CN102329743A - Industrial Saccharomyces cerevisiae strain with low glycerol synthesis and high alcohol tolerance and application thereof - Google Patents

Abstract

The invention provides an industrial Saccharomyces cerevisiae strain with low glycerol synthesis and high alcohol tolerance, namely Saccharomyces cerevisiae FG1. The strain was collected in China Center for Type Culture Collection in Wuhan University, Wuhan 430072, China, on August 1, 2011, and the collection number is CCTCC No: M2011274. The invention provides an excellent industrial Saccharomyces cerevisiae engineering strain with few byproducts and high alcohol tolerance and application thereof, and a method for improving various production properties of the industrial strain, namely the combination of genetic and metabolic engineering and complete genome rearrangement. By the method, various production properties of the Saccharomyces cerevisiae strain such as conversion rate of sugar alcohol, tolerance, fermentation rate and the like can be improved; the improved strain can be used for fermentation production of industrial high-gravity alcohol, the energy consumption is reduced, and the production cost is reduced; and the method can also be used for improving the properties of other industrial microorganisms.

Description

Low glycerine is synthetic, the industrial saccharomyces cerevisiae bacterial strain and the application thereof of high alcohol tolerance

(1) technical field

The present invention relates to that the low glycerine of a strain is synthetic, industrial saccharomyces cerevisiae bacterial strain---yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) FG1 and the application thereof of high alcohol tolerance; And the method for a kind of integrator gene metabolic engineering and full genome rearrangement structure industrial saccharomyces cerevisiae engineering bacteria.

(2) background technology

Alcohol thick mash fermentation is exactly the high gravity fermentation in the fermenting process in simple terms, and being in particular in to produce has following characteristics: 1, high wine part; 2, high osmotic pressure; 3, high yeast number.With regard to ethanol produce, the boundary of different material, different times thick mash fermentation exists tangible difference; The general differentiation as follows: starchy material: ethanol concn is at 14～16% (V/V), and molasses raw material: ethanol concn is at 10～12% (V/V).

Realization alcohol thick mash fermentation technology can greatly improve plant factor, reduces process water, reduce boiling distillation energy consumption and production cost, to the efficient that improves alcohol production and economic and social benefit has important practical significance and using value.Compare with conventional zymamsis, yeast saccharomyces cerevisiae not only is faced with more severe environment-stress (height oozes, high alcohol is coerced etc.) in the thick mash fermentation process, also can reduce the glucose ethanol conversion because of increasing of generating of by products such as glycerine, acetate.Therefore, for reaching the requirement of suitability for industrialized production to technical indicators such as fermentation rate, ethanol production and sugar alcohol transformation efficiencys, seed selection by product industrial saccharomyces cerevisiae bacterial strain synthetic low and that have high patience is the key point that breaks through the thick mash fermentation technical bottleneck.

Glycerine is main by product in the fermentation by saccharomyces cerevisiae production ethanol process, consumes total carbon source of 4%～10% approximately, is used to generate ethanol like these carbon sources, and need not to increase cost and can increase production 1,300,000,000 liters of ethanol global every year.At present; Studied more thorough to yeast cell glycerine pathways metabolism; Applying gene metabolic engineering technology is modified and is transformed glycerine synthetic relevant gene and approach, can effectively reduce the synthetic of glycerine, during the fermentation high sugar, high concentration ethanol etc. is coerced the decline of factor tolerance but transform bacterial strain; Poor growth, and then cause bad phenomenon such as fermentation rate reduction, fermentation period prolongation and ethanol production reduction.To one or a small amount of gene regulating, machine-processed known proterties; Gene metabolic engineering method is to be easier to directly feasibility is rational tactful; But for the complex character that relates to a plurality of genes and regulated and control network thereof (like fermentation proterties such as fermentation rate, tolerances); Gene metabolic engineering technology is difficult to produce a desired effect, even can cause the degeneration decay of bacterial strain key property.At present; Existing Application Research is based on the blindness breeding technique of full genomic level---full genome rearrangement; Effective tolerances such as the anti-acetate of improved strain, resisting high-concentration ethanol, but that other production performances such as the sugar alcohol transformation efficiency of transforming bacterial strain improve is not remarkable, and along with the increase of mash fermentability carbon source; By product is synthetic also to be increased, and has greatly limited the raising of ethanol production.

In sum, can improved strain proterties in a certain respect though use single breeding technique, be difficult to the comprehensive strain excellent of obtained performance, and cause producing the degeneration decay of bacterial strain key property easily.To fundamentally break through the thick mash fermentation technical bottleneck; The comprehensive good Wine brewing yeast strain of obtained performance still need combine different industrial micro breeding strategy advantages, realizes good and bad complementary; The improvement of industrial producing strain is carried out in integrated innovation more effectively and quickly.

(3) summary of the invention

The purpose of this invention is to provide a strain and have synthetic industrial saccharomyces cerevisiae engineering strain and the application thereof with high alcohol tolerance of low by product, and the method for a kind of integrator gene metabolic engineering and the complete multiple production performance of genome rearrangement technique improvement bacterial strain.

The technical scheme that the present invention adopts is:

The low glycerine of one strain is synthetic, industrial saccharomyces cerevisiae bacterial strain---yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) FG1 of high alcohol tolerance; Be preserved in Chinese typical culture collection center, address: China, Wuhan; Wuhan University; 430072, deposit number: CCTCC No:M 2011274, preservation date: on August 1st, 2011.

The invention still further relates to said yeast saccharomyces cerevisiae CCTCC No:M 2011274 and produce the application in the alcohol at the microbiological industry thick mash fermentation.

Concrete, said being applied as: yeast saccharomyces cerevisiae CCTCC No:M 2011274 is seeded to the corn mash fermented liquid with the double-enzyme method preparation, 30～35 ℃ of fermentation 60～80h, fermentation ends secondary fermentation liquid obtains alcohol through separation and purification.

Concrete; Said corn mash fermented liquid can be by conventional double-enzyme method preparation, and specific as follows among the present invention: get Semen Maydis powder, the water that adds 2～3 times of quality is sized mixing; Be heated with stirring to 50～70 ℃; Add high temperature resistant AMS 10U～30U/g Semen Maydis powder, gelatinization liquefaction ℃ is carried out in mixing post-heating to 80～90,100～110 ℃ of insulation 0.5～2h; The gelatinization wine with dregs is cooled to 50～70 ℃, adds saccharifying enzyme 100U～300U/g Semen Maydis powder, and 55～60 ℃ of saccharification 0.5～1.0h promptly get said corn mash fermented liquid.

The invention still further relates to the method for a kind of integrator gene metabolic engineering and full genome rearrangement structure industrial saccharomyces cerevisiae engineering bacteria, said method comprises:

(1) induce Wine brewing yeast strain to produce spore, the separation and purification haploid strains is identified haploid strains mating type, and the haploid strains Y2 of haploid strains Y1 and mating type α that chooses mating type a is as starting strain;

(2) the gene FPS1 of the encoding glycerol translocator of knock-out bacterial strain Y1 and bacterial strain Y2 respectively then; And in the FPS1 site integrative gene expression becomes the glyceraldehyde 3-phosphate dehydro-genase (GAPN) of suis (Streptococcus mutans), monoploid Y1 and Y2 are adopted G418 respectively ^rAnd Zeo ^rThe resistance marker screening obtains monoploid engineering strain: G418 ^rResistant strain YFG1 (MATa, the fps1 Δ:: PGKp-gapN) and Zeo ^rResistant strain YFG2 (MAT α, the fps1 Δ:: PGKp-gapN);

(3) use ultraviolet and EMS that bacterial strain YFG1 and bacterial strain YFG2 are carried out mutagenesis respectively, obtain four sudden change storehouses: YFG1 ultraviolet mutagenesis storehouse, YFG1 EMS mutagenesis storehouse, YFG2 ultraviolet mutagenesis storehouse and YFG2 EMS mutagenesis storehouse;

(4) the single rapidly bacterium colony of ethanol YPD plate screening growth with volumetric concentration 8% carries out full genome rearrangement of the first round; Reset son with the YPD plate screening that contains 300 μ g/mL G418 and 50 μ g/mL Zeocin; After inducing rearrangement to produce the spore broken wall; Carry out second with the single rapidly bacterium colony of the ethanol YPD plate screening of volumetric concentration 12% growth and take turns full genome rearrangement; Reset son with the YPD plate screening that contains 300 μ g/mLG418 and 50 μ g/mL Zeocin, through the thick mash fermentation test of simulation industrial raw material, obtain that low by product is synthetic, the industrial saccharomyces cerevisiae engineering strain of high sugar alcohol transformation efficiency and high ethanol production.

The aminoacid sequence GenBank NP 013057 of said glycerine translocator; Be AAA91091 GenBank number of the aminoacid sequence of the glyceraldehyde 3-phosphate dehydro-genase (GAPN) of said change suis (Streptococcus mutans).

The encoding sox GenBank NM 001181863 of said glycerine translocator; Be L38521 encoding sox GenBank number of the glyceraldehyde 3-phosphate dehydro-genase (GAPN) of said change suis (Streptococcus mutans).

Beneficial effect of the present invention is mainly reflected in: the invention provides a strain and has that by product is low, the good industrial saccharomyces cerevisiae engineering strain and the application thereof of high alcohol tolerance, and a kind of method that improves the multiple production performance of industrial strain---gene metabolic engineering and full genome rearrangement coupling; Utilization this method can improve the multiple production performances such as sugar alcohol transformation efficiency, patience, fermentation rate of Wine brewing yeast strain, and improved strain can be used for industrial performing thick mash alcohol fermentation production, reduces energy consumption, reduces production costs; This method also can be used for the improvement of other industrial microorganism performance.

(4) description of drawings

Fig. 1 is G418 ^rThe FPS1 that knocks out of resistance integrates gapN box synoptic diagram;

Fig. 2 is Zeo ^rThe FPS1 that knocks out of resistance integrates gapN box synoptic diagram;

Fig. 3 is full genome rearrangement schema; Wherein,

Be G418 ^rThe engineering strain YFG1 of resistance,

Be Zeo ^rThe engineering strain YFG2 of resistance;

Fig. 4 is the growth curve of bacterial strain under the ethanol stress conditions; (■,) control strain Y12; (●, zero) engineering strain YFG12; (▲, △) genetically engineered resets sub-FG1; Figure hollow core icon is 0% (v/v) ethanol stress conditions; Solid icon is 10% (v/v) ethanol stress conditions;

Fig. 5 is the thick mash fermentation performance test; (*) starting strain Z87; () control strain Y12; (zero) engineering strain YFG12; (▲) genetically engineered is reset sub-FG1.A: residual sugar; B: ethanol.

(5) embodiment

Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment 1: the acquisition of industrial saccharomyces cerevisiae glycerine metabolic engineering bacterial strain

1, the acquisition of monoploid starting strain

After 30 ℃ of YPD activation, switching goes into to produce the spore substratum with industrial saccharomyces cerevisiae Z87, cultivates 3～7 days at 26 ℃.Microscopically is observed and is collected thalline when its thecaspore generates; After the saline water washing 2 times; Add 700 μ L Tris-HCl (pH8.0; 0.01mol/L), 200 μ L 100mg/mL Snailase solution and 100 μ L 0.1mol/L mercaptoethanols, 120r/min cultivates 16h at 30 ℃, makes the ascus wall release spore that breaks.58 ℃ of deadly vegetative cells of pyroprocessing 15min, centrifugal collection spore, the YPD flat board is coated in dilution, cultivates 2～3 days for 30 ℃, and picking list bacterium colony produces the spore checking after the YPD slant activation, and the person confirms as haploid strains can not to produce the spore.

Bacterial strain to be measured and reference culture BY4741 (mating type α type) (ATCC 201388), BY4742 (mating type a type) (ATCC 201389) are inoculated in the YPD liquid nutrient medium respectively; 30 ℃, 200r/min overnight cultures are got strain cultured solution to be measured and are mixed with two kinds of reference culture nutrient solutions respectively, transfer in fresh YPD liquid nutrient medium; 30 ℃, 100r/min cultivation; Whether microscopy is observed in the culturing process has the dumb-bell shape cell to produce, centrifugal collection behind the 48h, and the dilution coating is produced spore and is cultivated basically; Cultivated 3～7 days for 26 ℃; Microscopy is observed and is had or not ascus to produce, and then thinks like the generation thecaspore and hybridizes successfully, can confirm this monoploid mating type: can be α with its mating type of bacterial strain of a type reference culture BY4742 hybridization; Can be a with its mating type of bacterial strain of α type reference culture BY4741 hybridization.Choose two strain haploid strains Y1 (mating type a type) and Y2 (mating type α type) the haploid strains that sets out, and Y1 and Y2 hybridization are obtained diploid bacterial strain Y12 as the experiment contrast bacterial strain as follow-up breeding.

2, the structure of yeast saccharomyces cerevisiae glycerine metabolic engineering bacterial strain

(1) utilizes the Lithium Acetate conversion method, with G418 ^rThe FPS1 that knocks out of resistance integrates gapN box (Fig. 1) and transforms haploid strains Y1 (mating type a type), obtains engineering haploid strains YFG1 with the YPD plate screening that contains 300 μ g/mL G418, genotype be (MAT a, the fps1 Δ:: PGKp-gapN);

(2) utilize the Lithium Acetate conversion method, with Zeo ^rThe FPS1 that knocks out of resistance integrates gapN box (Fig. 2) and transforms haploid strains Y2 (mating type α type), obtains engineering haploid strains YFG2 with the YPD that contains 50 μ g/mL Zeocin dull and stereotyped (pH7.0) screening, genotype be (MAT α, the fps1 Δ:: PGKp-gapN);

(3) YFG1 and YFG2 are hybridized; The two is after 30 ℃ of liquid YPD cultivate 24h; The YPD that contains 300 μ g/mL G418 and 50 μ g/mL Zeocin two anti-dull and stereotyped (pH7.0) is coated in mixed culture centrifugal collection after fresh liquid YPD cultivates 2 days, dilution, obtains diploid gene engineering strain YFG12 (MAT a/ α; The fps1 Δ:: PGKp-gapN, the fps1 Δ:: PGKp-gapN).

Wherein, The FPSA and the FPSB that knock out in the step (1) (2) in the FPS1 integration gapN box are that homology is integrated the district; Clone in yeast chromosomal dna; FPSA is that glycerine translocator encoding sox FPS1 ATG upstream from start codon-242 arrives-51 sequence fragments, and FPSB is 1906 to 2124 sequence fragments of glycerine translocator encoding sox FPS1; The PGK promotor (PGKp) and the PGK terminator (PGKt) that between FPSA and FPSB, insert are cloned in yeast chromosomal dna; Between promotor and terminator, insert the glyceraldehyde 3-phosphate dehydro-genase encoding sox gapN that clones in becoming the suis chromosomal DNA, and change the initiator codon TTG of gapN gene into ATG;

Wherein knock out FPS1 in the step (1) and integrate the G418 resistant gene (G418 in the gapN box ^r) as selection markers, clone in plasmid pUG6 (purchasing in Invitrogen).

Wherein knock out FPS1 in the step (2) and integrate the Zeocin resistant gene (Zeo in the gapN box ^r) as selection markers, clone in plasmid pPICZ α A (purchasing in Invitrogen).

Embodiment 2: engineering strain is implemented full genome rearrangement

The full genome rearrangement of flow implementation as shown in Figure 3, concrete steps are following:

1,1% (v/v) EMS mutagenic compound respectively to genetically engineered haploid strains YFG1 (MAT a, the fps1 Δ:: PGKp-gapN) and YFG2 (MAT α, the fps1 Δ:: PGKp-gapN) handle 30～120min; Every 30min sampling adds 5% (w/v) hypo-elimination EMS and pollutes; The centrifugal 5min of 4000rpm collects thalline, the saline water washed twice, and the gradient dilution coating contains 8% (v/v) alcoholic acid YPD plate; Cultivated the eugonic single bacterium colony of picking 3 days for 30 ℃;

2, be 40cm in distance; Power is respectively to genetically engineered haploid strains YFG1 (MAT a under the uv lamp of 15w; The fps1 Δ:: PGKp-gapN) and YFG2 (MAT α; The fps1 Δ:: PGKp-gapN) carry out ultraviolet mutagenesis, it is dull and stereotyped that every separated 1min sampling dilution coating contains 8% (v/v) alcoholic acid YPD plate, processing 5min.The treating processes all operations must carry out under red light, prevents the light reparation, and flat board is wrapped with the cloth of black, places 30 ℃ to cultivate the eugonic single bacterium colony of picking 3 days;

3, the mutagenic strain that step 1 and 2 is obtained is hybridized full genome rearrangement; Respectively behind 30 ℃ of activation 24h of liquid YPD; Mixed culture centrifugal collection after fresh liquid YPD cultivates 2 days, dilution are coated the YPD that contains 300 μ g/mL G418 and 50 μ g/mL Zeocin two anti-dull and stereotyped (pH7.0) and are cultivated for 30 ℃ and screened single bacterium colony in 3 days and be and reset son;

4, produce rearrangement 3～7 days of 26 ℃ of culturing step 3 pickings of spore substratum, behind the saline water washing thalline, add 700 μ L Tris-HCl (pH8.0; 0.01mol/L), 200 μ L 100mg/mL Snailase solution and 100 μ L 0.1mol/L mercaptoethanols, 120r/min cultivates 16h at 30 ℃, makes the ascus wall release spore that breaks; 58 ℃ of deadly vegetative cells of pyroprocessing 15min; Centrifugal collection spore, dilution are coated and are contained 12% (v/v) alcoholic acid YPD plate flat board, cultivate 3 days for 30 ℃; Behind the picking list bacterium colony, repeating step 3 carries out second and takes turns rearrangement;

5, the rearrangement of step 4 acquisition is sub, carry out the thick mash fermentation test, double-enzyme method preparation corn mash fermented liquid (containing glucose 250g/L approximately), rearrangement is sub by centrifugal collection behind 30 ℃ of cultivations of YPD 15h, by thalline OD after the inoculation ₆₀₀=1.0 amount is seeded to the Erlenmeyer flask that 260g corn mash fermented liquid is housed; 35 ℃ of fermentation 72h; Adopt porousness resin anion(R.A) post (Aminex HPX-87Hcolumn) through high-performance liquid chromatogram determination mash glucose and ethanol content; It is the highest to reset sub-FG1 (being CCTCC No:M 2011274) ethanol, and output reaches 117g/L, and residual sugar is lower than 5g/L.

Embodiment 3: the engineering strain performance test

1, the growth measurement under the high concentration ethanol stress conditions

Cultivate starting strain Z87, control strain Y12, engineering strain YFG12, genetically engineered and reset sub-FG1 with containing 30 ℃ of 0% and 10% (v/v) ethanol YPD liquid nutrient mediums respectively, different period sampling and measuring bacterial strain dry weights are calculated the bacterial strain maximum specific growth rate.As shown in Figure 4, containing under the 0% alcoholic acid YPD culture condition, four strain growths do not have significant difference, and lag phase is about 0.4h; Containing under 10% (v/v) alcoholic acid YPD culture condition, four bacterial strain lag phases all extend to about 10h, and genetically engineered resets that sub-FG1 growth is the fastest, and maximum specific growth rate improves 11.4% than Z87 and Y12, than YFG1 raising 21.9% (p＜0.05).

2, thick mash fermentation performance test

The simulation industrial raw material is reset sub-FG1 (being CCTCC No:M 2011274) to control strain Y12, engineering strain YFG12, genetically engineered and is carried out the thick mash fermentation test.Double-enzyme method preparation corn mash fermented liquid (containing glucose 250g/L approximately): get Semen Maydis powder 5kg; Adding 10L water sizes mixing; Be heated with stirring to 60 ℃; Add (20000U/mL) 3.8mL of high temperature resistant AMS (Henan Tianguan Enterprise Group Co), mixing post-heating to 90 ℃ carries out gelatinization liquefaction, 105 ℃ of insulation 1h; The gelatinization wine with dregs is cooled to 60 ℃, adds saccharifying enzyme (Henan Tianguan Enterprise Group Co) (10 ⁵U/mL) 11.3mL, behind 55 ℃ of insulation saccharification 1h, packing 260g to 500mL Erlenmeyer flask.

Bacterial strain is cultivated centrifugal collection behind the 15h by 30 ℃ of YPD, by inoculation back thalline OD ₆₀₀=1.0 amount is seeded to the Erlenmeyer flask that 200mL corn mash filtrating is housed, and 35 ℃ of fermentation 72h adopt porousness resin anion(R.A) post (Aminex HPX-87H column) through high-performance liquid chromatogram determination mash glucose, ethanol, glycerine and acetic acid content.Genetically engineered is reset sub-FG1 fermentation rate the fastest (Fig. 5), and fermentation 72h residual sugar is lower than 5g/L, has accomplished fermentation, and the YFG12 fermentation rate is slower, and have a relatively poor ethanol tolerance with it consistent.During fermentation ends 72h in the mash each component content see table 1: it is the highest to reset sub-FG1 ethanol; Improve 10.3%, 7.9% and 11.1% than starting strain Z87, control strain Y12 and engineering strain YFG12 respectively; The sugar alcohol transformation efficiency improves 3.3% than Z87 and Y12, does not have significant difference with YFG12; The glucose glycerol conversion yield of resetting sub-FG1 and YFG12 reduces by 14.7% than starting strain Z87, and comparison reduces by 15.5% according to bacterial strain Y12, and glucose acetate transformation efficiency reduces by 24.3% than starting strain Z87, and comparison reduces by 24.6% according to bacterial strain Y12.It is thus clear that under the thick mash fermentation condition, genetically engineered is reset sub-FG1, and to have glycerine, acetate by product synthetic low, fermentation rate is fast, and the sugar alcohol transformation efficiency is high, the high multiple good production performance of ethanol production.

Table 1: strain fermentation performance measurement

Claims (5)

1. industrial saccharomyces cerevisiae bacterial strain---yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) FG1 of synthetic, the high alcohol tolerance of the low glycerine of a strain; Be preserved in Chinese typical culture collection center, address: China, Wuhan; Wuhan University; 430072, deposit number: CCTCC No:M2011274, preservation date: on August 1st, 2011.

2. the said yeast saccharomyces cerevisiae CCTCC of claim 2 No:M 2011274 produces the application in the alcohol at the microbiological industry thick mash fermentation.

3. application as claimed in claim 3 is characterized in that said being applied as: yeast saccharomyces cerevisiae CCTCC No:M 2011274 is seeded to the corn mash fermented liquid, and 30～35 ℃ of fermentation 60～80h, fermentation ends secondary fermentation liquid obtains alcohol through separation and purification.

4. application as claimed in claim 4; It is characterized in that said corn mash fermented liquid makes as follows: get Semen Maydis powder; Add 2～3 times of quality water and size mixing, be heated with stirring to 50～70 ℃, add high temperature resistant AMS 10U～30U/g Semen Maydis powder; Gelatinization liquefaction ℃ is carried out in mixing post-heating to 80～90,100～110 ℃ of insulation 0.5～2h; The gelatinization wine with dregs is cooled to 50～70 ℃, adds saccharifying enzyme 100U～300U/g Semen Maydis powder, and 55～60 ℃ of saccharification 0.5～1.0h promptly get said corn mash fermented liquid.

5. an integrator gene metabolic engineering and full genome rearrangement make up the method for industrial saccharomyces cerevisiae engineering bacteria, and said method comprises:

(1) induce Wine brewing yeast strain to produce spore, the separation and purification haploid strains is identified haploid strains mating type, and the haploid strains Y2 of haploid strains Y1 and mating type α that chooses mating type a is as starting strain;

(2) the gene FPS1 of the encoding glycerol translocator of knock-out bacterial strain Y1 and bacterial strain Y2 respectively then; And in the FPS1 site integrative gene expression becomes the glyceraldehyde 3-phosphate dehydro-genase (GAPN) of suis (Streptococcus mutans), monoploid Y1 and Y2 are adopted G418 respectively ^rAnd Zeo ^rThe resistance marker screening obtains monoploid engineering strain: G418 ^rResistant strain YFG1 and Zeo ^rResistant strain YFG2;

(3) use ultraviolet and EMS that bacterial strain YFG1 and bacterial strain YFG2 are carried out mutagenesis respectively, obtain four sudden change storehouses: YFG1 ultraviolet mutagenesis storehouse, YFG1EMS mutagenesis storehouse, YFG2 ultraviolet mutagenesis storehouse and YFG2EMS mutagenesis storehouse;

(4) the single rapidly bacterium colony of ethanol YPD plate screening growth with volumetric concentration 8% carries out full genome rearrangement of the first round; Reset son with the YPD plate screening that contains 300 μ g/mL G418 and 50 μ g/mL Zeocin; After inducing rearrangement to produce the spore broken wall; Carry out second with the single rapidly bacterium colony of the ethanol YPD plate screening of volumetric concentration 12% growth and take turns full genome rearrangement; Reset son with the YPD plate screening that contains 300 μ g/mLG418 and 50 μ g/mL Zeocin, through the thick mash fermentation test of simulation industrial raw material, obtain that low by product is synthetic, the industrial saccharomyces cerevisiae engineering strain of high sugar alcohol transformation efficiency and high ethanol production.

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