CN101323838A - Recombinant distiller's yeast and use thereof in eutrit production - Google Patents
Recombinant distiller's yeast and use thereof in eutrit production Download PDFInfo
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- CN101323838A CN101323838A CNA2008101385646A CN200810138564A CN101323838A CN 101323838 A CN101323838 A CN 101323838A CN A2008101385646 A CNA2008101385646 A CN A2008101385646A CN 200810138564 A CN200810138564 A CN 200810138564A CN 101323838 A CN101323838 A CN 101323838A
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 66
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 title claims abstract description 37
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 8
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 claims abstract description 36
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 claims abstract description 36
- 229960002675 xylitol Drugs 0.000 claims abstract description 36
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- SRBFZHDQGSBBOR-QMKXCQHVSA-N alpha-L-arabinopyranose Chemical compound O[C@H]1CO[C@@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-QMKXCQHVSA-N 0.000 claims description 3
- 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 description 3
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- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 abstract description 7
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- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
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Images
Abstract
The invention relates to a recombinant Saccharomyces cerevisiae and application thereof in the production of xylitol, which pertains to the technical field of microbial engineering. A Saccharomyces cerevisiae GZ-5-T2 is preserved in the Common Microorganisms Center of China Committee for Culture Collection of Microorganisms on July 10, 2008, with the strain accession number of CGMCC No.2581. The strain can be applied to the production of the xylitol. The Saccharomyces cerevisiae preserved by the invention reduces the stable integration and high efficient expression of enzyme genes XYL1 in a chromosome rDNA area by xylose on the basis of keeping the original growth feature, thus obtaining the property of utilizing the xylose in the acidolysis solution of corncob to produce the xylitol.
Description
Technical field
The present invention relates to a kind of recombinant Saccharomyces cerevisiae and the application in producing Xylitol thereof, belong to the microbial engineering field.
Background technology
Xylitol is a kind of pentavalent alcohol, is one of intermediate product of xylose metabolism, and its chemical molecular formula is C
5H
12O
5, profile is a white crystals.As a kind of important function multi-sugar alcohol, Xylitol is extensively approved the good physiological function of human body, is mainly shown: prevention of dental caries, do not increase blood glucose value, bifidus factor effect, alleviate fat and improve liver function etc.Xylitol sugariness and sucrose are suitable, and its sugariness is higher at low temperatures, and it does not increase blood sugar, are the favorable substitutes of sucrose therefore.Because its special physiological function, Xylitol is widely used in industry such as food and medicine, and the international demand amount rises year by year.Though Xylitol is present among various plants such as careless tomb, plum, pears, birch etc. widely, quantity is considerably less, has only 0.014%-0.9%, can not satisfy the modern life people demand growing to Xylitol.
The production method of Xylitol can be divided into 3 kinds: biochemical extraction, chemical conversion and bio-transformation.The plant material that acid hydrolysis xylan content is higher as corn cob, bagasse, birch etc., can obtain a large amount of wood sugars.Present commercial Xylitol mostly is to adopt chemical transformation to produce, above-mentioned acid hydrolysis products is formed Xylitol behind hydrogenating reduction, but this method need be carried out under the condition of High Temperature High Pressure, therefore this method requires high to processing unit, the by product complicated component, thus the separation and purification difficulty caused.
The biotransformation method mild condition, normal temperature and pressure can carry out, and the metabolic efficiency height, and specificity is good, therefore relies on biotransformation method to utilize the Xylose reductase in the microorganism to produce Xylitol, is substituted chemistry synthetic feasible method.Some the natural yeast of wood sugar such as trunk pichia (Pichia stipitis) of utilizing can produce Xylitol in metabolic process, then exist many limitation but it is directly used in industrial production.
Yeast saccharomyces cerevisiae (Saccharomy cescerevisiae) is the security microorganism of generally acknowledging, is applied to the history of the existing more than one thousand years of food fermentation, and the zymotechnique maturation be the ideal industrial producing strain, but self does not have the ability of metabolism wood sugar.The first step of xylose metabolism is that Xylose reductase (xylose reductase) conversion wood sugar is an Xylitol.Stability and high efficiency is expressed Xylose reductase gene (XYL1) in the yeast saccharomyces cerevisiae industrial strain, is the elementary tactics that makes up the yeast saccharomyces cerevisiae microbial strain for xylitol production.
Contain toxicant and some other antinutritional factor such as furfural, phenol in the corncob acid hydrolysis solution, common yeast saccharomyces cerevisiae is difficult to well grow therein, therefore, obtains that a strain can adapt to corncob acid hydrolysis solution and the starting strain that can breed fast therein is particularly important.In addition, the pattern of foreign gene introducing yeast saccharomyces cerevisiae is also very crucial, and with the foreign gene poor stability that plasmid episomal is introduced as carrier, the then stability that is incorporated on the yeast chromosomal is better.The copy number of expression of exogenous gene level and its introducing has direct relation again, and therefore foreign gene high copy on yeast saccharomyces cerevisiae karyomit(e) being integrated is one of technological line of realizing foreign gene high-level stably express in yeast saccharomyces cerevisiae.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of recombinant Saccharomyces cerevisiae and the application in producing Xylitol thereof are provided.
A kind of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) GZ-5-T2, this bacterium is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 10th, 2008, and its culture presevation is numbered CGMCC No.2581.
Described yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) GZ-5-T2 karyomit(e) rDNA iteron has the Xylose reductase gene XYL1 in a plurality of pichias (Pichiastipitis) source, the introducing of this gene is to integrate in the homology in industrial producing strain GZ-5-T2 karyomit(e) rDNA district by height copy integrative vector pYMIC-xyl1 to realize, while is according to the requirement of stability, yeast saccharomyces cerevisiae rDNA sequence is analyzed, and the fragment that it is 2.2kb that selected rDNA upward crosses over 17S, 5.8S and three zone lengths of 26S is as integration site.
The application of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) GZ-5-T2 in producing Xylitol.
Preferably, described yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) GZ-5-T2 can be applied to the production of Xylitol as follows:
(1) uses lower concentration strong acid to be equipped with promotor, the hemicellulose in the corn cob meal thoroughly is converted into wood sugar, pectinose, glucose, get liquid glucose; Described lower concentration strong acid is the hydrochloric acid of 0.1～0.3wt%, and promotor is the iron trichloride of 0.01～0.04wt%;
(2) with gained liquid glucose in the step (1) through decolouring, electrodialysis depickling and ion exchange resin removal of impurities, fermentation substrate; In the described electrodialysis depickling, acid concentration is 0.04～0.12wt% in the sour water;
(3) adding mass volume ratio in the fermentation substrate that step (2) is made is 0.2～1.8% yeast powder, mass volume ratio is 0.2～1.8% peptone, the yeast saccharomyces cerevisiae of per-cent 5～15% inoculum sizes inoculation by volume GZ-5-T2,26～37 ℃ of temperature, pH is 4～7, the condition bottom fermentation of air flow 0.1～0.3vvm 60～120 hours, liquid just must ferment;
(4) the first liquid of the fermentation that step (3) is made promptly gets Xylitol by ion-exchange removal of impurities, vacuum concentration and Crystallization Separation.
Preferably, the yeast powder addition described in the step (3) is 0.5～1.5%, and the peptone addition is 0.5～1.5%.
Preferably, the inoculum size of the yeast saccharomyces cerevisiae GZ-5-T2 described in the step (3) is 8～12% of a volume ratio.
Preferably, the leavening temperature described in the step (3) is 28～32 ℃.
Preferably, the pH described in the step (3) is 4.5～6.5.
Preferably, the air flow described in the step (3) is 0.15～0.25vvm.
Preferably, the fermentation time described in the step (3) is 84～108 hours.
Aforesaid operations step and reagent are this area routine operation step and commercially available prod if no special instructions.
The content analysis of each composition is measured by high performance liquid chromatography (HPLC) in corncob acid hydrolysis solution, the tunning, and analytical column uses Aminex HPX-87H, and (moving phase is 5mmol/L H for Bio-Rad, USA) ion exchange column
2SO
4, flow velocity is 0.4mL/min, 55 ℃ of column temperatures.(Shimadzu Japan) detects with differential refraction detector RID-10A.
The present invention uses by genetic engineering modified yeast saccharomyces cerevisiae industrial producing strain---recombinant Saccharomyces cerevisiae GZ-5-T2, its starting strain GZ-4 obtains by screening on the corncob acid hydrolysis solution solid medium, can in corncob acid hydrolysis solution, well grow, effectively utilize various nutritive ingredients.Reorganization bacterium GZ-5-T2 is keeping on original growth characteristics basis, at the stable integration in karyomit(e) rDNA district and efficiently express, has obtained to utilize the character of xylose production Xylitol in the corncob acid hydrolysis solution by Xylose reductase gene XYL1.
The present invention is raw material with the corn cob, and corncob acid hydrolysis solution glucose content after treatment is at 4.0～8.0g/L, and wood sugar content is at 45～50g/L, fermentation back xylose utilization amount is higher than 40g/L, Xylitol output is higher than 25g/L, and the sugar alcohol transformation efficiency is higher than 60%, has the commercial application prospect.
Description of drawings
Fig. 1 is the physical map that makes up the employed recombinant plasmid pYMIC-xyl1 of recombinant Saccharomyces cerevisiae GZ-5-T2 bacterial strain.
Wherein: XYL1 has represented to connect the Xylose reductase gene of PGK promotor and terminator; RDNA represents the homology integration sequence; CUP I represents the cupric ion resistant gene.
Fig. 2 is the various composition HPLC analytical resultss in the different time fermented liquid in the recombinant Saccharomyces cerevisiae GZ-5-T2 fermented maize core acid hydrolysis solution process.
Wherein: ■ glucose; ● wood sugar; ▲ Xylitol.
Fig. 3 utilizes yeast saccharomyces cerevisiae GZ-5-T2 to produce the process flow sheet of Xylitol.
Embodiment
Below in conjunction with embodiment the present invention is further elaborated, but institute of the present invention protection domain is not limited only to this.
Bacterial strain recombinant Saccharomyces cerevisiae provided by the invention (Saccharomyces cerevisiae) GZ-5-T2, enclose microbial strains preservation management committee common micro-organisms center in being preserved on July 10th, 2008, preservation address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, postcode: 100080, its deposit number is CGMCC No.2581.
Embodiment 1
A kind of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) GZ-5-T2, this bacterium is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 10th, 2008, and its deposit number is CGMCC No.2581.Described yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) GZ-5-T2 karyomit(e) rDNA iteron has the Xylose reductase gene XYL1 in a plurality of pichias (Pichiastipitis) source, the introducing of this gene is to integrate in the homology in industrial producing strain GZ-5-T2 karyomit(e) rDNA district by height copy integrative vector pYMIC-xyl1 to realize, while is according to the requirement of stability, yeast saccharomyces cerevisiae rDNA sequence is analyzed, and the fragment that it is 2.2kb that selected rDNA upward crosses over 17S, 5.8S and three zone lengths of 26S is as integration site.
Embodiment 2
The construction process of yeast saccharomyces cerevisiae GZ-5-T2:
(1) use restriction enzyme HpaI with the restriction enzyme site place linearizing of recombinant plasmid pYMIC-xyl1 at 6453bp.
(2) utilize Lithium Acetate conversion method transformed saccharomyces cerevisiae industrial strain GZ-5, with the YEPD plate screening that the adds cupric ion bacterium GZ-5-T2 that obtains recombinating.
Wherein, PGK promotor (PGK promoter) and PGK terminator (PGK terminator) in the step (1) among the recombinant plasmid vector pYMIC-xyl1 are cloned in yeast chromosomal dna, insert between promotor and terminator and clone in the Xylose reductase gene XYL1 of trunk pichia chromosomal DNA;
Wherein the cupric ion resistant gene (CUP I) among the recombinant plasmid vector pYMIC-xyl1 is cloned in wine as selection markers and is spilt yeast chromosomal dna in the step (1).
Wherein the rDNA sequence among the recombinant plasmid vector pYMIC-xyl1 is integrated regional cloning in the yeast saccharomyces cerevisiae chromosomal DNA as homology in the step (1).
Wherein the screening of reorganization bacterium is transformant to be applied to contain 14mM/L cupric ion (Cu in the step (2)
2+) the YEPD flat board on, in the transformant that can grow, select the recombinant Saccharomyces cerevisiae bacterial strain.
Embodiment 3
The application of yeast saccharomyces cerevisiae GZ-5-T2 in producing Xylitol, concrete steps are as follows:
(1) uses the hydrochloric acid of 0.1wt% to be equipped with 0.01w% iron trichloride promotor, the hemicellulose in the corn cob meal thoroughly is converted into wood sugar, pectinose, glucose, get liquid glucose;
(2) with gained liquid glucose in the step (1) through decolouring, electrodialysis depickling and ion exchange resin removal of impurities, fermentation substrate; After the electrodialysis depickling in the sour water acid concentration reach 0.04wt%, reuse can be saved nearly 20% sour consumption;
(3) adding mass volume ratio in the fermentation substrate that step (2) is made is 0.5% yeast powder, mass volume ratio is 0.5% peptone, the yeast saccharomyces cerevisiae of per-cent 8% inoculum size inoculation by volume GZ-5-T2,26 ℃ of temperature, pH is 4.6, the condition bottom fermentation of air flow 0.1vvm 60 hours, liquid just must ferment;
(4) the first liquid of the fermentation that step (3) is made promptly gets Xylitol by ion-exchange removal of impurities, vacuum concentration and Crystallization Separation.
Through the various substrate product components of efficient liquid phase chromatographic analysis, the glucose utilization rate is 100%, and the xylose utilization rate is 81%, and Xylitol output is 22g/L, and the sugar alcohol transformation efficiency is 55%.
Embodiment 4
As the application of embodiment 3 described yeast saccharomyces cerevisiae GZ-5-T2 in producing Xylitol, difference is:
Use the hydrochloric acid of 0.2wt% to be equipped with 0.02wt% iron trichloride promotor in the step (1); In the step (2) after the electrodialysis depickling in the sour water acid concentration reach 0.08%, reuse can be saved nearly 25% sour consumption; The interpolation mass volume ratio is 1.0% yeast powder in the step (3), and mass volume ratio is 1.0% peptone, the yeast saccharomyces cerevisiae of per-cent 10% inoculum size inoculation by volume GZ-5-T2, fermentation condition is: 30 ℃ of leavening temperatures, pH is 5.6, air flow 0.20vvm, fermentation time 82 hours;
Through the various substrate product components of efficient liquid phase chromatographic analysis, the glucose utilization rate is 100%, and the xylose utilization rate is 83%, and Xylitol output is 25g/L, and the sugar alcohol transformation efficiency is 61%.
As the application of embodiment 3 described yeast saccharomyces cerevisiae GZ-5-T2 in producing Xylitol, difference is:
Use the hydrochloric acid of 0.3wt% to be equipped with 0.03wt% iron trichloride promotor in the step (1); In the step (2) after the electrodialysis depickling in the sour water acid concentration reach 0.12%, reuse can be saved nearly 30% sour consumption; The interpolation mass volume ratio is 1.2% yeast powder in the step (3), and mass volume ratio is 1.2% peptone, the yeast saccharomyces cerevisiae of per-cent 12% inoculum size inoculation by volume GZ-5-T2, fermentation condition is: 32 ℃ of leavening temperatures, pH is 6.2, air flow 0.25vvm, fermentation time 96 hours;
Through the various substrate product components of efficient liquid phase chromatographic analysis, the glucose utilization rate is 100%, and the xylose utilization rate is 84%, and Xylitol output is 23g/L, and the sugar alcohol transformation efficiency is 59%.
Claims (10)
1, a kind of yeast saccharomyces cerevisiae GZ-5-T2, this bacterium is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 10th, 2008, and its culture presevation is numbered CGMCC No.2581.
2, yeast saccharomyces cerevisiae GZ-5-T2 as claimed in claim 1, it is characterized in that, described yeast saccharomyces cerevisiae GZ-5-T2 karyomit(e) rDNA iteron has the Xylose reductase gene XYL1 in a plurality of pichias source, and the fragment that it is 2.2kb that selected rDNA upward crosses over 17S, 5.8S and three zone lengths of 26S is as integration site.
3, the application of a kind of yeast saccharomyces cerevisiae GZ-5-T2 as claimed in claim 1 in producing Xylitol.
4, as claim
Described application is characterized in that, described yeast saccharomyces cerevisiae GZ-5-T2 can be applied to the production of Xylitol as follows:
(1) uses lower concentration strong acid to be equipped with promotor, the hemicellulose in the corn cob meal thoroughly is converted into wood sugar, pectinose, glucose, get liquid glucose; Described lower concentration strong acid is the hydrochloric acid of 0.1～0.3wt%, and promotor is the iron trichloride of 0.01～0.04wt%;
(2) with gained liquid glucose in the step (1) through decolouring, electrodialysis depickling and ion exchange resin removal of impurities, fermentation substrate; In the described electrodialysis depickling, acid concentration is 0.04～0.12wt% in the sour water;
(3) adding mass volume ratio in the fermentation substrate that step (2) is made is 0.2～1.8% yeast powder, mass volume ratio is 0.2～1.8% peptone, the yeast saccharomyces cerevisiae of per-cent 5～15% inoculum sizes inoculation by volume GZ-5-T2,26～37 ℃ of temperature, pH is 4～7, the condition bottom fermentation of air flow 0.1～0.3vvm 60～120 hours, liquid just must ferment;
(4) the first liquid of the fermentation that step (3) is made promptly gets Xylitol by ion-exchange removal of impurities, vacuum concentration and Crystallization Separation.
5, application as claimed in claim 4 is characterized in that, the yeast powder addition described in the step (3) is 0.5～1.5%, and the peptone addition is 0.5～1.5%.
6, application as claimed in claim 4 is characterized in that, the inoculum size of the yeast saccharomyces cerevisiae GZ-5-T2 described in the step (3) is 8～12% of a volume ratio.
7, application as claimed in claim 4 is characterized in that, the leavening temperature described in the step (3) is 28～32 ℃.
8, application as claimed in claim 4 is characterized in that, the pH described in the step (3) is 4.5～6.5.
9, application as claimed in claim 4 is characterized in that, the air flow described in the step (3) is 0.15～0.25vvm.
10, application as claimed in claim 4 is characterized in that, the fermentation time described in the step (3) is 84～108 hours.
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Cited By (5)
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| CN103396956A (en) * | 2013-08-17 | 2013-11-20 | 湖北邦之德牧业科技有限公司 | Saccharomyces cerevisiae, screening and culture methods thereof and bean meal fermentation method thereof |
| CN105368732A (en) * | 2015-11-10 | 2016-03-02 | 四川大学 | Industrial saccharomyces cerevisiae strain capable of producing xylitol and construction method of industrial saccharomyces cerevisiae strain |
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| US10759727B2 (en) | 2016-02-19 | 2020-09-01 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
| TWI706037B (en) * | 2018-08-16 | 2020-10-01 | 行政院原子能委員會核能研究所 | A saccharomyces cerevisiae strain, a use thereof and a method for preparing xylitiol by the saccharomyces cerevisiae strain |
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| CN100390271C (en) * | 2005-02-25 | 2008-05-28 | 中国科学院微生物研究所 | Method of producing xylosic alcohol and its special baterial strain |
| CN1966694B (en) * | 2006-11-15 | 2010-05-12 | 山东大学 | Process for producing alcohol by co-fermentation of glucose and xylose |
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| CN103396956A (en) * | 2013-08-17 | 2013-11-20 | 湖北邦之德牧业科技有限公司 | Saccharomyces cerevisiae, screening and culture methods thereof and bean meal fermentation method thereof |
| CN103396956B (en) * | 2013-08-17 | 2018-07-31 | 湖北邦之德牧业科技有限公司 | A kind of saccharomyces cerevisiae and its screening and culturing method and the method for bean pulp fermentation |
| CN105368732A (en) * | 2015-11-10 | 2016-03-02 | 四川大学 | Industrial saccharomyces cerevisiae strain capable of producing xylitol and construction method of industrial saccharomyces cerevisiae strain |
| CN105368732B (en) * | 2015-11-10 | 2019-01-04 | 四川大学 | One plant of an industrial strain of S.cerevisiae strain for producing xylitol and construction method |
| US10759727B2 (en) | 2016-02-19 | 2020-09-01 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
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| CN107384815A (en) * | 2017-08-03 | 2017-11-24 | 山东大学 | One Accharomyces cerevisiae bacterial strain and its application in comprehensive utilization xylose mother liquid and xylose residue production xylitol |
| TWI706037B (en) * | 2018-08-16 | 2020-10-01 | 行政院原子能委員會核能研究所 | A saccharomyces cerevisiae strain, a use thereof and a method for preparing xylitiol by the saccharomyces cerevisiae strain |
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