CN103820347A - Industrial saccharomyces cerevisiae strain with acetic acid tolerance - Google Patents
Industrial saccharomyces cerevisiae strain with acetic acid tolerance Download PDFInfo
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- CN103820347A CN103820347A CN201410102837.7A CN201410102837A CN103820347A CN 103820347 A CN103820347 A CN 103820347A CN 201410102837 A CN201410102837 A CN 201410102837A CN 103820347 A CN103820347 A CN 103820347A
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Abstract
The invention relates to an industrial saccharomyces cerevisiae strain (4126-YHB1) with acetic acid tolerance, and belongs to the technical field of microbes. The strain is named Saccharomyces cerevisiae in a classified way; the registering number of the strain is CGMCC No.8725; the strain is collected in the China general microbiological culture collection center; the collection unit address is Beichen west road, No.1 yuan, 3, Chaoyang district, Beijing, and the collection date is On January 15, 2014. The invention discloses a gene engineering construction method of a recombination strain 4126-YHB1. The gene engineering construction method comprises the following steps: acquiring a gene; constructing a chromosomal integration vector; carrying out growth and fermentation experiments on the strain under the condition of acetic acid. Compared with a comparison strain for integrating an empty vector, the recombination strain 4126-YHB1 disclosed by the invention can be used for carrying out fast alcohol fermentation in a culture medium which contains 5 g/L of acetic acid, and tolerating the high-concentration acetic acid contained in a cellulose hydrolysate, and is a good strain which is subjected to the alcohol fermentation under the condition of environmental stress.
Description
Technical field
The present invention relates to an industrial strain of S.cerevisiae strain (4126-YHB1) that a strain has acetic acid tolerance, belong to microbial technology field.
Background technology
Because the margin of fossil oil constantly reduces and the factor such as unstable of International Crude Oil, domestic and international for need to day by day the increasing of renewable and clean energy resource, biofuel is the renewable energy source with good development prospect.Except having the general advantage of renewable energy source, biofuel can also alleviate by the caused series of problems of fossil oil, especially can reduce the caused Greenhouse effect of gaseous emission.In the eurypalynous biofuel of crowd, alcohol fuel has shown huge advantage (Applied Microbiology and Biotechnology, 2009,85:253-263).Ethanol is not only a kind of good fuel, is also a kind of good fuel qualities activator, has again the fabulous capability of antidetonance simultaneously.China has abundant cellulose biomass resource, if can fully effectively utilize these raw material production alcohol fuels, both can protection of the environment, can also solve utilize that starchy material produces that ethanol produces strive grain with people, the drawback of striving ground with grain.Utilize Mierocrystalline cellulose to produce ethanol for starting material and be conducive to economic Sustainable development, also can reduce the dependence of China for the Imported oil energy, ensure national energy security simultaneously.
In biorefinery of lignocellulose ethanol process, due to fine and close structure between Mierocrystalline cellulose, hemicellulose and xylogen in raw material, be unfavorable for that cellulase is to cellulosic hydrolysis, therefore, lignocellulose must be through pre-treatment to improve the accessibility of Mierocrystalline cellulose to cellulase before enzymic hydrolysis.Diluted acid and steam explosion pre-treatment are to study at present more pretreatment process, in diluted acid and steam explosion preprocessing process, due to acid and hot effect, in raw material there is degraded and Decomposition in part carbohydrate, produce a series of materials harmful to follow-up fermentation such as formic acid, acetic acid, wherein acetic acid is main fermentation inhibitor, higher (the biotechnology journal of content, 2014,30:368-380).The inhibitor that lignocellulose pre-treatment produces can affect the normal growth of yeast and follow-up fermenting process.Often cause sugared loss and the increase of production cost for reducing some detoxification strategies that the impact of inhibitor takes, affected the economy of producing.Therefore, seed selection has strong inhibitor tolerance, comprises that the Wine brewing yeast strain with strong acetic acid tolerance is very important for improving cellulosic ethanol productive rate.
By finding the gene relevant to cell stress tolerance, S. cervisiae is carried out to metabolic engineering, can improve the cytoactive of cell under environment-stress condition, thereby improve fermentation efficiency.YHB1 gene participates in flavohemoglobin (the Proceedings of the National Academy of Sciences that coding suppresses nitrogen protoxide toxicity; 1992; 89:5015-5019), flavohemoglobin can Cell protection be avoided nitrification and is coerced pressure.The cell that has knocked out YHB1 gene cannot be removed nitrogen protoxide; flavohemoglobin all can be avoided nitrogen protoxide and nitrate stress effect (Proceedings of the National Academy of Sciences by Cell protection under anaerobic and aerobic conditions; 2000,97:4672-4676).In addition, flavohemoglobin is not only coerced relevantly to nitrogenize, and it is also having remarkable effect (Journal of Biological Chemistry, 2005,280:7645-7653) eliminating aspect oxidative stress.But the research of this gene aspect ethanol and acetic acid tolerance is little, do not see the report that can improve acetic acid tolerance about the expression in this gene at present.
Summary of the invention
The object of the invention is to build a strain and have the recombinant Saccharomyces cerevisiae bacterial strain of acetic acid tolerance, thus can be under high-concentration acetic acid inhibition existence condition rapidly and efficiently carry out ethanol fermentation.
The present invention relates to the recombinant Saccharomyces cerevisiae bacterial strain (4126-YHB1) that a strain has acetic acid tolerance, this strain classification called after Saccharomyces cerevisiae, registering on the books of bacterial strain is numbered CGMCC No. 8725, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preservation date is on January 15th, 2014.
Described strain gene derives from laboratory pattern Saccharomyces Cerevisiae in S 288c, and the YHB1 gene that pcr amplification is obtained is connected to pHO composing type integrating expression vector, proceeds to industrial saccharomyces cerevisiae after linearizing, realizes integrative gene expression.
The described recombinant Saccharomyces cerevisiae bacterium (4126-YHB1) with resistance to acetic acid crosses expression flavohemoglobin YHB1 gene, and the GenBank accession number of YHB1 gene order is NC_001139.PGK1 promoter sequence GenBank accession number is FJ415226.1, and CYC1 terminator sequence GenBank accession number is EF210198.1.By genetic engineering means the HO of yeast integrative vector (NCBI:#AF324728, the David J.Stillman of Utah university of the U.S. is so kind as to give; Nucleic acids research, 2001,29:e59) basis on connect PGK1 strong promoter and CYC1 terminator forms pHO constitutive expression carrier (Applied Energy, 2012,110:33-40).Then YHB1 gene pcr amplification being obtained is connected between PGK1 promotor and CYC1 terminator, and after linearizing, electricity is transduceed in industrial saccharomyces cerevisiae 4126, inserts crossing of gene and expresses.
The present invention adopts the pHO constitutive expression carrier of yeast saccharomyces cerevisiae to carry out the integrative gene expression of recombinant bacterial strain, pHO carrier can carry out integrative gene expression in the HO site of yeast saccharomyces cerevisiae by the mode of homologous recombination, and this carrier contains Geneticin G418 resistance marker and can screen.
Useful achievement of the present invention is: recombinant Saccharomyces cerevisiae 4126-YHB1 of the present invention can contain under the condition of high-concentration acetic acid and carries out ethanol fermentation rapidly and efficiently in substratum, in the time carrying out cellulose alcoholic fermentation, can save the step of adjusting pH, directly under lower pH condition and under higher acetic acid existence condition, carry out ethanol fermentation.
Accompanying drawing explanation
Fig. 1 is the pcr amplification of YHB1 gene fragment.
Fig. 2 is that the enzyme of carrier is cut checking.
Fig. 3 is that the PCR of recombination yeast identifies.
Fig. 4 is containing the contrast yeast strain 4126-HO of empty carrier and the growth fraction of recombinant Saccharomyces cerevisiae 4126-YHB1 on the solid plate that contains ethanol and acetic acid.
Fig. 5 is contrast yeast 4126-HO and the growth result of recombinant Saccharomyces cerevisiae 4126-YHB1 in the liquid nutrient medium that contains 5g/L acetic acid.
Fig. 6 is contrast yeast 4126-HO and the ethanol fermentation result of recombinant Saccharomyces cerevisiae 4126-YHB1 in the liquid nutrient medium that contains 5g/L acetic acid.
Embodiment
Embodiment 1: structure and the conversion of crossing the recombinant Saccharomyces cerevisiae of expressing flavohemoglobin gene YHB1
The flavohemoglobin gene YHB1 sequence the present invention relates to comes from NCBI public database, and the GenBank accession number of this gene is NC_001139.The promotor of gene PGK1 promotor,, cuts the mode of connection and insert YHB1 gene between PGK1 promotor and CYC1 terminator as terminator with CYC1 terminator by enzyme.Then linear by restriction enzyme site Not I the plasmid enzyme restriction building, be transformed in industrial saccharomyces cerevisiae 4126 and express.
1.1 genes of brewing yeast group DNA extraction
(1) by centrifugal the yeast liquid of incubated overnight, 12000rpm, 2min, removes supernatant;
(2) add 480 μ L TE solution (pH8.0) to precipitation, 20 μ L N,O-Diacetylmuramidases (2mg/mL), put into 37 ℃ of shaking tables 1.5 hours after concussion mixes;
(3) add appropriate RNase A, again put into 37 ℃ of shaking tables 0.5 hour;
(4) from shaking table, take out, add 50 μ L20%SDS solution, 5 μ L Proteinase Ks (concentration is 20 μ g/mL) mix concussion, more than placing 1h in 55 ℃ of water-baths;
(5) the centrifugal liquid collecting adhering to that pipe is covered is to the pipe end; Add 500 μ L phenol: chloroform: second amylalcohol (25:24:1), after concussion mixes, centrifugal 10 minutes of 12000rpm, gets supernatant liquor, turns new pipe;
(6) add equal-volume Virahol, put into-20 ℃ of refrigerators more than 1 hour, precipitation DNA;
(7) centrifugal 10 minutes of 12000rpm, removes supernatant liquor, adds 1mL70% ethanol, washes precipitation 1-2 times, and centrifugal 8 minutes of 12000rpm, abandons supernatant;
(8) 37 ℃ add TE(pH8.0 after dry) 50 μ L dissolving DNAs ,-20 ℃ save backup.
1.2PCR amplification target gene band
Using S.cerevisiae S288c genomic dna as template amplification gene YHB1.Primer sequence is as follows:
PCR reaction system following (25 μ L):
PCR response procedures arranges:
After PCR finishes, whether product detects segment size with agarose gel electrophoresis and meets.
1.3 target fragment enzymes are cut
PCR product is carried out to purifying with DNA purification kit, and then enzyme is cut.Endonuclease reaction system is as follows:
37 ℃ of incubation reaction 2h.
Gel reclaims test kit and reclaims target DNA fragment, and-20 ℃ frozen for subsequent use.
1.4 are connected (25 μ L) with carrier
Carrier after the object fragment preparing is cut back to close with enzyme is connected.
Reaction system is as follows:
Reaction times 2-8 hour in connection instrument, temperature of reaction is 16 ℃
1.5 connect product transforms in bacillus coli DH 5 alpha
1.5.1 competent escherichia coli cell preparation
(1) inoculate bacillus coli DH 5 alpha in 10mL LB liquid nutrient medium, 37 ℃, 200rpm overnight incubation;
(2) in the bacterium liquid of the ratio switching incubated overnight of 1:100 to the fresh LB liquid nutrient medium of 50mL, 37 ℃, 200rpm cultivates 3-4h, to OD
600approximate 0.6;
(3) bacterium liquid is proceeded in the 50mL of precooling on ice centrifuge tube, place 30min on ice, 4 ℃, the centrifugal 5min of 4000rpm;
(4) abandon supernatant, with the 0.1mol/L CaCl of precooling
2solution 15mL suspension cell, places 30min on ice, and 4 ℃, the centrifugal 5min of 4000rpm;
(5) repeat above-mentioned steps;
(6) with the 0.1mol/L CaCl2 solution 2mL suspension cell of precooling, add 30% glycerine of 2mL precooling, mix gently, be distributed into the aliquot of 200 μ L ,-76 ℃ are frozen.
1.5.2 connect the conversion of product
(1) from-76 ℃ of refrigerators, get 200 μ L competent cells, melt on ice;
(2) 10 μ L are connected to products and add in competent cell, mix gently with liquid-transfering gun, place 30min on ice;
(3) competent cell is placed in to 42 ℃ of water-bath heat shocks 90 seconds, is then placed in rapidly the cooling 2min of ice bath;
(4) Xiang Guanzhong adds 800 μ L LB liquid nutrient mediums (not containing microbiotic), mixes rear 37 ℃ of shaking culture 1 hour;
(5) the centrifugal 5min of 4000rpm, remove supernatant, surplus solution is coated containing corresponding antibiotic screening dull and stereotyped upper (penbritin final concentration 100mg/mL), face up and place half an hour, after bacterium liquid dries completely, be inverted culture dish, cultivate 12-16 hour for 37 ℃;
(6) choosing bacterium colony identifies.
1.5.3 transformant plasmid extraction (solution preparation method used derives from Takara(Dalian) website)
(1) picking transforms being cloned in the fresh LB substratum that is added with corresponding microbiotic (penbritin final concentration 100mg/mL) on flat board, 37 ℃, 200rpm overnight incubation;
(2) get 3ml bacterium liquid in Eppendof pipe l.5mL, the centrifugal l min of 12000rpm, abandons supernatant, collecting precipitation thalline;
(3) precipitation is resuspended in the solution S olution I of 100 μ L precoolings on ice to vortex concussion;
(4) add 200 μ l Solution II, put upside down gently centrifuge tube 5 times, solution is mixed;
(5) add the solution S olution III of 150 μ L precoolings on ice, turn upside down for several times, centrifuge tube is placed on ice, place 5min;
(6) 4 ℃, the centrifugal 10min of 12000rpm, stays supernatant;
(7) add equal-volume (l) phenol of approximately 500 μ: chloroform: primary isoamyl alcohol (25:24:1), fully concussion, the centrifugal 10min of 12000rpm, transfers to supernatant in new pipe;
(8) add the Virahol of equal-volume precooling, turn upside down and fully mix, place 30min in-20 ℃, the centrifugal 10min of 12000rpm, abandons supernatant;
(9) by 1mL70% washing with alcohol precipitation, the centrifugal 2min of 12000rpm, removes supernatant liquor, can repeat this washing process, then places the residual alcohol that volatilizees at room temperature;
(10) add and contain in right amount the distilled water of RNaseA or TE solution dissolves plasmid DNA, 37 ℃ of digestion ,-20 ℃ are frozen or be directly used in subsequent experimental.
1.5.4PCR identify transformant
The plasmid of extraction is carried out to PCR reaction, to verify transformant.
Checking primer is as follows:
PCR reaction system following (25 μ L):
PCR response procedures arranges:
After PCR finishes, whether product detects segment size with agarose gel electrophoresis and meets.
The linearizing of 1.6 recombinant plasmids
Because recombinant plasmid needs just can proceed in yeast after linearizing, carry out integrative gene expression, so cut and plasmid is carried out to enzyme cut with Not I enzyme.Endonuclease reaction system is as follows:
37 ℃ of incubation reaction 2h.
Gel reclaims test kit and reclaims target DNA fragment, and-20 ℃ frozen for subsequent use.
Embodiment 2: contain the conversion of the industrial saccharomyces cerevisiae of flavohemoglobin YHB1 gene
Brewing yeast cell method for transformation is as follows:
The preparation of 2.1 yeast saccharomyces cerevisiae Electroporation-competent cells
(1) yeast-inoculated YPD substratum, 30 ℃, 150rpm cultivates 12-14 hour, YPD substratum (1% inoculation) incubated overnight of then transferring new;
(2) next day, is placed on ice at least 15min by culturing bottle, allows bacterium stop growing.By 50mL centrifuge tube, ultrapure water, the Sorbitol Solution USP of 1M is all placed on precooling on ice, in low-temperature condition;
(3) centrifugal collection thalline, mixes thalline (turn upside down and rock, do not blow and beat with liquid-transfering gun) gently with isopyknic ultrapure water, 3000g, and 5min, 4 ℃ of centrifugal collection thalline, abandon supernatant, repeat this step twice;
(4) clean bacterial sediment 2 times with the 1M Sorbitol Solution USP of 20mL precooling, use for the last time 0.5mL, the resuspended yeast cell of 1M Sorbitol Solution USP, is yeast competent cell.
2.2 electricity turn method and obtain transformant
(1) competent cell is divided and be filled in 1.5ml EP centrifuge tube, add 3-5 μ L linearization plasmid, mix gently with liquid-transfering gun, place on ice;
(2) 40ul competent cell and linearization plasmid mixed solution are added in 0.2 electric revolving cup to ice bath 5-10 minute;
(3) yeast parameter " fungi " is set, " Sc02 ", clicks Pulse;
(4) take out electric revolving cup, add rapidly the sorbyl alcohol of 1ml ice bath, blow and beat gently with rifle, transfer in 5ml sterilizing centrifuge tube, 30 ℃ leave standstill, hatch 5h;
(5) centrifugal 5 minutes of 3000g;
(6) concentrated, the YPD-G418 flat board that coating contains 300 μ g/ml;
(7) flat board is cultivated in 30 ℃ of incubators, to yeast transformant, clone grows;
(8) random 12 bacterium colonies of picking are cultivated in YPD liquid nutrient medium (containing the G418 microbiotic of 100 μ g/ml) again, then carry genomic dna, the integration of PCR checking gene.
The results are shown in Figure 3.
Embodiment 3: the dull and stereotyped growth fraction of recombinant Saccharomyces cerevisiae 4126-YHB1 under Different stress factor
(1) the recombination yeast control strain 4126-HO that contains empty carrier and recombinant Saccharomyces cerevisiae 4126-YHB1 are inoculated into and contain 50mL seed culture medium (20g/L glucose, 20g/L peptone, 10g/L yeast soaks powder) 250mL shaking flask in, 30 ℃, 150 revs/min, incubated overnight;
(2) repeating step (1);
(3) get respectively bacterium liquid, survey it at the light absorption value OD of 620nm place, then regulating OD with seed culture medium is all 0.3, and the inoculum size with 10% is inoculated into respectively (same to step (1)) in seed culture medium, cultivates 5h;
(4) get respectively bacterium liquid, survey it at the light absorption value OD of 620nm place, now OD value is all in 1.2 left and right, extremely identical by the distilled water adjusting OD value after sterilizing;
(5) the bacterium liquid after regulating is pressed to 10 times of gradient dilutions;
(6) 2 μ l point samples are respectively in concentration on the YPD flat board of 4.5g/L, 5g/L acetic acid and 8% ethanol (10g/L yeast soaks powder for 20g/L glucose, 20g/L peptone, 20g/L agar powder);
(7) in 30 ℃ of incubators, leave standstill and cultivate, after bacterium colony grows, observe and contrast and take pictures.
The results are shown in Figure 4.
Result: in Fig. 4, result can draw 4126-YHB1 and 4126-HO upgrowth situation indifference in common YPD, illustrated that expressing YHB1 gene can not affect the normal growth of bacterial strain.4126-YHB1 bacterial strain and 4126-HO bacterial strain be also basic indifference of upgrowth situation in 8% ethanol substratum, illustrates that expressing YHB1 gene did not affect the alcohol resistance of bacterial strain.But 4126-YHB1 bacterial strain and 4126-HO bacterial strain are grown all significantly better than control strain 4126-HO bacterial strain in the solid medium that contains 4.5g/L and 5g/L acetic acid, illustrate that expressing YHB1 gene had improved the tolerance of bacterial strain to acetic acid.
Embodiment 4: containing the contrast yeast 4126-HO of empty carrier and recombinant Saccharomyces cerevisiae 4126-YHB1 in the fermentation comparison containing in 5g/L acetic acid substratum
In order to verify the leavening property of recombinant Saccharomyces cerevisiae, investigate the leavening property of recombinant bacterium at the fermention medium that contains high-concentration acetic acid.
(1) contrast yeast strain 4126-HO control strain and recombinant Saccharomyces cerevisiae 4126-YHB1 are inoculated into and contain 50mL seed culture medium (20g/L glucose, 20g/L peptone, 10g/L yeast soaks powder) 250mL shaking flask in, 30 ℃, 150 revs/min, incubated overnight;
(2) repeating step (1);
(3) get respectively bacterium liquid, survey it at the light absorption value OD of 620nm place, then regulating OD with seed culture medium is all 0.3, and the inoculum size with 10% is inoculated into respectively (same to step (1)) in seed culture medium, cultivates 5h;
(4) get respectively bacterium liquid, survey it at the light absorption value OD of 620nm place, now OD value is all in 1.2 left and right, extremely identical by the distilled water adjusting OD value after sterilizing;
(5) be inoculated into fermention medium (10g/L yeast soaks powder for 100g/L glucose, 20g/L peptone, adds 5g/L acetic acid) with 10% inoculum size, at 30 ℃, under the condition of 150 revs/min, ferment relatively.
To unloaded yeast 4126-HO and recombinant Saccharomyces cerevisiae 4126-YHB1 sampling, get respectively part bacterium liquid microplate reader and measure light absorption value and determine the content (Fig. 5) of thalline in the identical time; After remaining bacterium liquid is centrifugal, get supernatant liquor detects residue glucose and ethanol content (Fig. 6) by high performance liquid chromatography, when the concentration of glucose in supernatant liquor is defined as fermentation ends during lower than 1g/L.
Result: as seen from Figure 5, along with the carrying out of fermentation, from 12h, the OD value of crossing expression strain 4126-YHB1 increases fast, in the time of 70 hours, the OD value of 4126-YHB1 strain growth is 2 times of 4126-HO bacterial strain, illustrates that expression strain 4126-YHB1 thalli growth in the fermention medium that contains 5g/L acetic acid was much better than the control strain 4126-HO that contains empty carrier.
By finding out in Fig. 6, cross the spending rate of expression strain 4126-YHB1 glucose and alcohol yied all higher than unloaded bacterial strain 4126-HO, in fermentation 58 hours time, cross expression strain 4126-YHB1 and consumed all glucose, and the consumption of control strain 4126-HO glucose is only less than half.The bacterial strain 4126-YHB1 that these presentation of results are crossed expression flavohemoglobin encoding gene YHB1 has higher acetic acid tolerance.
Claims (2)
1. a strain has an industrial strain of S.cerevisiae strain (Saccharomyces cerevisiae) of acetic acid tolerance, it is characterized in that: registering on the books of described bacterial strain is numbered CGMCC No.8725, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date is on January 15th, 2014.
2. an industrial strain of S.cerevisiae strain with acetic acid tolerance according to claim 1, it is characterized in that: described strain gene derives from laboratory pattern Saccharomyces Cerevisiae in S 288c, the YHB1 gene that pcr amplification is obtained is connected to pHO composing type integrating expression vector, after linearizing, proceed to industrial saccharomyces cerevisiae, realize integrative gene expression.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105154348A (en) * | 2015-08-05 | 2015-12-16 | 大连理工大学 | Method for improving tolerance of brewer's yeast to cellulose hydrolysate inhibitors |
| CN105586280A (en) * | 2014-10-22 | 2016-05-18 | 天津大学 | A use of inositol for enhancing bacterial strain tolerance, a use of genes, an expression vector, a use of the expression vector, a bacterial strain, and a use of the bacterial strain |
| CN105586281A (en) * | 2014-10-22 | 2016-05-18 | 天津大学 | A use of proline for enhancing bacterial strain tolerance, a use of genes, an expression vector, a use of the expression vector, a bacterial strain, and a use of the bacterial strain |
| CN107937297A (en) * | 2017-11-29 | 2018-04-20 | 大连理工大学 | Mortifier stress tolerance saccharomyces cerevisiae more than one plant and preparation method, application |
| CN110904123A (en) * | 2019-01-07 | 2020-03-24 | 广西科学院 | Gene for promoting saccharomyces cerevisiae to produce cane molasses alcohol with high yield and application of gene |
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| CN101434913A (en) * | 2008-12-12 | 2009-05-20 | 华东理工大学 | Wine brewing yeast strain and method for producing ethanol by efficient stalk fermentation |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105586280A (en) * | 2014-10-22 | 2016-05-18 | 天津大学 | A use of inositol for enhancing bacterial strain tolerance, a use of genes, an expression vector, a use of the expression vector, a bacterial strain, and a use of the bacterial strain |
| CN105586281A (en) * | 2014-10-22 | 2016-05-18 | 天津大学 | A use of proline for enhancing bacterial strain tolerance, a use of genes, an expression vector, a use of the expression vector, a bacterial strain, and a use of the bacterial strain |
| CN105586281B (en) * | 2014-10-22 | 2019-01-22 | 天津大学 | Proline is for enhancing purposes, the purposes of gene, expression vector of bacterial strain tolerance and application thereof, bacterial strain and application thereof |
| CN105586280B (en) * | 2014-10-22 | 2019-01-25 | 天津大学 | Inositol is for enhancing purposes, the purposes of gene, expression vector of bacterial strain tolerance and application thereof, bacterial strain and application thereof |
| CN105154348A (en) * | 2015-08-05 | 2015-12-16 | 大连理工大学 | Method for improving tolerance of brewer's yeast to cellulose hydrolysate inhibitors |
| CN105154348B (en) * | 2015-08-05 | 2018-05-04 | 大连理工大学 | It is a kind of to improve method of the saccharomyces cerevisiae to cellulosic hydrolysate mortifier tolerance |
| CN107937297A (en) * | 2017-11-29 | 2018-04-20 | 大连理工大学 | Mortifier stress tolerance saccharomyces cerevisiae more than one plant and preparation method, application |
| CN110904123A (en) * | 2019-01-07 | 2020-03-24 | 广西科学院 | Gene for promoting saccharomyces cerevisiae to produce cane molasses alcohol with high yield and application of gene |
| CN110904123B (en) * | 2019-01-07 | 2021-11-05 | 广西科学院 | Strain for promoting high-yield cane molasses alcohol fermentation and application thereof |
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