CN103114048A - Method for constructing industrial saccharomyces cerevisiae engineering bacteria by integrating metabolism and rearrangement of gene - Google Patents

Abstract

The invention provides a method for constructing industrial saccharomyces cerevisiae engineering bacteria by integrating metabolism and rearrangement of gene and further provides an excellent industrial saccharomyces cerevisiae engineering bacterial strain obtained by the method disclosed by the invention and provided with the advantages of low by-product and high alcohol resistance as well as an application of the excellent industrial saccharomyces cerevisiae engineering bacterial strain. Due to the adoption of the method disclosed by the invention, a plurality of production properties such as the sugar-alcohol conversion rate, the resistance and fermentation rate of the saccharomyces cerevisiae bacterial strain can be improved; the improved bacterial strain can be applied to the fermentation and the production of industrial thick mash alcohol, so that the energy consumption is reduced and the production cost is reduced; and the method disclosed by the invention can be used for improving properties of other industrial microorganisms.

Description

Integrator gene metabolism and the method for resetting the structure industrial yeast

The application is that application number 201110293244.X, name are called the dividing an application of patent of invention of " low glycerine is synthetic, an industrial strain of S.cerevisiae strain and the application thereof of high alcohol tolerance ".

(1) technical field

The present invention relates to a kind of method that integrator gene metabolic engineering and full genome rearrangement build industrial yeast.

(2) background technology

Alcohol thick mash fermentation is exactly the high gravity fermentation in 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 Alcohol Production, the boundary of different material, different times thick mash fermentation exists obvious difference; General differentiation is as follows: starchy material: ethanol concn is 14～16%(V/V), and molasses raw material: ethanol concn is 10～12%(V/V).

Realize the alcohol thick mash fermentation technology, can greatly improve plant factor, reduce 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 severeer 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 the by products such as glycerine, acetic acid.Therefore, for reaching suitability for industrialized production to the requirement of the technical indicators such as fermentation rate, ethanol production and sugar alcohol transformation efficiency, the synthetic low and an industrial strain of S.cerevisiae strain that have high patience of seed selection by product is the key point that breaks through the thick mash fermentation technical bottleneck.

Glycerine is main by product in fermentation by saccharomyces cerevisiae production ethanol process, approximately consumes total carbon source of 4%～10%, is used for generating ethanol as 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, research and comparison is thorough to yeast cell glycerol metabolism approach, applying gene metabolic engineering technology is modified and is transformed glycerine synthetic relevant gene and approach, can effectively reduce the synthetic of glycerine, but the transformation bacterial strain descends to stress factors tolerances such as high sugar, high concentration ethanols during the fermentation, poor growth, and then cause the bad phenomenon such as fermentation rate reduction, fermentation period prolongation and ethanol production reduction.For one or a small amount of gene regulating, the known proterties of mechanism, gene metabolic engineering method is to be easier to and direct feasibility rationality strategy, but for the complex character that relates to a plurality of genes and regulated and control network thereof (as fermentation proterties such as fermentation rate, tolerances), gene metabolic engineering technology is difficult to produce a desired effect, and even can cause the degeneration decay of bacterial strain key property.At present, the blindness breeding technique based on full genomic level is used in existing research---full genome rearrangement, the tolerances such as the anti-acetic acid of effective improved strain, resisting high-concentration ethanol, but other production performances such as the sugar alcohol transformation efficiency of transformation bacterial strain improve 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 although use single breeding technique, be difficult to obtain the comprehensive strain excellent of performance, and easily cause producing the degeneration decay of bacterial strain key property.To fundamentally break through the thick mash fermentation technical bottleneck, obtain the comprehensive good Wine brewing yeast strain of performance, still need in conjunction with different industrial micro breeding strategy advantages, realize 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

A kind of method that the purpose of this invention is to provide integrator gene metabolic engineering and the complete multiple production performance of genome rearrangement technique improvement bacterial strain.

The technical solution used in the present invention is:

A kind of integrator gene metabolic engineering and full genome rearrangement build the method for industrial yeast, and described method comprises:

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

(2) then distinguish the gene FPS1 of the encoding glycerol translocator of knock-out bacterial strain Y1 and bacterial strain Y2, and become the glyceraldehyde 3-phosphate dehydro-genase (GAPN) of suis (Streptococcus mutans) at FPS1 site integrative gene expression, monoploid Y1 and Y2 are adopted respectively G418 ^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 respectively ultraviolet and EMS to carry out mutagenesis to bacterial strain YFG1 and bacterial strain YFG2, obtain four mutation libraries: YFG1 ultraviolet mutagenesis storehouse, YFG1EMS mutagenesis storehouse, YFG2 ultraviolet mutagenesis storehouse and YFG2EMS mutagenesis storehouse;

(4) growing rapidly with the ethanol YPD plate screening of volumetric concentration 8%, single bacterium colony 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, with the growth of the ethanol YPD plate screening of volumetric concentration 12% rapidly single bacterium colony carry out second 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, thick mash fermentation test by the simulation industrial raw material, obtain low by product synthetic, the industrial yeast strain of high sugar alcohol transformation efficiency and high ethanol production.

The aminoacid sequence GenBank NP_013057 of described glycerine translocator; Be AAA91091 No. GenBank of the aminoacid sequence of the glyceraldehyde 3-phosphate dehydro-genase (GAPN) of described change suis (Streptococcus mutans).

The encoding gene GenBank NM_001181863 of described glycerine translocator; The encoding gene of the glyceraldehyde 3-phosphate dehydro-genase (GAPN) of described change suis (Streptococcus mutans) is L38521 No. GenBank.

The low glycerine of a strain that is obtained by aforesaid method synthesizes, an industrial strain of S.cerevisiae strain---yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) FG1 of high alcohol tolerance, the Classification And Nomenclature of proposing is: Saccharomyces cerevisiae FG1(MATa/a fps1 Δ:: PGK1-gapN fps1 Δ:: PGK1-gapN), be preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, 430072, deposit number: CCTCC No:M2011274, preservation date: on August 1st, 2011.

Described yeast saccharomyces cerevisiae CCTCC No:M 2011274 can be used for the microbiological industry thick mash fermentation and produces alcohol.Be specially: yeast saccharomyces cerevisiae CCTCC No:M 2011274 is seeded to the corn mash fermented liquid of preparing with double-enzyme method, 30～35 ℃ of fermentation 60～80h, fermentation ends secondary fermentation liquid obtains alcohol through separation and purification.

Concrete, the double-enzyme method preparation routinely of described corn mash fermented liquid, specific as follows in the present invention: as to 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 α-amylase 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 namely get described corn mash fermented liquid.

Beneficial effect of the present invention is mainly reflected in: the invention provides a strain and has that by product is low, good industrial yeast 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; Use this method can improve the multiple production performances such as sugar alcohol transformation efficiency, patience, fermentation rate of Wine brewing yeast strain, 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 schematic diagram;

Fig. 2 is Zeo ^rThe FPS1 that knocks out of resistance integrates gapN box schematic 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) the ethanol stress conditions; Solid icon is 10%(v/v) the 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

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

Embodiment 1: the acquisition of industrial saccharomyces cerevisiae glycerol metabolism engineering strain

1, the acquisition of monoploid starting strain

After 30 ℃ of YPD activation, switching enters 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 physiological saline washing 2 times, add 700 μ L Tris-HCl (pH8.0,0.01mol/L), 200 μ L100mg/mL helicase solution and 100 μ L0.1mol/L mercaptoethanols, 120r/min cultivates 16h at 30 ℃, makes the ascus wall release spore that breaks.58 ℃ of lethal vegetative cells of pyroprocessing 15min, centrifugal collection spore, dilution spread is dull and stereotyped in YPD, cultivates 2～3 days for 30 ℃, and picking list bacterium colony produces the spore checking after the YPD slant activation, and the person is defined as haploid strains can not to produce the spore.

bacterial strain to be measured and reference culture BY4741(mating type α type) (ATCC201388), BY4742(mating type a type) (ATCC201389) be inoculated in respectively the YPD liquid nutrient medium, 30 ℃, the 200r/min overnight incubation, getting strain cultured solution to be measured mixes with two kinds of reference culture nutrient solutions respectively, transfer in fresh YPD liquid nutrient medium, 30 ℃, 100r/min cultivates, whether in culturing process, microscopy is observed has the dumb-bell shape cell to produce, centrifugal collection after 48h, dilution spread produces spore and substantially cultivates, cultivated 3～7 days for 26 ℃, microscopy is observed and is had or not ascus to produce, as produce thecaspore and think and hybridize successfully, can determine 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 types) and Y2(mating type α type) as the haploid strains that sets out of follow-up breeding, and Y1 and Y2 hybridization are obtained diploid bacterial strain Y12 as the experiment contrast bacterial strain.

2, the structure of yeast saccharomyces cerevisiae glycerol metabolism engineering strain

(1) utilize 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), obtain 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) conversion haploid strains Y2(mating type α type), obtain 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, both after liquid YPD30 ℃ of cultivation 24h, mixed culture centrifugal collection after fresh liquid YPD cultivates 2 days, dilution spread is in the two anti-flat boards (pH7.0) of the YPD that contains 300 μ g/mL G418 and 50 μ g/mL Zeocin, obtain 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 step (1) (2) in FPS1 integration gapN box are the Homologous integration district, be cloned in yeast chromosomal dna, FPSA is that glycerine translocator encoding gene FPS1 ATG upstream from start codon-242 arrives-51 sequence fragments, and FPSB is 1906 to 2124 sequence fragments of glycerine translocator encoding gene FPS1; The PGK promotor (PGKp) and the PGK terminator (PGKt) that insert between FPSA and FPSB are cloned in yeast chromosomal dna, insert between promotor and terminator and be cloned in the glyceraldehyde 3-phosphate dehydro-genase encoding gene gapN that becomes the suis chromosomal DNA, and change the initiator codon TTG of gapN gene into ATG;

Wherein knock out the G418 resistant gene (G418 in FPS1 integration gapN box in step (1) ^r) as selection markers, be cloned in plasmid pUG6(and be purchased from Invitrogen).

Wherein knock out the Zeocin resistant gene (Zeo in FPS1 integration gapN box in step (2) ^r) as selection markers, be cloned in plasmid pPICZ α A(and be purchased from 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 as follows:

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

2, be 40cm in distance, power is respectively to genetically engineered haploid strains YFG1(MAT a under the ultraviolet lamp of 15w, the fps1 Δ:: PGKp-gapN) with YFG2(MAT α, the fps1 Δ:: PGKp-gapN) carry out ultraviolet mutagenesis, containing 8%(v/v every 1min sampling dilution spread) the YPD plate of ethanol is dull and stereotyped, processes 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, is placed in 30 ℃ and cultivates 3 days, the eugonic single bacterium colony of picking;

3, step 1 and 2 mutagenic strains that obtain are hybridized full genome rearrangement, respectively after liquid YPD30 ℃ of activation 24h, mixed culture centrifugal collection after fresh liquid YPD cultivates 2 days, dilution spread screened single bacterium colony in 3 days in 30 ℃ of cultivations of the two anti-flat boards (pH7.0) of the YPD that contains 300 μ g/mL G418 and 50 μ g/mL Zeocin and is rearrangement;

4, produce rearrangement 3～7 days of 26 ℃ of culturing step 3 pickings of spore substratum, after physiological saline washing thalline, add 700 μ L Tris-HCl (pH8.0,0.01mol/L), 200 μ L100mg/mL helicase solution and 100 μ L0.1mol/L mercaptoethanols, 120r/min cultivates 16h at 30 ℃, make the ascus wall release spore that breaks, 58 ℃ of lethal vegetative cells of pyroprocessing 15min, centrifugal collection spore, dilution spread is in containing 12%(v/v) the YPD plate of ethanol is dull and stereotyped, cultivated 3 days for 30 ℃, after 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 (approximately containing glucose 250g/L) is reset sub by centrifugal collection after YPD30 ℃ of cultivation 15h, by thalline OD after 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-87H column) through high-performance liquid chromatogram determination mash glucose and ethanol content, resetting sub-FG1(is CCTCC No:M 2011274) ethanol is the highest, 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

Respectively with contain 0% and 10%(v/v) 30 ℃ of ethanol YPD liquid nutrient mediums cultivate starting strain Z87, control strain Y12, engineering strain YFG12, genetically engineereds and reset sub-FG1, 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 YPD culture condition of 0% ethanol, four strain growths are without significant difference, and lag phase is the 0.4h left and right; Containing 10%(v/v) under the YPD culture condition of ethanol, four bacterial strain lag phases all extend to the 10h left and right, and it is the fastest that genetically engineered is reset sub-FG1 growth, maximum specific growth rate improves 11.4% than Z87 and Y12, improves 21.9%(p＜0.05 than YFG1).

2, thick mash fermentation performance test

It is CCTCC No:M 2011274 that the simulation industrial raw material is reset sub-FG1(to control strain Y12, engineering strain YFG12, genetically engineered) carry out the thick mash fermentation test.Double-enzyme method preparation corn mash fermented liquid (approximately containing glucose 250g/L): 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 α-amylase (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, after 55 ℃ of insulation saccharification 1h, packing 260g to 500mL Erlenmeyer flask.

Bacterial strain is cultivated centrifugal collection after 15h by 30 ℃ of YPD, by thalline OD after inoculation ₆₀₀=1.0 amount is seeded to the Erlenmeyer flask that 200mL corn mash filtrate 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 has been completed fermentation lower than 5g/L, and the YFG12 fermentation rate is slower, and to have a relatively poor ethanol tolerance consistent with it.During fermentation ends 72h in mash each component content see Table 1: reset sub-FG1 ethanol the highest, 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, with YFG12 without significant difference; The glucose glycerol conversion yield of resetting sub-FG1 and YFG12 reduces by 14.7% than starting strain Z87, and Y12 reduces by 15.5% than the contrast bacterial strain, and glucose acetic acid transformation efficiency reduces by 24.3% than starting strain Z87, and Y12 reduces by 24.6% than the contrast bacterial strain.As seen, under the thick mash fermentation condition, the sub-FG1 of genetically engineered rearrangement has glycerine, the acetic acid by product is synthetic low, and 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 (1)

1. an integrator gene metabolic engineering and full genome rearrangement build the method for industrial yeast, and described method comprises:

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

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

(3) use respectively ultraviolet and EMS to carry out mutagenesis to bacterial strain YFG1 and bacterial strain YFG2, obtain four mutation libraries: YFG1 ultraviolet mutagenesis storehouse, YFG1 EMS mutagenesis storehouse, YFG2 ultraviolet mutagenesis storehouse and YFG2 EMS mutagenesis storehouse;

(4) growing rapidly with the ethanol YPD plate screening of volumetric concentration 8%, single bacterium colony 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, with the growth of the ethanol YPD plate screening of volumetric concentration 12% rapidly single bacterium colony carry out second and take turns full genome rearrangement, reset son with the YPD plate screening that contains 300 μ g/mL G418 and 50 μ g/mL Zeocin, thick mash fermentation test by the simulation industrial raw material, obtain low by product synthetic, the industrial yeast strain of high sugar alcohol transformation efficiency and high ethanol production.

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