CN103820347B - One strain has an industrial strain of S.cerevisiae strain of Acetate tolerance - Google Patents
One strain has an industrial strain of S.cerevisiae strain of Acetate tolerance Download PDFInfo
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Abstract
One strain has an industrial strain of S.cerevisiae strain (4126-YHB1) of Acetate tolerance, belongs to microbial technology field.This strain classification called after Saccharomyces? cerevisiae, the numbering CGMCC that registers on the books of bacterial strain? 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.The invention discloses the genetically engineered construction process of recombinant bacterial strain 4126-YHB1, comprise the acquisition of gene, the structure of chromosomal integration vector, and growth under bacterial strain acetic acid condition and fermenting experiment.Compared with integrating the control strain of empty carrier, recombinant bacterial strain 4126-YHB1 can carry out quick ethanol fermentation in the substratum containing 5g/L acetic acid, can tolerate the acetic acid of cellulosic hydrolysate middle and high concentration, be the good bacterial strain of ethanol fermentation under environmental stress conditions.
Description
Technical field
The present invention relates to an industrial strain of S.cerevisiae strain (4126-YHB1) that a strain has Acetate tolerance, belong to microbial technology field.
Background technology
Margin due to fossil oil constantly reduces and the factors such as the instability of International Crude Oil, both at home and abroad for renewable and clean energy resource need day by day increase, biofuel is the renewable energy source with good development prospect.Except the general advantage with renewable energy source, biofuel can also alleviate the series of problems caused by fossil oil, especially can reduce the Greenhouse effect caused by gaseous emission.In the eurypalynous biofuel of crowd, alcohol fuel shows huge advantage (AppliedMicrobiologyandBiotechnology, 2009,85:253-263).A kind of excellent fuel is not only by ethanol, is also a kind of excellent fuel qualities activator, has again the fabulous capability of antidetonance simultaneously.China has abundant cellulose biomass resource, if these raw material production alcohol fuels fully can be effectively utilized, both can protection of the environment, can also solve utilize starchy material to produce ethanol produces strives grain with people, strive the drawback on 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 simultaneously, ensure the energy security of country.
In biorefinery of lignocellulose ethanol process, due to Mierocrystalline cellulose, structure fine and close between hemicellulose and xylogen in raw material, be unfavorable for that cellulase is to cellulosic hydrolysis, therefore, lignocellulose has to pass through pre-treatment to improve the accessibility of Mierocrystalline cellulose to cellulase before enzymic hydrolysis.Diluted acid and steam explosion pre-treatment are the pretreatment processs that research is more at present, in diluted acid and steam explosion preprocessing process, due to acid and hot effect, in raw material there is the effect of degraded Sum decomposition in portion carbohydrate, produce a series of material harmful to subsequent 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.Some the detoxification strategies taked for the impact reducing inhibitor often cause the loss of sugar and the increase of production cost, have impact on the economy of production.Therefore, seed selection has strong inhibitor tolerance, comprises the Wine brewing yeast strain with strong Acetate tolerance very important for raising cellulosic ethanol productive rate.
By finding the gene relevant to Cellular stress patience, metabolic engineering being carried out to S. cervisiae, the cytoactive of cell under environmental stress conditions can be improved, thus improve fermentation efficiency.YHB1 gene participates in the flavohemoglobin (ProceedingsoftheNationalAcademyofSciences that coding suppresses nitrogen protoxide toxicity; 1992; 89:5015-5019), flavohemoglobin can coerce pressure from nitrification by Cell protection.The cell having knocked out YHB1 gene cannot remove nitrogen protoxide; flavohemoglobin under anaerobic and aerobic conditions all can Cell protection from nitrogen protoxide and nitrate stress effect (ProceedingsoftheNationalAcademyofSciences; 2000,97:4672-4676).In addition, flavohemoglobin is not only coerced relevant to nitrogenize, and it also has remarkable effect (JournalofBiologicalChemistry, 2005,280:7645-7653) in elimination oxidative stress.But the research of this gene in ethanol and Acetate tolerance is little, does not see the report about can improve Acetate tolerance in the expression of this gene at present.
Summary of the invention
The object of the invention is to the recombinant Saccharomyces cerevisiae bacterial strain that structure one strain has Acetate tolerance, thus can 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 Acetate tolerance, this strain classification called after Saccharomycescerevisiae, registering on the books of bacterial strain is numbered CGMCCNo.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 obtained by pcr amplification is connected to pHO composing type integrating expression vector, proceeds to industrial saccharomyces cerevisiae, realize integrative gene expression after linearizing.
Described recombinant Saccharomyces cerevisiae bacterium (4126-YHB1) the process LAN flavohemoglobin YHB1 gene with resistance to acetic acid, the GenBank accession number of YHB1 gene order is NC_001139.PGK1 promoter sequence GenBank accession number is FJ415226.1, CYC1 terminator sequence GenBank accession number is EF210198.1.By genetic engineering means yeast HO integrative vector (NCBI:#AF324728, Utah university of U.S. DavidJ.Stillman is so kind as to give; Nucleicacidsresearch, 2001,29:e59) basis on connect PGK1 strong promoter and CYC1 terminator and form pHO constitutive expression carrier (AppliedEnergy, 2012,110:33-40).Then the YHB1 gene that pcr amplification obtains is connected between PGK1 promotor and CYC1 terminator, after linearizing, electricity is transduceed in industrial saccharomyces cerevisiae 4126, carries out the process LAN inserting gene.
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 by the mode of homologous recombination in the HO site of yeast saccharomyces cerevisiae, 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 in the medium containing the ethanol fermentation carried out under the condition of high-concentration acetic acid rapidly and efficiently, when carrying out cellulose alcoholic fermentation, the step of adjustment pH can be saved, 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 the digestion verification of carrier.
Fig. 3 is the PCR qualification of recombination yeast.
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 containing ethanol and acetic acid comparatively.
Fig. 5 is contrast yeast 4126-HO and the growth result of recombinant Saccharomyces cerevisiae 4126-YHB1 in the liquid nutrient medium containing 5g/L acetic acid.
Fig. 6 is the ethanol fermentation result of contrast yeast 4126-HO and recombinant Saccharomyces cerevisiae 4126-YHB1 in the liquid nutrient medium containing 5g/L acetic acid.
Embodiment
Embodiment 1: the structure of the recombinant Saccharomyces cerevisiae of process LAN flavohemoglobin gene YHB1 and conversion
The flavohemoglobin gene YHB1 sequence that the present invention relates to comes from NCBI public database, and the GenBank accession number of this gene is NC_001139.The promotor PGK1 promotor of gene, with CYC1 terminator as terminator, the mode of being cut connection by enzyme inserts YHB1 gene between PGK1 promotor and CYC1 terminator.Then pass through restriction enzyme site NotI linear for the plasmid enzyme restriction built, be transformed in industrial saccharomyces cerevisiae 4126 and express.
1.1 genes of brewing yeast group DNA extraction
(1) by centrifugal for the yeast liquid of incubated overnight, 12000rpm, 2min, remove supernatant;
(2) add 480 μ LTE solution (pH8.0) to precipitation, 20 μ L N,O-Diacetylmuramidases (2mg/mL), after concussion mixing, put into 37 DEG C of shaking tables 1.5 hours;
(3) add appropriate RNaseA, again put into 37 DEG C of shaking tables 0.5 hour;
(4) take out from shaking table, add 50 μ L20%SDS solution, 5 μ L Proteinase Ks (concentration is 20 μ g/mL) mixing concussion, places more than 1h in 55 DEG C of water-baths;
(5) centrifugal liquid collecting pipe being covered attachment is at the bottom of pipe; Add 500 μ L phenol: chloroform: second amylalcohol (25:24:1), after concussion mixing, centrifugal 10 minutes of 12000rpm, gets supernatant liquor, turns new pipe;
(6) add equal-volume Virahol, put into-20 DEG C of refrigerators more than 1 hour, precipitation DNA;
(7) centrifugal 10 minutes of 12000rpm, removes supernatant liquor, adds 1mL70% ethanol, wash precipitation 1-2 times, and centrifugal 8 minutes of 12000rpm, abandons supernatant;
TE(pH8.0 is added after (8) 37 DEG C of dryings) 50 μ L dissolving DNAs ,-20 DEG C save backup.
1.2PCR amplification target gene band
Using S.cerevisiaeS288c genomic dna as template amplification gene YHB1.Primer sequence is as follows:
PCR reaction system following (25 μ L):
PCR response procedures is arranged:
After PCR terminates, whether product agarose gel electrophoresis detects segment size and meets.
1.3 target fragment enzymes are cut
PCR primer DNA purification kit is carried out purifying, and then enzyme is cut.Endonuclease reaction system is as follows:
37 DEG C of incubation reaction 2h.
Gel reclaims test kit and reclaims target DNA fragment, and-20 DEG C frozen for subsequent use.
1.4 are connected with carrier (25 μ L)
Carrier after the object fragment prepared being 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 DEG C
1.5 connect product transforms in bacillus coli DH 5 alpha
1.5.1 competent escherichia coli cell preparation
(1) bacillus coli DH 5 alpha is inoculated in 10mLLB liquid nutrient medium, 37 DEG C, 200rpm overnight incubation;
(2) in 1:100 ratio switching incubated overnight bacterium liquid in the fresh LB liquid nutrient medium of 50mL, 37 DEG C, 200rpm cultivates 3-4h, to OD
600approximate 0.6;
(3) bacterium liquid is proceeded in the 50mL centrifuge tube of precooling on ice, place 30min on ice, 4 DEG C, the centrifugal 5min of 4000rpm;
(4) supernatant is abandoned, with the 0.1mol/LCaCl of precooling
2solution 15mL suspension cell, places 30min on ice, 4 DEG C, the centrifugal 5min of 4000rpm;
(5) above-mentioned steps is repeated;
(6) with the 0.1mol/LCaCl2 solution 2mL suspension cell of precooling, add 30% glycerine of 2mL precooling, mix gently, be distributed into the aliquot of 200 μ L ,-76 DEG C frozen.
1.5.2 the conversion of product is connected
(1) from-76 DEG C of refrigerators, 200 μ L competent cells are got, thawed on ice;
(2) 10 μ L being connected product adds in competent cell, mixes gently, place 30min on ice with liquid-transfering gun;
(3) competent cell is placed in 42 DEG C of water-bath heat shocks 90 seconds, is then placed in rapidly ice bath and cools 2min;
(4) Xiang Guanzhong adds 800 μ LLB liquid nutrient mediums (not containing microbiotic), mixes rear 37 DEG C of shaking culture 1 hour;
(5) the centrifugal 5min of 4000rpm, remove supernatant, surplus solution is coated containing corresponding antibiotic screening flat board 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 DEG C;
(6) choose bacterium colony to identify.
1.5.3 transformant plasmid extraction (solution preparation method used derives from Takara(Dalian) website)
(1) being cloned on picking transformation plate is added with in the fresh LB of corresponding microbiotic (penbritin final concentration 100mg/mL), 37 DEG C, 200rpm overnight incubation;
(2) get 3ml bacterium liquid in l.5mLEppendof pipe, the centrifugal lmin of 12000rpm, abandons supernatant, collecting precipitation thalline;
(3) be resuspended in the solution S olutionI of 100 μ L precoolings on ice by precipitation, vortex shakes;
(4) add 200 μ lSolutionII, put upside down centrifuge tube gently 5 times, solution is mixed;
(5) add the solution S olutionIII of 150 μ L precoolings on ice, turn upside down for several times, centrifuge tube is placed on ice, place 5min;
(6) 4 DEG C, the centrifugal 10min of 12000rpm, stays supernatant;
(7) add equal-volume (about 500 μ l) phenol: chloroform: primary isoamyl alcohol (25:24:1), fully shake, the centrifugal 10min of 12000rpm, supernatant is transferred in new pipe;
(8) add the Virahol of equal-volume precooling, fully mixing of turning upside down, places the centrifugal 10min of 30min, 12000rpm in-20 DEG C, abandons supernatant;
(9) by 1mL70% washing with alcohol precipitation, the centrifugal 2min of 12000rpm, removes supernatant liquor, can repeat this washing process, then place the residual alcohol that volatilizees at room temperature;
(11) add appropriate distilled water containing RNaseA or TE solubilize plasmid DNA, 37 DEG C of digestion ,-20 DEG C frozen or be directly used in subsequent experimental.
1.5.4PCR transformant is identified
The plasmid of extraction is carried out PCR reaction, to verify transformant.
Checking primer is as follows:
PCR reaction system following (25 μ L):
PCR response procedures is arranged:
After PCR terminates, whether product agarose gel electrophoresis detects segment size and meets.
The linearizing of 1.6 recombinant plasmids
Because recombinant plasmid just can proceed in yeast after needing linearizing, carry out integrative gene expression, enzyme is carried out to plasmid cut so cut with NotI enzyme.Endonuclease reaction system is as follows:
37 DEG C of incubation reaction 2h.
Gel reclaims test kit and reclaims target DNA fragment, and-20 DEG C frozen for subsequent use.
Embodiment 2: containing 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 DEG C, 150rpm cultivates 12-14 hour, YPD substratum (1% inoculation) incubated overnight of then transferring new;
(2) next day, culturing bottle is placed on ice at least 15min, allows bacterium stop growing.By 50mL centrifuge tube, ultrapure water, the Sorbitol Solution USP of 1M is all placed on precooling on ice, is in low-temperature condition;
(3) collected by centrifugation thalline, mixed gently by thalline (turn upside down and rock, do not blow and beat with liquid-transfering gun) with isopyknic ultrapure water, 3000g, 5min, 4 DEG C of collected by centrifugation thalline, abandon supernatant, repeat this step twice;
(4) clean bacterial sediment 2 times with the 1M Sorbitol Solution USP of 20mL precooling, use 0.5mL for the last time, the resuspended yeast cell of 1M Sorbitol Solution USP, is competent yeast cells.
2.2 electric robin obtain transformant
(1) competent cell is divided be filled in 1.5mlEP 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 the electric revolving cup of 0.2, 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 sterile centrifugation tube, 30 DEG C leave standstill, hatch 5h;
(5) centrifugal 5 minutes of 3000g;
(6) concentrated, the YPD-G418 of coating containing 300 μ g/ml is dull and stereotyped;
(7) cultivated in 30 DEG C of incubators by flat board, to yeast transformant, clone grows;
(8) random picking 12 bacterium colonies cultivate in YPD liquid nutrient medium (the G418 microbiotic containing 100 μ g/ml) again, then carry genomic dna, and PCR verifies the integration of gene.
The results are shown in Figure 3.
Embodiment 3: the plated growth of recombinant Saccharomyces cerevisiae 4126-YHB1 under Different stress factor compares
(1) the recombination yeast control strain 4126-HO containing empty carrier and recombinant Saccharomyces cerevisiae 4126-YHB1 is inoculated into containing 50mL seed culture medium (20g/L glucose, 20g/L peptone, 10g/L yeast leaching powder) 250mL shaking flask in, 30 DEG C, 150 revs/min, incubated overnight;
(2) repeating step (1);
(3) get bacterium liquid respectively, survey it at 620nm place light absorption value OD, then regulate OD to be all 0.3 with seed culture medium, the inoculum size with 10% is inoculated into (same to step (1)) in seed culture medium respectively, cultivates 5h;
(4) get bacterium liquid respectively, survey it at 620nm place light absorption value OD, now OD value is all about 1.2, regulates OD value to identical with the distilled water after sterilizing;
(5) 10 times of gradient dilutions are pressed to the bacterium liquid after adjustment;
(6) 2 μ l point samples are respectively (20g/L glucose, 20g/L peptone, 10g/L yeast leaching powder, 20g/L agar powder) on the YPD flat board of 4.5g/L, 5g/L acetic acid and 8% ethanol in concentration;
(7) quiescent culture in 30 DEG C of incubators, observes and contrasts and take pictures after bacterium colony grows.
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, illustrates that process LAN YHB1 gene can not affect the normal growth of bacterial strain.4126-YHB1 bacterial strain and 4126-HO bacterial strain be upgrowth situation also basic indifference in 8% ethanol substratum, illustrates that process LAN YHB1 gene does not affect the alcohol resistance of bacterial strain.But 4126-YHB1 bacterial strain and 4126-HO bacterial strain grow all significantly better than control strain 4126-HO bacterial strain in the solid medium containing 4.5g/L and 5g/L acetic acid, illustrate that process LAN YHB1 gene improves the tolerance of bacterial strain to acetic acid.
Embodiment 4: comparing containing the fermentation in 5g/L acetic acid substratum containing the contrast yeast 4126-HO of empty carrier and recombinant Saccharomyces cerevisiae 4126-YHB1
In order to verify the leavening property of recombinant Saccharomyces cerevisiae, investigate the leavening property of recombinant bacterium at the fermention medium containing high-concentration acetic acid.
(1) contrast yeast strain 4126-HO control strain and recombinant Saccharomyces cerevisiae 4126-YHB1 are inoculated into containing 50mL seed culture medium (20g/L glucose, 20g/L peptone, 10g/L yeast leaching powder) 250mL shaking flask in, 30 DEG C, 150 revs/min, incubated overnight;
(2) repeating step (1);
(3) get bacterium liquid respectively, survey it at 620nm place light absorption value OD, then regulate OD to be all 0.3 with seed culture medium, the inoculum size with 10% is inoculated into (same to step (1)) in seed culture medium respectively, cultivates 5h;
(4) get bacterium liquid respectively, survey it at 620nm place light absorption value OD, now OD value is all about 1.2, regulates OD value to identical with the distilled water after sterilizing;
(5) be inoculated into fermention medium (10g/L yeast leaching powder, adds 5g/L acetic acid for 100g/L glucose, 20g/L peptone) with the inoculum size of 10%, at 30 DEG C, carry out fermentation under the condition of 150 revs/min and compare.
In the identical time, unloaded yeast 4126-HO and recombinant Saccharomyces cerevisiae 4126-YHB1 is sampled, get part bacterium liquid microplate reader respectively and measure light absorption value to determine the content (Fig. 5) of thalline; Supernatant liquor detects residue glucose and ethanol content (Fig. 6) by high performance liquid chromatography is got, when the concentration of glucose in supernatant liquor is defined as fermentation ends lower than during 1g/L after remaining bacterium liquid is centrifugal.
Result: as seen from Figure 5, along with the carrying out of fermentation, from 12h, the OD value of process LAN bacterial strain 4126-YHB1 increases fast, at 2 times that the OD value of the 70 little strain growths of 4126-YHB1 is constantly 4126-HO bacterial strain, illustrate that process LAN bacterial strain 4126-YHB1 thalli growth in the fermention medium containing 5g/L acetic acid is much better than the control strain 4126-HO containing empty carrier.
As can be seen from Fig. 6, the spending rate of process LAN bacterial strain 4126-YHB1 glucose and alcohol yied are all higher than unloaded bacterial strain 4126-HO, constantly little in fermentation 58, process LAN bacterial strain 4126-YHB1 has consumed all glucose, and the consumption of control strain 4126-HO glucose is only less than half.These results illustrate that the bacterial strain 4126-YHB1 of process LAN flavohemoglobin encoding gene YHB1 has higher Acetate tolerance.
Claims (2)
1. a strain have Acetate tolerance an industrial strain of S.cerevisiae strain (
saccharomycescerevisiae), it is characterized in that: registering on the books of described bacterial strain is numbered CGMCCNo.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 Acetate tolerance according to claim 1, is characterized in that: the process LAN of described bacterial strain
yHB1pcr amplification, in laboratory pattern Saccharomyces Cerevisiae in S 288c, obtains by gene source
yHB1gene is connected to pHO composing type integrating expression vector, proceeds to industrial saccharomyces cerevisiae, realize integrative gene expression after linearizing.
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| 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 |
| CN107937297B (en) * | 2017-11-29 | 2021-04-20 | 大连理工大学 | Multi-inhibitor stress tolerance saccharomyces cerevisiae, and preparation method and application thereof |
| 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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