 1st to 289 Nucleotide of 5 ' end, sequence table, from the 290th to 887 Nucleotide of 5 ' end, be the sequence 2 of PGK promotor, second gene, sequence table from the sequence 2 of the ADH1 terminator shown in the 2691st to 3236 Nucleotide of 5 ' end, sequence table from the ADH1 promotor shown in the 3237th to 4643 Nucleotide of 5 ' end; The expression cassette of the 3rd gene is from 5 ' end to 3 ' end is followed successively by following element: the sequence 3 of sequence table from the sequence 3 of the ADH1 promotor shown in the 1st to 1407 Nucleotide of 5 ' end, the 3rd gene, sequence table from the sequence 3 of the Histidine terminator shown in the 2224th to 2424 Nucleotide of 5 ' end, sequence table from the homology arm second shown in the 2425th to 3148 Nucleotide of 5 ' end;
The expression cassette of described xylose isomerase gene is as shown in the sequence 1 of sequence table; The expression cassette of described xylulokinase gene is as shown in the sequence 2 of sequence table; The expression cassette of described G418 gene is as shown in the sequence 3 of sequence table.
2. the method for claim 1, is characterized in that: described a plurality of foreign genes also comprise nadh oxidase gene and tryptophane gene; Described nadh oxidase gene is as shown in the sequence 9 of sequence table; Protein shown in the sequence 10 of described tryptophane gene coded sequence table; Described nadh oxidase gene is if the sequence 7 of sequence table is from as shown in the 1779th to 3119 Nucleotide of 5 ' end; Described tryptophane gene is if the sequence 8 of sequence table is from as shown in the 444th to 1118 Nucleotide of 5 ' end;
The expression cassette of described nadh oxidase gene is specifically as shown in the sequence 7 of sequence table; The expression cassette of described tryptophane gene is specifically as shown in the sequence 8 of sequence table;
Described host microorganism is the recombinant microorganism that method prepares described in claim 1.
3. the recombinant microorganism that described in claim 1 or 2, method obtains.
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| WO2004099381A2 (en) * | 2003-05-02 | 2004-11-18 | Cargill Dow Llc | Genetically modified yeast species and fermentation processes using genetically modified yeast |
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| WO2004099381A2 (en) * | 2003-05-02 | 2004-11-18 | Cargill Dow Llc | Genetically modified yeast species and fermentation processes using genetically modified yeast |
| CN101016551A (en) * | 2007-02-01 | 2007-08-15 | 南京师范大学 | Method of introducing a plurality of DNA fragments simultaneously into DNA vector |
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