CN103805673A - Method of producing stalk ethanol by means of mixed fermentation of transgenetic yeast - Google Patents
Method of producing stalk ethanol by means of mixed fermentation of transgenetic yeast Download PDFInfo
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- CN103805673A CN103805673A CN201410071176.6A CN201410071176A CN103805673A CN 103805673 A CN103805673 A CN 103805673A CN 201410071176 A CN201410071176 A CN 201410071176A CN 103805673 A CN103805673 A CN 103805673A
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 90
- 238000000855 fermentation Methods 0.000 title claims abstract description 45
- 230000004151 fermentation Effects 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 39
- 241000235060 Scheffersomyces stipitis Species 0.000 claims abstract description 46
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- 239000008103 glucose Substances 0.000 claims abstract description 45
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- 102000004190 Enzymes Human genes 0.000 claims abstract description 25
- 229940088598 enzyme Drugs 0.000 claims abstract description 25
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- 150000004823 xylans Chemical class 0.000 claims abstract description 21
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 19
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- 239000010902 straw Substances 0.000 claims description 28
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- 238000004519 manufacturing process Methods 0.000 abstract description 16
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- 239000002994 raw material Substances 0.000 abstract description 8
- 230000015556 catabolic process Effects 0.000 abstract description 5
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- 241000192263 Scheffersomyces shehatae Species 0.000 abstract description 2
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- 230000001737 promoting effect Effects 0.000 abstract 1
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- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 20
- 210000005253 yeast cell Anatomy 0.000 description 18
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- 238000012408 PCR amplification Methods 0.000 description 9
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- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 9
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 241000235647 Pachysolen tannophilus Species 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 238000010564 aerobic fermentation Methods 0.000 description 2
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
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- 238000011081 inoculation Methods 0.000 description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
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- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method of producing stalk ethanol by means of mixed fermentation of transgenetic yeast. The transgenetic yeast can respectively degrade cellulose and xylan of stalks to glucose and xylose and ferment glucose and xylose to ethanol without additionally providing cellulase and xylanase. Degradation of cellulose and xylan and fermentation are synchronously carried out. The method disclosed by the invention has the advantages that by means of transgenetic saccharomyces cerevisiae, transgenetic pichia stipitis and transgenetic candida shehatae, synchronous enzyme hydrolysis-fermentation of a stalk cellulose raw material is realized to produce ethanol without additionally adding cellulase and xylanase, thereby greatly lowering the production cost of stalk cellulose ethanol and promoting the progress of replacing petroleum fuel by the stalk cellulose ethanol.
Description
Technical field
The present invention relates to fermentation engineering and genetically engineered field, especially use the mixed fermentation such as transgenosis yeast saccharomyces cerevisiae and transgenosis trunk pichia spp, by cellulose raw materials such as stalks through same stage enzymatic hydrolysis-zymotechnique, be degraded to the method that then glucose, wood sugar are converted into ethanol, described recombinant Saccharomyces cerevisiae can be secreted three kinds of cellulases, transgenosis trunk pichia spp, transgenosis shehatae candida can secrete 3 kinds of zytases, can utilize wood-sugar fermentation to produce ethanol.And above-mentioned bacterial strains is through screening domestication, better to the tolerance of ethanol, acetic acid, furfural.
Background technology
Along with fossil fuel becomes exhaustion by shortage, energy dilemma is the common issue of facing mankind.In June, 2006, China puts into effect " Renewable Energy Development special fund management tentative method ", clearly gives special assistance to the bio-ethanol fuel that development is produced with non-grain resources such as stalks.Exploitation substitutes grain resource producd fibers ethanol, is to solve the high fundamental solution of fuel ethyl hydrate raw material cost.
Chinese patent application 200880119451.X discloses the bacterium of glucose fermentation, seminose and wood sugar simultaneously and has used this bacterium to produce the method for bio-ethanol, but, this technique can not directly be used stalk, can only utilize glucose, wood sugar, seminose, stalk is converted into glucose, and its cost is much larger than being ethanol by conversion of glucose.In addition, the ethanol tolerance of bacterial classification used is low, causes the ethanol content of technique low, and mean concns is only 3.83%.
Chinese patent application 201110135607.7, the processing method of utilizing low-temperature cellulase simultaneous saccharification and fermentation to produce ethanol is disclosed, but, owing to having used cellulase, cause production cost to increase substantially, and, wood sugar can not be utilized, make stalk ethanol conversion yield low, lack the market competitiveness.
Chinese patent application 200710012382.X, discloses a kind of method of enzymolysis straws for producing fuel alcohol, adds the microbial compound enzyme bacterium liquid of stalk weight 1-5%, and available Mierocrystalline cellulose, hemicellulose in stalk are decomposed, and makes it be converted into sugar.But this technology has only been used yeast saccharomyces cerevisiae, can only utilize glucose, stalk overall utilization rate is low; In addition, because composite enzyme bacterium liquid efficiency used is low, need fermentation to reach 10-15 days, growth cycle is long, has increased considerably production cost, has reduced competitive power.
Chinese patent application 201210540604.6, discloses and has a kind ofly prepared the method for bio-ethanol so that alkali is fluffy as raw material, and this technology can only be utilized glucose, and has used low-temperature cellulase.Production cost is increased considerably.
Chinese patent application 201110460248.2, discloses the fermentation process of Mierocrystalline cellulose yeast, two bacterial classification yeast saccharomyces cerevisiae composition and cellulosic ethanol, and owing to having used plurality of enzymes, production cost is high.And yeast saccharomyces cerevisiae used, can only utilize glucose, can not utilize wood sugar, stalk overall utilization rate is low.
The patent ZL200710107687.9 that the inventor proposes, discloses a kind of method of utilizing recombinant Saccharomyces cerevisiae production of cellulosic ethanol, still, the method can only be utilized glucose, can not utilize wood sugar, and, only expressed a kind of zytase, thus can not by degradable xylan be wood sugar.These deficiencies, have limited the competitive power of the method.
At present, produce that straw ethanol cost high subject matter is cellulase and zytase production cost is high, wood sugar does not utilize, yeast is low etc. to the toxic substance tolerance in stalk pre-treatment, need to address these problems, could significantly reduce production costs, make straw ethanol there is the market competitiveness.
Summary of the invention
The object of the present invention is to provide a kind of Mierocrystalline cellulose, xylan that can directly transform stalk is glucose, wood sugar the technique that is ethanol by their fermentations, do not need to provide in addition cellulase, zytase, bacterial strain uses therefor has higher tolerance to acetic acid, furfural in stalk pre-treatment degradation solution, and product ethanol is also had to higher tolerance.
For example, with transgenosis yeast saccharomyces cerevisiae and genetically modified yeast (pichia stipitis) mixed fermentation that utilizes wood sugar, and the cellulose raw material such as same stage enzymatic hydrolysis-fermented stalk produces the technology of ethanol, can realize above-mentioned technique.
Described transgenosis yeast saccharomyces cerevisiae can be secreted three kinds of cellulases: endoglucanase, cellobiohydrolase, beta-glucan glycosides enzyme.The described genetically modified yeast that utilizes wood sugar can be secreted circumscribed β-Isosorbide-5-Nitrae-zytase inscribe β-Isosorbide-5-Nitrae-zytase and xylan glycosides enzyme, and has the ability of utilizing wood sugar to be converted into ethanol.
In the plant celluloses such as stalk, the content of Mierocrystalline cellulose, hemicellulose and xylogen is than being 4:3:3, and the major ingredient of hemicellulose is xylan, is about 85～90%, and xylan hydrolysis product is wood sugar.Alcohol is produced in the wood-sugar fermentation making full use of in cellulosic material, can make the output of the zymamsis of stalk plant fiber element raw material increase by 25% on original basis.Therefore, to utilize wood-sugar fermentation to produce ethanol be to increase substantially stalk overall utilization rate, determine that stalk plant fiber resource produces one of economically viable key factor of ethanol to yeast.
At present found to utilize the bacterial classification of wood sugar to have kind more than 200, but bacterial classification that can xylose fermentation ethanol is with regard to tens kinds, and the bacterial classification that is considered to have most industrial value be shehatae candida (
candida shehatae), pachysolen tannophilus (
pachys-olen tannophilus), pichia stipitis (
pichia stipitis)
Being glucose and xylose by the Mierocrystalline cellulose in stalk and xylan degrading, need cellulase and zytase, and the cost of enzyme accounting for straw ethanol cost >70%, is the key factor of restriction straw ethanol commercialization process.The synergy of cellulase, zytase, removes after hemicellulose parcel, and thoroughly decomposition of cellulose, is degraded to fermentable sugars to greatest extent by it.Produce straw ethanol, conventionally will carry out pre-treatment to remove the parcel of hemicellulose and xylogen, to improve the transformation efficiency of Mierocrystalline cellulose, hemicellulose, in stalk pre-treatment, can produce acetic acid, furfural etc. to yeast toxic substance.Therefore,, in order to reduce straw ethanol cost, the present invention proposes following measures:
(1) in fermenting process, glucose and xylose is all converted into ethanol simultaneously, is expected to improve output 25%, thus the bacterial strain mixed fermentation that can utilize glucose and xylose should be adopted, thus glucose and xylose is all converted into ethanol;
(2) adopt can eccrine fiber the strain fermentation of element enzyme, zytase, thereby save high enzyme cost.Cellulose enzyme gene and xylan gene can be proceeded to corresponding yeast by genetically engineered, obtain can eccrine fiber the yeast of element enzyme and zytase; Along with engineered technical development, foreign gene is expressed in the yeast such as yeast saccharomyces cerevisiae, shehatae candida, pichia stipitis, can realize.
(3) good, the tolerance acetic acid of seed selection tolerance ethanol, bacterial strain that furfural is good, thus higher alcohol concn can be obtained, and significantly reduce stalk pre-treatment degradation solution detoxification cost, and fermentation efficiency is high.Tame by screening, can obtain such bacterial strain.
Cellulase is a kind of multi-component prozyme, comprises endoglucanase (EG), and 1,4-BETA-D-glucancellobio-hydrolase (or claim cellobiohydrolase) (CHB) and 3 kinds of main ingredients such as beta-glucan glycosides enzyme (BG).Be hydrolyzed in the process of glucose at natural cellulose, must rely on the synergy of 3 kinds of enzymes just can complete.Yeast saccharomyces cerevisiae can not degraded cellulose, can only utilize glucose.In yeast saccharomyces cerevisiae, to proceed to endoglucanase, 1,4-BETA-D-glucancellobio-hydrolase and 3 kinds of enzyme genes of beta-glucan glycosides enzyme, thereby build can eccrine fiber the yeast saccharomyces cerevisiae of element enzyme, it can be glucose by cellulose conversion, and then fermentation is ethanol.Save thus high cellulase cost.
Equally, zytase is also a kind of multi-component prozyme, comprises circumscribed β-Isosorbide-5-Nitrae-zytase inscribe β-Isosorbide-5-Nitrae-zytase and xylan glycosides enzyme, is in wood sugar process by xylan degrading, also needs the synergy of 3 kinds of enzymes just can complete.
Shehatae candida, pichia stipitis etc. can utilize wood sugar, are ethanol by wood-sugar fermentation, are therefore the desirable strain that wood sugar is converted into ethanol.But they can only utilize wood sugar, and can not utilize xylan, after xylan degrading need to being wood sugar, can utilize.If by circumscribed β-1,4-zytase inscribe β-1,4-zytase and xylan glycosides enzyme are expressed in pichia stipitis, shehatae candida, make it have the ability that degradation of xylan is wood sugar, transgenosis trunk pichia spp, shehatae candida can be just wood sugar by xylan degrading, and then converted in-situ is ethanol, save thus high zytase cost.
By above-mentioned transgenosis yeast saccharomyces cerevisiae (expression cellulase) and transgenosis trunk pichia spp, shehatae candida (expressed xylanase), mixed culture, can directly use stalk fermentation to produce ethanol, and utilize glucose and xylose to be converted into ethanol simultaneously.
Yeast saccharomyces cerevisiae used and pichia stipitis, shehatae candida, should pass through domestication breeding, obtain tolerating all good bacterial strains of ethanol, acetic acid, furfural, make it in stalk pre-treatment degradation solution, have higher tolerance, can bring into play preferably producing and ethanol ability.
Therefore, three cellulose enzyme genes are proceeded in yeast saccharomyces cerevisiae simultaneously, build a kind of saccharomyces cerevisiae engineered yeast that can simultaneously secrete three kinds of cellulases; Three xylanase genes are proceeded in pichia stipitis (or shehatae candida) simultaneously, build a kind of pichia stipitis (or shehatae candida) engineering bacteria that can simultaneously secrete three kinds of zytases, directly the Mierocrystalline cellulose in stalk, xylan are converted into glucose, wood sugar, and are fermented for ethanol simultaneously.For reducing straw ethanol production cost, promote that the process of industrialization of straw ethanol is significant.
Technical scheme of the present invention is as follows:
1, yeast saccharomyces cerevisiae and pichia stipitis (or shehatae candida) screening domestication
Respectively yeast saccharomyces cerevisiae and pichia stipitis (or shehatae candida) are screened to domestication with steam puffed stalk pretreatment fluid, screen the bacterial strain that ethanol tolerance level is high simultaneously.Tame by screening, obtain yeast saccharomyces cerevisiae and pichia stipitis (or shehatae candida) bacterial strain that acetic acid, furfural, ethanol are tolerated.
2, build transgenosis yeast saccharomyces cerevisiae
(1) build recombinant expression vector structure
[0005]glucose incision enzyme gene eg1, cellobiose hydrolase gene cbh1 are connected with suitable carrier and promotor with beta-glucan glycoside enzyme gene bglc, are built into three DNA recombinant expression carriers;
(2) electrotransformation transformed saccharomyces cerevisiae;
(3) carry out the screening of positive transformant
Utilize recon itself with antibiotics resistance gene or the selective medium of amino acid, uridylic defect screen.Secondly, carry out PCR detection, Southern blot to the further evaluation and screening of the bacterial strain of selecting, finishing screen is selected the yeast saccharomyces cerevisiae positive transformant that simultaneously contains three cellulose enzyme genes, can secrete 3 kinds of cellulases.
3, build transgenosis trunk pichia spp (or shehatae candida etc.) yeast
(1) build recombinant expression vector structure
[0006]circumscribed β-Isosorbide-5-Nitrae-zytase inscribe β-Isosorbide-5-Nitrae-zytase is connected with suitable carrier and promotor with xylan glycosides enzyme, is built into three DNA recombinant expression carriers;
(2) electrotransformation transforms pichia spp (or shehatae candida) yeast;
(3) carry out the screening of positive transformant
Utilize recon itself with antibiotics resistance gene or the selective medium of amino acid, uridylic defect screen.Secondly, carry out PCR detection, Southern blot to the further evaluation and screening of the bacterial strain of selecting, finishing screen is selected pichia stipitis (or shehatae candida) positive transformant that simultaneously contains three xylanase genes, can secrete 3 kinds of zytases.
4, transgenosis yeast saccharomyces cerevisiae, pichia stipitis (or shehatae candida) mix with stage enzymatic hydrolysis producing ethanol by efficient stalk fermentation
[0008]apply above-mentioned transgenosis yeast saccharomyces cerevisiae, transgenosis trunk pichia spp (or shehatae candida), take cellulosics such as stalks as raw material, adopt the quick-fried pretreated straw of vapour, carry out same stage enzymatic hydrolysis-fermentation, by stalk transforming glucose, wood sugar, and then original position fermentation is ethanol.Due to the ratio of temperature, pH, stalk concentration, transgenosis yeast saccharomyces cerevisiae and transgenosis trunk pichia spp (or shehatae candida), nitrogenous source, incubation time etc. all have considerable influence to alcohol yied, fermenting speed, so, the test design that need to be optimized by response surface method, to determine preferably transformation fermentation processing condition.And, owing to being mixed culture, the requirement difference of 2 primary yeasts to fermentation condition, so, need design by optimization Test, obtain comprehensively processing condition preferably of 2 primary yeasts.
The invention has the advantages that:
(1) transgenosis yeast saccharomyces cerevisiae of the present invention can be secreted three kinds of cellulases outside born of the same parents simultaneously, i.e. endoglucanase (EG), cellobiohydrolase (CHB) and beta-glucan glycosides enzyme (BG); Transgenosis trunk pichia spp of the present invention (or shehatae candida), can secrete three kinds of zytases outside born of the same parents simultaneously, it is circumscribed β-1,4-zytase inscribe β-1,4-zytase and xylan glycosides enzyme, not needing provides enzyme in addition, thereby significantly reduces the production cost of straw ethanol.Meanwhile, because cellulase and xylan act on simultaneously, can significantly provide the transformation efficiency of Mierocrystalline cellulose, xylan, improve alcohol getting rate.
(2) heavy transgenosis yeast saccharomyces cerevisiae of the present invention, transgenosis trunk pichia spp (or shehatae candida) can directly change into glucose by Mierocrystalline cellulose, and xylan is converted into wood sugar, glucose, xylan in-situ transesterification is changed into ethanol simultaneously;
(3) one recombination yeast mixed fermentation of the present invention, do not need to add in addition enzyme, directly transform the method for the cellulose raw material production ethanol such as stalk, can significantly reduce the production cost of straw ethanol, the process that promotes straw ethanol petroleum replacing fuel, has broad application prospects.
The imagination of this technique, is to have experienced lot of experiments, and creative work, just obtains.
Below in conjunction with specific embodiment, the invention will be further described.
embodiment 1 yeast saccharomyces cerevisiae and pichia stipitis (or shehatae candida) screening domestication
Respectively yeast saccharomyces cerevisiae and pichia stipitis (or shehatae candida) are screened to domestication with steam puffed stalk pretreatment fluid, screen the bacterial strain that ethanol tolerance level is high simultaneously.Tame by screening, obtain yeast saccharomyces cerevisiae and pichia stipitis (or shehatae candida) bacterial strain that acetic acid, furfural, ethanol are tolerated.
?specific experiment condition is as follows:
The quick-fried condition of vapour, temperature 160-200 ℃, time, 3-20 minute, pressure, 1.1-1.8MP, because the quick-fried condition of vapour all has impact to Mierocrystalline cellulose, xylan degrading and transformation efficiency and acetic acid, furfural generation, used, be optimized by response surface method, having obtained the preferably quick-fried condition of vapour is, the time: 5-8 minute; Pressure: 1.3-1.5MP; Temperature: 165-180
℃.
Acclimation and screening obtains Wine brewing yeast strain kdn-6, kdn-31, kdn-59, pichia stipitis bacterial strain kds-3, kds-9, kds-75, shehatae candida bacterial strain kdx-10, kdx-13, kdx-21 well-grown in the quick-fried pretreatment fluid of vapour, ethanol tolerance is all greater than to 15%, and glucose, xylose utilization rate are all greater than 89 %..
embodiment 2 builds transgenic fibre element enzyme yeast saccharomyces cerevisiae 1
Carrier pYEX-BX(7.1kb) (containing Ampr, tri-selective markers of URA3 and leu2), glyceraldehyde 3-phosphate dehydro-genase gene promoter (GAPDH
p) and terminator (GAPDH
t), signal coding sequence adopts the signal peptide sequence (XYNSEC) of the xylanase gene in T. reesei, builds recombinant expression vector I, its expressed sequence frame is GAPDH
p-XYNSEC-cbh1-GAPDH
t-GAPDH
p-XYNSEC-eg1-GAPDH
t-GAPDH
p– GLUSEC-bglc-GAPDH
t, called after pYEX-BX-GAPDH-EchBl.Adopt electric shock conversion method that the recombinant expression vector building is transformed into yeast saccharomyces cerevisiae kdn-6; According on proceeded to recombinant expression vector with selected marker, yeast cell after transforming is coated on the substratum that contains ammonia benzyl mycin or kantlex or on auxotrophic solid medium flat board, if transformed successfully, yeast cell will obtain antibiotics resistance or auxotroph resistance, on corresponding selective medium, will grow the single bacterium colony that contains recon.Then, upgrading grain carries out pcr amplification, and double digestion detects in transformant whether contain eg1 simultaneously, tri-cellulose enzyme genes of cbh1 and bglc, and finishing screen is selected the yeast saccharomyces cerevisiae positive transformant that simultaneously contains three kinds of cellulose enzyme genes, called after zkdn-6.
embodiment 3 cellulase transgenosis yeast saccharomyces cerevisiaes 2
Carrier pYEX-BX(7.1kb) (containing Ampr, tri-selective markers of URA3 and leu2), phosphoglyceric kinase gene promotor (PGK
p) and terminator (PGK
t), signal coding sequence adopts the signal peptide sequence (XYNSEC) of the xylanase gene in T. reesei, builds recombinant expression vector II, its expressed sequence frame is PGK
p-XYNSEC-cbh1-PGK
t-PGK
p-XYNSEC-eg1-PGK
t-PGK
p-PGK
p-GLUSEC-bglc-PGK
t, called after pYEX-BX-PGK-EchBl; Adopt electric shock conversion method that the recombinant expression vector building is transformed into yeast saccharomyces cerevisiae kdn-59; According on proceeded to recombinant expression vector with selected marker, yeast cell after transforming is coated on the substratum that contains ammonia benzyl mycin or kantlex or on auxotrophic solid medium flat board, if transformed successfully, yeast cell will obtain antibiotics resistance or auxotroph resistance, on corresponding selective medium, will grow the single bacterium colony that contains recon.Then, upgrading grain carries out pcr amplification, and double digestion detects in transformant whether contain eg1 simultaneously, tri-cellulose enzyme genes of cbh1 and bglc, and finishing screen is selected the yeast saccharomyces cerevisiae positive transformant that simultaneously contains three kinds of cellulose enzyme genes, called after zkdn-59.
embodiment 4 cellulase transgenosis yeast saccharomyces cerevisiaes 3
Carrier Yeplac195(5.24kb) (containing two selective markers of Kanr and URA3), glyceraldehyde 3-phosphate dehydro-genase gene promoter (GAPDH
p) and terminator (GAPDH
t), the glucose amylase gene signal peptide (GLUSEC) of Rhizopusoryzae, build recombinant expression vector III, its expressed sequence frame is GAPDH
p-XYNSEC-cbh1-GAPDH
t-GAPDH
p-XYNSEC-eg1-GAPDH
t-GAPDH
p– GLUSEC-bglc-GAPDH
t, called after Yeplac-GAPDH-EchBl.Adopt electric shock conversion method that the recombinant expression vector building is transformed into yeast saccharomyces cerevisiae kdn-31; According on proceeded to recombinant expression vector with selected marker, yeast cell after transforming is coated on the substratum that contains ammonia benzyl mycin or kantlex or on auxotrophic solid medium flat board, if transformed successfully, yeast cell will obtain antibiotics resistance or auxotroph resistance, on corresponding selective medium, will grow the single bacterium colony that contains recon.Then, upgrading grain carries out pcr amplification, and double digestion detects in transformant whether contain eg1 simultaneously, tri-cellulose enzyme genes of cbh1 and bglc, and finishing screen is selected the yeast saccharomyces cerevisiae positive transformant that simultaneously contains three kinds of cellulose enzyme genes, zkdn-31.
embodiment 5 builds transgenosis zytase pichia stipitis 1
Carrier pYEX-BX(7.1kb) (containing Ampr, tri-selective markers of URA3 and leu2), glyceraldehyde 3-phosphate dehydro-genase gene promoter (GAPDH
p) and terminator (GAPDH
t), signal coding sequence adopts the signal peptide sequence (XYNSEC) of the xylanase gene in T. reesei, builds recombinant expression vector I.Adopt electric shock conversion method that the recombinant expression vector building is transformed into pichia stipitis bacterial strain kds-3; According on proceeded to recombinant expression vector with selected marker, yeast cell after transforming is coated on the substratum that contains ammonia benzyl mycin or kantlex or on auxotrophic solid medium flat board, if transformed successfully, yeast cell will obtain antibiotics resistance or auxotroph resistance, on corresponding selective medium, will grow the single bacterium colony that contains recon.Then, upgrading grain carries out pcr amplification, and double digestion detects in transformant whether contain three kinds of xylanase genes simultaneously, and finishing screen is selected the pichia stipitis positive transformant that simultaneously contains three kinds of xylanase genes, called after zkds-3.
embodiment 6 builds transgenosis zytase pichia stipitis 2
Carrier pYEX-BX(7.1kb) (containing Ampr, tri-selective markers of URA3 and leu2), phosphoglyceric kinase gene promotor (PGK
p) and terminator (PGK
t), signal coding sequence adopts the signal peptide sequence (XYNSEC) of the xylanase gene in T. reesei, builds recombinant expression vector II, adopts electric shock conversion method that the recombinant expression vector building is transformed into pichia stipitis bacterial strain kds-9; According on proceeded to recombinant expression vector with selected marker, yeast cell after transforming is coated on the substratum that contains ammonia benzyl mycin or kantlex or on auxotrophic solid medium flat board, if transformed successfully, yeast cell will obtain antibiotics resistance or auxotroph resistance, on corresponding selective medium, will grow the single bacterium colony that contains recon.Then, upgrading grain carries out pcr amplification, and double digestion detects in transformant whether contain three kinds of xylanase genes simultaneously, and finishing screen is selected the pichia stipitis positive transformant that simultaneously contains three kinds of xylanase genes, called after zkds-9.
embodiment 7 builds transgenosis zytase pichia stipitis 3
Carrier Yeplac195(5.24kb) (containing two selective markers of Kanr and URA3), glyceraldehyde 3-phosphate dehydro-genase gene promoter (GAPDH
p) and terminator (GAPDH
t), the glucose amylase gene signal peptide (GLUSEC) of Rhizopusoryzae, build recombinant expression vector III.Adopt electric shock conversion method that the recombinant expression vector building is transformed into pichia stipitis bacterial strain kds-75; According on proceeded to recombinant expression vector with selected marker, yeast cell after transforming is coated on the substratum that contains ammonia benzyl mycin or kantlex or on auxotrophic solid medium flat board, if transformed successfully, yeast cell will obtain antibiotics resistance or auxotroph resistance, on corresponding selective medium, will grow the single bacterium colony that contains recon.Then, upgrading grain carries out pcr amplification, and double digestion detects in transformant whether contain three kinds of xylanase genes simultaneously, and finishing screen is selected the pichia stipitis positive transformant that simultaneously contains three kinds of xylanase genes, zkds-75.
embodiment 8 builds transgenosis zytase shehatae candida 1
Carrier pYEX-BX(7.1kb) (containing Ampr, tri-selective markers of URA3 and leu2), glyceraldehyde 3-phosphate dehydro-genase gene promoter (GAPDH
p) and terminator (GAPDH
t), signal coding sequence adopts the signal peptide sequence (XYNSEC) of the xylanase gene in T. reesei, builds recombinant expression vector I.Adopt electric shock conversion method that the recombinant expression vector building is transformed into shehatae candida bacterial strain kdx-10; According on proceeded to recombinant expression vector with selected marker, yeast cell after transforming is coated on the substratum that contains ammonia benzyl mycin or kantlex or on auxotrophic solid medium flat board, if transformed successfully, yeast cell will obtain antibiotics resistance or auxotroph resistance, on corresponding selective medium, will grow the single bacterium colony that contains recon.Then, upgrading grain carries out pcr amplification, and double digestion detects in transformant whether contain three kinds of xylanase genes simultaneously, and finishing screen is selected the pichia stipitis positive transformant that simultaneously contains three kinds of xylanase genes, called after zkd-10.
embodiment 9 builds transgenosis zytase shehatae candida 2
Carrier pYEX-BX(7.1kb) (containing Ampr, tri-selective markers of URA3 and leu2), phosphoglyceric kinase gene promotor (PGK
p) and terminator (PGK
t), signal coding sequence adopts the signal peptide sequence (XYNSEC) of the xylanase gene in T. reesei, builds recombinant expression vector II; Adopt electric shock conversion method that the recombinant expression vector building is transformed into shehatae candida bacterial strain kdx-13; According on proceeded to recombinant expression vector with selected marker, yeast cell after transforming is coated on the substratum that contains ammonia benzyl mycin or kantlex or on auxotrophic solid medium flat board, if transformed successfully, yeast cell will obtain antibiotics resistance or auxotroph resistance, on corresponding selective medium, will grow the single bacterium colony that contains recon.Then, upgrading grain carries out pcr amplification, and double digestion detects in transformant whether contain three kinds of xylanase genes simultaneously, and finishing screen is selected the pichia stipitis positive transformant that simultaneously contains three kinds of xylanase genes, called after zkdx-13.
embodiment 10 builds transgenosis zytase shehatae candida 3
Carrier Yeplac195(5.24kb) (containing two selective markers of Kanr and URA3), glyceraldehyde 3-phosphate dehydro-genase gene promoter (GAPDH
p) and terminator (GAPDH
t), the glucose amylase gene signal peptide (GLUSEC) of Rhizopusoryzae, build recombinant expression vector III.Adopt electric shock conversion method that the recombinant expression vector building is transformed into shehatae candida bacterial strain kdx-21; According on proceeded to recombinant expression vector with selected marker, yeast cell after transforming is coated on the substratum that contains ammonia benzyl mycin or kantlex or on auxotrophic solid medium flat board, if transformed successfully, yeast cell will obtain antibiotics resistance or auxotroph resistance, on corresponding selective medium, will grow the single bacterium colony that contains recon.Then, upgrading grain carries out pcr amplification, and double digestion detects in transformant whether contain three kinds of xylanase genes simultaneously, and finishing screen is selected the pichia stipitis positive transformant that simultaneously contains three kinds of xylanase genes, zkdx-21.
embodiment 11 transgenosis yeast saccharomyces cerevisiaes, pichia stipitis mixed fermentation maize transformation Ethanol Production Process with Straw
(1) stalk pre-treatment
[0009]get maize straw 200g, pulverize, cross 20 mesh sieves, the quick-fried pre-treatment of vapour, steam heating 1.5min reaches 1.45MP to pressure, maintain 7min, cooling, to pressure be 2 bar, take out stalk, with 50 ° of C water washings 5 times, filter, obtain 120g maize straw after treatment, for subsequent use.
(2) spawn culture
[0010]bacterial strain: transgenosis yeast saccharomyces cerevisiae zkdn-31, pichia stipitis zkds-3.
Slant medium composition (g/L) and culture condition: glucose, 20; Yeast extract, 3; Agar, 20; Wort, 3; PH 6.5,30 ± 1 ° of C of temperature
Seed culture medium composition (g/L) and culture condition: glucose, 30; Yeast extract, 1; Corn steep liquor, 2; (NH
4) SO
4, 7.5; K
2hPO
4, 3.5; MgSO
47H
2o, 0.75; CaCl
22H
2o, 1M citrate buffer solution, 6.5,38 ° of C of pH, cultivate 30 h, rotating speed 150 rpm
Inoculum size: by every 100ml conversion fluid inoculation 1.0g thalline (in dry mycelium), 2 primary yeasts, cultivate respectively separately.
(3) stalk is converted into ethanol
[0011]fermented liquid composition (g/l): yeast extract, 0.2; Corn steep liquor, 2.5; (NH4)
2sO4,7.5; K
2hPO
4, 3.5; Urea, 1; MgSO
47H
2o, 0.75; CaCl
22H
2o, 1; Pretreated stalk, 170,0.05M citrate buffer solution, puts in 250 ml shaking flasks, with 2 M NaOH tune pH 6.6.
After fermented liquid sterilizing, add transgenosis yeast saccharomyces cerevisiae, pichia stipitis bacterial classification to amount to 1.0g(in dry mycelium), transgenosis yeast saccharomyces cerevisiae zkdn-6 is 1:0.8 with the ratio of pichia stipitis zkds-9.0.1 g Tween 80 (g/l), fermented liquid cumulative volume is 100ml, at 38 ° of C, fermentation 48-60h, alcohol concn reaches 140.7g/L, glucose, xylose utilization rate >89%.
Due to the ratio of temperature, pH, stalk concentration, transgenosis yeast saccharomyces cerevisiae and transgenosis trunk pichia spp (or shehatae candida), nitrogenous source, incubation time etc. all have considerable influence to alcohol yied, fermenting speed, so, the test design that need to be optimized by response surface method, to determine preferably transformation fermentation processing condition.And, owing to being mixed culture, the requirement difference of 2 primary yeasts to fermentation condition, so, need design by optimization Test, obtain comprehensively processing condition preferably of 2 primary yeasts.
The condition of the present embodiment is carries out obtaining after a large amount of optimization Test.
embodiment 12 transgenosis yeast saccharomyces cerevisiaes, pichia stipitis mixed fermentation maize transformation Ethanol Production Process with Straw
(1) stalk pre-treatment
[0012]5 tons of maize straws, pulverize, and cross 20 mesh sieves, the quick-fried pre-treatment of vapour, steam heating 1.5min reaches 1.35MP to pressure, maintains 5.5min, cooling, to pressure be 2 bar, take out stalk for subsequent use.
(2) spawn culture
[0013]bacterial strain: transgenosis yeast saccharomyces cerevisiae zkdn-6, pichia stipitis zkds-9.
Slant medium composition (g/L) and culture condition: glucose, 20; Yeast extract, 3; Agar, 20; Wort, 3; PH 6.5,30 ± 1 ° of C of temperature
Seed culture medium composition (g/L) and culture condition: glucose, 50; Yeast extract, 1; Corn steep liquor: 2; (NH
4) SO
4, 7.5; K
2hPO
4, 3.5; MgSO
47H
2o, 0.75; CaCl
22H
2o, 1M citrate buffer solution, 6.5,36 ° of C of pH, cultivate 30 h, rotating speed 150 rpm
Inoculum size: 10%, seed liquor/fermented liquid, volume ratio, multistage seed liquor is cultivated, and transgenosis yeast saccharomyces cerevisiae zkdn-6 is 1:1 with the ratio of pichia stipitis zkds-9.
(3) stalk is converted into ethanol
[0014]fermented liquid composition (g/l): yeast extract, 0.3; Corn steep liquor, 3; (NH4)
2sO4,7.5; K
2hPO
4, 3.5; Urea, 1; MgSO
47H
2o, 0.75; CaCl
22H
2o, 1; The quick-fried pretreated stalk of vapour, batch feeding drops into, and maintenance concentration is 200-230g/L, and 0.05M citrate buffer solution, with 2 M NaOH tune pH 6.6.0.
At 30M
3industrial fermentation tank ferments, and after fermented liquid sterilizing, adds transgenosis yeast saccharomyces cerevisiae, trunk to finish red yeast-like fungi kind, inoculum size: 8%, and seed liquor/fermented liquid, volume ratio, transgenosis yeast saccharomyces cerevisiae zkdn-6 is 1:1 with the ratio of pichia stipitis zkds-9.At 36 ° of C, fermentation 48-55h, alcohol concn reaches 150.1g/L, glucose, xylose utilization rate >90%.
The present embodiment is the test of pilot scale, is amplified to 30M by embodiment 11
3industrial fermentation tank, is that laboratory lab scale is amplified to pilot scale, and technical difficulty is very large, experiences a large amount of tests, is not that normal experiment work can solve.
embodiment 13 transgenosis yeast saccharomyces cerevisiaes, shehatae candida mixed fermentation transform rice straw and produce ethanol
(1) stalk pre-treatment
[0015]get straw 150g, pulverize, cross 20 mesh sieves, the quick-fried pre-treatment of vapour.Take out stalk, with 50 ° of C water washings 5 times, filter, obtain 105g rice straw after treatment, for subsequent use.
(2) spawn culture
[0016]bacterial strain: transgenosis yeast saccharomyces cerevisiae zkdn-6, shehatae candida z kdx-10.
Slant medium composition (g/L) and culture condition: glucose, 20; Yeast extract, 3; Agar, 20; Wort, 3; PH 6.5,30 ± 1 ° of C of temperature
Seed culture medium composition (g/L) and culture condition: glucose, 50; Yeast extract, 2; Corn steep liquor, 2; (NH
4) SO
4, 7.5; K
2hPO
4, 3.5; MgSO
47H
2o, 0.75; CaCl
22H
2o, 1M citrate buffer solution, 6.5,30 ° of C of pH, cultivate 30 h, rotating speed 150 rpm
Inoculum size: amount to 1.0g thalline (in dry mycelium) by every 100ml conversion fluid inoculation, transgenosis yeast saccharomyces cerevisiae zkdn-6 is 1:0.92 with the ratio of shehatae candida z kdx-10.
(3) stalk is converted into ethanol
[0017]fermented liquid composition (g/l): yeast extract, 0.5; Corn steep liquor, 2.5; (NH4)
2sO4,7.5; K
2hPO
4, 3.5; Urea, 1; MgSO
47H
2o, 0.75; CaCl
22H
2o, 1; Pretreated rice straw, 165,0.05M citrate buffer solution, puts in 250 ml shaking flasks, with 2 M NaOH tune pH6. 5.0.
After fermented liquid sterilizing, border on transgenic yeast, inoculum size: 8%, seed liquor/fermented liquid, volume ratio, transgenosis yeast saccharomyces cerevisiae zkdn-6 is 1:0.92 with the ratio of shehatae candida z kdx-10.0.1 g Tween 80 (g/l), fermented liquid cumulative volume is 100ml, at 30 ° of C, aerobic fermentation 24-48 h, alcohol concn reaches 138.2g/L, glucose, xylose utilization rate >92%.
embodiment 14 transgenosis yeast saccharomyces cerevisiaes, pichia stipitis mixed fermentation maize transformation Ethanol Production Process with Straw
(1) stalk pre-treatment
[0018]get 5000 tons of maize straws, pulverize, cross 20 mesh sieves, the quick-fried pre-treatment of vapour, steam heating 1.5min reaches 1.35MP to pressure, maintains 5min, cooling, to pressure be 2 bar normal pressures, take out stalk for subsequent use.
(2) spawn culture
[0019]bacterial strain: transgenosis yeast saccharomyces cerevisiae zkdn-59, pichia stipitis zkds-75.
Slant medium composition (g/L) and culture condition: glucose, 20; Yeast extract, 3; Agar, 20; Wort, 3; PH 6.5,30 ± 1 ° of C of temperature
Seed culture medium composition (g/L) and culture condition: glucose, 50; Yeast extract, 1; Corn steep liquor: 2; (NH
4) SO
4, 7.5; K
2hPO
4, 3.5; MgSO
47H
2o, 0.75; CaCl
22H
2o, 1M citrate buffer solution, 6.5,35 ° of C of pH, cultivate 30 h, rotating speed 150 rpm
Inoculum size: 10%, seed liquor/fermented liquid, volume ratio, transgenosis yeast saccharomyces cerevisiae zkdn-59 is 1:0.9 with the ratio of pichia stipitis zkds-75, multistage seed liquor is cultivated.
(3) stalk is converted into ethanol
[0019]fermented liquid composition (g/l): yeast extract, 0.3; Corn steep liquor, 3; (NH4)
2sO4,7.5; K
2hPO
4, 3.5; Urea, 1; MgSO
47H
2o, 0.75; CaCl
22H
2o, 1; Pretreated stalk, batch feeding drops into, and maintenance concentration is 180-210g/L, and 0.05M citrate buffer solution, with 2 M NaOH tune pH 6.60.
At 300M
3industrial fermentation tank ferments, and after fermented liquid sterilizing, adds transgenosis yeast saccharomyces cerevisiae, trunk to finish red yeast-like fungi kind, inoculum size: 8%, and seed liquor/fermented liquid, volume ratio, transgenosis yeast saccharomyces cerevisiae zkdn-59 is 1:0.9 with the ratio of pichia stipitis zkds-75.At 35 ° of C, anaerobic fermentation 40-50h, alcohol concn reaches 150.3g/L,, glucose, xylose utilization rate >93%.
?the present embodiment is plant-scale test, is amplified to 300M by embodiment 12
3industrial fermentation tank, is to be amplified to batch production scale from pilot scale, and work difficulty is very large, experiences a large amount of tests, is not that normal experiment work can solve.
embodiment 15 transgenosis yeast saccharomyces cerevisiaes and shehatae candida mixed fermentation maize transformation Ethanol Production Process with Straw
(1) stalk pre-treatment
[0020]5000 tons of maize straws, pulverize, and cross 20 mesh sieves, the quick-fried pre-treatment of vapour, steam heating 1.5min reaches 1.35MP to pressure, maintains 5min, cooling, to pressure be 2 bar normal pressures, take out stalk for subsequent use.
(2) spawn culture
[0021]bacterial strain: transgenosis yeast saccharomyces cerevisiae zkdn-31, shehatae candida z kdx-13.
Slant medium composition (g/L) and culture condition: glucose, 20; Yeast extract, 3; Agar, 20; Wort, 3; PH 6.5,30 ± 1 ° of C of temperature
Seed culture medium composition (g/L) and culture condition: glucose, 50; Yeast extract, 1; Corn steep liquor: 2; (NH
4) SO
4, 7.5; K
2hPO
4, 3.5; MgSO
47H
2o, 0.75; CaCl
22H
2o, 1M citrate buffer solution, 6.5,353 ° of C of pH, cultivate 30 h, rotating speed 150 rpm
Inoculum size: 10%, seed liquor/fermented liquid, volume ratio, transgenosis yeast saccharomyces cerevisiae zkdn-59 is 1:0.8 with the ratio of pichia stipitis zkds-75.Multistage seed liquor is cultivated.
(3) stalk is converted into ethanol
[0022]fermented liquid composition (g/l): yeast extract, 0.3; Corn steep liquor, 3; (NH4)
2sO4,7.5; K
2hPO
4, 3.5; Urea, 1; MgSO
47H
2o, 0.75; CaCl
22H
2o, 1; Pretreated stalk, batch feeding drops into, and maintenance concentration is 150-180g/L, and 0.05M citrate buffer solution, with 2 M NaOH tune pH 6.2.
At 300M
3industrial fermentation tank ferments, and after fermented liquid sterilizing, adds transgenosis yeast saccharomyces cerevisiae, trunk to finish red yeast-like fungi kind, inoculum size: 8%, and seed liquor/fermented liquid, volume ratio, transgenosis yeast saccharomyces cerevisiae zkdn-59 is 1:0.8 with the ratio of pichia stipitis zkds-75.At 33 ° of C, aerobic fermentation 35-45h, alcohol concn reaches 150.3g/L, glucose, xylose utilization rate >92%.
By embodiment 14, to the present embodiment, experimental size does not change, and just bacterial strain has changed, so work difficulty is relatively little.
Claims (2)
1. a method of utilizing transgenic yeast mixed fermentation to produce straw ethanol, it is characterized in that transgenic yeast can be degraded to the Mierocrystalline cellulose of stalk, xylan respectively glucose, wood sugar and their original positions are fermented for ethanol, do not need to provide in addition cellulase, zytase, Mierocrystalline cellulose, xylan degrading are synchronizeed and are carried out with fermentation.
2. method described in claim 1, is characterized in that transgenic yeast is transgenosis yeast saccharomyces cerevisiae, transgenosis trunk pichia spp, transgenosis shehatae candida; Transgenosis yeast saccharomyces cerevisiae has proceeded to endoglucanase, cellobiohydrolase and beta-glucan glycoside enzyme gene simultaneously, can secrete endoglucanase, cellobiohydrolase and beta-glucan glycosides enzyme; Transgenosis trunk pichia spp, transgenosis shehatae candida have all proceeded to circumscribed β-1 simultaneously, 4-zytase inscribe β-1,4-zytase and xylan glycoside enzyme gene, can secrete circumscribed β-1,4-zytase inscribe β-1,4-zytase and xylan glycosides enzyme, transgenosis trunk pichia spp, transgenosis shehatae candida can utilize wood-sugar fermentation ethanol.
3. method described in claim 1, it is characterized in that transgenosis yeast saccharomyces cerevisiae and transgenosis trunk pichia spp, or with the mixed fermentation of transgenosis shehatae candida, fermentation condition is: transgenosis yeast saccharomyces cerevisiae and pichia stipitis or transgenosis shehatae candida are for it than being 1:0.8-1, leavening temperature is 30-38 ° of C, and fermentation time is 24-60h, and the quick-fried pretreated stalk batch feeding of vapour drops into fermentor tank, keeping stalk concentration is 150-230g/L, and pH is 6.2-6.6; Fermented liquid composition (g/l): yeast extract, 0.3; Corn steep liquor, 2.5-3; (NH4)
2sO4,7.5; K
2hPO
4, 3.5; Urea, 1; MgSO
47H
2o, 0.75; CaCl
22H
2o, 1.
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| CN110055184A (en) * | 2018-12-28 | 2019-07-26 | 吉林中粮生化有限公司 | Saccharomyces cerevisiae, comprising its microorganism formulation and using its produce ethyl alcohol method |
| CN110055184B (en) * | 2018-12-28 | 2022-06-28 | 吉林中粮生化有限公司 | Saccharomyces cerevisiae, microbial preparation comprising same, and method for producing ethanol using same |
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