CN103122323A - Quick-fermentation bread yeast strain and breeding method thereof - Google Patents
Quick-fermentation bread yeast strain and breeding method thereof Download PDFInfo
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- CN103122323A CN103122323A CN2012105063879A CN201210506387A CN103122323A CN 103122323 A CN103122323 A CN 103122323A CN 2012105063879 A CN2012105063879 A CN 2012105063879A CN 201210506387 A CN201210506387 A CN 201210506387A CN 103122323 A CN103122323 A CN 103122323A
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Abstract
The invention discloses a quick-fermentation bread yeast strain and a breeding method thereof. The bread yeast strain is Saccharomyces cerevisiae Sy-5-11L with the storage number of CGMCC No 5637. The fermentation capacity of the no-sugar dough is remarkably improved on the condition of not affecting other fermentation performances of the strain; and 950 mL of CO2 is generated one hour before the fermentation of the no-sugar dough. Compared with the parent strain, the fermentation capacity is improved by 22.58% and the repression degree of the glucose is reduced by 17.05%. The repression factor coding gene MIG1 with the repression action in the strain bread yeast CICC 31616 glucose is removed, and a strong promoter PGK1 is selected to over-express maltase coding gene MAL6S to achieve the breeding method of the bread yeast strain. The bread yeast strain obtained by the breeding method has no special requirements to the fermentation equipment and fermentation conditions, and the equipment and the conditions of a general factor can be applicable, and so the bread yeast strain has an extensive application prospect.
Description
[technical field]:
The invention belongs to technical field of bioengineering, relate to the breeding of industrial microorganism, especially strain accelerated fermentation baker's yeast bacterial classification and a selection thereof.
[background technology]:
Steamed bun, steamed twisted roll etc. are Chinese most typical bread dough steamed wheaten foods, and there is the population of half left and right in China take wheaten food as main, and are mostly the plain doughs such as steamed bun fermentation class wheaten food, and the north approximately has 70% whole meal flour to be used for making steamed bun according to statistics.The essence of panary fermentation is under the effect of various enzymes, starch is changed into fermentability monose and disaccharide, wherein be mainly maltose, then change into carbonic acid gas through the fermentative action of yeast, make dough expand and generate other fermented material, make bread increase the process of local flavor, dough maturity.
In plain doughs, maltose is main fermentable sugars, so the metabolic capacity of maltose affects aerogenesis speed and the fermentation capacity of bread yeast.For common bread yeast, glucose is the preferential carbon source of utilizing of yeast, under glucose exists, utilizes the needed enzyme of maltose to be suppressed, and this phenomenon is called as carbon source and suppresses or glucose repression.Therefore, in plain doughs, the inspiration speed of common bread yeast is slower.
The MIGI transcription repression factor M ig1 that encodes, Mig1 is one and contains two C
2H
2The albumen of zinc fingers can combine with the polygenic promotor of being permitted that is subjected to glucose repression, exercises its transcription repression effect.In order to reduce the reptation behavior of glucose, accelerate bread yeast to the speed of utilizing of maltose, knocking out the MIGI gene will be an effective way.Yeast utilizes maltose to need three genoid products: maltose permease (by the MALxT genes encoding) (x represents among five unconnected MAL sites), maltin (by the MALxS genes encoding), and positive regulator (by the MAlxR genes encoding).This laboratory early-stage Study result proves, the key enzyme in the maltose metabolic process is maltin.Therefore, utilize ability in order to improve bread yeast maltose, can realize by crossing the work of expression maltin encoding gene raising bread yeast maltin enzyme.
[summary of the invention]:
The purpose of this invention is to provide strain accelerated fermentation baker's yeast bacterial classification and a selection thereof.
Bread yeast bacterial classification provided by the invention is the bread yeast bacterial strain with quick fermentation performance, is specially yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) SY-5-11L.This bacterium is preserved in Chinese microbial preservation management committee's common micro-organisms center on December 22nd, 2011, and (be called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No 5637.
Described bread yeast bacterial strain is compared with parent strain in the impregnable situation of other leavening property, and per hour aerogenesis increases 175mL when plain doughs ferments, and fermentation time shortens 15min, and the glucose repression degree has reduced by 17.05%.
Described bread yeast bacterial strain specifically can be by knocking out starting strain yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC31616 (Chinese industrial microbial strains preservation administrative center, the public can obtain starting strain CICC31616 by this preservation administrative center) the part or all of sequence of repressor encoding gene MIGI in resistance to glucose, and select simultaneously strong promoter PGK1 to cross expression maltin encoding gene MAL6S and realize.
Above-mentioned knock out and cross express and can obtain with conventional method.These methods have many bibliographical informations, as JosephSambrook etc., " molecular cloning experiment guide " second edition, Science Press, 1995.Also can build transgenation and cross the yeast of expressing with other method known in the art.This by knock out with genome on the bacterial strain that obtains of integrative gene expression gene be difficult for producing reverse mutation and expressing gene is lost, the stable Billy of bacterial strain is higher with the strain stability that the methods such as free expression on point mutation and plasmid build, and more is conducive to industrial applications.
The method of the above-mentioned bread yeast bacterial strain of structure provided by the present invention is, the recombination box that the pcr amplification recombinant plasmid is obtained imports in the bread yeast starting strain and obtains recombinant bacterium by homologous recombination.
Described recombinant plasmid pUC-PMKAB, to knock out the glucose repression factor code gene of bread yeast fully and cross simultaneously the recombinant plasmid of expressing the maltin encoding gene, described glucose repression factor code gene is the MIGI gene, and the maltin encoding gene is the MAL6S gene.
On described recombinant plasmid without any auxotroph selection markers.
The Gene ID of described glucose repression factor code gene MIG1 is: 852848; The GI of maltin encoding gene MAL6S is: 171888.
The present invention provides a kind of gene order of identifying described accelerated fermentation baker's yeast bacterial strain that is specifically designed to simultaneously, this gene order is the fragment take described quick bread yeast strain gene group as template amplification with Y1-U/Y1-D and two pairs of primers of Y2-U/Y2-D respectively, and its sequence is as shown in sequence table 1 and sequence table 2.
Advantage of the present invention and positively effect:
The one, the glucose repression factor code gene that the bread yeast bacterial strain of seed selection of the present invention knocks out is 100% disappearance, be difficult for producing reverse mutation, cross simultaneously and express coding maltin gene by integrating realization on genome, be difficult for losing in strain passage, and the KanMX resistant gene in recombinant bacterial strain knocks out, and has guaranteed the security of bacterial strain and leavened prod; The 2nd, the bread yeast bacterial strain of seed selection of the present invention not only partial solution except the checking of glucose, and has improved maltose and has utilized ability, can accelerate the speed of sending out that rises of plain doughs, and other leavening property does not have considerable change.The engineering bacteria that screening obtains does not have particular requirement to fermentation equipment and condition, and equipment and the condition of general factory all can be used, thereby have wide practical use.
[description of drawings]:
Fig. 1 is the Technology Roadmap that recombinant bacterial strain builds.
Fig. 2 is pUC-PMKAB plasmid construction process.
Fig. 3 is the regrouping process of plasmid.
Accelerated fermentation baker's yeast of the present invention is specially yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) SY-5-11L, be preserved in Chinese microbial preservation management committee's common micro-organisms center (abbreviation CGMCC on December 22nd, 2011, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No 5637.
[embodiment]:
Method in following embodiment if no special instructions, is ordinary method.
Embodiment 1: the seed selection of accelerated fermentation baker's yeast bacterial strain
(1) structure of recombinant bacterial strain
The structure flow process of recombinant bacterial strain as shown in Figure 1.Amphiploid starting strain CICC31616 generates a type and α type haploid strains 70a and 17 α through haploidization, method by twice homologous recombination builds the restructuring haploid strains, remove the KanMX resistant gene and hybridize this haploid strains, generating amphiploid recombinant bacterial strain SY-5-11L.
The structure flow process of recombinant plasmid pUC-PMKAB as shown in Figure 2.By the pcr amplification technology, MIG1 gene both sides are used for that homologous recombination knocks out the sequence MA of this gene and MB fragment and from the KanMX resistant gene amplification of pUG6 plasmid, and take the pUC19 plasmid as carrier with these three fragments according to being linked in sequence of MA-KanMX-MB, obtain the pUC-MKAB plasmid; The MAL6S gene is connected with the pUC-PGK that contains strong promoter, and resulting PGKP-MAL6s-PGKT (PM) fragment and above-mentioned plasmid are linked in sequence according to MA-KanMX-PM-MB's, builds the pUC-PMKAB recombinant plasmid.
The PMKAB regrouping process as shown in Figure 3.Carry out pcr amplification take plasmid pUC-PMKAB as template, obtain MA-KanMX-PM-MB recombination box.Transform the recombination box by Lithium Acetate and enter the yeast haploid cell, the homologous sequence homologous recombination by MIG1 gene both sides on MA and MB fragment and yeast chromosomal copies thereby be incorporated on yeast chromosomal and with karyomit(e).After transforming, by G418 resistance screening recon, KanMX-PM fragment homologous recombination has been replaced the MIG1 gene on the yeast chromosomal, thereby realizes that knocking out fully of MIG1 gene utilize strong promoter PGK1 to cross expression MAL6S simultaneously.
Utilize the method for homologous recombination to knock out respectively a type of bread yeast CICC31616 and the glucose repression factor code gene MIG1 of α type haploid strains 70a and 17 α, and cross simultaneously expression maltin encoding gene MAL6S, obtain a type and α type monoploid recombinant bacterial strain Δ M::M-a and Δ M::M-α.Resistant gene KanMX in the monoploid recombinant bacterial strain is removed, and hybridization obtains amphiploid recombinant bacterial strain SY-5-11L.
(2) specific sequence of engineering strain
Contain two sections specific sequences in the engineering strain SY-5-11L karyomit(e) that obtains, carry out identification of strains after can checking order by pcr amplification.
The primer sequence of specific fragment amplification is:
Y1-U：5’-GCATTGTGGAGGAAAGTAAGTG-3’
Y1-D：5’-ACGCTACCTTTGCCATGTTTCA-3’
Y2-U：5’-ATTTCTATGTTCGGGTTC-3’
Y2-D：5’-CAACCAACCACCTTAACT-3’
The gene order of two sections specific fragments is seen sequence table 1 and sequence table 2.
Embodiment 2: the experiment of accelerated fermentation baker's yeast strain fermentation
(1) transformant and monoploid parent's fermenting experiment
The restructuring monoploid of monoploid parent 70a and 17 α and correspondence thereof is carried out plain doughs fermentation and simulation panary fermentation simultaneously, measure the strain fermentation performance and also calculate the glucose repression degree, the results are shown in Table 1.Result shows, 1h aerogenesis 750mL before the panary fermentation of parent 70a, and 80min fermentation ends, glucose repression degree are 54.88%; 1h aerogenesis 925mL before the haploid panary fermentation of its restructuring, 68min fermentation ends, glucose repression degree are 30.91%, the monoploid Δ M::M-a that namely recombinates than the panary fermentation of parent 70a before the 1h aerogenesis increase 175mL, fermentation time shortens 12min, and the glucose repression degree reduces by 23.97%.1h aerogenesis 925mL before the panary fermentation of parent 17 α, the 67min fermentation ends, the glucose repression degree is 49.81%, 1h aerogenesis 1050mL before the haploid panary fermentation of its restructuring, 57min fermentation ends, glucose repression degree are 29.25%, the monoploid Δ M::M-α that namely recombinates than the panary fermentation of parent 17 α before the 1h aerogenesis increase 125mL, fermentation time shortens 10min, and the glucose repression degree reduces by 20.56%.Found out by result, the MIG1 gene lacks the combination of expressing Mig1 albumen capable of blocking and maltin promotor with the strong promoter of maltin fully, and then weaken to a certain extent resistance to glucose, and the speed of utilizing of maltose has been accelerated in the expression of crossing of maltin, thereby has saved the panary fermentation time.
Table 1 haploid strains Dough fermentation ability and glucose repression degree
Annotate: shown in data be the mean value of three parallel test results.
(2) amphiploid recombinant bacterium and amphiploid parent's fermenting experiment
With monoploid recombinant bacterial strain Δ M::M-a and Δ M::M-α hybridization, obtain amphiploid recombinant bacterial strain SY-5-11L.Amphiploid recombinant bacterial strain and amphiploid parent are carried out plain doughs fermentation and simulation panary fermentation, measure the leavening property of bacterial strain and calculate the glucose repression degree, the results are shown in Table 2.As shown in Table 2,1h aerogenesis 775mL before the fermentation of parent CICC31616 plain dough, 80min finishes fermentation, the glucose repression degree is 55.87%, 1h aerogenesis 950mL before the plain dough fermentation of amphiploid recombinant bacterial strain, and 65min finishes fermentation, the glucose repression degree is 38.82%, be the amphiploid recombinant bacterial strain than 1h fecund gas 175mL before the plain dough fermentation of amphiploid parent BY-14, fermentation time shortens 15min, and the glucose repression degree has reduced by 17.05%.
Table 2 amphiploid bacterial strain Dough fermentation ability and glucose repression degree
Annotate: shown in data be the mean value of three parallel test results.
Claims (2)
1. a strain accelerated fermentation baker's yeast bacterial classification, be specially Saccharomyces cerevisiae SY-5-11L, and preserving number is CGMCC No 5637.
2. the selection of an accelerated fermentation baker's yeast bacterial classification claimed in claim 1, it is characterized in that, by repressor encoding gene MIGI in the disappearance resistance to glucose, cross simultaneously expression maltin encoding gene MAL6S, obtain accelerated fermentation baker's yeast bacterial classification claimed in claim 1.
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| CN104073449A (en) * | 2014-07-14 | 2014-10-01 | 天津科技大学 | Fast-fermenting bread yeast strain and construction method thereof |
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| CN102199556A (en) * | 2011-04-15 | 2011-09-28 | 天津科技大学 | Saccharomyces cerevisiae genetic engineering bacteria with high ester yield and construction method thereof |
| CN102604849A (en) * | 2012-03-23 | 2012-07-25 | 天津科技大学 | Saccharomyces cerevisiae engineering bacterial strain capable of efficiently using lactose to produce fuel ethanol |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102199556A (en) * | 2011-04-15 | 2011-09-28 | 天津科技大学 | Saccharomyces cerevisiae genetic engineering bacteria with high ester yield and construction method thereof |
| CN102604849A (en) * | 2012-03-23 | 2012-07-25 | 天津科技大学 | Saccharomyces cerevisiae engineering bacterial strain capable of efficiently using lactose to produce fuel ethanol |
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| 《J Ind Microbiol Biotechnol》 20120606 Xi Sun et al Enhanced leavening properties of baker's yeast overexpressing MAL62 with deletion of MIG1 in lean dough 材料与方法部分、表1、表3、图1-3 1-2 第39卷, * |
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| XI SUN ET AL: "Enhanced leavening properties of baker’s yeast overexpressing MAL62 with deletion of MIG1 in lean dough", 《J IND MICROBIOL BIOTECHNOL》, vol. 39, 6 June 2012 (2012-06-06) * |
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| CN104073449A (en) * | 2014-07-14 | 2014-10-01 | 天津科技大学 | Fast-fermenting bread yeast strain and construction method thereof |
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