CN101914461B - Low-yield higher-alcohol saccharomyces cerevisiae engineering bacterium and construction method thereof - Google Patents
Low-yield higher-alcohol saccharomyces cerevisiae engineering bacterium and construction method thereof Download PDFInfo
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Abstract
The invention discloses low-yield high-alcohol saccharomyces cerevisiae engineering bacterium and a construction method thereof. The saccharomyces cerevisiae AY-20 engineering bacterium has the preservation number of CGMCC No.3930. Under the condition that other fermenting performances are not influenced, the content of the converted sub strain is respectively reduced by 55.19 percent and 34.43 percent compared with isobutanol and isoamyl alcohol of a parent strain, and the content of total higher alcohol is reduced by 35.01 percent. The invention can be realized by inserting a nucleotide sequence into start bacterium of saccharomyces cerevisiae 2.1190 or deleting partial or complete amino acid transaminase coding gene (BAT2). If the BAT2 is deleted by 100 percent, the back mutation is not easy to generate and a KanMX resistance gene in a recombinant strain is eliminated so as to ensure the safety of the strain and fermenting products. The screened engineering bacterium has no special requirements on the fermenting equipments and conditions and can be used by equipments and conditions in a general wine factory, so the invention has wide application potentials and can bring remarkable economic benefit for industrial production of a while spirit factory.
Description
[technical field]:
The invention belongs to technical field of bioengineering, relate to the breeding of industrial microorganism, especially a kind of saccharomyces cerevisiae engineered yeast of low yield higher alcohols and construction process thereof.
[background technology]:
Liquor is Chinese distinctive liquor, and unique flavor is liked deeply.Higher alcohols is one of staple of liquor flavor material, it to the local flavor that forms wine with impel that the wine body is plentiful, dense to play an important role.The soft coordination of mouthfeel, the wine body that suitable higher alcohols content and the eurythmy between the various higher alcohols can make liquor plentiful mellow and full, taste is unique; If but the liquor higher alcohols content is lower, it is dull thin that vinosity will become; And when too high levels, can lack of proper care with component proportionss such as acid, esters, make liquor produce different assorted flavor.In addition, higher alcohols is harmful material, and is all strong than ethanol to people's toxicity and anesthetic action.Therefore, the content that must control higher alcohols is in suitable scope.
China is higher with the liquor higher alcohols content that the fermenting raw materials of richs in protein such as corn is produced at present, and the higher alcohols total content is usually more than 300mg/L (or 300mg/kg, in isopropylcarbinol and primary isoamyl alcohol) after the fermentation ends.Except that in still-process, adopting the suitable operation of leaving out the beginning and the end, great majority are the growing amounts that reduce higher alcohols through the control zymotechnique aborning, and higher alcohols content is not higher than the hygienic standard of 200mg/L when guaranteeing that reaching liquor dispatches from the factory.Though many RRs about the middle-and-high-ranking pure growing amount of reduction fermenting process are arranged both at home and abroad, and these reports are laboratory result, also have with a certain distance from practical application.The subject matter that exists has: the one, and the method for control production technique reduces the growing amount of higher alcohols; Because the growth of every batch of bacterial classification and fermentation state are different; Will adjust zymotechnique accordingly; Therefore, if there is not the very abundant and employee conscientious of experience, this red-tape operati is difficult to the requirement that reaches predetermined; The 2nd, though obtained the bacterial strain (higher alcohols content can reduce the bacterial strain about 30%) of some low yield higher alcoholss, these bacterial strains are normally auxotrophic, only are applicable to laboratory study to be unfavorable for suitability for industrialized production.At present, it is the first that China's liquor YO occupies the whole world, exceeded 7,000,000 tons, accounts for 40% of world's ardent spirit ultimate production.But the middle-and-high-ranking pure growing amount of fermenting process is high, has increased the weight of the production cost of enterprise, has restricted the development of enterprise.Therefore, it is higher fundamentally to solve higher alcohols content, still will make up the low-yield higher-alcohol saccharomyces cerevisiae engineering bacterium strain, and this is the key point that reduces the middle-and-high-ranking pure growing amount of ethanol produce process, also is the developing direction of industrial micro breeding.
In the yeast saccharomyces cerevisiae alcoholic fermentation process, two higher alcohols pathways metabolisms are arranged, be respectively carbohydrate metabolism route of synthesis and amino acid catabolic pathway (Ehrlich approach).Adopting the molecular breeding technology to weaken or suppresses regulation and control higher alcohols synthetic key enzyme activity, thereby cut off or weaken the higher alcohols pathways metabolism, is reduction higher alcohols growing amount effective measures.The catabolic the first step of yeast saccharomyces cerevisiae branched-chain amino acid is transamination, and it is to be regulated and control by the amino acid transaminase of BAT1 and BAT2 genes encoding, wherein, and BAT1 genes encoding plastosome transaminase, BAT2 genes encoding tenuigenin transaminase.Show that according to the research report branched-chain amino acid transaminase of BAT2 genes encoding plays important effect in the generative process of higher alcohols (particularly isopropylcarbinol and primary isoamyl alcohol).Therefore, in order to reduce the growing amount of the middle-and-high-ranking alcohol of fermenting process, knock out that higher alcohols is had the amino acid transaminase encoding sox (BAT2) of material impact will be a valid approach.
[summary of the invention]:
The objective of the invention is to solve fermentative prodn liquor process higher alcohols content problem of higher, a kind of low-yield higher-alcohol saccharomyces cerevisiae engineering bacterium and construction process thereof are provided.
Saccharomyces cerevisiae engineered yeast provided by the invention is the saccharomyces cerevisiae engineered yeast (Saccharomyces cerevisiae) with low yield higher alcohols performance, is specially AY-20.This bacterium has been preserved in Chinese microbial preservation common micro-organisms center (the abbreviation CGMCC of management committee on June 21st, 2010; The address is: No. 3, Da Tun road, Chaoyang District, BeiJing, China city first); Preserving number is CGMCC No 3930, suggestion classification called after Saccharomyces Cerevisiae in S accharomyces cerevisiae.
Said engineering bacteria is under the impregnable situation of other leavening property, and the transformant bacterial strain has reduced by 55.19%, 34.43% respectively than isopropylcarbinol, the primary isoamyl alcohol content of parent strain, and total higher alcohols content has reduced by 35.01%.
Said saccharomyces cerevisiae engineered yeast specifically can be through to the bacterium yeast saccharomyces cerevisiae that sets out (Saccharomyces cerevisiae) 2.1190 (China Committee for Culture Collection of Microorganisms, the public can obtain starting strain 2.1190 through this preservation management committee).Nucleotide sequence of middle insertion is perhaps realized through partial or complete amino acid transaminase encoding sox (BAT2) sequence in the disappearance yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 2.1190.
Above-mentioned insertion or disappearance etc. can use conventional knockout technique to obtain.These methods have many bibliographical informations, like JosephSambrook etc., " molecular cloning experiment guide " second edition, Science Press, 1995.Also available other method known in the art makes up the yeast of transgenation.Wherein more excellent is to obtain through knocking out the amino acid transaminase gene fully.This sequence is the encoding sequence of disappearance BAT2 100%.This through knocking out the difficult generation of the bacterial strain reverse mutation that the amino acid transaminase encoding sox obtains fully, The stability of strain Billy is higher with the strain stability that methods such as point mutation make up, and more helps industrial applications.
The method of the above-mentioned saccharomyces cerevisiae engineered yeast of structure provided by the present invention is that the recombination box that the pcr amplification recombinant plasmid is obtained imports in the saccharomyces cerevisiae engineered yeast through homologous recombination acquisition reorganization bacterium.
Described recombinant plasmid pUC-BBAK is the recombinant plasmid that can knock out the amino acid transaminase encoding sox of yeast saccharomyces cerevisiae fully, and said amino acid transaminase encoding sox is the BAT2 gene.
No any auxotrophy selection markers on the said recombinant plasmid.
The sequence number of said amino acid transaminase encoding sox (BAT2) is: 853613.
The present invention provides a kind of gene order that is specifically designed to the saccharomyces cerevisiae engineered yeast of identifying said low yield higher alcohols simultaneously; This gene order is to be primer with PK-U and PK-D; Saccharomyces cerevisiae engineered yeast strain gene group with said low yield higher alcohols is a template; The amplified fragments order-checking is a specific sequence, shown in sequence table 1.
Advantage of the present invention and positively effect:
The one, the yeast amino acid transaminase encoding sox that the recombinant Saccharomyces cerevisiae engineering bacteria that the present invention makes up is knocked out is 100% disappearance; Be difficult for producing reverse mutation; And the KanMX resistant gene in the recombinant bacterial strain knocks out, and has guaranteed the security of bacterial strain and leavened prod; The 2nd, the recombinant Saccharomyces cerevisiae engineering bacteria that the present invention makes up can reduce the growing amount of higher alcohols, 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 the equipment and the condition of general brewery all can be used, thereby have wide practical use.
[description of drawings]:
Fig. 1 is the technological line figure that engineering bacteria makes up
Fig. 2 is a pUC-BBAK plasmid construction process
Fig. 3 is the regrouping process of BAT2.
Recombinant Saccharomyces cerevisiae engineering bacteria of the present invention (Saccharomyces cerevisiae) is specially AY-20; Be preserved in Chinese microbial preservation common micro-organisms center (the abbreviation CGMCC of management committee on June 21st, 2010; The address is: No. 3, Da Tun road, Chaoyang District, BeiJing, China city first); Preserving number is CGMCC No 3930, suggestion classification called after Saccharomyces Cerevisiae in S accharomyces cerevisiae.
[embodiment]:
Method among the following embodiment if no special instructions, is ordinary method.
Embodiment 1: the structure of low-yield higher-alcohol saccharomyces cerevisiae engineering bacterium
(1) structure of engineering strain
The structure flow process of engineering strain is as shown in Figure 1.Amphiploid 2.1190 generates haploid strains a-8 and α-22 through haploidization, through the method structure genetically engineered haploid strains of twice homologous recombination, removes the KanMX resistant gene and also hybridizes this haploid strains, generates the amphiploid engineering strain.
The structure flow process of recombinant plasmid pUC-BBAK is as shown in Figure 2.Through the pcr amplification technology; BAT2 gene both sides are used for that homologous recombination knocks out sequence B A and the BB fragment of this gene and from the KanMX resistant gene amplification of pUG6 plasmid; And be carrier with these three fragments according to being linked in sequence of BA-KanMX-BB with the pUC19 plasmid, make up the pUC-BBAK recombinant plasmid.
The BAT2 regrouping process is as shown in Figure 3.With plasmid pUC-BBAK is that template is carried out pcr amplification, obtains BA-KanMX-BB recombination box.Electricity transforms the recombination box and gets into the yeast haploid cell, through the homologous sequence homologous recombination of BAT2 gene both sides on BA and BB fragment and the yeast chromosomal, duplicates thereby be incorporated on the yeast chromosomal and with karyomit(e).Transform the back through G418 resistance screening recon, KanMX fragment homologous recombination has been replaced the BAT2 gene on the yeast chromosomal, thereby realizes knocking out fully of this gene.
Utilize the method for twice homologous recombination to knock out the haploid strains a-8 of yeast saccharomyces cerevisiae 2.1190 and the amino acid transaminase gene of α-22 (BAT2 gene) fully, obtain B-a and B-α bacterial strain.Resistant gene in the transformant (KanMX) is removed, and hybridization obtains amphiploid heterozygote AY-20.
(2) specific sequence of engineering strain
Contain one section specific sequence in the engineering strain AY-20 karyomit(e) that obtains, can be through carrying out identification of strains after the pcr amplification order-checking.
The primer sequence of specific fragment amplification is:
PK-U：5’-CTTCGTATAATGTATGCTATACGAA-3’
PK-D：5’-TTCTGGATACTTTTTTCTTACTTGC-3’
The gene order of this specific fragment is seen sequence table 1.
Embodiment 2: saccharomyces cerevisiae engineered yeast strain fermenting experiment
(1) transformant and monoploid parent's fermenting experiment
Parent a-8 and α-22 and corresponding BAT2 gene thereof are lacked transformant fully carry out alcohol thick mash fermentation simultaneously, measure strain fermentation performance and higher alcohols growing amount after the fermentation ends, the result sees table 1.The result shows; The isopropylcarbinol of parent a-8, primary isoamyl alcohol and higher alcohols content are respectively 70.37mg/L and 208.33mg/L and 312.93mg/L; Isopropylcarbinol, primary isoamyl alcohol and higher alcohols total content that its BAT2 gene lacks transformant fully are respectively 35.37mg/L, 137.62mg/L and 204.13mg/L, and promptly the a-8 transformant has reduced by 49.74%, 33.94% and 34.77% respectively than isopropylcarbinol, primary isoamyl alcohol and the higher alcohols of parent a-8; The isopropylcarbinol of parent α-22, primary isoamyl alcohol and higher alcohols content are respectively 63.16mg/L, 221.09mg/L and 320.12mg/L; Isopropylcarbinol, primary isoamyl alcohol and higher alcohols total content that its BAT2 gene lacks transformant fully are respectively 29.52mg/L, 143.29mg/L and 206.92mg/L, and promptly the transformant of α-22 has reduced by 53.26%, 35.19% and 35.36% respectively than isopropylcarbinol, primary isoamyl alcohol and the higher alcohols of α-22.Find out by the result; The BAT2 gene lacks blocking-up fully or has seriously weakened the activity of branched-chain amino acid transaminase; And then reduce Xie Ansuan and reduce higher alcohols to the conversion of primary isoamyl alcohol to isopropylcarbinol and leucine; It is about 52% that the growing amount of isopropylcarbinol has been reduced, and it is about 34% that the growing amount of primary isoamyl alcohol has reduced, and the higher alcohols total content has reduced about 35%.
Table 1 bacterial strain higher alcohols growing amount and leavening property
Annotate: shown in data be three parallel test results' MV.
(2) heterozygote and amphiploid parent's fermenting experiment
The transformant B-a of a-8 and the transformant B-α of α-22 are hybridized, obtain hybridizing sub-AY-20.Heterozygote and amphiploid parent 2.1190 are carried out alcohol thick mash fermentation, measure the leavening property and the higher alcohols growing amount of bacterial strain after the fermentation ends, the result sees table 2.Can know by table 2; Parent 2.1190 isopropylcarbinol, primary isoamyl alcohol and higher alcohols total content are respectively 72.66mg/L and 213.92mg/L and 317.62mg/L; The isopropylcarbinol of heterozygote, primary isoamyl alcohol and higher alcohols total content are respectively 32.56mg/L, 140.26mg/L and 206.44mg/L, and promptly heterozygote has reduced by 55.19%, 34.43% and 35.01% respectively than amphiploid parent 2.1190 isopropylcarbinol, primary isoamyl alcohol and higher alcohols total content.
The leavening property of table 2 hybridization and the growing amount of higher alcohols
Annotate: shown in data be three parallel test results' MV.
Claims (2)
1. the yeast saccharomyces cerevisiae of a low yield higher alcohols (Saccharomyces cerevisiae) engineering bacteria is specially AY-20, and preserving number is CGMCC No 3930, suggestion classification called after Saccharomyces Cerevisiae in S accharomyces cerevisiae.
2. the construction process of the saccharomyces cerevisiae engineered yeast of the described low yield higher alcohols of claim 1; It is characterized in that; Through knocking out the amino acid transaminase encoding sox in the yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 2.1190 fully; Obtain the saccharomyces cerevisiae engineered yeast of the described low yield higher alcohols of claim 1, this bacterial strain is the encoding sequence of disappearance BAT2 100%.
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| CN105385615A (en) * | 2015-12-28 | 2016-03-09 | 天津科技大学 | Saccharomyces cerevisiae strain with high yield of ester and low yield of higher alcohol as well as building and application of saccharomyces cerevisiae strain |
| CN106834148B (en) * | 2017-01-20 | 2020-03-03 | 吉林大学 | Beer yeast capable of reducing content of higher alcohol and application thereof |
| CN107858368A (en) * | 2017-06-30 | 2018-03-30 | 天津科技大学 | The Wine brewing yeast strain and its construction method of one plant of appropriate production higher alcohol |
| CN109136254B (en) * | 2018-07-10 | 2021-06-04 | 天津科技大学 | Efficient saccharomyces cerevisiae traceless gene knockout method and application thereof |
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| CN101696392A (en) * | 2009-10-30 | 2010-04-21 | 湖南农业大学 | Saccharomyces cerevisiae strain and application in preparation of purple yarn yellow wine |
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